Fig. 16.1
Antihypertensive, antithrombotic, and antiatherosclerotic effects of endothelial nitric oxide synthase (eNOS). The eNOS enzyme in endothelial cells (ECs) can be activated by shear stress or agonists such as bradykinin (BK) and vascular endothelial growth factor (VEGF). Endothelial NO diffuses into the blood and inhibits platelet aggregation and adhesion, as well as leukocyte adhesion to the vascular endothelium and leukocyte migration into the vascular wall. NO diffused into smooth muscle cells (SMC) induces vasodilation and prevents SMC proliferation
NO produced by eNOS can diffuse from endothelial cells into the underlying smooth muscle cells (SMCs), and can induce vasodilation by stimulating NO-sensitive guanylyl cyclase. Endothelial NO can also diffuse into the blood and inhibit platelet aggregation and adhesion (Fig. 16.1). In addition to these antihypertensive and antithrombotic actions, eNOS-derived NO also possesses multiple antiatherosclerotic properties, including prevention of leukocyte adhesion to the vascular endothelium and leukocyte migration into the vascular wall, inhibition of low-density lipoprotein (LDL) oxidation, and inhibition of vascular SMC proliferation [49, 51]. Genetic depletion of eNOS exacerbates diet-induced atherosclerosis in the apolipoprotein E-knockout (ApoE-KO) mouse model. The blood pressure of eNOS knockout mice is approximately 30 % higher than that of wild-type animals [58]. Recent studies suggest that eNOS is also involved in mitochondrial biogenesis, anti-aging effects, and extension of lifespan in mammals [11, 65].
16.2.2 Inducible NOS (iNOS)
iNOS is normally absent in the vasculature. However, under conditions of inflammation, sepsis, or oxidative stress, iNOS expression can be induced in the endothelium, the media, and/or the adventitia of blood vessels [25, 69].
In contrast to the regulated production of NO by eNOS, iNOS may generate large amounts of NO over long periods of time if substrate and co-factors are not limited. This excessive NO production by iNOS leads to vascular dysfunction, evident as impairment of both vasoconstriction and endothelium-dependent vasorelaxation. Several mechanisms have been proposed by which iNOS impairs contractile responses [105], including continuous activation of the soluble guanylyl cyclase [25], abnormal vascular calcium regulation [10], and oxidative modification of catecholamines [88]. In parallel, the endothelium-dependent, NO-mediated vasodilation response (e.g. to acetylcholine or bradykinin) is impaired by iNOS. This may be the result of reduced NO production from eNOS [25, 40] or enhanced inactivation of eNOS-derived NO by superoxide [100]. Tetrahydrobiopterin (BH4) is an essential co-factor for NO production by NOS enzymes. iNOS expressed in the endothelium competes with eNOS for BH4 and reduces NO production from eNOS by limiting BH4 availability for eNOS [25]. The continuous generation of NO by iNOS induced in the vascular wall can impair the signal transduction cascade that links activation of endothelial receptors to the calcium–calmodulin-dependent activation of eNOS [40]. Moreover, the reduction of endothelium-dependent relaxation may be mediated in part by reduced reactivity of SMCs to NO [17]. The consequence of such dysregulations (impaired vasomotor reactivity to both vasoconstrictor and vasodilator agonists) can be seen, for example, in septic shock. Septic shock is characterized by massive arteriolar vasodilatation, hypotension, and microvascular damage largely mediated by iNOS. Inappropriate vasodilation, abnormal regulation of blood flow to organs, myocardial suppression, and interference with cellular respiration all contribute to hypotension and mortality in septic shock [22, 58].
The induction of iNOS in the vasculature is also associated with enhanced formation of peroxynitrite [59, 100, 106], a key pathogenic mechanism in conditions such as septic shock, stroke, myocardial infarction, chronic heart failure, diabetes, and atherosclerosis [57, 66]. iNOS is present in human atherosclerosis plaque. Genetic deletion of iNOS reduces atherosclerosis in ApoE-KO mice [43]. iNOS also contributes to tissue damage after cerebral ischemia. Inhibition of iNOS by selective pharmacologic inhibitors [34], or gene deletion of iNOS [33], reduces brain damage .
16.2.3 Neuronal NOS (nNOS)
nNOS plays an important role in blood vessels independently of its effects in the central nervous system [61]. nNOS is expressed in perivascular nerve fibers as well as in the vascular wall [84, 96]. Using the selective nNOS inhibitor S-methyl-L-thiocitrulline (SMTC), it has been found that nNOS inhibition leads to a dose-dependent reduction in basal forearm blood flow in human subjects [86]. Importantly, the acetylcholine- [86] and flow-induced [85] vasodilation, effects that are mediated by eNOS, is not affected by SMTC. A similar situation has been observed in human coronary circulation, where SMTC causes a significant reduction in basal coronary blood flow without any effect on increases in flow evoked by intracoronary substance P [85]. In addition, nNOS may play a role in the regulation of mental stress-induced vasodilatation [86] and in the cutaneous blood flow increase in response to whole-body heat stress [39].
In addition to these human studies, animal experiments suggest an atheroprotective role of nNOS in the vasculature. Gene deletion of nNOS in ApoE-KO mice accelerates atherosclerotic plaque formation in the aortic root and descending thoracic aorta [45, 80]. An increase in mortality has also been observed nNOS/ApoE double knockout mice after 24 weeks of Western-type diet [45]. These results are compatible with earlier studies showing accelerated neointimal formation and constrictive vascular remodeling in nNOS-KO mice in a carotid artery ligation model [64, 96].
16.3 Molecular Mechanisms of eNOS Uncoupling
Under physiological conditions, eNOS produces NO, which represents a key element in the vasoprotective function of the endothelium [22, 51, 52]. However, under pathological conditions associated with oxidative stress, eNOS may become dysfunctional [21, 53, 54]. Oxidative stress contributes markedly to endothelial dysfunction , primarily due to rapid oxidative inactivation of NO by excess superoxide. In a second step, the persisting oxidative stress renders eNOS uncoupled (i.e. uncoupling of O2 reduction from NO synthesis), such that it no longer produces NO but superoxide.
Numerous mechanisms have been proposed to play a role in eNOS uncoupling [22, 51]. Among these, depletion of BH4, an essential co-factor for the eNOS enzyme, is likely to be a major cause for eNOS uncoupling and endothelial dysfunction. Superoxide can modestly, and peroxynitrite strongly, oxidize BH4, leading to BH4 deficiency [48]. ApoE-KO mice show increased oxidative degradation of BH4 and eNOS uncoupling in cardiovascular tissues [1, 103, 104]. Evidence for BH4 deficiency and eNOS uncoupling has been obtained in patients with endothelial dysfunction resulting from hypercholesterolemia [93] or diabetes mellitus [28], and in chronic smokers [97].
Another important cause of eNOS uncoupling is a deficiency of L-arginine due to upregulation of arginase expression/activity. In humans and mammals, there are two isoforms of arginases: arginase I (Arg-I) and arginase II (Arg-II), which are encoded by two separate genes [109]. Both Arg-I and Arg-II have been found in the vasculature, with a dependency on species and cell type [70]. Studies performed with vascular endothelial cells suggest that these two isoforms share similar functions, i.e. metabolizing L-arginine to urea and L-ornithine, whereby an upregulation of Arg-I and/or Arg-II limits L-arginine bioavailability for NO production, leading to endothelial dysfunction [109]. Selective endothelial overexpression of Arg-II induces endothelial dysfunction and hypertension and enhances atherosclerosis in mice [99]. Aging is associated with upregulation of Arg-II, enhanced vascular arginase activity, and eNOS uncoupling [41, 111]. The uncoupling of eNOS under conditions of aging can be reversed by arginase inhibitors [41] or by genetic deletion of Arg-II [111]. The expression/activity of vascular arginases is enhanced by a variety of stimuli [70], including angiotensin II [87] or high glucose [76], thrombin [62], and oxidized LDL [79].
Uncoupling of eNOS is a crucial mechanism contributing significantly to endothelial dysfunction and atherogenesis. It not only reduces NO production but also potentiates the pre-existing oxidative stress. The overproduction of reactive oxygen species (ROS) [e.g. superoxide and subsequently peroxynitrite] by uncoupled eNOS in turn enhances oxidation of BH4 and upregulation of arginase expression/activity [8], creating a vicious circle .
16.4 Uncoupling of eNOS in Diabetes Mellitus
The production of eNOS-derived NO is reduced in diabetes and insulin resistance. The implicated mechanisms include inhibition of eNOS activity by post-translational modification (i.e. enhancement of protein kinase C [PKC]-mediated phosphorylation of eNOS at threonine 495 [94], reduction in phosphorylation of eNOS at serine 1177 [15], and increase in O-linked N-acetylglucosamine modification of eNOS [15]) and eNOS uncoupling due to deficiency of BH4 or L-arginine [50].
16.4.1 BH4 Deficiency in Diabetes
In the rat model of streptozotocin-induced type 1 diabetes mellitus, eNOS is uncoupled in blood vessels [29]. The major cause for this eNOS uncoupling is BH4 oxidation due to PKC-mediated activation and upregulation of NADPH oxidase [29]. Indeed, BH4 oxidation and BH4 deficiency is evident in streptozotocin-treated mice [2], as well as in streptozotocin-treated diabetic spontaneously hypertensive rats [19].
Diabetes-induced ROS may cause proteasomal degradation of guanosine 5ʹ-triphosphate cyclohydrolase-1 (GCH1), a rate-limiting enzyme in the synthesis of BH4 [108], which may subsequently lead to BH4 deficiency [81, 101, 108]. In addition, S-glutathionylation may represent another important trigger of eNOS uncoupling in the setting of type 1 diabetes [81].
In spontaneously diabetic db/db mice, an animal model of type 2 diabetes that lacks the leptin receptor, the acetylcholine-induced relaxation of isolated small mesenteric arteries is reduced. Although the absolute vascular BH4 level is unchanged, a relative BH4 deficiency is evident due to enhanced BH4 oxidation and a low BH4:BH2 ratio [67]. Endothelial dysfunction can be improved by acute ex vivo incubation with BH4 or with sepiapterin, a stable precursor of BH4 [68]. Fructose-fed rats show insulin resistance with endogenous hyperinsulinemia. Aortic BH4 contents of fructose-fed rats are decreased, whereas the levels of BH2 are increased. Impaired endothelial function can be reversed by ex vivo preincubation of aortic strips with BH4 [90] or by oral BH4 supplementation [91]. These results indicate that BH4 deficiency represents a major cause for endothelial dysfunction in type 2 diabetes or insulin resistance (Fig. 16.2).
Fig. 16.2
Uncoupling of endothelial NOS (eNOS) in cardiovascular disease. Cardiovascular risk factors such as hypertension, hypercholesterolemia, or diabetes mellitus lead to superoxide production by eNOS (‘eNOS uncoupling’) through two major mechanisms—deficiency of the co-factor tetrahydrobiopterin (BH 4 ) or the substrate L-arginine. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived superoxide (O 2 ●−) may react with nitric oxide (NO), resulting in peroxynitrite (ONOO−). ONOO− oxidizes BH4 to dihydrobiopterin (BH 2 ). BH4 deficiency can be exacerbated by downregulation of GTP cyclohydrolase-1 (GCH1, the rate-limiting enzyme for BH4 de novo biosynthesis) or dihydrofolate reductase (DHFR, the enzyme required for BH4 regeneration from BH2). L-arginine deficiency is caused by upregulation of arginase expression and/or activity. Uncoupled eNOS produces reactive oxygen species (e.g. superoxide and subsequently peroxynitrite) , which in turn oxidize BH4 and increase arginase expression and activity, creating a vicious circle
16.4.2 L-Arginine Deficiency in Diabetes
The reduced NO production from eNOS under conditions of diabetes may also be caused by L-arginine deficiency due to induction of arginases. High glucose upregulates Arg-I in (bovine and murine) endothelial cells by stimulating the RhoA-ROCK pathway [76, 110], whereas persistent insulin stimulation (mimicking the hyperinsulinemia condition) in human endothelial cells upregulates the expression and activity of Arg-II via a signaling cascade involving SHP2 phosphorylation and p38 mitogen-activated protein kinase (MAPK) activation [23]. Increased arginase expression/activity decreases L-arginine bioavailability for eNOS on the one hand, and on the other, L-arginine depletion may also lead to eNOS uncoupling (Fig. 16.2).
In aortas from streptozotocin-induced diabetic rats [76] or mice [75, 110], Arg-I expression is enhanced, which is associated with endothelial dysfunction. Aortic Arg-II expression is either barely detectable (rats) or not changed by streptozotocin-induced diabetes (mice). Diabetes-induced coronary vascular dysfunction involves increased arginase activity, with a major involvement of Arg-I [75, 76]. Enhanced arginase activity in the aorta or coronary artery of streptozotocin-induced diabetic animals is believed to induce eNOS uncoupling [75, 76]. Diabetic Arg-I+/−Arg-II−/− mice exhibit less arginase activity/expression and less endothelial dysfunction than diabetic wild-type or Arg-I+/+Arg-II−/− mice, indicating that Arg-I is likely to be the primary arginase isoform involved in type 1 diabetes-induced vascular dysfunction [75]. In contrast, Arg-II seems to be the primary arginase isoform upregulated in corpus cavernosum tissue of streptozotocin-treated mice, and Arg-II deletion improves corpora cavernosal relaxation in type 1 diabetes [95].
In type 2 diabetic Goto-Kakizaki rats, coronary artery microvascular dysfunction is associated with increased Arg-II expression. Arginase inhibition restores coronary microvascular function in type 2 diabetic rats by a mechanism related to increased utilization of arginine by NOS and increased NO bioavailability [24].
In patients with type 2 diabetes mellitus, plasma arginase activity is elevated [37]. An upregulation of Arg-I in coronary arterioles of patients with (type 1 or type 2) diabetes mellitus has been shown to contribute to reduced NO production and consequently diminished vasodilation [4]. Arginase inhibition markedly improves endothelium-dependent vasodilatation in the forearm of patients with type 2 diabetes and coronary artery disease, whereas it does not affect endothelial function in healthy controls [89]. This observation indicates a functional role of arginase contributing to endothelial dysfunction in patients with diabetes.
16.5 Uncoupling of eNOS in Hypertension
Endothelial dysfunction and eNOS uncoupling has been demonstrated in different types of hypertension, including animal models of genetic hypertension (spontaneously hypertensive rats, SHR), angiotensin II-induced hypertension, and deoxycorticosterone acetate (DOCA)-salt hypertension (summarized in Li and Forstermann [50]).
16.5.1 BH4 Deficiency in Hypertension
BH4 deficiency has been shown to be a major mechanism for eNOS uncoupling in DOCA-salt hypertension. NADPH oxidase-mediated oxidation of BH4 is evident in the aorta of DOCA-salt hypertensive mice as BH4 oxidation and BH4 deficiency are absent in mice lacking p47phox, a critical component of the NADPH oxidase enzyme complex. Oral BH4 treatment reverses eNOS uncoupling and prevents hypertension development in DOCA-salt mice [47]. Consistently, endothelium-specific overexpression of GCH1 attenuates blood pressure progression in this model of salt-sensitive, low-renin hypertension [16]. In contrast, administration of sepiapterin is not effective in recoupling eNOS in DOCA-salt hypertension. Because of downregulation of endothelial sepiapterin reductase, sepiapterin cannot be converted to BH4 [112].
In spontaneously hypertensive rats, the expression of vascular NADPH oxidase components is enhanced [50]. BH4 content is decreased and eNOS in an uncoupled state in SHR aorta [56]. Suppression of NADPH oxidase with gp91ds-tat decreases ROS production in SHR to the level of control Wistar-Kyoto rats (WKY) [113]. Pharmacological reversal of eNOS uncoupling by midostaurin [56] or resveratrol [5] results in a reduction in blood pressure in spontaneously hypertensive rats.
Angiotensin II-induced hypertension is also associated with eNOS uncoupling [63]. Angiotensin II activates vascular PKC, which leads to enhanced expression [63] and activity [6] of vascular NADPH oxidase (Fig. 16.2). Indeed, NADPH oxidases play a crucial role in angiotensin II-induced hypertension. Angiotensin II-induced elevation in blood pressure and production of superoxide are significantly blunted in mice deficient of the NADPH oxidase components Nox1 [60] or p47phox [46], and are potentiated in mice with Nox1 overexpression [13].
BH4 deficiency represents a major cause for angiotensin II-induced eNOS uncoupling. Angiotensin II reduces the expression of GCH1 and thus BH4 de novo synthesis [82], as well as that of dihydrofolate reductase (DHFR) [7], which catalyzes the regeneration of BH4 from its oxidized form, BH2. Oral supplementation with BH4 restores NO/cGMP signaling in small arteries, and attenuates angiotensin II-induced hypertension in rats [36].
16.5.2 L-Arginine Deficiency in Hypertension
In addition to BH4 deficiency, the endothelial dysfunction in hypertension may be partially attributable to reduced NO production by eNOS because of L-arginine deficiency. Upregulation of arginase expression/activity in blood vessels has been observed in spontaneously hypertensive rats [12], mineralocorticoid-salt hypertensive rats [74], and Dahl rats with salt-induced hypertension [35]. The mechanisms underlying the upregulation of arginase in hypertension are not completely understood. Angiotensin II has been shown to increase endothelial arginase activity/expression through AT1 receptors and subsequent activation of RhoA/ROCK/p38 MAPK pathways, leading to endothelial dysfunction [87]. Upregulation of arginase expression/activity is of functional relevance to blood pressure development. Selective endothelial overexpression of Arg-II induces endothelial dysfunction and hypertension in mice [99]. Treatment with arginase inhibitors improves vascular function and lowers blood pressure in spontaneously hypertensive rats [3].
In patients with hypertension, reflex cutaneous vasodilatation is augmented by arginase inhibitors via skin microdialysis catheters [30]. Antihypertensive treatment with the angiotensin-converting enzyme (ACE) inhibitor lisinopril reduced erythrocyte arginase activity in patients with arterial hypertension [42].
16.6 Uncoupling of eNOS in Atherosclerosis
In 1995, it was shown for the first time that LDL enhances superoxide production from eNOS [72]. Thereafter, evidence for eNOS uncoupling has been obtained in hypercholesterolemic mice [48] and patients [93].
16.6.1 BH4 Deficiency in Atheorsclerosis
Hypercholesterolemic ApoE-KO mice show increased oxidative degradation of BH4 [1], leading to eNOS uncoupling [1, 103, 104]. Supplementation with BH4 restores endothelial dysfunction in hypercholesterolemic patients [93]. BH4 deficiency due to oxidative stress is likely to be a major cause for eNOS uncoupling under these conditions. Diet-induced hypercholesterolemia in rabbits is associated with enhanced NADPH oxidase activity and reduced superoxide dismutase (SOD) activity in the vasculature [98]. Oxidized LDL enhances endothelial superoxide production by stimulating NADPH oxidase [27, 92]. Native LDL and oxidized LDL may also activate endothelial xanthine oxidase [92].
It should be noted that eNOS uncoupling is not an all-or-none phenomenon. Rather, uncoupled eNOS molecules and coupled eNOS proteins may exist in the same cell at the same time [50, 53]. Because of relative BH4 deficiency, part of the eNOS molecules become uncoupled, while others may still remain coupled. In the hypercholesterolemic ApoE-KO mice, for instance, both superoxide and NO production by eNOS are detectable [71]. Moreover, the damaging effects of superoxide produced by uncoupled eNOS do not overwhelm the protective role of eNOS-derived NO, at least in this mouse model of atherosclerosis [71]. For this reason, genetic deletion of eNOS [9, 44, 71] or pharmacological inhibition [38] of both the coupled and uncoupled eNOS, accelerates atherosclerosis development in ApoE-KO mice.
16.6.2 L-Arginine Deficiency in Atherosclerosis
In addition to BH4 deficiency, upregulation of arginase expression and/or activity has been shown to contribute to reduced endothelial NO production in experimental models of atherosclerosis, including the ApoE-KO mice (Arg-II) [18, 78] and in hyperlipidemic rabbits (Arg-I and Arg-II) [26]. The aortic arginase activity in ApoE-KO mice is significantly reduced after the removal of the endothelium, suggesting an important contribution from endothelial cells [78]. The functional relevance of arginase upregulation in atherosclerosis has been shown in ApoE-KO mice. Selective endothelial overexpression of Arg-II induces endothelial dysfunction and enhances atherosclerosis in mice [99]. Chronic treatment with an arginase inhibitor for 4 or 8 weeks reduces aortic plaque burden in ApoE-KO mice [78].
The RhoA/ROCK pathway seems to play a central role in the upregulation of arginase expression and activity under conditions of atherosclerosis (Fig. 16.2). Thrombin enhances the activity of Arg-II in human umbilical vein endothelial cells (HUVECs; Arg-I is not detectable in these cells) without changing the protein level of the enzyme [62]. This effect is associated with an upregulation of RhoA and is preventable by statins or ROCK inhibitors [62]. Oxidized LDL not only stimulates arginase enzymatic activity (after 5 min) but also enhances Arg-II protein levels (after 12 h) [79]. The activation of Arg-II by oxidized LDL is mediated by the lectin-like oxidized LDL (LOX-1) receptor and subsequent RhoA/ROCK-dependent microtubule depolymerization, leading to a dissociation of Arg-II from the mitochondria to the cytosol where it limits NO synthesis by eNOS [77]. In bovine aortic endothelial cells, oxidative species (such as peroxynitrite and hydrogen peroxide) increase Arg-I expression and activity through PKC-dependent activation of the RhoA-ROCK pathway [8].
In humans with hypercholesterolemia, the reduced skin blood flow responses are associated with enhanced expression and activity of Arg-I and Arg-II [31]. Statin treatment has no effect of the expression levels of the arginase enzymes but restores the functional vasodilator properties by inhibiting arginase activity [31].
16.7 Pharmacological Prevention of eNOS Uncoupling
Because of eNOS uncoupling under conditions of cardiovascular disease , pharmacological upregulation of eNOS expression alone will not be beneficial because this would lead to enhanced ROS production by the uncoupled eNOS. In fact, many cardiovascular diseases are associated with compensatory upregulation of eNOS expression; however, the compensation is futile, or even harmful, because the eNOS is in an uncoupled state [51, 55].
Therefore, it is essential to prevent eNOS uncoupling, or reverse an existing eNOS uncoupling, for the treatment of cardiovascular disease. Fortunately, eNOS uncoupling is a reversible event. With the growing understanding of molecular mechanisms underlying eNOS uncoupling, numerous pharmacological strategies to prevent eNOS uncoupling have been successfully tested in animal models. These include the ACE inhibitors, AT1 receptor blockers (ARBs), 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins), the third-generation β-blocker nebivolol, the organic nitrate pentaerythritol tetranitrate (PETN), the plant polyphenolic phytoalexin resveratrol, and some small-molecular-weight eNOS transcription enhancers (for details see our recent review articles [50, 53, 54]). These compounds prevent BH4 oxidation by inhibiting NADPH oxidase expression/activity. Some drugs additionally improve BH4 regeneration from BH2 by upregulating DHFR (e.g. ARBs and PETN), or enhance eNOS enzymatic activity (e.g. ARBs, statins, nebivolol, resveratrol) [50, 53]. Statins [31, 62, 76, 77] and ACE inhibitors [42] also improve eNOS functionality by inhibiting arginase activity. The improvement of NO bioavailability represents part of the pleiotropic effects of these drugs that contribute to their therapeutic benefit.
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