Nitric Oxide in Vascular Damage and Regeneration

Nitric Oxide in Vascular Damage and Regeneration

A. Nitric Oxide and Cardiovascular Function Chapter 20 Nitric Oxide in Vascular Damage and Regeneration Claudio Napoli,1 Lilach O. Lerman,2 Maria Lu...

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A. Nitric Oxide and Cardiovascular Function Chapter

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Nitric Oxide in Vascular Damage and Regeneration Claudio Napoli,1 Lilach O. Lerman,2 Maria Luisa Balestrieri,1 and Louis J. Ignarro3 Department of General Pathology, Excellence Research Center on Cardiovascular Disease and Division of Clinical Pathology, and Department of Biochemistry and Biophysics, 1st School of Medicine, II University of Naples, Naples, Italy 2 Department of Internal Medicine, Divisions of Nephrology and Hypertension, Mayo College School of Medicine Mayo Clinic, Rochester, Minnesota, USA 3 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, USA

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Summary Nitric oxide (NO) controls vasorelaxation, endothelial regeneration, inhibition of platelet adhesion, and leukocyte chemotaxis. NO deficiency, critical in the development of atherosclerosis and renovascular diseases, occurs through reduced expression and activity of NO synthase, decreased levels or impaired utilization of L-arginine, and enhanced degradation of NO by oxidation-sensitive mechanisms. Genetic manipulation of NO synthase provides important insights into the pathogenic pathway of vascular diseases. Results from pre-­clinical and clinical studies suggest that modulation of oxidation-sensitive mechanisms and augmentation of NO production through the administration of L-arginine and antioxidants improve the neovascularization following bone marrow cell therapy or gene therapy. Moreover, nitrite infusion represents a promising NOgenerating approach which offers the potential to modulate vascular function during ischemia. Here, molecular mechanisms of NO signaling in vascular damage are presented with emphasis on the promising findings in the field of NO and vascular regeneration. Key words: nitric oxide, nitric oxide synthase, cardiovascular diseases, renovascular diseases, L-arginine.

Nitric oxide signaling in the artery wall Nitric oxide (NO) acts as a key signaling messenger in the cardiovascular system (Ignarro et al., 1999). Adequate levels of this gaseous molecule are important to preserve normal ­vascular physiology. Established regulatory functions are controlled by NO (Napoli and Ignarro, 2001; Napoli et al., 2006; Rabelink and Luscher, 2006). Indeed, NO participates in

Nitric Oxide: Biology and Pathobiology, second edition Copyright © 2009 2010 by Academic Press. Inc. All rights of reproduction in any form reserved.

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the control of hemostasis, fibrinolysis, presentation of histocompatibility antigens, vascular tone and proliferation of vascular smooth muscle cells (VSMC), homeostasis of blood ­pressure, and interaction of leukocytes and platelets with the arterial wall (Napoli et al., 2006; Napoli and Ignarro, 2001; Rabelink and Luscher, 2006). Diminished NO bioavailability and abnormalities in NO-dependent signaling are among the central factors of vascular disease, although it is unclear whether they are a cause of or a result of endothelial dysfunction or, as is more likely, both events. Altered NO bioavailability causes endothelial dysfunction, thus increasing susceptibility to atherosclerotic diseases, diabetes mellitus, hypertension, hypercholesterolemia, congestive heart failure, thrombosis, and stroke (Napoli and Ignarro, 2001; Napoli et al., 2006). NO is produced by a family of NO synthase (NOS) enzymes (Napoli and Ignarro, 2001; Napoli et al., 2006), of which three main isoforms, identified in human beings and other organisms, are expressed in different cell types (Napoli and Ignarro, 2001; Napoli et al., 2006) (Table 1): neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). nNOS, also known as NOS1, and eNOS (or NOS3) are constitutive enzymes regulated by calcium and calmodulin and by post-translational modifications. The third isoform, iNOS (or NOS2), regulated by cytokine stimulation, produces larger amounts of NO compared to the other two isoforms. The three NOS isoforms have similar enzymatic mechanisms that involve electron transfer for oxidation of the terminal guanidino nitrogen of L-arginine. These enzymes all require cofactors for proper function, including tetrahydrobiopterin (BH4), nicotinamide-adenine-­dinucleotide phosphate (NADPH), flavin

Table 1   Characteristics of NOS isoforms 630

eNOS

iNOS

nNOS

Other names

NOS-3, NOSIII, Type III NOS

NOS-2, NOSII, Type II NOS

NOS-1, NOSI, Type I NOS

Function

Vasodilation, Defense against modulation of pathogens, platelet aggregation, inflammation modulation of leukocyte–endothelial interactions

Signal transduction, neurotransmission, toxicity at high levels

Human chromosomal 7q35–7q36 location

17cen-q11.2

12q24.2-12q24.3

Human gene structure

26 exons

26 exons

29 exons

Site of expression

Endothelial cells, smooth muscle, liver

Macrophages, smooth muscle

Neurons, smooth muscle, skeletal muscle

Subcellular localization

Caveolae

Soluble

Neuromuscular junction, sarcoplasmic reticulum

Ca2 dependency

Ca2 dependent

Ca2 independent

Ca 2 dependent

Covalent modifications

Phosphorylation, myristoylation, palmitoylation

Phosphorylation

Protein–protein interaction

hsp90, caveolin

hsp90, caveolin

Chapter 20 NO in Vascular Damage and Regeneration

adenine dinucleotide (FAD), and flavin mononucleotide (FMN). Genetic disruption of each NOS isoform provided useful tools to complement other approaches investigating the multiple roles of NO in the cardiovascular system. Indeed, lack of eNOS causes hypertension, endothelial dysfunction, and a severe response to vascular injury, cerebral ischemia, and diet-induced atherosclerosis, whereas lack of the nNOS is linked to a less severe outcome in response to cerebral ischemia but increased diet-induced atherosclerosis (Liu et al., 2008). Finally, mice lacking the iNOS isoform show reduced hypotension to septic shock (Liu et al., 2008). The physiologic target of NO is soluble guanylate cyclase (Napoli and Ignarro, 2001; Napoli et al., 2006). NO activates guanylate cyclase by binding to its heme moiety, resulting in increased cGMP levels. In the vasculature, cGMP mediates NO-dependent relaxation of VSMC, resulting in vasodilation. Similarly, NO produced as a neurotransmitter in the gastrointestinal, urinary, and respiratory tract mediates smooth muscle relaxation by increasing cGMP production. These effects are likely mediated by the phosphorylation of downstream proteins by cGMP-dependent protein kinases, including myosin light chain (Napoli and Ignarro, 2001; Napoli et al., 2006). Another target for NO is sulfhydryl groups on proteins, to form nitrosothiol (SNOs) compounds. Findings aimed to define the mechanisms of NO-mediated protein modification, the identity and function of the modified proteins, and the significance of these changes in vascular disease have been extensively reviewed (Handy and Loscalzo, 2006; Liu et al., 2008; Napoli and Ignarro, 2001; Napoli et al., 2006). Alterations in endogenous S-nitrosylated proteins that include eNOS, beta-actin, vinculin, diacylglycerol kinase-alpha, GRP78, extracellular signal-regulated kinase 1, and transcription factor nuclear factor-kappaB (NF-B) may underlie the adverse effect of hyperglycemia on the vasculature, such as endothelial dysfunction and the development of diabetic vascular complications (Wadham et al., 2007). Interestingly, in vitro studies indicate that these changes can be completely reversed by inhibition of superoxide production, suggesting a key role for oxidative stress in the regulation of S-nitrosylation under hyperglycemic conditions (Wadham et al., 2007). NO and/or SNOs can prevent the loss of beta-adrenergic receptor signaling in vivo and regulation occurs through modulation of G-protein-coupled receptor kinase 2 (GRK2) (Whalen et al., 2007). Indeed, in both cells and tissues, GRK2 was S-nitrosylated by SNOs, and Cys340 of GRK2 has been identified as a principal locus of inhibition by S-nitrosylation (Whalen et al., 2007). Moreover, regulation of cytokineinduced S-nitrosylation of p65 by NOS2 delineates a novel mechanism by which NOS2 modulates NF-B activity and regulates gene expression in inflammation (Kelleher et al., 2007). Hemoglobin (Hb), which may serve as a natural carrier an SNO derivative (SNO-Hb), is formed in vivo and circulating concentrations are capable of dilating blood vessels. A number of novel reactions of NO, nitrite, and SNO that produce SNO-Hb in situ (Allen and Piantadosi, 2006; Doctor et al., 2005; Sonveaux et al., 2005) and result in release of NO bioactivity under hypoxic conditions (Liu et al., 2008; Rabelink and Luscher, 2006) have been described. Levels of SNO-Hb are altered in several disease states characterized by disorders in tissue oxygenation (Crawford et al., 2004; McMahon et al., 2005; Pawloski et al., 2005) and NO bioactivity is depleted in banked blood, impairing the vasodilatory response to hypoxia. These findings suggest that SNO-Hb repletion may improve transfusion efficacy. NO S-nitrosylated the ryanodine receptor on the sarcolemmal membrane and critical residues in the N-ethylmaleimide-sensitive factor, which is important to the regulation of exocytosis. Moreover, NO mediated regulation of endocytic vesicles suggests a mechanism by which the trafficking of receptors may be regulated, and suggests that cellular entry of pathogenic microbes and viruses is facilitated by S-nitrosylation of dynamin (Wang et al., 2006). Mechanisms underlying some of the toxicity of NO include reaction with superoxide anion to form peroxynitrite anion and activation of the enzyme poly-ADP ribose polymerase, resulting in depletion of cellular energy stores (Napoli and Ignarro, 2001; Napoli et al., 2006; Radovits et al., 2007). Pharmacological modulation of NO and combined therapy

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with antioxidants and L-arginine may restore endothelial dysfunction, inhibit oxidationsensitive mechanisms, reduce atherogenesis, and improve bone marrow cell-mediated vascular repair (Napoli et al., 2006; Napoli et al., 2007a,b). Some pharmacological agents exert direct beneficial effects on endothelium (Furchgott, 1999; Ignarro et al., 2002; Napoli and Ignarro, 2003), while others elicit their actions by improving the deleterious oxidation-sensitive mechanisms leading to vascular dysfunction and atherosclerosis (de Nigris et al., 2003; Napoli and Lerman, 2001). Here, we will update the field of NO in vascular damage and regeneration, describing novel scientific evidence from the latest genetic manipulation of the NOS isoform.

NO in vascular dysfunction An early event in the pathophysiology of atherosclerosis is the impairment of endothelial function. Diminished levels of bioavailable NO, one of the hallmarks of endothelial dysfunction, occur through several potential mechanisms, such as reduced eNOS expression levels, eNOS enzymatic activity, and NO bioavailability (Liu et al., 2008; Napoli and Ignarro, 2001; Napoli et al., 2006) (Fig. 1). Endothelial dysfunction is associated with an increase in ROS production in the vasculature, which occurs via activation of the NAD(P)H oxidase(s) in endothelial, vascular smooth muscle, or adventitial cells, or via the enzyme xanthine oxidase (Ginnan et al., 2008; Kagota et al., 2007; Napoli et al., 2006). Activation of the endothelial cell NADPH oxidase and formation of peroxynitrite during angiotensin II-induced mitochondrial dysfunction modulates endothelial NO and superoxide generation (Doughan et al., 2008).

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The angiotensin receptor antagonist, telmisartan, is able to prevent endothelial dysfunction by decreasing aortic levels of the protein expression of gp91, a component of NADPH oxidase, thus reducing the formation of peroxynitrite and the consequent decrease of active NO leading to attenuation of endothelium-dependent relaxation (Kagota et al., 2007). Increased reaction rates with superoxide determine a decrease in NO bioavailabilty and production of reactive nitrogen/oxygen species that induce protein nitration (Napoli and Ignarro, 2001; Napoli et al., 2006). Indeed, superoxide radical reacts with NO to form peroxynitrite. This compound possesses the same biologic activity as NO at very low concentrations. However, at high levels, peroxynitrite is toxic as it forms the cytotoxic peroxynitrous acid, as well as inducing protein modification by nitration of amino acids (Napoli and Ignarro, 2001; Napoli et al., 2006). Besides the key role of the decreased NO bioactivity in endothelial dysfunction and vascular damage, NOS itself is capable of producing reactive oxygen species (ROS) (Satoh et al., 2005) in the absence of substrate, L-arginine, or cofactors such as BH4. BH4, whose synthesis is rate limited by GTP cyclohydrolase, is a particularly important cofactor, because in its absence electron transport through eNOS can become ‘uncoupled’, resulting in generation of superoxide anion (Fig. 1) (Ignarro and Napoli, 2004; Satoh et al., 2005). Clinically, a decrease in bioavailable NO in the presence of coronary heart disease (CHD) risk factors is linked to an altered coronary vasodilator response to acetylcholine challenge (Napoli and Ignarro, 2001; Napoli et al., 2006;). Moreover, recent findings indicate that the local enhancement of oxidative stress during coronary endothelial dysfunction in humans plays a role in the reduction of NO bioavailability (Lavi et al., 2008; Schiffrin, 2008). Overall, changes in eNOS mRNA or protein expression levels may contribute to endothelial dysfunction. These conditions determine a reduced level of eNOS activity (Bechara et al., 2007), L-arginine and eNOS cofactor availability (FAD, FMN, NADPH, and BH4) (Cosentino et al., 1998), altered eNOS phosphorylation at S1179 (Dimmeler et al., 1999; Fulton et al., 1999) dimerization, and intracellular localization to caveolae mediated by caveolin and hsp90 (Shaul, 2002) (Fig. 1).

Chapter 20 NO in Vascular Damage and Regeneration

Figure 1 Mechanisms of eNOS regulation in vascular damage and regeneration. In vascular disease states such as diabetes, hypertension, and hypercholesterolemia, superoxide production by oxidases is markedly increased. Several mechanisms can account for endothelial dysfunction and vascular damage, including: (I) decreased substrate availability; (II) changes in eNOS mRNA or protein levels; (III) decreased cofactor availability; (IV) improper subcellular localization; (V) abnormal phosphorylation; (VI) scavenging of NO by superoxide (O2) to form peroxynitrite anion (ONOO); and (VII) KLF2 regulation. Peroxynitrite and other reactive oxygen species oxidize BH4, via the BH3 radical to BH2 and biopterin, which reduces the bioavailability of BH4 and promotes eNOS uncoupling. This form of eNOS no longer produces NO, but instead generates superoxide. KLF2 is inhibited by the inflammatory cytokine IL-1 and is inducted by laminar shear stress in endothelial cells. Overexpression of KLF2 strongly induces eNOS expression and may inhibit the proinflammatory cytokine-dependent induction of VCAM-1 and endothelial adhesion molecule E-selectin. L-arginine supplementation exerts a beneficial effect on endothelial NO production during vascular regeneration by enhancing the neovascularization capacity of autologous BMC transplantation and the number of EPC that can incorporate into the sites of vascular damage. Abbreviations: ADMA, asymmetric dimethylarginine; BH4, tetrahydrobiopterin; SOD, superoxide dismutase; PKG, protein kinase G; eNOS, endothelial nitric oxide synthase; TM, thrombomodulin; VCAM-1, vascular cell adhesion molecule; IL-1, interleukin-1; 1 KLF2, Krüppel-like transcription factor 2; BMC, bone marrow cell; EPC, endothelial progenitor cells.

NO and renovascular disease All three isoforms of NOS are expressed in the kidney, with two of them (eNOS and nNOS) expressed constitutively (Kone, 2004). As in most other organ systems, eNOS is expressed mainly in vascular endothelial cells, although some eNOS expression has also been detected in tubular segments like the inner medullary collecting duct or the thick ascending limb of the loop of Henle (Table 2). nNOS is predominantly expressed in the macula densa but is also reportedly expressed in specialized neurons within renal vessels and in several tubular segments. Protein expression of iNOS is mostly induced by proinflammatory stimuli, and is then expressed in infiltrating macrophages as well as VSMC, although constitutive expression of iNOS mRNA has been detected in the thick ascending limb of the loop of Henle. In line with the ubiquitous distribution of NOS in the kidney, NO plays a number of physiological roles in the regulation of renal function (Fig. 2). In the afferent and efferent arterioles

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Table 2   Relative distribution of NOS isoform expression in the normal kidney Kidney structure

NOS1

NOS2

NOS3

Macula densa







Intra-renal arteries

 (neurons)

/ (VSMC)

 (EC)

Intra-renal veins







Vasa recta







Mesangial cells



/

Proximal tubule

/





Inner medullary thin limb







Medullary thick ascending limb

/

*



  inner medullary







  outer medullary







  cortical







Collecting duct:

NOS1: neuronal NOS (nNOS); NOS2: inducible NOS (iNOS); NOS3: endothelial NOS (eNOS); EC: endothelial cells; VSMC: vascular smooth muscle cells. * only RNA of NOS2 expressed constitutively.

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NO participates in regulation of renal blood flow and glomerular filtration rate, balancing the effects of angiotensin II, and in conjunction with prostaglandins it modulates renal blood flow autoregulation. NO released by nNOS in the macula densa plays a critical role in modulating the tubuloglomerular feedback mechanism, which links distal sodium and solute delivery to changes in glomerular filtration rate. Importantly, NO regulates renal sodium and fluid reabsorption and excretion both by modulating tubular sodium transport and by mediating the pressure natriuresis mechanisms (Herrera and Garvin, 2005). In addition, NO also attenuates renal vasoconstriction induced by renal sympathetic nerves. Therefore, NO plays a pivotal role in regulating renal hemodynamics and function, and thereby extracellular volume and systemic blood pressure. Because of its central participation in regulation of renal function (Fig. 2), it is not surprising that a NO deficiency has been linked to various forms of renal disease (Baylis, 2008). Experimental evidence has shown that chronic inhibition of NOS leads to development of hypertension, renal ischemia, tubulointerstitial damage, and eventually glomerulosclerosis. Clinical studies have further demonstrated deficiency of NO in patients with chronic kidney disease or end-stage renal disease (Fliser et al., 2005). Similar to other vascular systems, decreases in NO bioavailability are associated with renal endothelial dysfunction, and hence loss of many protective activities of the endothelium on prevention of thrombosis, fibrosis, endothelial barrier function, platelet adherence, etc. In addition, down-regulation of nNOS interferes with the tubuloglomerular feedback mechanism. With little counterbalance for angiotensin II action renal ischemia may ensue, with a decrease in renal blood flow and progression of renal disease (Patzak and Persson, 2007). In contrast, iNOS expression seems to be up-regulated in various forms of renal disease, such as mesangial proliferative glomerulonephritis. In animal models of early renovascular disease, down-regulated eNOS

Chapter 20 NO in Vascular Damage and Regeneration

Figure 2 Regulation of renal hemodynamics and function by NO. NO derived from eNOS regulates renal blood flow and glomerular filtration rate by maintaining a balance with other autocoids to modulate the vascular tone of the afferent and efferent arterioles, and modulates the vascular tone of other vessels within the kidney. NO released by nNOS in the macula densa adjusts glomerular filtration rate to distal sodium delivery. In addition, NO regulates tubular sodium transport and offsets renal vasoconstriction induced by renal sympathetic nerves.

e­ xpression in intra-renal vessels was accompanied by up-regulated iNOS expression in both VSMC and renal tubules (Chade et al., 2002; Stulak et al., 2001). This altered expression is likely at least partly responsible for both endothelial dysfunction, reflected in impaired responses to vasodilators, as well as epithelial dysfunction, which exhibits as decreased sodium reabsorption and increased sodium excretion potentially linked to the increased iNOS expression in the renal tubules. Blood pressure in renovascular hypertension is also strongly modulated by NO, and revascularization has been shown to effectively improve endothelial function in these patients (Higashi et al., 2002). A pioneering study by Goldblatt et al. (1934) showed that obstruction of one renal artery leads to a decrease in renal blood flow in the obstructed kidney and an increase in systemic arterial pressure. Since then, clipping one renal artery has become a useful and accepted maneuver in order to manipulate renal perfusion on both the clipped and non-clipped kidney, and to study mechanisms responsible for regulating renal hemodynamics and function, as well as arterial hypertension. This model also provides an outstanding opportunity to examine the role of NO in regulation of renal function under different pathophysiological conditions. Indeed, an elegant series of seminal studies by Sigmon and Beierwaltes (1994a,b, 1998) have explored the role of NO in regulation of renal hemodynamics using a rat model of renal artery stenosis and infusion of the NO synthesis inhibitor N(G)-nitro L-arginine methyl ester (L-NAME). They have shown that in the clipped kidney the response to L-NAME is gradually diminished as the degree of stenosis progresses from mild to severe. In contrast, the non-clipped kidney shows exaggerated responses to NO synthesis ­inhibition,

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almost regardless of the degree of stenosis in the clipped kidney. When L-NAME was infused and renal perfusion pressure was controlled, renal blood flow fell in both kidneys, by 52% in the non-clipped kidney but only 15% in the clipped kidney. These observations suggest that NO maintains the perfusion of the non-clipped kidneys exposed to hypertension, while in the stenotic kidney its effect in maintaining renal perfusion diminishes as the relative stenosis becomes greater. The changes in the clipped kidney were attributed to the fact that the obstruction to the perfusion decreases shear stress and thereby the stimulus for NO synthesis, and allows angiotensin II-mediated renal vasoconstriction to predominate. Local shear stress in the clipped kidney was then perceived as the local regulator of NO synthesis. The systemic response of blood pressure to L-NAME was prominent, arguing against endothelial dysfunction in this model and possibly representing a compensatory response to the increase in total peripheral resistance caused by angiotensin II. These responses were observed both in the early phase of renovascular hypertension (4 weeks after clipping) as well as in the chronic phase (13–16 weeks after clipping). Tokuyama et al. (2002) subsequently demonstrated in a dog model that within 4 weeks of moderate unilateral renal artery stenosis NO contributes predominantly to the homeostasis of the renal circulation, while when the ischemia becomes severe, prostaglandins are major determinants of renal function and maintain renal hemodynamics (Tokuyama et al., 2002). Therefore, NO plays an important role in maintaining renal hemodynamics during renal artery stenosis; however, its role shifts as the stenosis progresses with prostaglandin assuming an important role in the clipped kidney.

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In humans with renal artery stenosis, Wierema et al. (2001) showed that NO was not available in stenotic kidneys with low-grade stenosis but was increased in non-stenotic kidneys, in which the level was comparable to that in patients with essential hypertension. Injection of N(G)-monomethyl L-arginine (L-NMMA) into the non-stenotic kidney induced a 40% decrease in renal blood flow, whereas injection of L-NMMA into the stenotic kidney did not induce a significant change in renal blood flow. In the non-stenotic kidney, a compensatory increase in NO-mediated vasodilatation appears to compensate for the decreased perfusion of the stenotic kidney in order to sustain overall renal function. In bilateral renal artery stenosis in patients, because of the lost ability of one kidney to compensate for the other, as both renal arteries became stenotic they showed increased dependence on NO and a marked vasoconstriction response to L-NMMA, suggesting higher availability of NO in both kidneys (Wierema et al., 2001). Notably, high level of angiotensin II correlated with greater NO synthase activity in stenotic kidneys, but inversely in the non-stenotic kidneys. These differences could be related to different levels of expression or density of angiotensin II receptors in both kidneys. Interestingly, the patients had mild-to-moderate degrees of stenosis and this effect was apparent even with low-grade stenosis. Therefore, in renovascular disease the compensatory mechanism for a decrease in renal perfusion is at least partly regulated by NO and angiotensin II. Because the non-stenotic kidney seems to be particularly dependent on NO for regulation of renal blood flow and for compensation for the decrease in renal blood flow of the stenotic kidney, it may be particularly vulnerable to conditions which are associated with a concurrent decrease in NO availability, such as hypercholesterolemia, pre-existing kidney disease, and prolonged hypertension. Atherosclerosis is often the underlying etiology and complicates renal artery stenosis in humans, and experimental models with both renal artery stenosis and atherosclerosis may closely mimic the human condition of atherosclerotic renal artery stenosis. Early atherosclerosis induces renal vascular dysfunction and decreased expression of eNOS, while the expression of iNOS increases (Stulak et al., 2001). Indeed, coexisting renal hypoperfusion and hypercholesterolemia, as surrogate for early atherosclerosis, have been shown to elicit in stenotic kidneys glomerular endothelial dysfunction, detected by the responses of glomerular filtration rate to the prototypical endothelium-dependent vasodilator acetylcholine (Chade et al., 2002). Such alterations in glomerular function reflect an early

Chapter 20 NO in Vascular Damage and Regeneration

pathophysiological mechanism that may accompany early tubular dysfunction in humans at risk for essential hypertension (O’Connor et al., 2001). Similarly, in the non-stenotic kidney concurrent hypercholesterolemia and hypertension also exacerbated renal endothelial dysfunction (Rodriguez-Porcel et al., 2001), suggesting that superimposed endothelial injury can disrupt the delicate balance achieved by the non-stenotic kidney and its attempt to maintain overall renal hemodynamic function and regulation of systemic blood pressure. This may also be the reason for the observation that, in contrast to early studies in rats, studies have shown that human renovascular hypertension does induce systemic endothelial dysfunction, which is reversible upon endovascular intervention (Higashi et al., 2002). In addition, due to the diverse expression of the different isoforms of NOS in the kidney, its participation in the pathophysiology of renovascular disease is complex. The renal tubules are distinctly vulnerable to hypoxia and inflammation. In contrast to the decrease in endothelial-derived NO, a marked increase in iNOS expression along the nephron characterizes experimental renovascular disease, and leads to decreases in tubular filtrate concentration capacity, a measure of intrinsic renal damage. A dysfunction of the tubules can be expressed in natriuretic responses and decreased fluid and sodium reabsorption. Therefore, the complex roles of NO in the kidney put it in a key position to regulate diverse aspects of renal hemodynamics and function, both in health and in disease.

Clinical measurements of endothelial function Assessment of endothelial function is a useful diagnostic and prognostic tool (Kasprzak et al., 2006). The pathophysiology of endothelial dysfunction includes abnormal vasomotor function, proinflammatory and prothrombotic state. A number of laboratory markers have been used as indicators of abnormal function of the endothelium, such as E-selectin, ICAM1, VCAM-1, CD40L, CRP, Il-1, TNF-, IFN-g, MCP-1, von Willebrand factor, t-PA, PAI-1, microalbuminuria, and tests of apoptosis (Agnoletti et al., 1999; Ochodnicky et al., 2006; Szmitko et al., 2003; Widlansky et al., 2003). The clinical utility of these measurements is limited due to their non-specific character. However, they offer a complement to the imaging assessment of endothelial function allowing insight into endothelial physiology. An augmented apoptotic rate is possibly involved in endothelial denudation and subsequent dysfunction. Proapoptotic activity of serum, i.e. evaluation of neurohormones, cytokines and second messengers, can be studied in endothelial cell lines in vitro (Kasprzak et al., 2006). Although findings cannot be fully extrapolated to in vivo clinical conditions, they show interaction between the bloodstream and human endothelium. NO, the most widely used clinical marker of endothelial function, is determined by measuring the vasomotor response to physiological and pharmacological stimuli. Quantitative coronary angiography after intraarterial infusion of acetylcholine is an in situ and relatively operator-independent test used for the assessment of endothelial function in coronary arteries. Possible ‘hot’ sites at risk of atherosclerosis are indicated by vasoconstriction or no changes in the arterial diameter after acetylcholine stimulation (Schachinger et al., 2000). Cardiovascular events can be independently predicted by assessment of coronary vasoreactivity using acetylcholine, cold pressor testing and increased blood flow as shown in 147 patients, of which 57% showed angiographic evidence of atherosclerosis (Schachinger et al., 2000). Angiographically smooth epicardial coronary arteries can be associated with endothelial dysfunction, which predicts a worse long-term outcome in a low-risk population (Halcox et al., 2002; Schindler et al., 2003). Indeed, a larger study of 308 patients monitored after intracoronary acetylcholine indicated that epicardial and microvascular coronary endothelial dysfunction independently predicted cardiovascular events in patients with and without CHD. Event-free survival was significantly related to endothelial function when only unpredictable acute cardiovascular events, such as death, myocardial infarction, stroke, and unstable angina, were analyzed (Halcox et al., 2002). Another large clinical study evaluated

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forearm blood flow in response to acetylcholine in 281 patients with angiographically documented CHD, and results indicated that patients who experienced cardiovascular events had a lower vasodilator response to acetylcholine (Heitzer et al., 2001). Non-invasive measurements of vascular disease are more promising methods for wide application in clinical practice. High-resolution ultrasound imaging of brachial artery flow-mediated dilation (FMD) is a non-invasive, sensitive, and reproducible ultrasonic measurement of endothelial function (Uehata et al., 1997), which has been shown to correlate with invasive testing of coronary artery endothelial function (Anderson et al., 1995; Takase et al., 1998). FMD has been most commonly studied in the brachial artery (Smith et al., 2000) and is widely used and accepted as a marker of endothelial function. As a safe and quick method, FMD can be conducted among children and young adults, especially if repeated measurements are required (Corretti et al., 2002). The occlusion-induced production of reactive hyperemia to promote endothelial NO-dependent vasodilation (i.e. flow-mediated) evaluates the shear stress mechanism of NO production. The primary hemodynamic determinant of FMD is the induced wall shear stress following transient ischemia (inflation and deflation of a sphygmomanometer cuff). The few technical and interpretive limitations include the not well defined normal value range, because of an inverse relation between the degree of vasodilation and baseline brachial diameter (Corretti et al., 2002). This may preclude comparison of vasodilator responses between individuals with different baseline diameters. Indeed, subjects with small-sized brachial arteries may have normal arterial dilation, even in the presence of endothelial dysfunction. On the other hand, patients with very large brachial arteries appear to show impairment of FMD despite normal endothelial function (Heitzer et al., 2001). 638

Mean FMD values can vary considerably across studies in similar populations: in CHD ranging from 1.3 to 14%, in diabetes mellitus from 0.75 to 12%, and in healthy volunteers from 0.2 to 19.2% (Bots et al., 2005). Such differences are, in part, due to methodological variations, including arterial occlusion times, positioning of the occlusion cuff, and the time point of vessel measurement following cuff release (Berry et al., 2000). A direct vasodilator effect of ischemia on the brachial artery during upper arm occlusion is also possible (Corretti et al., 2002). The time of day chosen is critically important for FMD measurement. Indeed, in healthy humans endothelial function is reduced in the early morning (6am) compared with measurements obtained before sleep (9pm) and later in the day (11am) (Otto et al., 2004). FMD at 6am (4.4%) was markedly decreased, compared with the measurements at 9pm (7.5%) and 11am (7.7%). Specific mechanisms responsible for the morning-related endothelial impairment are unknown and this phenomenon still needs to be recognized in clinical studies. A study of 73 patients who underwent cardiac catheterization due to chest pain and the assessment of brachial artery FMD by ultrasound revealed that cardiovascular events occurred more often in patients with impaired (10%) versus preserved (10%) FMD (Neunteufl et al., 2000). Limitations of this study are represented by relatively small sample size, heterogeneous mix of stable and unstable patients, and dominance of revascularization procedures as adverse outcome. FMD may be particularly useful to identify a subgroup of patients with peripheral arterial disease (PAD) at very high risk and, more importantly, to improve the prognostic value of the ankle–brachial pressure index. A study conducted on 131 patients with PAD of the lower limbs showed that reduced brachial artery vasoreactivity was an independent predictor for increased cardiovascular risk (Brevetti et al., 2003). Moreover, results indicated that the median FMD was significantly lower in patients with an event than in those without (Brevetti et al., 2003). There is substantial evidence suggesting an improvement in FMD

Chapter 20 NO in Vascular Damage and Regeneration

following exercise training in healthy and clinical populations (Gokce et al., 2002), with a subsequent reduction in cardiovascular disease risk. Furthermore, 45 min of moderate intensity (acute) exercise has been shown to improve FMD and other parameters of endothelial function (Camsarl et al., 2003; Gill et al., 2004). FMD response to moderate intensity acute exercise seems to be reproducible even if heart rate, blood pressure, and sympathetic activity may influence measurements (Harris et al., 2007). The FMD response to acute exercise is enhanced in active men who are overweight, whereas inactive men who are overweight exhibit an attenuated response (Harris et al., 2008). Finally, reactive hyperemia peripheral arterial tonometry (RH-PAT) is another non-invasive and operator-independent tool for measurement of endothelial function. This technique is used to assess peripheral microvascular endothelial function by measuring changes in digital pulse volume during reactive hyperemia. Normal response is characterized by a distinct increase in the signal amplitude after cuff release, compared to baseline. Digital hyperemic response is attenuated in patients with established coronary endothelial dysfunction, suggesting a role for RH-PAT as a non-invasive, bedside test to identify patients with this disorder (Bonetti et al., 2004).

NOS competitive inhibitors and atherosclerosis Decreased NO bioavailability and impairment of the NOS isoform-dependent pathways, which normally suppress the processes leading to the development of atherosclerotic plaques, drive disruption of the non-thrombogenic intimal surface and promotion of platelet adhesion and aggregation, as well as deposition of platelets on the abnormal endothelial surface. Additional abnormalities in the coagulation system take part in the establishment of atherosclerosis (reviewed in Napoli et al., 2006; de Nigris et al., 2003; Ignarro and Napoli, 2004). Analogues of L-arginine such as L-NMMA and asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of NOS, have been used to inhibit NOS. Patients with hypercholesterolemia and atherosclerotic vascular disease show elevated plasma levels of ADMA which correlate with the severity of endothelial dysfunction and of cardiovascular risk factors (Dayoub et al., 2008; Juonala et al., 2007; Kals et al., 2007, Maas et al., 2008; Napoli et al., 2004a; Okyay et al., 2007; Selcuk et al., 2007) (Fig. 1). The inhibitory effect of ADMA on endothelial function has been shown not only to involve NO-mediated endothelium-dependent vasodilatation but also the endothelium-derived hyperpolarizing factor-mediated pathways in hypertensive animals and humans (Napoli et al., 2004a). In apparently healthy subjects without symptomatic coronary or peripheral vascular disease, the plasma ADMA level varied with age, blood pressure, glucose tolerance, and with carotid intima-media thickness (Miyazaki et al., 1999). An acute infusion of ADMA (2.0–10 mol/L) to healthy subjects caused a significant decrease in plasma cGMP concentration and produced a significant decrease in cardiac output (Kielstein et al., 2004). A study by Cardounel et al. (2007) provided insights into the open question as to whether the small change in plasma level of ADMA in patients with coronary artery disease is sufficient to alter significantly NO production and contribute to the development of coronary artery disease. Indeed, ADMA is preferentially taken up by endothelial cells and the intracellular level of ADMA is 5–10 times that of the extracellular level. Also, kinetic studies showed that the KM of eNOS for L-arginine is 3.14 mol/L and the Ki for ADMA to inhibit NOS is 0.9 mol/L (Cardounel et al., 2007). Therefore, a small change in the plasma level of ADMA would presumably have a large effect on the intracellular level of ADMA and on NO production. Moreover, plasma levels of ADMA are negatively associated with basal forearm blood flow in peritoneal dialysis patients and also with vasoconstriction induced by its analogue L-NMMA (Mittermayer et al., 2005). Using L-NMMA, it was shown that there was a loss of basal and flow-mediated NO production in the coronary arteries (Tousoulis et al., 1997b). The response to L-NMMA indicated that the endothelium holds basal NO production in the presence of atherosclerotic disease.

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Blockade of a biological process by L-NAME, a specific NOS inhibitor, and competition with this effect by an excess of L-arginine, provide very strong evidence for the role of endogenous NO as an important progression factor of atherosclerosis. Apo E-deficient mice (ApoE-/-) treated with L-NAME showed significant inhibition of NO-mediated vascular responses and a significant increase in the aortic atherosclerotic plaque/surface area (Kauser et al., 2000). On the contrary, L-arginine treatment had no influence on endothelial function and did not alter lesion size (Kauser et al., 2000). The damaged NO-mediated responses are critical for the progression of atherosclerosis in mice and correlate with a rapid increase in atherosclerotic lesion size. The mechanism responsible for vascular endothelial dysfunction and cardiovascular remodeling induced by NO deficiency following long-term treatment with L-NAME involves the apoptosis signal-regulating kinase-1 by reducing eNOS activity and disrupting the eNOS dimer (Yamashita et al., 2007). Advanced glycation end products (AGEs), senescent macroprotein derivatives closely linked to the development of diabetic atherosclerosis (Cantero et al., 2007; Goldin et al., 2006; Napoli et al., 2006), accelerate the development of diabetic atherosclerosis via iNOS and heme oxygenase-1 induction (Sumi and Ignarro, 2004). The stimulation of RAW 264.7 cells with AGEs leads to increased heme oxygenase-1 and iNOS protein expression, and nitrite accumulation via regulation of p42/44 and p38 mitogen-activated protein kinase (Sumi and Ignarro, 2004). Another essential mechanism in diabetic atherosclerosis is represented by up-regulation of the ligand for the AGE receptor via angiotensin II type I receptor stimulation (Ihara et al., 2007).

NOS knockout mice in the pathobiology of NO and atherosclerosis 640

Inhibition of NOS by L-arginine analogs has yielded a considerable amount of important information. One potential limitation of pharmacologic inhibitors, however, is that they may inhibit more than one NOS isoform. Genetic manipulation complements pharmacologic approaches because its specificity is at the genetic level. eNOS gene transfer ameliorates response to acetylcholine in atherosclerotic rabbit aortic rings (Mozes et al., 1998) and decreases adhesion molecule expression and infiltration of inflammatory cells in carotid arteries of cholesterol-fed rabbits (Qian et al., 1999). Gene transfer of eNOS, but not eNOS plus iNOS, regressed atherosclerosis in rabbits (Hayashi et al., 2004). Moreover, anti-monocyte chemoattractant protein-1 (anti-MCP-1) gene therapy blocked the development of vascular medial thickening and the recruitment of monocyte into the coronary vessels in a rat model with chronic inhibition of endothelial NO synthesis (Egashira et al., 2000), indicating that MCP-1 is necessary for the development of medial thickening. Another approach is represented by the manipulation of genes encoding the NOS enzymes to generate knockout mice of a particular NOS gene (eNOS, iNOS, and nNOS) (Liu et al., 2008). Results obtained with pharmacologic agents were confirmed by those obtained with eNOS knockout (eNOS/) mice. Indeed, eNOS/ mice show significantly greater neointima formation after cuff injury than wild-type mice (Moroi et al., 1998) indicating that deficiency in the amount of available NO in the vessel wall is responsible for the increase of neointimal formation in response to vascular injury. ApoE/eNOS double knockout mice (ApoE//eNOS/) on a Western diet show a higher development of atherosclerosis and greater size of atherosclerotic lesion areas than ApoE/ (Kuhlencordt et al., 2001a). In ApoE//eNOS/ mice, lesion area increased compared to ApoE/ mice after 16 weeks of ‘Western-type’ diet (Kuhlencordt et al., 2001b). This was accompanied by evidence of coronary artery disease, left ventricular dysfunction, aortic aneurysm, and aortic dissection. Moreover, ApoE//eNOS/ mice are hypertensive and the pharmacological control of blood pressure was ineffective in preventing accelerated atherosclerosis and development of aortic aneurysms (Chen et al., 2001). Unexpectedly, the aortic lesions of eNOS/

Chapter 20 NO in Vascular Damage and Regeneration

mice fed an atherogenic diet (15% fat, 1.25% cholesterol, and 0.5% sodium cholate for 12 weeks) were smaller than those in the wild-type control group of mice (Shi et al., 2002). This reduction was ascribed to the absence of eNOS-mediated low density lipoprotein (LDL) oxidation and was not related to the plasma lipid levels and susceptibility of LDL to oxidation (Shi et al., 2002; Napoli, 2003). Several studies have implicated dysfunctional eNOS as a common pathogenic pathway in diabetic vascular complications (Cai et al., 2005; Luo et al., 2004; Satoh et al., 2005). Further studies in eNOS/ diabetic mice model confirmed a vital role for eNOS-derived NO in the pathogenesis of diabetic nephropathy. Diabetic eNOS/ mice developed hypertension, albuminuria, and renal insufficiency with arteriolar hyalinosis, mesangial matrix expansion, mesangiolysis with microaneurysms, gracilis muscle arterioles, and Kimmelstiel-Wilson nodules (Nakagawa et al., 2007; Zhao et al., 2006). Glomerular and peritubular capillaries are increased with endothelial proliferation and vascular endothelial growth factor (VEGF) expression. Moreover, inhibition of eNOS predisposes mice to classic diabetic nephropathy through a mechanism likely due to VEGF-NO uncoupling with excessive endothelial cell proliferation (Nakagawa et al., 2007). In addition, decreased levels of eNOS-derived NO induce albuminuria and accelerate hypertension and glomerular basal membrane thickening in diabetic mice resistant to nephropathy (Kanetsuna et al., 2007). Chronic deficiency of NOS expression or activity may augment the contractions in response to endothelin-1 (ET-1), as demonstrated by evidence that in wild-type mice the ET-1induced aorta contraction consistently increases when all NOS isoforms are inhibited with N(G)-nitro-l-arginine (Lamping and Faraci, 2003). Moreover, in eNOS/ mice contractions in response to ET-1 increase two-fold compared to wild-type mice whereas N(G)-nitro-larginine has no effect (Lamping and Faraci, 2003). This evidence may partially explain the proatherogenic role of ET-1. In addition, transgenic mice that overexpress human ET with additional knockout of eNOS (ET//eNOS/) show a further enhancement of blood pressure as compared to eNOS/ mice (Quaschning et al., 2007). Arterioles of eNOS/ mice express high levels of cyclooxygenase (COX)-2 protein, together with an up-regulation of COX-2 gene expression, suggesting that COX-2-derived prostaglandins are the mediators responsible for maintenance of the flow-induced dilation observed in arterioles of eNOS/ mice (Sun et al., 2006). Among the NOS isoforms, deletion of nNOS prevents impaired vasodilation in septic mouse skeletal muscle, suggesting its involvement in the arteriolar hyporesponsiveness to acetylcholine in septic skeletal muscle (Lidington et al., 2007). Similarly to eNOS, nNOS plays atheroprotective roles (Kuhlencordt et al., 2006). Indeed, the mortality rates of ApoE//nNOS/ mice are higher compared to ApoE/ mice (Kuhlencordt et al., 2006). In contrast, ApoE//iNOS/ mice show significantly smaller lesion areas compared to ApoE/ mice (Kuhlencordt et al., 2001a). Moreover, the reduction in atherosclerosis in double knockout animals is associated with decreased plasma levels of lipoperoxides, suggesting that a reduction in iNOS-mediated oxidative stress provides the protection from lesion formation in double knockout animals (Kuhlencordt et al., 2001a). More recently, it has been shown that iNOS and not eNOS is involved in the control of ET-1-induced prostacyclin release and related inhibition of platelet aggregation in the murine models eNOS/ and iNOS/ (Carrier et al., 2007). Results from this study indicate that systemically administered ET-1 triggers a dose-dependent inhibition of platelet aggregation and an increase in plasma levels of 6-keto prostaglandin F(1alpha) in wild-type and eNOS/ but not in iNOS/ mice. Finally, ET-1 significantly increases 8-isoprostane plasma levels in wild-type but not in iNOS/ mice (Carrier et al., 2007). Although singly eNOS/ mice manifest accumulation of cardiovascular risk factors that mimic human metabolic syndrome, and although it is well established that eNOS

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exerts antiarteriosclerotic effects, singly eNOS/ mice do not spontaneously develop ­arteriosclerotic/atherosclerotic vascular lesion formation. This inconsistency may be due to a compensatory mechanism by other NOS that are not disrupted genetically. The critical role of the endogenous NOS system in the maintenance of cardiovascular and metabolic homeostasis has been recently demonstrated using a mice model deficient in all NOS isoforms (n/i/eNOS/ mice). Indeed, genetic disruption of the three NOS isoforms induces spontaneous myocardial infarction associated with multiple cardiovascular risk factors of metabolic origin in mice in vivo (Nakata et al., 2008). In addition, the n/i/eNOS/ mice develop visceral obesity, hypertension, hypertriglyceridemia, and impaired glucose tolerance, a typical phenotype of metabolic syndrome in humans (Nakata et al., 2008). Importantly, n/i/eNOS/ mice show activation of the renin–angiotensin system. Long-term oral administration of an angiotensin II type 1 receptor blocker suppressed the formation of coronary arteriosclerotic lesion and the occurrence of spontaneous myocardial infarction. Moreover, an improved prognosis for those mice and amelioration of the metabolic abnormalities were also observed (Nakata et al., 2008).

NO and oxidation-sensitive mechanisms A broad range of oxidation-dependent mechanisms have been extensively reviewed (de Nigris et al., 2003; Ignarro and Napoli, 2004; Napoli and Lerman, 2001; Napoli et al., 2006). Lipids with atherogenic properties, like oxidized low density lipoproteins (oxLDL), play critical roles in endothelial dysfunction participating in human early atherogenesis. Indeed, oxLDL induce monocyte adhesion to the endothelium, and migration and proliferation of smooth muscle cells, cause injury to cells, interfere with NO release, and promote procoagulant properties of vascular cells (Fig. 1). oxLDL are responsible for eNOS uncoupling and local depletion of the L-arginine substrate determining an increase in superoxide radical production. 642

Moreover, oxLDL increase the availability and activity of arginase II (at both transcriptional and post-translational levels), reciprocally decrease NOx production, and contribute to impaired vascular NO signaling. The mechanism for the activation of arginase within endothelial cells involves dissociation of arginase II from microtubules, a key mechanism in early arginase II activation. This activation is inhibited by the microtubule-stabilizing agent epothilone B and augmented by the microtubule-depolymerizing agent nocodazole. In addition, oxLDL induce arginase II mRNA transcription and a subsequent increase in protein levels (Ryoo et al., 2006). Native LDL significantly reduces intracellular NOHA (N(G)hydroxy-L arginine) levels, but the magnitude of inhibition was significantly greater with oxLDL, consistent with reduced endothelial NO production. Inhibition of NOS or L-arginine transport can, individually or in combination, explain these observations. Interestingly, it has been shown that eNOS enzymatic activity but not its subcellular distribution is negatively affected by native LDL (Ji et al., 2004; Pritchard et al., 2002). All NOS isoforms, under specific conditions such as a lack of essential cofactors, can form superoxide. Identification of lipooxygenase (LOX)-1 as the major receptor for oxLDL in endothelial cells has provided a new clue to the mechanisms involved in oxLDL accumulation in the vessel wall. This receptor, by facilitating the uptake of oxLDL, induces endothelial dysfunction and mediates numerous oxLDL-induced proatherogenic effects (Biocca et al., 2008). Besides endothelial cells, LOX-1 is also expressed by smooth muscle cells and macrophages. In these cells, LOX-1 may function as a scavenger receptor and promote foam cell formation. Another mechanism, also at least partly involving the LOX-1 receptor, is the impact of oxLDL on ADMA. oxLDL can consistently increase the concentration of ADMA in endothelial cells. This leads to an up-regulation of arginine N-methyltransferase expression which generates ADMA (Monsalve et al., 2007; Tan et al., 2007) (Fig. 1). In endothelial cells, ADMA increases oxidative stress, and in activated macrophages it up-regulates the expression of LOX-1, thus creating a vicious circle.

Chapter 20 NO in Vascular Damage and Regeneration

Interestingly, arterial segments from different regions can be affected by physiological ­differences (D’Armiento et al., 2001; Napoli et al., 1997, 1999). An example of this is the impairment of contraction and endothelium-dependent relaxation determined by oxLDL in carotid but not in basilar artery (Napoli et al., 1997), indicating that endothelial resistance to oxidative injury can represent the protection of intracranial arteries from atherosclerosis. Moreover, a large number of oxidation-sensitive apoptotic signaling factors can interact with NO in the arterial wall (Napoli and Lerman, 2001; Napoli et al., 2002). Finally, the balance between oxidative stress and NO bioactivity is critical in the development of atherogenesis, demonstrating that the L-arginine hypothesis and the concept of increased oxidative stress do not exclude each other but can coexist. NO also plays a critical role in regulation of renal physiology (Fig. 2) and in several pathways implicated in progression of renal damage in renal artery stenosis. In renal hypoperfusion a mixture of adaptive responses, and tubular and endothelial cell damage and repair events take place, and may result in tubulointerstitial fibrosis, vascular sclerosis, or glomerulosclerosis. Renal functional impairment is often triggered by endothelial injury, reduced NO bioavailability, increased vasoconstrictor activity, and increased generation of ROS (Lerman et al., 2001; Stulak et al., 2001), resulting in a high prevalence of renal vasoconstrictors and growth factors. Generation of ROS in the kidney is mediated mainly by radical-producing enzyme systems such as NAD(P)H oxidase, xanthine oxidase, and uncoupled eNOS. ROS interact with and decrease the availability of NO, resulting in formation of the pro-oxidant peroxynitrite, and impairing intra-renal vascular, glomerular, and tubular function. ROS thereby modulate renal microvascular function and contribute to the enhanced renal vascular tone, sensitivity to vasoconstrictors, endothelial dysfunction, and tubuloglomerular feedback. With the blunted buffering effect of vascular NO, elevated levels of intra-renal vasoconstrictors like angiotensin II and ET-1 prevail, leading to vasoconstriction and impairing both renal hemodynamics and function (Schnackenberg et al., 2000). A decrease in endogenous NO also leads to loss of both its anti-thrombotic protection and inhibition of fibrosis-related responses to injury. Renal structural injury in human renovascular disease develops in several phases, in which glomerulosclerosis is a late event that results from long duration and co-morbid conditions. The earliest pathologic feature in renal ischemia is tubulointerstitial injury, which involves activation of inflammatory mediators like iNOS and redox-sensitive transcription factors, possibly through ROS release (Zhen et al., 2008). Cellular activation is followed by release of cytokines and growth factors, which in turn modulate renal tissue responses to ischemia, like collagen deposition, extracellular matrix turnover, and fibrosis. In fact, it has long been recognized that glomerulosclerosis and atherosclerosis share similar pathophysiological mechanisms (Keane et al., 1988). Renal atherosclerosis may contribute to the evolution of renal damage both directly through deposition of atherogenic lipoproteins, and indirectly by decreasing NO bioavailability and increasing oxidative stress. Atherosclerosis, the main etiology of human renovascular disease, is also characterized by increased levels of oxLDL, which is cytotoxic to renal mesangial, epithelial, and endothelial cells (Meier et al., 2007). Moreover, activation of the renin–angiotensin system, the hallmark of renovascular disease, contributes to its pathophysiology by stimulating renal production of ROS (Sachse and Wolf, 2007), LDL oxidation, and LOX-1 expression, possibly mediated via ET-1 (Chade et al., 2003). This mechanism also contributes to renal microvascular remodeling and loss in the stenotic kidney (Zhu et al., 2004), which may blunt the ability of the kidney to recover following revascularization of the stenotic renal artery. The contribution of disrupted NO/ROS balance to renal injury in renovascular disease is underscored by the observation that L-arginine supplementation increased NO end products in the earliest phase of renal ischemia/reperfusion injury, and prevented eNOS uncoupling and induction of iNOS and LOX-1 (Kosaka et al., 2003). Moreover, experimental chronic

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antioxidant supplementation improved renal hemodynamics and decreased oxidative stress, inflammation, and fibrosis in the ischemic kidney (Chade et al., 2003), mainly by increased renal expression of eNOS and decreased expression of NAD(P)H-oxidase, nitrotyrosine, and iNOS, suggesting decreased superoxide abundance and inflammation (Chade et al., 2004a,b). Importantly, excessive oxidative stress is involved, at least in part, in impaired systemic endothelium-dependent vasodilatation (Higashi et al., 2002) and sustaining (Lerman et al., 2001) renovascular hypertension. Hence, the pivotal role of NO in regulation of renal function and structural integrity renders it a critical modulator of the kidney in health and in disease. Most experimental and clinical evidence indicates clearly that strategies capable of increasing NO bioavailability are effective in reducing renal injury and restoring its function.

eNOS polymorphisms eNOS gene polymorphisms are risk factors that contribute to endothelial dysfunction and atherosclerosis in many cardiovascular events (Napoli et al., 2006). The polymorphisms in exon 7 (894 G→T) and in the promoter region (T-786→C) of the eNOS gene are linked to functional changes in the endothelium and carotid intima-media thickness, low modulation of the predisposition to abdominal aortic aneurysm (reviewed in Napoli and Ignarro, 2007; Napoli et al., 2006), and could be a risk factor for angiographic CHD and recent myocardial infarction (Napoli et al., 2006).

644

To date, evidence for an association between polymorphisms in heart disease-related genes and early onset of a first myocardial infarction is still limited. Multivariate regression analysis was used to evaluate age at onset of a first myocardial infarction in relationship to individual single-nucleotide polymorphisms in a cohort of 814 patients enrolled in the THROMBO (Thrombogenic Factors and Recurrent Coronary Events) Study (Morray et al., 2007). Patients with high-risk genotype eNOS E298D showed age at onset of a first myocardial infarction to be 3.5 years (P  0.02), earlier than for non-carriers of the genotype. High-risk genotypes of the eNOS E298D polymorphism were significantly associated with onset of a first myocardial infarction at age 50 years (adjusted odds ratio (OR) 2.15, P  0.01). Analysis of the GluAsp or AspAsp genotype of the Glu298Asp polymorphism in 337 Japanese patients with diabetes indicated that this polymorphism is significantly associated with ischemic heart disease (IHD) (Tamemoto et al., 2008). Among 337 subjects analyzed for the eNOS gene polymorphisms, the first group was composed of 45 patients with the GluAsp and 5 with the AspAsp genotype (GluAsp or AspAsp group), and the second group contained the remaining subjects. In the first group, 16 subjects (32%) showed IHD. Among 287 subjects of the second group with the GluGlu genotype, 38 (13.2%) showed IHD. The number of subjects with IHD was significantly greater in the GluAsp or AspAsp group than in the GluGlu group (P  0.0006). Several studies have evaluated the relationship between common variants of the eNOS gene and the risk of CHD (Jaramillo et al., 2008; Kim et al., 2007; Tamemoto et al., 2008) with results sometimes conflicting. In the evaluation of the association between presence of CHD documented by angiography and the 786TC polymorphism of the eNOS gene in 112 unrelated Chilean patients with CHD and 109 controls, the frequency of the CC homozygous genotype for the 786TC polymorphism was 6% in CHD patients and 4% in the control group (Jaramillo et al., 2008). However, the genotype distribution and allele frequencies were not significantly different between the two groups (P  NS). Moreover, the odds ratio for CHD associated with the C variant failed to reach statistical significance (OR  1.03; 95% confidence interval (CI) 0.60–1.76, P  NS), suggesting that the 786TC polymorphism of the eNOS gene is not associated with CHD in these Chilean individuals.

Chapter 20 NO in Vascular Damage and Regeneration

In a case-control study performed to evaluate the association between the eNOS 786TC, 4a4b, or 894GT polymorphism and CHD in 147 Korean patients and 222 healthy controls, the eNOS 786TC (OR  1.61; 95% CI  0.97–2.69), 894GT (OR  1.12; 95% CI  0.65–1.92) and 4a4b (OR  1.44, 95% CI  0.87–2.39) polymorphisms were not an independent predisposition factor to CHD (Kim et al., 2007). However, a subgroup analysis adjusted with various cardiovascular risk factors confirmed positive association of the 786TC polymorphism in CHD patients with hypertension and a smoking history, and also a significant association of the intron 4 genotypes with a smoking history, but no significance was found for the eNOS polymorphisms of 894GT upon any risk adjustment. The distribution of heterozygotes (786TC, 894GT, and 4a4b) and variant homozygotes for the 786C, 894T, and intron 4a alleles of eNOS in the Koreans was found to be significantly lower than in Caucasian populations (Kim et al., 2007). Similarly, a systematic examination of the associations of eight variants of the eNOS gene (two potentially functional variants, 786TC and Glu298Asp, and six tagging single nucleotide polymorphisms) with CHD risk in a large cohort of diabetic patients suggests that 786TC, Glu298Asp, and an intron 8 polymorphism of the eNOS gene are potentially involved in the atherogenic pathway among diabetic men in the US (Zhang et al., 2006). Indeed, among 861 diabetic men (97% Caucasian), 220 developed CHD. Genotype distributions of 786TC and Glu298Asp polymorphisms were not significantly different between case and control subjects, and CHD risk was significantly higher among men with the variant allele at the rs1541861 locus (intron 8 A/C) than men without it (adjusted OR 1.5; 95% CI  1.1–2.1). Moreover, among control subjects, plasma soluble vascular cell adhesion molecule concentrations were significantly higher among carriers of this allele (P  0.019) and carriers of the variant allele of 786TC (P  0.010) or the Glu298Asp polymorphism (P  0.002), compared with non-carriers. In patients with diabetes mellitus, the Asp298 allele of the eNOS gene is significantly associated with impaired coronary collateral development (Gulec et al., 2008). Patients with poor collaterals are more likely to have diabetes mellitus (P 0.001) and unstable angina pectoris (P  0.014), and to carry the Asp298 variant (P  0.02) (Gulec et al., 2008). Multivariate analysis demonstrates that Asp298 allele carriers are 1.7 times more likely to have poor collaterals than patients with the GluGlu genotype (95% CI  1.09–2.69; P  0.024). Moreover, a significant interaction between diabetes mellitus and the eNOS Glu298Asp poly­ morphism in the analysis of collateral development was reported (Gulec et al., 2008). The T(786)C single-nucleotide polymorphism of the eNOS gene implies blunted endotheliumdependent vasodilation in hypertensive patients and correlates with multivessel CHD. This polymorphism is associated with changes in markers of oxidative stress in high-risk white patients referred for coronary angiography (Rossi et al., 2006). Determination of eNOS T(786)C by melting curve analysis of amplicons from allele-specific fluorescence resonance energy transfer probes in patients of the GENICA (Genetic and Environmental Factors in Coronary Atherosclerosis) Study showed that there was a significant impact of the T(786)C eNOS genotype on cardiovascular death-free (P  0.0102) survival (Rossi et al., 2006). Moreover, an additive effect of eNOS gene polymorphisms (G894T, T  786C, and 27-bp repeat in intron 4) contributes to the severity of atherosclerosis in patients on dialysis (Spoto et al., 2007).

Molecular mechanisms regulating eNOS The molecular mechanisms regulating eNOS involve both genomic and non-genomic pathophysiological mechanisms (Napoli and Ignarro, 2001; Napoli et al., 2006). Indeed, transcription and nuclear factors, such as the AP-1 complex, NF-B, and interleukin (IL)-6, can bind the eNOS promoter gene (de Nigris et al., 2003). Physiological shear stress increases eNOS abundance, which is decreased by LDL, angiotensin II, and tumor necrosis factor (TNF) .

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eNOS is a tightly coupled enzyme system that may be easily dysregulated by perturbations in availability of substrates and cofactors as well as by competitive inhibitors such as ADMA (Napoli and Ignarro, 2001; Napoli et al., 2006). The increased superoxide production and NO conversion to peroxynitrite, following eNOS uncoupling, determines a reduction in eNOS bioactivity. A positive effect of peroxisome proliferator-activated receptors (PPARs) on eNOS occurs during eNOS recoupling with BH4, L-arginine, and antioxidants (Napoli and Ignarro, 2001; Napoli et al., 2006). Other compounds that regulate eNOS activity via genomic and non-genomic mechanisms are angiotensin-converting enzyme inhibitors, statins, angiotensin II receptor blockers, and calcium channel blockers (Napoli and Ignarro, 2001; Napoli et al., 2006).

646

The suppression of inflammatory signaling pathways by PPAR- activation provides an additional mechanism whereby fenofibrate, a specific PPAR- agonist, could influence eNOS bioactivity (Goya et al., 2004). Fenofibrate seems to increase the mRNA expression, protein level, and enzyme activity of eNOS in a dose-dependent manner. However, the eNOS promoter sequence does not possess a PPAR response element indicating that fenofibrate did not enhance eNOS promoter activity (Goya et al., 2004). Moreover, in mRNA stability assays, fenofibrate increases the half-life of eNOS mRNA. The observation that PPAR- agonists stabilize mRNA levels is unexpected and raises the question of whether these effects occur via PPAR-. Further studies employing gene knockdown technology and/or in vivo analysis of PPAR--deficient mice are required to address this point (Goya et al., 2004). PPAR- activation can also have direct antiatherogenic effects on the different cell types of the vascular wall by decreasing the expression of adhesion molecules, tissue factor, IL-6, and endothelin-1 (Israelian-Konaraki et al., 2005). Latest evidence of how PPAR transcription factors may modulate different steps of atherosclerosis development and progression, and the therapeutic potential of PPAR ligands has been recently reviewed elsewhere (Bouhlel et al., 2008). Over the last few years, there has been a considerable amount of research implicating Krüppel-like transcription factor 2 (KLF2) as a key regulator of the endothelial proinflammatory pathways (Atkins and Jain, 2007) (Fig. 1). Several key factors involved in maintenance of an antithrombotic endothelial surface are differentially regulated by KLF2. Indeed, in endothelial cells KLF2 mediates the beneficial effects of statins, is induced by laminar shear stress, and up-regulates eNOS expression and inhibits induction of vascular cell adhesion molecule-1 (VCAM-1) and endothelial adhesion molecule E-selectin (Napoli et al., 2006). In another study, KLF2 was found to strongly induce thrombomodulin and eNOS expression, and reduce plasminogen activator inhibitor-1 (PAI-1) expression (Lin et al., 2005b) (Fig. 1). In situ hybridization approaches revealed that KLF2 expression is limited to the endothelial layer of the human aorta and, importantly, KLF2 expression is decreased at branch points (SenBanerjee et al., 2004). More recently, KLF2 has been implicated in the activator protein 1 (AP-1) pathway (Boon et al., 2007; Fledderus et al., 2007) and in the inhibition of transforming growth factor (TGF)-ß signaling via two distinct mechanisms (Boon et al., 2007). Using overexpression and knockdown studies, KLF2 was shown to induce Smad7, subsequently suppressing Smad2 phosphorylation and Smad3/4-dependent transcriptional activation. In addition, KLF2 simultaneously inhibits the TGF-ß signaling cofactor AP-1 (Boon et al., 2007). Increased levels of phosphorylated nuclear activating transcription factor 2 (ATF2), a heterodimeric component of AP-1, are typical of human endothelial cells overlying ­atherosclerotic plaques (Boon et al., 2007). Using knockdown and overexpression studies, shear stress suppressed nuclear levels of activated ATF2 by inhibiting its nuclear translocation via KLF2. Studies by SenBanerjee et al. (2004) were the first to identify KLF2 as a potent inducer of eNOS expression and activity. eNOS promoter deletion and mutational analysis revealed a single KLF2 site to be critical for the ability of KLF2 to bind and activate the eNOS promoter

Chapter 20 NO in Vascular Damage and Regeneration

via recruitment of the transcriptional coactivator cAMP response element-binding protein (CBP/p300). Many studies confirmed the ability of KLF2 to induce eNOS and inhibit the expression of genes regulating vessel tone, such as endothelin, angiotensin-converting enzyme, and adrenomedullin (Dekker et al., 2005, 2006; Parmar et al., 2006). Moreover, KLF2 is able to induce C-natriuretic peptide and arginosuccinate synthase, a limiting enzyme in eNOS substrate bioavailability (Goodwin et al., 2004; Parmar et al., 2005). Loss-of-function studies with siRNA approaches validate the effects of KLF2 in reducing the expression of eNOS and C-natriuretic peptide under basal and flow conditions (Dekker et al., 2005; Parmar et al., 2006). Post-translational modification of eNOS is critically important in the homeostasis of NO, mainly by regulating the subcellular localization of the enzyme. The deacylation/reacylation reaction of eNOS in the plasmalemmal caveolae regulates NO production (Hayashi et al., 2007; Lim et al., 2007; Napoli et al., 2006). Acylation targets the localization of eNOS to plasmalemmal caveolae, a site where the enzyme activity is inhibited through association with caveolin. The increase of cytosolic [Ca2] in response to the activation of cell surface receptors by acetylcholine is followed by the binding of calmodulin to eNOS, the dissociation of the enzyme from caveolin, and the production of NO (Napoli et al., 2006). Association with caveolin-1 maintains inactive eNOS in caveolae (Cao et al., 2003; GarciaCardena et al., 1998). eNOS activity can be increased by Ca2/calmodulin and binding to HSP90, which facilitates the phosphorylation of eNOS by forming a ternary complex with eNOS and Akt, and dynamin-2, which regulates eNOS activity through the binding of its proline-rich domain to the FAD domain of eNOS (Cao et al., 2003; Garcia-Cardena et al., 1998). A novel mechanism of eNOS activation and NO production in endothelial cells involves caveolae-mediated endocytosis induced by the 60-kDa albumin-binding glycoprotein gp60 (Maniatis et al., 2006). Inhibition of endocytosis resulted in marked impairment of NO production. eNOS activity induced by gp60 is mediated by Gß activation of downstream Src, Akt, and PI3K pathways. As caveolae internalization is a constitutive process in endothelial cells, this mechanism of NO production may be important in regulating basal NO-dependent vasomotor tone (Maniatis et al., 2006).

NO and polyphenols Polyphenols, a class of phytochemical dietary antioxidants relatively abundant in the diet, possess important biological properties, including protection against cholesterol oxidation and development of atherosclerosis (Ignarro et al., 2007). These compounds, abundant in cereals, chocolate, dry legumes, fruits, and plant-derived beverages such as fruit juices, tea, coffee, and red wine, induce a modification of the redox status of the cell and trigger redoxdependent reactions by interacting directly with cell receptors or enzymes (Manach et al., 2004; Scalbert et al., 2005). Consistent data derived from in vitro animal studies or epidemiological studies described the protective effect of polyphenol consumption against cardiovascular diseases (Arts and Hollman, 2005; Bea et al., 2006; Djoussé and Gaziano, 2007; Ignarro et al., 2007). Pomegranate juice (PJ), rich in ellagic acid, ellagitannins, punicic acid, flavonoids, anthocyanidins, anthocyanins, and estrogenic flavonols and flavones, has been shown to reduce the severity of atherosclerosis by reducing oxidative stress and by increasing NO production and action (Aviram et al., 2000; de Nigris et al., 2005; Ignarro et al., 2007; Kaplan et al., 2001; Rosenblat et al., 2003). Prolonged supplementation of hypercholesterolemic LDL receptor-deficient (LDLR-/-) mice with PJ attenuated the perturbed shear stress-induced proatherogenic disequilibrium by reducing macrophage foam cell formation, oxidation-specific epitopes, and lesion area in atherosclerotic lesion-prone regions. The therapeutic benefits of PJ can be attributed to several mechanisms. Among these, an increased eNOS activity and a decrease in redox-sensitive transcription factors (ELK-1 and p-JUN) (de Nigris et al., 2005)

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section III Principles of Pathobiology

participate in the molecular mechanism responsible for its antiatherogenic properties. The enhancement of eNOS bioactivity is also determined by the ability of PJ to revert the down-regulation of eNOS expression induced by oxLDL (de Nigris et al., 2006). In addition, the antioxidant activity of PJ determines a marked protection of NO against oxidative destruction, resulting in augmentation of the biological actions of NO (Ignarro et al., 2006). Similarly, other pomegranate derivations rich in polyphenolic antioxidants, such as pomegranate by-product, which includes the whole pomegranate fruit left after juice preparation, and pomegranate fruit extract, improved the redox status of arterial cells and positively influenced arterial reactivity, vascular expression of eNOS, and NOx levels, most likely by both increasing NO production and preventing its degradation (de Nigris et al., 2007a; Rosenblat et al., 2006;). Several in vitro and in vivo studies have elucidated the potential anticancer properties of the pomegranate (Bell and Hawthorne, 2008; Heber, 2008). Indeed, several pomegranate extracts (juice, seed oil, peel) potently inhibit prostate cancer cell invasiveness and proliferation, cause cell cycle disruption, induce apoptosis, and inhibit tumor growth (Bell and Hawthorne, 2008; Heber, 2008).

648

Modulation of NO levels is also a critical step in the beneficial effects exerted by polyphenols contained in red wine or grape extracts, such as the prevention of endothelial dysfunction, reduction of susceptibility of LDL to oxidation, and platelet aggregation (Aviram and Fuhrman, 1998; Hayek et al., 1997; Ignarro et al., 2007; Leikert et al., 2002; López-Sepúlveda et al., 2008; Madeira et al., 2009). A direct effect of red wine on monocytes for the release of NO, demonstrated with an in vitro model of human peripheral blood mononuclear cells, suggests that flavonoids and resveratrol, once absorbed at intestinal level, enter the circulation and trigger monocytes for NO production via iNOS (Magrone et al., 2007). Administration of red wine to hypertensive rats prevented endothelial dysfunction through the induction of vascular NADPH oxidase and preservation of arterial NO availability (Sarr et al., 2006). In platelets, resveratrol stimulates human platelet NO production by enhancing platelet NOS activity and phosphorylation of protein kinase B, an activator of the eNOS (Gresele et al., 2008). The increase in eNOS in response to red wine involves several polyphenolic compounds. However, a major contribution comes from trans-resveratrol and lesser contributions from cinnamic and hydroxycinnamic acids, cyanidin, and some phenolic acids (Wallerath et al., 2005). In endothelial cells, red wine polyphenols activate NO production via redox-sensitive activation of the PI3-kinase/Akt pathway which, in turn, causes phosphorylation of eNOS (Ndiaye et al., 2005). More recently, it has been demonstrated that resveratrol, at concentrations compatible with oral consumption, determines an increase in estrogen receptor-caveolin-1-Src interaction in lipid rafts/caveolae leading to NO production through a G-protein-coupled mechanism (Klinge et al., 2008). Up-regulation of eNOS expression levels in the aortic lesion areas of LDLR-/- mice was observed after chronic oral administration of a moderate amount of red wine with high resveratrol content (Napoli et al., 2008a). The increase of eNOS protein levels in the atherosclerotic lesion areas was accompanied by an increase in p-JUN and a decrease in ELK-1. Other important beneficial effects of polyphenols linked to the NO pathway are the up-regulation of eNOS aortic expression and amelioration of endothelial progenitor cell (EPC) number and functional activity in C57BL/6 J mice during physical training (Napoli et al., 2008a; Balestrieri et al., 2008a) and in ApoE-/- mice after hindlimb ischemia (Lefèvre et al., 2007). These in vivo observations support in vitro evidence of the beneficial effect of red wine and resveratrol in the modulation of EPC levels (Balestrieri et al., 2008b,c). Indeed, red wine polyphenols and pure resveratrol alone prevented the reduction of human EPC number induced by treatment with TNF- or high glucose concentrations, through the enhancement of NOx levels, inhibition of p38 phosphorylation, and up-regulation of SIRT1 expression levels (Balestrieri et al., 2008b,c).

Chapter 20 NO in Vascular Damage and Regeneration

NO and vascular regeneration Pre-clinical studies L-arginine administered to hypercholesterolemic rabbits induces an increase in apoptotic macrophages in the intimal lesions and determines regression of the atherosclerotic plaque (Napoli, 2003). This evidence, along with the powerful anti-atherogenic characteristics of NO, supports the hypothesis that NO is a promising candidate to induce regression of the plaque by triggering apoptosis. This suggests that manipulation of the NOS pathway may well represent a therapeutic approach to resolving the inflammatory response in the vessel wall (Napoli, 2003). In a murine model of unilateral hindlimb ischemia, autologous bone marrow cells (BMCs) together with metabolic intervention significantly ameliorated ischemia-induced angiogenesis in C57BL/6 J (Napoli et al., 2005), hypercholesterolemic (de Nigris et al., 2007c), and diabetic (Sica et al., 2006) mice by modulation of cellular oxidation-selective mechanisms and augmentation of NO production. This beneficial effect is amplified by metabolic treatment (vitamin C, vitamin E, and L-arginine), which induces vascular protection, at least in part, through the NO pathway, and reduces systemic oxidative stress. These results demonstrate that this combined approach is superior to BMC therapy alone. Moreover, a long-term combined beneficial effect of L-arginine treatment is also observed on atherosclerosis in hypercholesterolemic mice during graduated physical training (Napoli et al., 2004b). BMC therapy alone and, more consistently, in combination with metabolic treatment improved the functional activities of bone marrow-derived EPC by decreasing cellular senescence and increasing CXCR4 expression levels (de Nigris et al., 2007b). Short-term supplementation with L-arginine potentiates the effects of moderate physical exercise on EPC and VEGF levels, supporting the evidence that L-arginine positively modulates EPC levels (Fiorito et al., 2008). These effects could be ascribed to increased recruitment of EPC from the bone marrow vascular niche which represents a potential therapeutic target for PAD. A recent study showed that targeting the vascular niche with parathyroid hormone administered in combination with granulocyte-colony stimulating factor (G-CSF) improved stem-cell-based therapy in an experimental murine model of hindlimb ischemia (Napoli et al., 2008c). The protective vascular effect was achieved through amplification of the beneficial effect of G-CSF, at least partly via the NO pathway and a reduction in macrophage activation (Napoli et al., 2008c). Indeed, following mobilization induced by G-CSF treatment, treatment with parathyroid hormone determined increases in blood flow, capillary density, EPC, and nitrite/nitrate release. At the same time reduced apoptosis, fibrosis, oxidative stress, and inflammation in ischemic muscles was observed (Napoli et al., 2008c). Combined antioxidant and gene therapy has also been reported for middle-cerebral artery occlusion in the rat (Baker et al., 2007). Rats that 3 days before occlusion received L-arginine, vitamin E, and gene therapy with virus-mediated overexpression of tissue inhibitors of matrix metalloproteinases (TIMPs) showed smaller brain lesions than control rats, rats pre-treated with anti-oxidant therapy alone, and rats pre-treated with gene therapy alone. After ischemia, the activation of matrix metalloproteinases (MMPs) seems to play a pivotal role in causing brain damage. Therefore, the induction of TIMP overexpression might reduce ischemic damage by reducing MMP activity or by activating neuroprotective signals. Beneficial effects can be explained by inhibition of radical production and increase of radical degradation by endogenous antioxidants. The combined treatment of BMCs with TIMPs and metabolic supplementation had further beneficial neuroprotective effects, improving histological and functional outcome in brain ischemia. This benefit seems to be related to BMCs homing to the brain, enhanced neurogenesis/angiogenesis, NO bioactivity, and decreased systemic oxidative stress and MMP activity (Baker et al., 2007). Pre-clinical models show that stem cells or progenitor cells can be administered in acute and chronic renal failure diseases and lead to improvement in renal function and structure. Renovascular disease, by increasing blood pressure, mobilizes endogenous endothelial

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section III Principles of Pathobiology

650

­ rogenitor cells from the bone marrow of mice, in a fashion dependent on NAD(P)H p ­oxidase p47 and involving stromal cell-derived factor and MMP in the bone marrow (Salguero et al., 2008). Nevertheless, in view of the chronic and progressive nature of renal damage in this disease, the number or function of endogenous progenitor or multipotent stem cells available for renal repair is evidently insufficient to address its need. Furthermore, similar to other cardiovascular risk factors, progression of chronic renal disease in humans is associated with a decrease in the number of circulating progenitors. Therefore, exogenous delivery of several types of stem or progenitor cells has been utilized in an attempt to mitigate the consequences of acute or chronic renal failure. Initially, bone marrow-derived stem cells have been administered, but experimental evidence argued against a significant role of unfractionated BMCs in replacing epithelial cells during renal injury (Lin et al., 2005a). On the other hand, a sub-population of these cells, mesenchymal stem cells, appeared to be effective in stimulation of tubular cell growth in mice with kidney injury (Imberti et al., 2007), possibly by exerting an immunomodulatory and anti-inflammatory effect, as well as by paracrine secretion of angiogenic growth factors (Togel et al., 2005). Mesenchymal stem cells appear to be particularly effective in models of acute tubular injury, but less so in models of glomerular injury (Ninichuk et al., 2006). This raises the possibility that different populations of cells could be selectively effective in different disease models. Furthermore, resident renal stem cells have been identified in both animal and human kidneys, some in renal tubules (Oliver et al., 2004) and others in parietal epithelial cells in the urinary pole of Bowman’s capsule (Sagrinati et al., 2006). Injection of such cells a short time (4–20 h) after chemical injury significantly ameliorates the extent of acute renal failure in mice (Mazzinghi et al., 2008). In addition, fetal kidneys contain cells which are progenitors of tubular cells and podocytes, and eventually almost disappear during renal maturation. Administration of these immature cells is followed by incorporation in an engraftment in renal tubules of nude mice with acute renal failure (Lazzeri et al., 2007). However, the possible beneficial effect of EPC (such as autologous cells isolated from peripheral blood) has not been fully explored in models of kidney disease. A recent study showed that intra-renal infusion of autologous EPC restored renal function in an experimental model of chronic renal arterial stenosis (RAS) (Chade et al., 2009). EPC stimulated the proliferation of new vessels and accelerated their maturation and stabilization. Moreover, intra-renal infusion of autologous EPC attenuated renal microvascular remodeling and fibrosis in RAS, thus helping to preserve the blood supply, hemodynamics, and function of the RAS kidney (Chade et al., 2009). Regression of intra-renal microvessels accompanies many forms of renal disease, like diabetes and aging, and microvascular remodeling correlates with the development of renal scarring (Kang et al., 2002). Microvascular loss also contributes to renal dysfunction observed in chronic renovascular disease (Chade et al., 2006; Zhu et al., 2004). Therefore, it is not unlikely that delivery of EPC with angiogenic potential could enhance renal vascularization and prevent microvascular regression and progression of renal disease, especially if the microenvironment is modulated to attenuate oxidation processes, increase NO availability, and allow cellular engraftment (Baker et al., 2007). Yet the efficacy of concurrent metabolic treatment to enhance the engraftments or outcomes of progenitor stem cells in the kidney is yet to be shown. Studies are needed to determine the feasibility of this approach, and to establish whether renal repair requires exogenous and/or intrinsic renal progenitor cell populations, which type of cells are needed, and by which mechanisms they exert their beneficial effects. Indeed, preliminary studies suggest that prolonged treatment with lipoprotein A1 mimetic peptides, which protect LDL against oxidation, decreases oxLDL levels, increases eNOS expression in EPC, and prevents loss of renal microvessels (Peterson et al., 2007). Treatment with erythropoietin, a key molecule in vascular repair, has also been shown to enhance proliferation and differentiation of circulating bone marrow-derived EPC in renal

Chapter 20 NO in Vascular Damage and Regeneration

patients (Bahlmann et al., 2003). Considering the importance of endothelium in models of acute and chronic ischemic renal injury, endothelial repair or replacement would likely facilitate amelioration of renal injury. Delivery of stem cells in pre-clinical models of renal failure thus can lead to improvement in renal function and structure, but different types may be appropriate for different phases of renal disease. Both autologous and allogenic mesenchymal stem cells have been shown to be safe and effective in acute renal injury (Togel et al., 2009). The deterioration of renal function in humans following acute injury or during exposure to chronic risk factors suggests that the population of stem cells available for kidney repair is insufficient for adequate regeneration. The success of cell transplantation renal injury in increasing life expectancy and decreasing renal injury in animal models of acute and chronic renal failure encourages us to continue and pursue this potentially effective therapy, with the caveat that it may need to be tailored for specific disease models.

Clinical studies Early clinical studies describing the effect of L-arginine supplementation, extensively reviewed (Cylwik et al., 2005; Napoli et al., 2006), indicate its clinical beneficial effects in patients with cardiovascular diseases. Some clinical studies on NO and vascular regeneration are shown in Table 3. L-Arginine administration partially restores endothelium-dependent vasodilation in hypercholesterolemia (Creager et al., 1992; Drexler et al., 1991), dilates coronary stenosis in patients with CHD (Tousoulis et al., 1997a), and improves coronary smallvessel function patients with endothelial dysfunction and non-obstructive CHD (Lerman et al., 1998). Oral 6-week application of L-arginine (5.6 or 12.6 g/day) in patients with heart failure causes a drop in arterial blood pressure (Rector et al., 1996), whereas the administration of L-arginine (21 g for 3 days) to young men with CHD determined no changes in arterial pressure, despite the fact that the brachial artery was dilated, and the dilatation could be endothelium dependent (Adams et al., 1997). In addition, treatment with L-arginine has been reported to be effective in reducing blood pressure, particularly in patients with gestational hypertension (Facchinetti et al., 2007), and to improve endothelial function, oxidative stress, and adipokine release in obese type 2 diabetic patients with insulin resistance (Lucotti et al., 2006). Intracoronary infusion of L-arginine (40 mg/min for 14 min) in patients with hyperlipidemia and cardiac transplant recipients (Berkenboom et al., 1999) attenuated serotonin-induced constriction in the hyperlipidemic group but not in the transplant recipients. Moreover, sequential intracoronary infusions of L-arginine in patients with CHD determined an improvement in abnormal microvascular responses to sympathetic activation (Gellman et al., 2004) and dilatation of coronary segments and stenosis (Tousoulis et al., 2003), but did not increase the magnitude of the response to atrial pacing in proximal and distal segments and in coronary stenosis. It has been demonstrated that L-arginine administered intravenously is effective in patients with obliterative atheromatosis of the lower limbs (Boger et al., 1998). Clinical improvement was manifested in the extension of painless intermittent claudication distance, shortening of pain regression after walking the maximum distance, improvement of lower limb blood supply and increase in ankle–arm pressure ratio (Boger et al., 1998). A marked reduction in diastolic and systolic pressure and an increased blood flow in the femoral artery in patients with critical limb ischemia were observed even after a single intravenous infusion of L-arginine at a dose of 30 g for 60 min. In the NO-PAIN (Nitric Oxide in Peripheral Arterial Insufficiency) randomized clinical trial, 133 subjects with intermittent claudication due to PAD received oral L-arginine (3 g/day) for 6 months (Wilson et al., 2007). The change at 6 months in the absolute claudication distance, assessed by the Skinner-Gardner treadmill protocol, was the primary end point of this study. Results showed a significant increase in plasma L-arginine levels. However, a reduction or no improvement was observed when NO

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section III Principles of Pathobiology

Table 3   Published clinical studies on NO and vascular function Condition

Intervention

Clinical outcome

Reference

Hypercho­ lesterolemia

i.v. administration of L-arginine

Improvement of endotheliumdependent vasodilation

Creager et al., 1992

L-arginine infusion

Improvement of coronary microcirculation

Drexler et al., 1991

Intracoronary infusion of L-arginine

Improvement of abnormal microvascular function

Gellman et al., 2004

L-arginine infusion

Dilatation of coronary stenosis

Tousoulis et al., 1997a

Oral supplementation of L-arginine

Reduced arterial blood pressure

Rector et al., 1996

Intracoronary infusion of L-arginine

Dilatation of coronary segments

Tousoulis et al., 2003

Oral supplementation of L-arginine

Ineffective in influencing endothelial function

Blum et al., 2000b

Oral supplementation of L-arginine

Improvement of small-vessel coronary endothelial function

Lerman et al., 1998

Gestational hypertension

Oral supplementation of L-arginine

Reduced blood pressure, improved endothelial function

Facchinetti et al., 2007

Type 2 diabetes

Oral supplementation of L-arginine

Improved endothelial function, oxidative stress, and adipokine release

Lucotti et al., 2006

Congestive heart failure

Oral supplementation of L-arginine

Ineffective in influencing endothelial function

Chin-Dusting et al., 1996

Cardiac transplant

Intracoronary infusion of L-arginine

Attenuation of serotonin-induced constriction

Berkenboom et al., 1999

MI

Oral supplementation of L-arginine

No improvements of vascular stiffness measurements or ejection fraction

Schulman et al., 2006

Oral supplementation of L-arginine

Beneficial nonsignificant trend towards reduction of major clinical events

Bednarz et al., 2005

i.v. administration of L-arginine

Extension of painless intermittent claudication distance, improvement of lower limb blood supply

Boger et al., 1998

CHD

652

PAD

(Continued)

Chapter 20 NO in Vascular Damage and Regeneration

Table 3   Continued Condition

Intervention

Clinical outcome

Reference

Oral supplementation of L-arginine

No significant improvements of claudication distance

Wilson et al., 2007

Oral supplementation of L-arginine

Increase in walking distance

Oka et al., 2005

Oral supplementation of L-arginine, Vitamine C and E

Improved neovascularization capacity of autologous BMC transplantation

Napoli et al., 2008b

i.v., intravenous; CHD, coronary heart disease; PAD, peripheral arterial disease; MI, myocardial infarction; BMC, bone marrow cell.

availability was measured. This included flow-mediated vasodilation, vascular ­compliance, plasma and urinary nitrogen oxides, and plasma citrulline formation. Moreover, the improvement in the L-arginine-treated group was significantly less than that in the placebo group (28.3% vs 11.5%; P  0.024), even if absolute claudication distance improved in both L-arginine- and placebo-treated patients (Wilson et al., 2007). A trend of a greater increase in walking distance and walking speed in the group treated with L-arginine (3 g/day) was observed in a pilot study conducted to establish the lowest oral dose of L-arginine effective in patients with PAD and intermittent claudication (Oka et al., 2005). A more recent controlled study evaluated the neovascularization capacity of autologous BMC transplantation alongside treatment with vitamin C, vitamin E, and L-arginine (Napoli et al., 2008b). Eighteen patients with PAD (advanced III/IV Fontaine stages) were enrolled in a pilot clinical study, and a group of 18 patients under maximal drug therapy who refused BMC therapy served as control. BMCs were infused in the leg arteries of the BMC-treated group at time 0 and after 45 days. In addition, patients received daily antioxidants and L-arginine 30 days after the first BMC dose. Therapeutic neoangiogenesis was estimated by angiography and laser Doppler\capillaroscopy, and results showed an improvement in the ankle brachial index (ABI: 0.1) in 10 patients at 3 months and in 12 patients at 12–18 months. Ischemic ulcers improved in 13 patients (after 6–12 months). A significant increase in the mean maximum walking distance was observed at 3 months and was sustained up to 18 months. Only 2 BMC-treated patients underwent amputation compared to 10 patients of the control group (55.6 vs 13.3%; P  0.014). Thus, intra-arterial autologous BMC therapy combined with antioxidants and L-arginine is safe and effective in patients with advanced atherosclerotic PAD with positive effects until 18 months (Napoli et al., 2008b). Long-term oral administration of L-arginine in patients with congestive heart failure, stable angina pectoris and healed myocardial infarction, CHD, and in postmenopausal women was ineffective in influencing endothelial function (Blum et al., 2000a,b; Chin-Dusting et al., 1996). Results from a clinical trial conducted on 153 patients showed no significant changes in vascular and ventricular function (Schulman et al., 2006). A possible explanation lies in the inhibition of ADMA catabolism by L-arginine, supported by a recent study on the effect of insulin infusion in healthy adults (Eid et al., 2007). Results demonstrate that insulin reduced both ADMA and L-arginine plasma levels, but the ratio of L-arginine/ADMA remained unchanged with no effect on NO production and forearm blood flow. In the multicenter, randomized, double-blind, placebo-controlled ARAMI pilot performed on 792 patients with acute myocardial infarction, oral L-arginine supplementation (3.0 t.i.d p.o. for

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section III Principles of Pathobiology

30 days), although well tolerated, showed a beneficial non-significant trend towards reduction of major clinical events (Bednarz et al., 2005). Other results from studies concerning the effect of oral L-arginine supplementation on the cardiovascular system in humans have been summarized by Preli et al. (2002). Among 17 human studies, 5 showed no vascular health benefit from oral L-arginine supplementation and 12 demonstrated that oral L-arginine supplementation decreased platelet aggregation and adhesion and monocyte adhesion, and improved endothelium-dependent vasodilation. Clinical studies on healthy volunteers provided a novel important insight into the role of NO and vascular function. It has been proposed that nitrite functions as a physiological regulator of vascular function and endocrine NO homeostasis and can be considered a therapeutically active metabolite of the organic nitrates (Dejam et al., 2007). In this study, 20 healthy volunteers with a mean age of 28  3 years, a height of 172  7 cm, and a weight of 71  7 kg were recruited. Sodium nitrite, along with saline to maintain a total infusion volume of 120 mL/h, was infused at doses from 0, 7, 14, 28, and 55 to 110 mg  kg1  min1. Each dose was infused for 5 min, and during the infusion blood flow was measured at 15-s intervals in both arms, i.e. ipsilateral and contralateral to the infusion site (Fig. 3). After each dose was infused, blood was drawn from both veins to measure plasma and whole blood nitrite, and the infusion was paused briefly to determine blood pressure and heart rate before initiating the next dose of nitrite (Dejam et al., 2007). Results of this study indicate that nitrite, at physiological concentrations, is a potent systemic vasodilator, and this effect is temporally associated with intra-erythrocytic reduction of nitrite to NO by deoxyhemoglobin (Dejam et al., 2007). Thus, nitrite can be considered a circulating endocrine NO-generating molecule able to regulate basal vascular function. Its therapeutic use offers the potential to modulate vascular tone and cellular function during ischemia and infarction, and to bypass nitrate tolerance (Dejam et al., 2007). 654

Recently, a study challenged the notion that basal vascular NO generation is derived from eNOS. Intra-arterial infusion of S-methyl-L-thiocitrulline (SMTC), a nNOS inhibitor that has a 17-fold selectivity over eNOS, was used in 48 healthy male volunteers (28  1.1 years of age) to investigate the role of nNOS-derived NO in the local regulation of basal blood flow and acetylcholine-mediated vasodilation in vivo (Seddon et al., 2008). Local infusion of SMTC into the brachial artery of healthy males resulted in a significant dose-dependent

Nitrite infusion

Ipsilateral

Contralateral

Measurements • forearm blood flow • plasma nitrite • whole blood nitrite • blood pressure

Figure 3 Nitrite infusion in humans. Schematic protocol of nitrite infusion performed at doses from 0, 7, 14, 28, and 55 to 110 g  kg1  min1, as described by Dejam et al. (2007). After each dose was infused, blood was drawn from both veins to measure plasma and whole blood nitrite, blood pressure, and heart rate. Forearm blood flow was measured in the opposite arms.

Chapter 20 NO in Vascular Damage and Regeneration

reduction in basal blood flow. The reduction in resting blood flow induced by SMTC at an estimated local concentration of 1.25–10 mmol/L was completely abolished in the presence of excess L-arginine but was unaffected by D-arginine, which indicates that the effects of SMTC were mediated by stereospecific inhibition of the L-arginine/NO pathway. Thus, NO generation by nNOS plays a role in the regulation of basal vasomotor tone and in the control of blood pressure. Instead, eNOS-generated NO facilitates dynamic alterations in blood flow distribution and possesses antiatherosclerotic effects at endothelium level (Seddon et al., 2008).

Conclusions and road ahead It is clear from the evidence brought forth that NO is a critical modulator of vascular disease via its effects on vascular tone, endothelial regeneration, attenuation of inflammation, and inhibition of platelet adhesion. Reduced bioavailability of NO, due to impaired release or enhanced degradation, underlies many forms of human disease. Its central position in the regulation of organ physiology and molecular signaling generates the impetus to augment its levels in an attempt to interfere with the pathophysiological cascade that leads to tissue dysfunction and destruction. Although our understanding of the function of this molecule has increased tremendously over the past few years, future studies need to further establish the specific role of the NOS isoforms in regulation of function and in disease process, and explore interventions to selectively increase their expression and function. Obviously, NO regulates not only vascular function, but also many levels of parenchymal function in organs like the kidney, liver, brain, and lung. Preferably, modulation of NO availability by increased release or decreased degradation should be done in a spatially specific manner that would correspond to areas in which a specific isoform plays a regulatory role. Indeed, it is likely that several concurrent interventions would be necessary to guarantee that the delicate balance that characterizes NO availability and function is maintained.

Acknowledgments Joint studies from the writing group were supported in part by grants from the PRIN MIUR 2006 code 0622153_002 “Meccanismi fisiopatologici di danno vascolare/trombotico ed angiogenesi” (C.N.), Funds from Regione Campania- del. N° 2308 (29/12/07)- BURC N°6-2008 “Valutazione innovativa di biomarkers implicati nella vulnerabilità tromboembolica della placca aterosclerotica” (C.N.), and research grants from the Mayo Clinic Foundation (L.O.L.).

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