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2017; 3(7):38–46


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Implication of eNOS Uncoupling in Cardiovascular Disease


Ning Xia1, Ulrich Förstermann1, and Huige Li1,2,3

1Department of Pharmacology, Johannes Gutenberg University Medical Center, Mainz, Germany; 2Center for Translational Vascular Biology (CTVB), Johannes Gutenberg University Medical Center, Mainz, Germany; 3German Center for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Mainz, Germany

Correspondence: (H.L.)

Xia N et al. Reactive Oxygen Species 3(7):38–46, 2017; ©2017 Cell Med Press

(Received: December 16, 2016; Accepted: December 23, 2016)

ABSTRACT | Under physiological conditions, nitric oxide (NO) is produced in the vasculature mainly by the endothelial nitric oxide synthase (eNOS). Endothelial NO relaxes blood vessels, inhibits platelet activity, and protects against atherosclerosis. Under pathological conditions such as hypertension, diabetes, and hypercholesterolemia, eNOS may become uncoupled. Uncoupled eNOS generates superoxide at the expense of NO and contributes substantially to oxidative stress and endothelial dysfunction. Major mechanisms of eNOS uncoupling include deficiency of the eNOS cofactor tetrahydrobiopterin, deficiency of the eNOS substrate L-arginine, and eNOS S-glutathionylation. Reversal of eNOS uncoupling may represent a feasible strategy for the prevention and treatment of cardiovascular diseases.

KEYWORDS | Cardiovascular disease; eNOS uncoupling; Nitric oxide; Oxidative stress; Reactive oxygen species

ABBREVIATIONS | ACE, angiotensin-converting enzyme; ApoE-KO, apolipoprotein E-knockout; ARB, angiotensin AT1 receptor blocker; BH2, 7,8-dihydrobiopterin; BH4, tetrahydrobiopterin; CAD, coronary artery disease; DHRF, dihydrofolate reductase; eNOS, endothelial nitric oxide synthase; LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein; PETN, pentaerythritol tetranitrate; ROS, reactive oxygen species; STZ, streptozotocin


1. The Phenomenon of eNOS Uncoupling

2. Molecular Mechanisms of eNOS Uncoupling

3. Uncoupling of eNOS in Cardiovascular Disease

3.1. Hypertension

3.2. Hypercholesterolemia

3.3. Diabetes Mellitus

4. Therapeutic Strategies

5. Conclusion


Under physiological conditions, nitric oxide (NO) is produced in the blood vessel mainly by the endothelial NO synthase (eNOS) [1‒3]. Endothelial NO processes multiple vasoprotective properties, including vasodilation, inhibition of platelet aggregation and adhesion, and anti-atherosclerotic effects [1‒3]. In addition, eNOS-derived NO has antioxidant activities by abating Fenton-type reactions, terminating radical chain reactions, and inhibiting peroxidases and oxidases through S-nitrosylation of allosteric thiols [4].

Under pathological conditions associated with oxidative stress, however, eNOS may become dysfunctional, producing superoxide at the expense of NO. This phenomenon is referred to as eNOS uncoupling [5, 6]. Uncoupling of eNOS is not an all-or-none phenomenon. Rather, uncoupled and coupled eNOS proteins may exist in the same cell at the same time [6, 7], as shown in the hypercholesterolemic apolipoprotein E-knockout (ApoE-KO) mice [8]. In this murine model of atherosclerosis, the protective role of NO derived from coupled eNOS overwhelms the detrimental effect of superoxide produced by uncoupled eNOS [8]. This may be an explanation for the observations that genetic deletion of eNOS  [8‒10] and pharmacological inhibition [11] of (both the coupled and the uncoupled) eNOS accelerate atherosclerosis development, despite the existence of eNOS uncoupling in these animals.


Numerous mechanisms have been implicated in eNOS uncoupling [2, 3, 12]. Among these, depletion of tetrahydrobiopterin (BH4), an essential cofactor for the eNOS enzyme, is likely to play a major role in eNOS uncoupling and endothelial dysfunction. Peroxynitrite and superoxide can oxidize BH4 to 7,8-dihydrobiopterin (BH2), leading to BH4 deficiency [13]. BH2 competes with BH4 for eNOS binding, but has no cofactor activity. BH2 can be reduced back to BH4 by the enzyme dihydrofolate reductase (DHFR) [1, 2].

Another important cause of eNOS uncoupling is a deficiency of eNOS substrate L-arginine, mostly due to upregulation of arginase expression/activity. Arginases metabolize L-arginine to urea and L-ornithine [14]. The expression/activity of vascular arginases is enhanced by diverse stimuli [15], including angiotensin II [16], high glucose [17], thrombin [18], oxidative species [19], and oxidized low-density lipoprotein (oxLDL) [20]. An increased arginase expression/activity decreases L-arginine bioavailability for eNOS, and can lead to eNOS uncoupling.

Recently, S-glutathionylation has been identified as another crucial mechanism for eNOS uncoupling [21]. S-Glutathionylation is a posttranslational modification in which a glutathione tripeptide is reversibly bound to the protein via the formation of a disulfide bond with a protein thiol [12]. S-Glutathionylation of cysteine residues in the reductase domain (Cys689 and Cys908) [21] shifts eNOS from an NO-generating enzyme to a superoxide producer. This mechanism has been implicated in eNOS uncoupling under conditions of aging [22], ambient ultrafine particles exposure [23], organic nitrate-induced endothelial dysfunction [24, 25], angiotensin II-induced vascular dysfunction [26‒28], in vessels of hypertension rats [21], in hyperglycemia and experimental diabetes [29, 30] and in response to carbamylated LDL [31]. It is noteworthy that eNOS uncoupling induced by BH4 deficiency and by S-glutathionylation is mechanistically independent of each other. However, they are functionally linked and act in concert to regulate NO or superoxide production by eNOS [32]. BH4 deficiency leads to superoxide production by the oxygenase domain which in turn decreases the ratio of reduced form of glutathione (GSH) to glutathione disulfide (GSSG) and thereby initiates eNOS S-glutathionylation and eNOS uncoupling. Vice versa, S-glutathionylation triggers superoxide production from the reductase domain which then oxidizes BH4, and thus results in the superoxide production from the oxygenase domain [32].

The consequences of eNOS uncoupling are reduced NO production and augmentation of pre-existing oxidative stress by overproduction of reactive oxygen species (ROS) such as superoxide and subsequently peroxynitrite. This potentiation then leads to enhanced oxidation of BH4, upregulation of arginase expression/activity, and S-glutathionylation of eNOS, creating a vicious circle (Figure 1). Hence, eNOS uncoupling is a key mechanism in and contributes substantially to endothelial dysfunction and cardiovascular disease.

FIGURE 1. Uncoupling of eNOS in cardiovascular disease. Cardiovascular risk factors such as hypertension, hypercholesterolemia and diabetes mellitus promote superoxide production by eNOS (eNOS uncoupling) through three major mechanisms: depletion of the eNOS cofactor tetrahydrobiopterin (BH4), depletion of the eNOS substrate L-arginine, and eNOS S-glutathionylation. NADPH oxidase-derived reactive oxygen species (e.g., superoxide and subsequently peroxynitrite) oxidize BH4 leading to BH4 deficiency. L-Arginine deficiency is caused by upregulation of arginase expression/activity. Oxidative stress-induced reduction in GSH:GSSG ratio favors eNOS S-glutathionylation. Uncoupled eNOS produces reactive oxygen species, which in turn oxidize the BH4, increase arginase expression and activity, and enhance eNOS S-glutathionylation, creating a vicious circle. GSH and GSSG denote reduced form of glutathione and glutathione disulfide, respectively.


All established cardiovascular risk factors, such as hypertension, hypercholesterolemia, and diabetes mellitus, enhance oxidative stress and induce eNOS uncoupling [6, 33].

3.1. Hypertension

Uncoupled eNOS contributes substantially to vascular oxidative stress in hypertension [6, 33]. BH4 deficiency, L-arginine deficiency, and S-glutathionylation have been shown as molecular mechanisms for eNOS uncoupling in animal models of hypertension, including angiotensin II-induced hypertension, spontaneously hypertensive rats (an animal model of genetic hypertension) and deoxycorticosterone acetate-salt (DOCA-salt) hypertension. The deficiency of BH4 has been attributed to NADPH oxidase-mediated of BH4 oxidation [34] and to reduced BH4 recycling from BH2 due to a downregulation of endothelial dihydrofolate reductase (DHFR) [35]. L-Arginine deficiency in hypertension models is likely to result from an upregulation of arginase expression/activity in blood vessels [36‒38]. Uncoupling of eNOS by S-glutathionylation is evident in angiotensin II-induced hypertension [26, 27] and in vessels of hypertension rats [21]. Reversal of eNOS uncoupling reduces blood pressure in hypertensive animals [39] or contributes to blood pressure reduction by some antihypertensive drugs [28].

In hypertensive patients, intra-arterial infusion of BH4 augments forearm blood flow response to acetylcholine [40]. Oral BH4 administration improves endothelial function and reduces blood pressure in human subjects with essential hypertension [41], indicating the relevance of BH4 deficiency and eNOS uncoupling in human hypertension.

3.2. Hypercholesterolemia

Both native LDL and oxLDL have been shown to stimulate superoxide/peroxynitrite production, and to uncouple eNOS [42, 43]. ROS production from uncoupled eNOS has been shown in LDL-treated endothelial cells, in hypercholesterolemic ApoE-KO mice [44], and in hypercholesterolemic patients [45]. Hypercholesterolemia leads to BH4 oxidation and BH4 deficiency [44]. In addition, L-arginine deficiency also represents a cause of eNOS uncoupling in hypercholesterolemia. An upregulation of arginase expression and/or activity has been shown in ApoE-KO mice [46, 47] and in hyperlipidemic rabbits [48]. The aortic arginase activity in ApoE-KO mice is significantly reduced after the removal of the endothelium, suggesting an important contribution of endothelial cells [46]. The functional relevance of arginase upregulation in atherosclerosis has been shown in ApoE-KO mice. Selective endothelial overexpression of arginase 2 induces endothelial dysfunction and enhances atherosclerosis in mice [49]. Treatment with an arginase inhibitor for 4 or 8 weeks reduces aortic plaque burden in ApoE-KO mice [46].

Vascular (but not plasma) BH4 content has been shown to be an important determinant of eNOS uncoupling and superoxide production in vessels isolated from patients with coronary artery disease (CAD) [50]. BH4 restores endothelial function in patients with hypercholesterolemia [45]. Serum levels of carbamylated LDL are elevated in patients with CAD [51, 52] and carbamylated LDL-induced eNOS uncoupling by S-glutathionylation has been recently proposed to be a molecular mechanism contributing to the pathogenesis of atherosclerosis [31].

3.3. Diabetes Mellitus

Uncoupling of eNOS has been observed in streptozotocin (STZ)-induced type 1 diabetes mellitus [53]. The underlying mechanisms involve NADPH oxidase-mediated BH4 oxidation. Indeed, BH4 oxidation and BH4 deficiency are evident in STZ-treated mice [54] and rats [55]. In addition, diabetes also causes BH4 deficiency by reducing BH4 synthesis. Enhanced ROS production in diabetes accelerates proteasomal degradation of guanosine 5′-triphosphate cyclohydrolase 1 (GCH1), a rate-limiting enzyme in the biosynthesis of BH4 [29, 56, 57]. Moreover, eNOS S-glutathionylation represents another important mechanism of eNOS uncoupling in the setting of type 1 diabetes [29].

In mouse models of type 2 diabetes, a relative BH4 deficiency is evident due to enhanced BH4 oxidation and a low BH4:BH2 ratio [58‒60]. The increased levels of angiotensin II in diabetic patients may additionally reduce DHFR expression and decrease BH4 recycling from BH2 [35].

L-Arginine deficiency and eNOS uncoupling have also been documented in rodent models of type 1 [17, 61‒63] as well as type 2 diabetes [64]. High glucose and persistent insulin stimulation upregulate arginase expression in endothelial cells [17, 62, 65].

In patients with type 2 diabetes mellitus, the reduced forearm blood flow response to acetylcholine is significantly improved by BH4, an effect that can be blocked by NOS inhibition [66]. In contrast, BH4 has no effect in healthy controls [66]. These results indicate that BH4 deficiency and eNOS uncoupling play a role in diabetes mellitus-induced vascular dysfunction.

Plasma arginase activity is elevated in patients with type 2 diabetes mellitus [67]. An upregulation of arginase 1 in coronary arterioles of patients with (type 1 or type 2) diabetes mellitus has been shown to contribute to the reduced NO production and consequently diminished vasodilation [68]. Arginase inhibition markedly improves endothelium-dependent vasodilation in the forearm of patients with type 2 diabetes mellitus and CAD whereas it does not affect endothelial function in healthy controls [69]. This observation indicates a functional role of arginase contributing to endothelial dysfunction in patients with diabetes.


Uncoupling of eNOS plays a crucial role in endothelial dysfunction. On this account, it is an important objective of cardiovascular disease treatment to prevent eNOS uncoupling or to reverse an existing eNOS uncoupling. Since molecular mechanisms underlying eNOS uncoupling are more and more understood, various pharmacological approaches, which aim at the prevention of eNOS uncoupling, have been successfully studied in animal models [6, 7]. Examples are angiotensin-converting enzyme (ACE) inhibitors, angiotensin AT1-receptor blockers (ARBs), the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins), the organic nitrate pentaerythritol tetranitrate (PETN), and the plant polyphenolic phytoalexin resveratrol (for details see our recent review articles [6, 7, 33]). These compounds prevent BH4 oxidation partly by inhibiting NADPH oxidase expression or activity. Some of these compounds, such as ARB and PETN, also upregulate DHFR and hereby enhance BH4 regeneration from BH2 [6, 7]. Others, such as statins [17, 18, 70, 71], ACE inhibitors [72], and ARBs [16], additionally inhibit arginase activity which in turn leads to an improved eNOS functionality. It has been demonstrated that ACE inhibitors [28], ARBs [24], and PETN [29] additionally prevent eNOS S-glutathionylation. The enhanced NO bioavailability, which comes along with these treatment approaches, is part of the pleiotropic effects that contribute to their therapeutic benefit.

The health impact of long-term L-arginine supplementation is currently under debate [14]. Two clinical studies have shown that chronic L-arginine supplementation is not beneficial and can even be potentially harmful [73, 74]. In contrast, small-scale “proof-of-concept” clinical studies have shown that local administration of arginase inhibitors improves vascular function in aged humans [75] as well as in patients with CAD and type 2 diabetes mellitus [69], heart failure [76], and hypertension [77]. Larger clinical studies with systemic arginase inhibition are warranted [15, 78].


Uncoupling of eNOS represents a major mechanism for the reduced NO production, enhanced oxidative stress, and endothelial dysfunction in cardiovascular disease. Reversal of eNOS uncoupling may represent a feasible strategy for the prevention and treatment of cardiovascular diseases.


Original work from the authors’ laboratory contributing to this review was supported by the DFG (LI-1042/1-1 and LI-1042/3-1), and intramural fund (Stufe I) of the Johannes Gutenberg University Medical Center, Mainz, Germany.


  1. Li H, Forstermann U. Nitric oxide in the pathogenesis of vascular disease. J Pathol 2000; 190(3):244‒54. doi: 10.1002/(SICI)1096-9896(200002)190:3<244::AID-PATH575>3.0.CO;2-8.
  2. Li H, Forstermann U. Prevention of atherosclerosis by interference with the vascular nitric oxide system. Curr Pharm Des 2009; 15(27):3133‒45.
  3. Forstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J 2012; 33(7):829‒37, 37a-37d. doi: 10.1093/eurheartj/ehr304.
  4. Hare JM, Stamler JS. NO/redox disequilibrium in the failing heart and cardiovascular system. J Clin Invest 2005; 115(3):509‒17. doi: 10.1172/JCI24459.
  5. Forstermann U, Munzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation 2006; 113(13):1708‒14. doi: 10.1161/CIRCULATIONAHA.105.602532.
  6. Li H, Forstermann U. Uncoupling of endothelial NO synthase in atherosclerosis and vascular disease. Curr Opin Pharmacol 2013; 13(2):161‒7. doi: 10.1016/j.coph.2013.01.006.
  7. Li H, Forstermann U. Pharmacological prevention of eNOS uncoupling. Curr Pharm Des 2014; 20(22):3595‒606. doi: 10.2174/13816128113196660749
  8. Ponnuswamy P, Schrottle A, Ostermeier E, Gruner S, Huang PL, Ertl G, et al. eNOS protects from atherosclerosis despite relevant superoxide production by the enzyme in apoE mice. PLoS One 2012; 7(1):e30193. doi: 10.1371/journal.pone.0030193.
  9. Kuhlencordt PJ, Gyurko R, Han F, Scherrer-Crosbie M, Aretz TH, Hajjar R, et al. Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase double-knockout mice. Circulation 2001; 104(4):448‒54.
  10. Chen J, Kuhlencordt PJ, Astern J, Gyurko R, Huang PL. Hypertension does not account for the accelerated atherosclerosis and development of aneurysms in male apolipoprotein e/endothelial nitric oxide synthase double knockout mice. Circulation 2001; 104(20):2391‒4.
  11. Kauser K, da Cunha V, Fitch R, Mallari C, Rubanyi GM. Role of endogenous nitric oxide in progression of atherosclerosis in apolipoprotein E-deficient mice. Am J Physiol Heart Circ Physiol 2000; 278(5):H1679‒85.
  12. Zweier JL, Chen CA, Druhan LJ. S-Glutathionylation reshapes our understanding of endothelial nitric oxide synthase uncoupling and nitric oxide/reactive oxygen species-mediated signaling. Antioxid Redox Signal 2011; 14(10):1769‒75. doi: 10.1089/ars.2011.3904.
  13. Laursen JB, Somers M, Kurz S, McCann L, Warnholtz A, Freeman BA, et al. Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation 2001; 103(9):1282‒8.
  14. Yang Z, Ming XF. Arginase: the emerging therapeutic target for vascular oxidative stress and inflammation. Front Immunol 2013; 4:149. doi: 10.3389/fimmu.2013.00149.
  15. Pernow J, Jung C. Arginase as a potential target in the treatment of cardiovascular disease: reversal of arginine steal? Cardiovasc Res 2013; 98(3):334‒43. doi: 10.1093/cvr/cvt036.
  16. Shatanawi A, Romero MJ, Iddings JA, Chandra S, Umapathy NS, Verin AD, et al. Angiotensin II-induced vascular endothelial dysfunction through RhoA/Rho kinase/p38 mitogen-activated protein kinase/arginase pathway. Am J Physiol Cell Physiol 2011; 300(5):C1181‒92. doi: 10.1152/ajpcell.00328.2010.
  17. Romero MJ, Platt DH, Tawfik HE, Labazi M, El-Remessy AB, Bartoli M, et al. Diabetes-induced coronary vascular dysfunction involves increased arginase activity. Circ Res 2008; 102(1):95‒102. doi: 10.1161/CIRCRESAHA.107.155028.
  18. Ming XF, Barandier C, Viswambharan H, Kwak BR, Mach F, Mazzolai L, et al. Thrombin stimulates human endothelial arginase enzymatic activity via RhoA/ROCK pathway: implications for atherosclerotic endothelial dysfunction. Circulation 2004; 110(24):3708‒14. doi: 10.1161/01.CIR.0000142867.26182.32.
  19. Chandra S, Romero MJ, Shatanawi A, Alkilany AM, Caldwell RB, Caldwell RW. Oxidative species increase arginase activity in endothelial cells through the RhoA/Rho kinase pathway. Br J Pharmacol 2012; 165(2):506‒19. doi: 10.1111/j.1476-5381.2011.01584.x.
  20. Ryoo S, Lemmon CA, Soucy KG, Gupta G, White AR, Nyhan D, et al. Oxidized low-density lipoprotein-dependent endothelial arginase II activation contributes to impaired nitric oxide signaling. Circ Res 2006; 99(9):951‒60. doi: 10.1161/01.RES.0000247034.24662.b4.
  21. Chen CA, Wang TY, Varadharaj S, Reyes LA, Hemann C, Talukder MA, et al. S-Glutathionylation uncouples eNOS and regulates its cellular and vascular function. Nature 2010; 468(7327):1115‒8. doi: 10.1038/nature09599.
  22. Oelze M, Kroeller-Schoen S, Steven S, Lubos E, Doppler C, Hausding M, et al. Glutathione peroxidase-1 deficiency potentiates dysregulatory modifications of endothelial nitric oxide synthase and vascular dysfunction in aging. Hypertension 2014; 63(2):390‒6. doi: 10.1161/HYPERTENSIONAHA.113.01602.
  23. Du Y, Navab M, Shen M, Hill J, Pakbin P, Sioutas C, et al. Ambient ultrafine particles reduce endothelial nitric oxide production via S-glutathionylation of eNOS. Biochem Biophys Res Commun 2013; 436(3):462‒6. doi: 10.1016/j.bbrc.2013.05.127.
  24. Knorr M, Hausding M, Kroller-Schuhmacher S, Steven S, Oelze M, Heeren T, et al. Nitroglycerin-induced endothelial dysfunction and tolerance involve adverse phosphorylation and S-Glutathionylation of endothelial nitric oxide synthase: beneficial effects of therapy with the AT1 receptor blocker telmisartan. Arterioscler Thromb Vasc Biol 2011; 31(10):2223‒31. doi: 10.1161/ATVBAHA.111.232058.
  25. Oelze M, Knorr M, Kroller-Schon S, Kossmann S, Gottschlich A, Rummler R, et al. Chronic therapy with isosorbide-5-mononitrate causes endothelial dysfunction, oxidative stress, and a marked increase in vascular endothelin-1 expression. Eur Heart J 2013; 34(41):3206‒16. doi: 10.1093/eurheartj/ehs100.
  26. Kroller-Schon S, Steven S, Kossmann S, Scholz A, Daub S, Oelze M, et al. Molecular mechanisms of the crosstalk between mitochondria and NADPH oxidase through reactive oxygen species-studies in white blood cells and in animal models. Antioxid Redox Signal 2014; 20(2):247‒66. doi: 10.1089/ars.2012.4953.
  27. Kossmann S, Hu H, Steven S, Schoenfelder T, Fraccarollo D, Mikhed Y, et al. Inflammatory monocytes determine endothelial nitric-oxide synthase uncoupling and nitro-oxidative stress induced by angiotensin II. J Biol Chem 2014; 289(40):27540‒50. doi: 10.1074/jbc.M114.604231.
  28. Galougahi KK, Liu CC, Gentile C, Kok C, Nunez A, Garcia A, et al. Glutathionylation mediates angiotensin II-induced eNOS uncoupling, amplifying NADPH oxidase-dependent endothelial dysfunction. J Am Heart Assoc 2014; 3(2):e000731. doi: 10.1161/JAHA.113.000731.
  29. Schuhmacher S, Oelze M, Bollmann F, Kleinert H, Otto C, Heeren T, et al. Vascular dysfunction in experimental diabetes is improved by pentaerithrityl tetranitrate but not isosorbide-5-mononitrate therapy. Diabetes 2011; 60(10):2608‒16. doi: 10.2337/db10-1395.
  30. Karimi Galougahi K, Liu CC, Garcia A, Gentile C, Fry NA, Hamilton EJ, et al. Beta3 Adrenergic stimulation restores nitric oxide/redox balance and enhances endothelial function in hyperglycemia. J Am Heart Assoc 2016; 5(2). doi: 10.1161/JAHA.115.002824.
  31. Speer T, Owala FO, Holy EW, Zewinger S, Frenzel FL, Stahli BE, et al. Carbamylated low-density lipoprotein induces endothelial dysfunction. Eur Heart J 2014; 35(43):3021‒32. doi: 10.1093/eurheartj/ehu111.
  32. Crabtree MJ, Brixey R, Batchelor H, Hale AB, Channon KM. Integrated redox sensor and effector functions for tetrahydrobiopterin- and glutathionylation-dependent endothelial nitric-oxide synthase uncoupling. J Biol Chem 2013; 288(1):561‒9. doi: 10.1074/jbc.M112.415992.
  33. Li H, Horke S, Forstermann U. Oxidative stress in vascular disease and its pharmacological prevention. Trends Pharmacol Sci 2013; 34(6):313‒9. doi: 10.1016/
  34. Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, et al. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest 2003; 111(8):1201‒9.
  35. Chalupsky K, Cai H. Endothelial dihydrofolate reductase: critical for nitric oxide bioavailability and role in angiotensin II uncoupling of endothelial nitric oxide synthase. Proc Natl Acad Sci USA 2005; 102(25):9056‒61. doi: 10.1073/pnas.0409594102.
  36. Demougeot C, Prigent-Tessier A, Bagnost T, Andre C, Guillaume Y, Bouhaddi M, et al. Time course of vascular arginase expression and activity in spontaneously hypertensive rats. Life Sci 2007; 80(12):1128‒34. doi: 10.1016/j.lfs.2006.12.003.
  37. Rodriguez S, Richert L, Berthelot A. Increased arginase activity in aorta of mineralocorticoid-salt hypertensive rats. Clin Exp Hypertens 2000; 22(1):75‒85.
  38. Johnson FK, Johnson RA, Peyton KJ, Durante W. Arginase inhibition restores arteriolar endothelial function in Dahl rats with salt-induced hypertension. Am J Physiol Regul Integr Comp Physiol 2005; 288(4):R1057‒62. doi: 10.1152/ajpregu.00758.2004.
  39. Li H, Witte K, August M, Brausch I, Godtel-Armbrust U, Habermeier A, et al. Reversal of endothelial nitric oxide synthase uncoupling and up-regulation of endothelial nitric oxide synthase expression lowers blood pressure in hypertensive rats. J Am Coll Cardiol 2006; 47(12):2536‒44. doi: 10.1016/j.jacc.2006.01.071.
  40. Higashi Y, Sasaki S, Nakagawa K, Fukuda Y, Matsuura H, Oshima T, et al. Tetrahydrobiopterin enhances forearm vascular response to acetylcholine in both normotensive and hypertensive individuals. Am J Hypertens 2002; 15(4 Pt 1):326‒32.
  41. Porkert M, Sher S, Reddy U, Cheema F, Niessner C, Kolm P, et al. Tetrahydrobiopterin: a novel antihypertensive therapy. J Hum Hypertens 2008; 22(6):401‒7. doi: 10.1038/sj.jhh.1002329.
  42. Pritchard KA, Jr., Groszek L, Smalley DM, Sessa WC, Wu M, Villalon P, et al. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ Res 1995; 77(3):510‒8.
  43. Stepp DW, Ou J, Ackerman AW, Welak S, Klick D, Pritchard KA, Jr. Native LDL and minimally oxidized LDL differentially regulate superoxide anion in vascular endothelium in situ. Am J Physiol Heart Circ Physiol 2002; 283(2):H750‒9. doi: 10.1152/ajpheart.00029.2002.
  44. Xia N, Daiber A, Habermeier A, Closs EI, Thum T, Spanier G, et al. Resveratrol reverses endothelial nitric-oxide synthase uncoupling in apolipoprotein E knockout mice. J Pharmacol Exp Ther 2010; 335(1):149‒54. doi: 10.1124/jpet.110.168724.
  45. Stroes E, Kastelein J, Cosentino F, Erkelens W, Wever R, Koomans H, et al. Tetrahydrobiopterin restores endothelial function in hypercholesterolemia. J Clin Invest 1997; 99(1):41‒6. doi: 10.1172/JCI119131.
  46. Ryoo S, Gupta G, Benjo A, Lim HK, Camara A, Sikka G, et al. Endothelial arginase II: a novel target for the treatment of atherosclerosis. Circ Res 2008; 102(8):923‒32. doi: 10.1161/CIRCRESAHA.107.169573.
  47. Erdely A, Kepka-Lenhart D, Salmen-Muniz R, Chapman R, Hulderman T, Kashon M, et al. Arginase activities and global arginine bioavailability in wild-type and ApoE-deficient mice: responses to high fat and high cholesterol diets. PLoS One 2010; 5(12):e15253. doi: 10.1371/journal.pone.0015253.
  48. Hayashi T, Esaki T, Sumi D, Mukherjee T, Iguchi A, Chaudhuri G. Modulating role of estradiol on arginase II expression in hyperlipidemic rabbits as an atheroprotective mechanism. Proc Natl Acad Sci USA 2006; 103(27):10485‒90. doi: 10.1073/pnas.0603918103.
  49. Vaisman BL, Andrews KL, Khong SM, Wood KC, Moore XL, Fu Y, et al. Selective endothelial overexpression of arginase II induces endothelial dysfunction and hypertension and enhances atherosclerosis in mice. PLoS One 2012; 7(7):e39487. doi: 10.1371/journal.pone.0039487.
  50. Antoniades C, Shirodaria C, Crabtree M, Rinze R, Alp N, Cunnington C, et al. Altered plasma versus vascular biopterins in human atherosclerosis reveal relationships between endothelial nitric oxide synthase coupling, endothelial function, and inflammation. Circulation 2007; 116(24):2851‒9. doi: 10.1161/CIRCULATIONAHA.107.704155.
  51. Ok E, Basnakian AG, Apostolov EO, Barri YM, Shah SV. Carbamylated low-density lipoprotein induces death of endothelial cells: a link to atherosclerosis in patients with kidney disease. Kidney Int 2005; 68(1):173‒8. doi: 10.1111/j.1523-1755.2005.00391.x.
  52. Wang Z, Nicholls SJ, Rodriguez ER, Kummu O, Horkko S, Barnard J, et al. Protein carbamylation links inflammation, smoking, uremia and atherogenesis. Nat Med 2007; 13(10):1176‒84. doi: 10.1038/nm1637.
  53. Hink U, Li H, Mollnau H, Oelze M, Matheis E, Hartmann M, et al. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res 2001; 88(2):E14‒22.
  54. Alp NJ, Mussa S, Khoo J, Cai S, Guzik T, Jefferson A, et al. Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I overexpression. J Clin Invest 2003; 112(5):725‒35. doi: 10.1172/JCI17786.
  55. Faria AM, Papadimitriou A, Silva KC, Lopes de Faria JM, Lopes de Faria JB. Uncoupling endothelial nitric oxide synthase is ameliorated by green tea in experimental diabetes by re-establishing tetrahydrobiopterin levels. Diabetes 2012; 61(7):1838‒47. doi: 10.2337/db11-1241.
  56. Wenzel P, Daiber A, Oelze M, Brandt M, Closs E, Xu J, et al. Mechanisms underlying recoupling of eNOS by HMG-CoA reductase inhibition in a rat model of streptozotocin-induced diabetes mellitus. Atherosclerosis 2008; 198(1):65‒76. doi: 10.1016/j.atherosclerosis.2007.10.003.
  57. Xu J, Wu Y, Song P, Zhang M, Wang S, Zou MH. Proteasome-dependent degradation of guanosine 5′-triphosphate cyclohydrolase I causes tetrahydrobiopterin deficiency in diabetes mellitus. Circulation 2007; 116(8):944‒53. doi: 10.1161/CIRCULATIONAHA.106.684795.
  58. Pannirselvam M, Simon V, Verma S, Anderson T, Triggle CR. Chronic oral supplementation with sepiapterin prevents endothelial dysfunction and oxidative stress in small mesenteric arteries from diabetic (db/db) mice. Br J Pharmacol 2003; 140(4):701‒6. doi: 10.1038/sj.bjp.0705476.
  59. Pannirselvam M, Verma S, Anderson TJ, Triggle CR. Cellular basis of endothelial dysfunction in small mesenteric arteries from spontaneously diabetic (db/db -/-) mice: role of decreased tetrahydrobiopterin bioavailability. Br J Pharmacol 2002; 136(2):255‒63. doi: 10.1038/sj.bjp.0704683.
  60. Shinozaki K, Nishio Y, Okamura T, Yoshida Y, Maegawa H, Kojima H, et al. Oral administration of tetrahydrobiopterin prevents endothelial dysfunction and vascular oxidative stress in the aortas of insulin-resistant rats. Circ Res 2000; 87(7):566‒73.
  61. Romero MJ, Iddings JA, Platt DH, Ali MI, Cederbaum SD, Stepp DW, et al. Diabetes-induced vascular dysfunction involves arginase I. Am J Physiol Heart Circ Physiol 2012; 302(1):H159‒66. doi: 10.1152/ajpheart.00774.2011.
  62. Yao L, Chandra S, Toque HA, Bhatta A, Rojas M, Caldwell RB, et al. Prevention of diabetes-induced arginase activation and vascular dysfunction by Rho kinase (ROCK) knockout. Cardiovasc Res 2013; 97(3):509‒19. doi: 10.1093/cvr/cvs371.
  63. Toque HA, Tostes RC, Yao L, Xu Z, Webb RC, Caldwell RB, et al. Arginase II deletion increases corpora cavernosa relaxation in diabetic mice. J Sex Med 2011; 8(3):722‒33. doi: 10.1111/j.1743-6109.2010.02098.x.
  64. Gronros J, Jung C, Lundberg JO, Cerrato R, Ostenson CG, Pernow J. Arginase inhibition restores in vivo coronary microvascular function in type 2 diabetic rats. Am J Physiol Heart Circ Physiol 2011; 300(4):H1174‒81. doi: 10.1152/ajpheart.00560.2010.
  65. Giri H, Muthuramu I, Dhar M, Rathnakumar K, Ram U, Dixit M. Protein tyrosine phosphatase SHP2 mediates chronic insulin-induced endothelial inflammation. Arterioscler Thromb Vasc Biol 2012; 32(8):1943‒50. doi: 10.1161/ATVBAHA.111.239251.
  66. Heitzer T, Krohn K, Albers S, Meinertz T. Tetrahydrobiopterin improves endothelium-dependent vasodilation by increasing nitric oxide activity in patients with type II diabetes mellitus. Diabetologia 2000; 43(11):1435‒8. doi: 10.1007/s001250051551.
  67. Kashyap SR, Lara A, Zhang R, Park YM, DeFronzo RA. Insulin reduces plasma arginase activity in type 2 diabetic patients. Diabetes Care 2008; 31(1):134‒9. doi: 10.2337/dc07-1198.
  68. Beleznai T, Feher A, Spielvogel D, Lansman SL, Bagi Z. Arginase 1 contributes to diminished coronary arteriolar dilation in patients with diabetes. Am J Physiol Heart Circ Physiol 2011; 300(3):H777‒83. doi: 10.1152/ajpheart.00831.2010.
  69. Shemyakin A, Kovamees O, Rafnsson A, Bohm F, Svenarud P, Settergren M, et al. Arginase inhibition improves endothelial function in patients with coronary artery disease and type 2 diabetes mellitus. Circulation 2012; 126(25):2943‒50. doi: 10.1161/CIRCULATIONAHA.112.140335.
  70. Ryoo S, Bhunia A, Chang F, Shoukas A, Berkowitz DE, Romer LH. OxLDL-dependent activation of arginase II is dependent on the LOX-1 receptor and downstream RhoA signaling. Atherosclerosis 2011; 214(2):279‒87. doi: 10.1016/j.atherosclerosis.2010.10.044.
  71. Holowatz LA, Santhanam L, Webb A, Berkowitz DE, Kenney WL. Oral atorvastatin therapy restores cutaneous microvascular function by decreasing arginase activity in hypercholesterolaemic humans. J Physiol 2011; 589(Pt 8):2093‒103. doi: 10.1113/jphysiol.2010.203935.
  72. Kosenko E, Tikhonova L, Suslikov A, Kaminsky Y. Impacts of lisinopril and lisinopril plus simvastatin on erythrocyte and plasma arginase, nitrite, and nitrate in hypertensive patients. J Clin Pharmacol 2012; 52(1):102‒9. doi: 10.1177/0091270010388647.
  73. Schulman SP, Becker LC, Kass DA, Champion HC, Terrin ML, Forman S, et al. L-Arginine therapy in acute myocardial infarction: the Vascular Interaction With Age in Myocardial Infarction (VINTAGE MI) randomized clinical trial. JAMA 2006; 295(1):58‒64. doi: 10.1001/jama.295.1.58.
  74. Wilson AM, Harada R, Nair N, Balasubramanian N, Cooke JP. L-Arginine supplementation in peripheral arterial disease: no benefit and possible harm. Circulation 2007; 116(2):188‒95. doi: 10.1161/CIRCULATIONAHA.106.683656.
  75. Holowatz LA, Thompson CS, Kenney WL. L-Arginine supplementation or arginase inhibition augments reflex cutaneous vasodilatation in aged human skin. J Physiol 2006; 574(Pt 2):573‒81. doi: 10.1113/jphysiol.2006.108993.
  76. Quitter F, Figulla HR, Ferrari M, Pernow J, Jung C. Increased arginase levels in heart failure represent a therapeutic target to rescue microvascular perfusion. Clin Hemorheol Microcirc 2013; 54(1):75‒85. doi: 10.3233/CH-2012-1617.
  77. Holowatz LA, Kenney WL. Up-regulation of arginase activity contributes to attenuated reflex cutaneous vasodilatation in hypertensive humans. J Physiol 2007; 581(Pt 2):863‒72. doi: 10.1113/jphysiol.2007.128959.
  78. Caldwell RB, Toque HA, Narayanan SP, Caldwell RW. Arginase: an old enzyme with new tricks. Trends Pharmacol Sci 2015; 36(6):395‒405. doi: 10.1016/