Author's Accepted Manuscript
Oxidative Stress and Endothelial Dysfunction: Clinical Evidence and Therapeutic Implications Yukihito Higashi MD, PhD, FAHA, FJSH, Tatsuya Maruhashi MD, PhD, Kensuke Noma MD, PhD, Yasuki Kihara MD, PhD
www.elsevier.com/locate/tcm
PII: DOI: Reference:
S1050-1738(13)00164-3 http://dx.doi.org/10.1016/j.tcm.2013.12.001 TCM5982
To appear in: trends in cardiovascular medicine
Cite this article as: Yukihito Higashi MD, PhD, FAHA, FJSH, Tatsuya Maruhashi MD, PhD, Kensuke Noma MD, PhD, Yasuki Kihara MD, PhD, Oxidative Stress and Endothelial Dysfunction: Clinical Evidence and Therapeutic Implications, trends in cardiovascular medicine, http://dx.doi.org/ 10.1016/j.tcm.2013.12.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Oxidative Stress and Endothelial Dysfunction: Clinical Evidence and Therapeutic Implications Brief title: Antioxidant Property and Endothelial Function 1,2
Yukihito Higashi, MD, PhD, FAHA, FJSH; 3Tatsuya Maruhashi, MD, PhD; 1,2
1
Kensuke Noma, MD, PhD; 3Yasuki Kihara, MD, PhD
Department of Cardiovascular Regeneration and Medicine, Research Institute for
Radiation Biology and Medicine (RIRBM), Hiroshima University, Hiroshima, Japan 2
Division of Regeneration and Medicine, Medical Center for Translational and Clinical Research, Hiroshima University Hospital, Hiroshima University, Hiroshima, Japan 3
Department of Cardiovascular Medicine, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan Address for correspondence: Yukihito Higashi, MD, PhD, FAHA Department of Cardiovascular Regeneration and Medicine,
Research Institute for Radiation Biology and Medicine (RIRBM), Hiroshima University 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan Phone: +81-82-257-5831
Fax: +81-82-257-5831
E-mail: [email protected] Total word: 1733 (abstract: 154; 4 sentences) Number of Figure: 2; Number of Table: 1 Number of Reference: 39 Abstract An imbalance of nitric oxide (NO) and reactive oxygen species (ROS), so-called “oxidative stress”, may promote endothelial dysfunction, leading to cardiovascular complications. Activation of nicotinamide-adenine dinucleotide phosphate oxidase, xanthine oxidase, cyclooxygenase, and mitochondrial electron transport, inactivation of the antioxidant system and uncoupling of endothelial NO synthase lead to oxidative stress along with an increase in ROS production and decrease in ROS degradation. Although experimental studies, both in vitro and in vivo, have shown a critical role of
2
oxidative stress in endothelial dysfunction under the condition of excessive oxidative stress, there is little information on whether oxidative stress is really involved in endothelial function in humans. In a clinical setting, we showed an association between oxidative stress and endothelial function, especially in patients with renovascular hypertension as a model of increased oxidative stress and in patients with Gilbert syndrome as a model of decreased oxidative stress, through an increase in the antioxidant property unconjugated bilirubin. Introduction A healthy endothelium maintains vascular function and structure by balance of vasodilation and vasoconstriction, growth inhibition and growth promotion, antithrombosis and prothrombosis, anti-inflammation and proinflammation, and also antioxidation and prooxidation (Vallance et al. 1989; Vanhoutte 1989). Endothelial dysfunction is the initial step in the pathogenesis of arteriosclerosis, resulting in cardiovascular complications (Ross 1999). Several lines of evidence have clearly shown an association between coronary or peripheral endothelial function and cardiovascular events (Heitzer et al. 2001; Schachinger et al. 2000). Endothelial dysfunction is independently associated with cardiovascular events (Lerman and Zeiher 2005). The mechanisms for oxidative stress-induced endothelial dysfunction in cardiovascular diseases have been postulated. However, it is unclear whether these mechanisms work in humans. Renovascular hypertension is caused by renal artery stenosis, leading to stimulation of the renin-angiotensin system and increased production of its main active peptide, angiotensin II (Ang II), resulting in production of reactive oxygen species (ROS). Patients with renovascular hypertension are ideal models for determining how endothelium-dependent vasodilation is affected by excess Ang II and an Ang II-related increase in oxidative stress. In addition, it has been shown that although bilirubin at a high concentration acts as a cytotoxic metabolite, bilirubin, particularly unconjugated bilirubin, at a low concentration is a potent endogenous antioxidant (Kapitulnik 2004). Patients with Gilbert syndrome have mild unconjugated hyperbilirubinemia (Gilbert 1901). Therefore, Gilbert syndrome is an ideal model for determining how endothelium-dependent vasodilation is affected by bilirubin-related decrease in oxidative stress.
3
In this review, we take particular note of recent findings and the relationships between antioxidant property, oxidative stress, and endothelial function in humans. Is oxidative stress really involved in endothelial function in humans? In various pathophysiological states, activation of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase (XO), cyclooxygenase, and uncoupled endothelial nitric oxide synthase (eNOS) with tetrahydrobiopterin (BH4) oxidation and mitochondrial electron transport as well as inactivation of the antioxidant system, including superoxide dismutase, glutathione peroxidase and catalase, lead to an increase in ROS production and decrease in ROS degradation. (Cai and Harrison 2000; Rodriguez-Crespo and Gerber 1996; Kaufman 1993). An imbalance of reduced production of NO or increased production of ROS promotes endothelial dysfunction, leading to remodeling, platelet aggregation, loss of vasodilation, inflammation, and smooth muscle cell growth (Touyz 2004; Guzik et al. 2000; Sowers 2002). ROS directly inhibit NO activity (Cai and Harrison 2000). In addition, ROS activate the PI3K/ras/Akt/MAPK pathway, related to redox transcriptional factors, leading to redox gene expression, which results in inhibition of eNOS mRNA expression and eNOS activity. Indeed, several lines of evidence have shown, both in vitro and in vivo, the critical role of oxidative stress in endothelial function under the condition of excessive oxidative stress (Cai and Harrison 2000; Münzel et al. 2010; Virdis et al. 2013). However, the condition of excessive oxidative stress in experimental studies may have no application in humans, even in patients with cardiovascular disease. Patients with renovascular hypertension: NADPH oxidase NADPH oxidase is a multisubunit complex composed of cytosolic components, such as p47phox, p67phox and Rac 1, and membrane-spanning components, such as p22phox and gp91phox. NADPH oxidase should be the most important source of ROS in the vasculature (Dzau 2001). In an atherosclerotic state, Ang II-induced activation of NADPH oxidase is one of the major sources of ROS (Cai and Harrison 2000; Touyz 2004; Guzik et al. 2000; Dzau 2001). Upregulation of p22phox mRNA expression is a key component of Ang II-induced NADPH oxidase activation, and increased expression levels of other components also play an important role in NADPH oxidase under pathological conditions (Cai and Harrison 2000). Patients with renovascular hypertension have excess Ang II and Ang II-related increase in oxidative stress (Figure 1) (Higashi et al. 2002a). Renal angioplasty
4
decreased
plasma
renin
activity,
plasma
AngII
concentration,
and
serum
malondialdehyde-modified low-density lipoprotein (MDA-LDL) concentration and urinary
8-hydroxy-2’-deoxyguanosine
(8-OHdG)
excretion
in
patients
with
renovascular hypertension. After renal angioplasty, forearm blood flow response to acetylcholine was enhanced in these patients. Co-infusion of the antioxidant vitamin C augmented acetylcholine-induced vasodilation before angioplasty but not after angioplasty. The increase in maximal acetylcholine-induced vasodilation was associated with the decrease in urinary excretion of 8-OHdG and the decrease in serum concentration of MDA-LDL. These findings suggest that endothelial function is impaired in relation to the severity of oxidative stress in a clinical setting. Patients with Gilbert syndrome: bilirubin It is well known that ascorbic acid (vitamin C), urobilinogen, glutathione, tocopherol (vitamin E) ubiqunone (coenzyme Q10), -carotene, and lycopene are endogenous antioxidant properties in humans (Table 1). Unconjugated bilirubin is also a potent antioxidant (Stocker et al. 1987; Abraham and Kappas; 2008). Bilirubin is an end product of the heme oxygenase-1 (HO-1)/Bach1 pathway (Figure 2) (Muto et al. 2004; Sun et al. 2002). In brief, in the heme oxygenase-1 (HO-1) promoter region, there are two enhancer sequences called E1 and E2. In the basal state, HO-1 expression is turned off as Bach1 binds to these enhancer sequences, forming heterodimers with small Maf proteins. HO-1 activators including Nrf2 are not accessible to E1 and E2 because the Bach1 heterodimer has higher affinity to the sequence. In cases of cellular injury, including oxidative stress, it is thought that intracellular heme level is increased. These hemes strongly associate with Bach1, causing the Bach1 heterodimer to dissociate from DNA, which in turn allows transcriptonal activators to access E1 and E2, and thereby HO-1 expression is activated. Inducible HO-1 catalyzes the cleavage of heme to free iron, carbon monoxide and biliverdin. Biliverdin is rapidly converted to unconjugated bilirubin by biliverdin reductase. Unconjugated bilirubin is changed to the conjugated form of bilirubin through the uridine-diphosphate-glucuronosyltransferase isoform 1A (UGT1A). In patients with Gilbert syndrome, enzymatic activity of UGT1A1 is reduced by mutation of its gene, which is located on human chromosome 2 (Bosma et al. 1995). Therefore, conversion of unconjugated bilirubin to conjugated bilirubin is decreased, resulting in hyperbilirubinemia. Therefore, Gilbert syndrome is an ideal model for
5
determining how endothelium-dependent vasodilation is affected by bilirubin-related decrease in oxidative stress. The incidence of Gilbert syndrome in the general population is 5% to 10% (Owens and Evans 1975; Sieg et al. 1987). We evaluated the role of oxidative stress in endothelial function in patients with Gilbert syndrome under normal conditions without cardiovascular risk factors (Maruhashi et al. 2012). A total of 108 young men with Gilbert syndrome without cardiovascular risk factors and 108 age-matched healthy men (normal controls) were enrolled. Gilbert syndrome was diagnosed by the following criteria: mild unconjugated hyperbilirubinemia (1.2 to 6.0 mg/dL), normal values of hepatic biochemical tests, absence of liver disease or overt hemolysis, UGT1A activity of less than 35% of normal, and results of real-time PCR detection of UGT1A*28 mutation (Lin et al. 2006). In control subjects, the upper limit of the normal range for serum concentrations of bilirubin was 1.0 mg/dL. Serum concentration of bilirubin was significantly higher in patients with Gilbert syndrome than in control subjects. Serum concentration of MDALDL and urinary excretion of 8-OHdG were significantly lower in patients with Gilbert syndrome than in control subjects. There was no significant difference in other parameters between the two groups. Flow-mediated vasodilation (FMD) was significantly greater in patients with Gilbert syndrome than in normal control subjects. FMD correlated with serum concentrations of bilirubin and MDA-LDL and with urinary excretion of 8-OHdG in patients with Gilbert syndrome but not in control subjects. In addition, serum concentration of bilirubin correlated with MDA-LDL and urinary excretion of 8-OHdG in these patients. Patients with Gilbert syndrome had low levels of oxidative stress associated with hyperbilirubinemia and enhancement of endothelium-dependent vasodilation. These beneficial effects on the vasculature may contribute to reduced prevalence of vascular complications in atherosclerotic patients with Gilbert syndrome compared with that in atherosclerotic patients without Gilbert syndrome (Inoguchi et al. 2007). It is likely that superoxide plays an important role in vascular function even under a normal condition without cardiovascular risk factors. XO/uric acid In humans, the antioxidant uric acid circulates in greater concentrations than all other antioxidants (Table 1). Uric acid is an end product from xanthine through XO, which is one of the enzymatic sources of ROS, in higher mammals. Several investigators have shown that an XO inhibitor improves endothelial function in patients with
6
hyperuricemia, probably through a decrease in XO-induced ROS production (Yiginer et al. 2008; George et al. 2006). On the other hand, it has been reported that uric acid enhances the production of ROS by activation of the local renin-angiotensin system, particularly Ang II, in human endothelial vascular cells (Yu et al. 2010). Thus, uric acid is thought to mediate endothelial dysfunction by generating oxidative stress once absorbed into endothelial cells. Interestingly, although uric acid at a concentration of over 6 mg/dL induced generation of ROS and senescence and apoptosis of endothelial cells, uric acid at a concentration of 3 mg/dL had no effect on endothelial cells (Vítek et al. 2002). These findings suggest that XO is an exacerbating factor of endothelial dysfunction and that there may be a certain threshold for uric acid levels before proatherogenic properties become apparent. eNOS uncoupling/BH4 BH4 is an allosteric factor in the coupling of the oxidase and reductase domains of eNOS (Rodriguez-Crespo et al. 1996). While eNOS activation at optimal levels of BH4 leads to production of NO, under conditions of BH4 deficiency, electron flow from the reductase domain to the oxidase domain is diverted to molecular oxygen rather than to L-arginine, a substrate of NO, leading to eNOS uncoupling, which results in generation of ROS rather than NO (Kuzkaya et al. 2003). In addition, it has been reported that degradation of BH4 by ROS is associated with down-regulation of eNOS (Stoes et al. 1997). In clinical studies, supplementation of BH4 improved endothelial function in smokers, in patients with diabetes, hypertension, hypercholesterolemia, and chronic heart failure, and in elderly subjects (Setoguchi et al. 2003; Higashi et al. 2002; Higashi et al. 2006; Hayden and Tyagi 2003; Moat and McDowell 2005). Infusion of a NOS inhibitor abolished the BH4-induced enhancement of forearm vasorelaxation evoked by acetylcholine in patients with hypertension (Higashi et al. 2002b). These findings suggest that deficiency and/or oxidation of BH4 may contribute to endothelial dysfunction.
Clinical perspectives Oxidative stress plays a critical role in endothelial function and atherosclerosis in humans. Oxidative stress-related endothelial dysfunction should be a therapeutic target for atherosclerosis. Future studies are needed to evaluate the direct effects of exogenous and endogenous antioxidants on endothelial function and the prevention of
7
cardiovascular morbidity and mortality for long-term periods. Acknowledgments This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, Japan Heart Foundation Grant for Research on Hypertension and Metabolism, and a Grant for Research Foundation for Community Medicine.
Conflict of Interest None. Figure Legends Figure 1. Endothelial function and oxidative stress in patients with renovascular hypertension. (A) Circulating angiotensin II levels before and after angioplasty. (B) Circulating oxidative stress markers, urinary excretion of 8-hydroxy-2’-deoxyguanosine (8-OHdG) and serum malondialdehyde-modified low-density lipoprotein (MDA-LDL) concentration before and after angioplasty. (C) Effects of concomitant administration of the antioxidant vitamin C on forearm blood flow response to acetylcholine administration before and after angioplasty. (D) Correlations of the maximal response of forearm blood flow to administration of acetylcholine with urinary excretion of 8OHdG and serum MDA-LDL concentration before and after angioplasty. Modified from Ref. (Higashi et al. 2002a). Figure 2. Heme oxygenase-1 (HO-1)/Bach1/bilirubin pathway with and without oxidative stress. Unconjugated bilirubin is changed to the conjugated form of bilirubin through the uridine-diphosphate-glucuronosyltransferase isoform 1A (UGT1A). In patients with Gilbert syndrome, enzymatic activity of UGT1A1 is reduced by mutation of its gene.
8
References Abraham NG, Kappas A. Pharmacological and clinical aspects of heme oxygenase. Pharmacol Rev. 2008; 60: 79-127. Bosma PJ, Chowdhury JR, Bakker C, et al. The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert's syndrome. N Engl J Med. 1995; 333: 1171-1175. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000; 87: 840-844. Dzau VJ. Theodore Cooper Lecture: tissue angiotensin and pathobiology of vascular disease: a unifying hypothesis. Hypertension. 2001; 37: 1047-1052. George J, Carr E, Davies J, et al. High-dose allopurinol improves endothelial function by profoundly reducing vascular oxidative stress and not by lowering uric acid. Circulation. 2006; 114: 2508-2516. Gilbert A, Lereboullet P. La cholémie simple familiale. Semaine Med. 1901; 21: 241243. Guzik TJ, West NE, Black E, et al. Vascular superoxide production by NAD(P)H oxidase: association with endothelial dysfunction and clinical risk factors. Circ Res. 2000; 86: E85-E90. Hayden MR, Tyagi SC. Is type 2 diabetes mellitus a vascular disease (atheroscleropathy) with hyperglycemia a late manifestation? The role of NOS, NO, and redox stress. Cardiovasc Diabetol. 2003; 2: 1-10. Heitzer T, Schlinzig T, Krohn K, et al. Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation. 2001; 104: 2673-8. Higashi Y, Sasaki S, Nakagawa K, et al. Endothelial function and oxidative stress in renovascular hypertension. N Engl J Med. 2002a; 346: 1954-1962. Higashi Y, Sasaki S, Nakagawa K, et al. Tetrahydrobiopterin Improves Impaired Endothelium-Dependent Vasodilation in Patients with Essential Hypertension. Am J Hypertens. 2002b; 15: 326-332. Higashi Y, Sasaki S, Nakagawa K, et al. Tetrahydrobiopterin improves aging-related impairment of endothelium-dependent vasodilation through increase in nitric oxide production and decrease in reactive oxygen species. Atherosclerosis. 2006; 186: 390-395. Inoguchi T, Sasaki S, Kobayashi K, et al. Relationship between Gilbert syndrome and prevalence of vascular complications in patients with diabetes. JAMA. 2007; 298: 1398–1400. Kapitulnik J. Bilirubin: an endogenous product of heme degradation with both cytotoxic and cytoprotective properties. Mol Pharmacol. 2004; 66: 773-779. Kaufman S. New tetrahydrobiopterin-dependent systems. Annu Rev Nutr. 1993; 13: 261-286. Kuzkaya N, Weissmann N, Harrison DG, et al. Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase. J Biol Chem. 2003; 278: 22546-22554. Lerman A, Zeiher AM. Endothelial function: cardiac events. Circulation. 2005; 111: 363-368. Lin JP, O'Donnell CJ, Schwaiger JP, et al. Association between the UGT1A1*28 allele, bilirubin levels, and coronary heart disease in the Framingham Heart Study. Circulation. 2006; 114: 1476–1481. Maruhashi T, Soga J, Idei N, et al. Hyperbilirubinemia, augmentation of endothelial
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function and decrease in oxidative stress in Gilbert syndrome. Circulation. 2012; 126: 598-603. Moat SJ, McDowell IF. Homocysteine and endothelial function in human studies. Semin Vasc Med. 2005; 5: 172-182. Münzel T, Gori T, Bruno RM, et al. Is oxidative stress a therapeutic target in cardiovascular disease? Eur Heart J. 2010; 31: 2741-2748. Muto A, Tashiro S, Nakajima O, et al. The transcriptional programme of antibody class switching involves the repressor Bach2. Nature. 2004; 429: 566-571. Owens D, Evans J. Population studies on Gilbert’s syndrome. J Med Gebet. 1975; 12: 152-156. Rodriguez-Crespo I, Gerber NC, Ortiz de Montellano PR. Endothelial nitric-oxide synthase. J Biol Chem. 1996; 271: 11462-11467. Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med. 1999; 340: 115-126. Schachinger V, Britten MB, Zeiher AM. Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease. Circulation. 2000; 101: 1899-1906. Setoguchi S, Hirooka Y, Eshima K, et al. Tetrahydrobiopterin improves impaired endothelium-dependent forearm vasodilation in patients with heart failure. J Cardiovasc Pharmacol. 2003; 39: 363-368. Sieg A, Arab L, Schlierf G, et al. Prevalence of Gilbert's syndrome in Germany. Dtsch Med Wochenschr. 1987; 112: 1206-1208. Sowers JR. Hypertension, Angiotensin II, and Oxidative Stress. N Engl J Med. 2002; 346: 1999-2001. Stocker R, Yamamoto Y, McDonagh AF, et al. Bilirubin is an antioxidant of possible physiological importance. Science. 1987; 235: 1043–1046. Stoes E, Kastelein J, Cosentino F, et al. Tetrahydrobiopterin restores endothelial function in hypercholesterolemia. J Clin Invest. 1997; 99: 41-46. Sun J, Hoshino H, Takaku K, et al. Hemoprotein Bach1 regulates enhancer availability of heme oxygenase-1 gene. EMBO J. 2002; 21: 5216-5224. Touyz RM. Reactive oxygen species, vascular oxidative stress, and redox signaling in hypertension: what is the clinical significance? Hypertension. 2004; 44: 248-252. Vallance P, Collier J, Moncada S. Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet. 1989; 2: 997-1000. Vanhoutte PM. Endothelium and control of vascular function: state of the art lecture. Hypertension. 1989; 13: 658-667. Virdis A, Bacca A, Colucci R, et al. Endothelial dysfunction in small arteries of essential hypertensive patients: role of cyclooxygenase-2 in oxidative stress generation. Hypertension. 2013; 62: 337-344. Vítek L, Jirsa M, Brodanová M, et al. Gilbert syndrome and ischemic heart disease: a protective effect of elevated bilirubin levels. Atherosclerosis. 2002; 160: 449-456. Yiginer O, Ozcelik F, Inanc T, et al. Allopurinol improves endothelial function and reduces oxidant-inflammatory enzyme of myeloperoxidase in metabolic syndrome. Clin Res Cardiol. 2008; 97: 334-340. Yu MA, Sanchez-Lozada LG, Johnson RJ, et al. Oxidative stress with an activation of the renin-angiotensin system in human vascular endothelial cells as a novel mechanism of uric acid-induced endothelial dysfunction. J Hypertens. 2010; 28: 1234-1242.
10
Table 1. Circulating Antioxidant in Humans Antioxidant properties Water-soluble Uric acid Ascorbic acid (vitamin C) Bilirubin (unconjugated) Urobilinogen Glutathione Lipid-soluble Tocopherol (vitamin E) Ubiqunone (coenzyme Q10) -carotene Lycopene
Concentration of plasma (mol/L)
Antioxidant proteins Enzyme Superoxide dismutase Catalase Glutathione peroxidase Non-enzyme Albumin Transferrin Iron-binding proteins/copper-binding proteins
300 50 15 10 2 30 2 0.5 0.5
C
A
Forearm b blood flow (mL/min/100 0 mL tissue)
Angiotensin III (pg/mL)
0
40 35 30 25 20 15 10 5
50 45
0
5
10
15
20
25
30
Before
After
P < 0.05
Baseline
15
30
Acetylcholine (µg/min)
7.5
P < 0.0001
Before angioplasty g p y Before angioplasty + vitamin C After angioplasty After angioplasty + vitamin C
P < 0.05
D
B
Serum MDA-L LDL (U/L) Max response tto acetylcholine (mL/min/100 0 mL tissue)
Figure 1
15
20
25
30
35
40 Urinary 8-OHdG excretion (ng/mg cr.)
0
0
30
10
10 0
r = - 0.51 P = 0.004
40
50
0
10
5
After
After angioplasty Before angioplasty
Before
5
10
15
20
25
30
20
0
Control
P < 0.05
20
30
40
50
0
30
60
90
120
150
180
P < 0.05 Urinary 8-OHdG G (µg/mg Cr.)
0
50
Control
200 Serum MDA-LDL (U/L)
150
250
r = - 0.39 P = 0.02
After angioplasty g p y Before angioplasty
After
P < 0.05
Before
100
P < 0.05
E2
small Maf
E2
Nrf2
Oxidative stress (yes)
small Maf
Bach1
Oxidative stress (no)
Figure 2
E1
E1
Heme
HO-1
HO-1
HO-1
Gilbert syndrome
Fe
CO
Biliverdin
Biliverdin reductase
Bilirubin (conjugated)
UGT1A
Bilirubin (unconjugated)
Antioxidant Anti-inflammation
Oxidative Stress and Endothelial Dysfunction: Clinical Evidence and Therapeutic Implications Yukihito Higashi MD, PhD, FAHA, FJSH, Tatsuya Maruhashi MD, PhD, Kensuke Noma MD, PhD, Yasuki Kihara MD, PhD
www.elsevier.com/locate/tcm
PII: DOI: Reference:
S1050-1738(13)00164-3 http://dx.doi.org/10.1016/j.tcm.2013.12.001 TCM5982
To appear in: trends in cardiovascular medicine
Cite this article as: Yukihito Higashi MD, PhD, FAHA, FJSH, Tatsuya Maruhashi MD, PhD, Kensuke Noma MD, PhD, Yasuki Kihara MD, PhD, Oxidative Stress and Endothelial Dysfunction: Clinical Evidence and Therapeutic Implications, trends in cardiovascular medicine, http://dx.doi.org/ 10.1016/j.tcm.2013.12.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Oxidative Stress and Endothelial Dysfunction: Clinical Evidence and Therapeutic Implications Brief title: Antioxidant Property and Endothelial Function 1,2
Yukihito Higashi, MD, PhD, FAHA, FJSH; 3Tatsuya Maruhashi, MD, PhD; 1,2
1
Kensuke Noma, MD, PhD; 3Yasuki Kihara, MD, PhD
Department of Cardiovascular Regeneration and Medicine, Research Institute for
Radiation Biology and Medicine (RIRBM), Hiroshima University, Hiroshima, Japan 2
Division of Regeneration and Medicine, Medical Center for Translational and Clinical Research, Hiroshima University Hospital, Hiroshima University, Hiroshima, Japan 3
Department of Cardiovascular Medicine, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan Address for correspondence: Yukihito Higashi, MD, PhD, FAHA Department of Cardiovascular Regeneration and Medicine,
Research Institute for Radiation Biology and Medicine (RIRBM), Hiroshima University 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan Phone: +81-82-257-5831
Fax: +81-82-257-5831
E-mail: [email protected] Total word: 1733 (abstract: 154; 4 sentences) Number of Figure: 2; Number of Table: 1 Number of Reference: 39 Abstract An imbalance of nitric oxide (NO) and reactive oxygen species (ROS), so-called “oxidative stress”, may promote endothelial dysfunction, leading to cardiovascular complications. Activation of nicotinamide-adenine dinucleotide phosphate oxidase, xanthine oxidase, cyclooxygenase, and mitochondrial electron transport, inactivation of the antioxidant system and uncoupling of endothelial NO synthase lead to oxidative stress along with an increase in ROS production and decrease in ROS degradation. Although experimental studies, both in vitro and in vivo, have shown a critical role of
2
oxidative stress in endothelial dysfunction under the condition of excessive oxidative stress, there is little information on whether oxidative stress is really involved in endothelial function in humans. In a clinical setting, we showed an association between oxidative stress and endothelial function, especially in patients with renovascular hypertension as a model of increased oxidative stress and in patients with Gilbert syndrome as a model of decreased oxidative stress, through an increase in the antioxidant property unconjugated bilirubin. Introduction A healthy endothelium maintains vascular function and structure by balance of vasodilation and vasoconstriction, growth inhibition and growth promotion, antithrombosis and prothrombosis, anti-inflammation and proinflammation, and also antioxidation and prooxidation (Vallance et al. 1989; Vanhoutte 1989). Endothelial dysfunction is the initial step in the pathogenesis of arteriosclerosis, resulting in cardiovascular complications (Ross 1999). Several lines of evidence have clearly shown an association between coronary or peripheral endothelial function and cardiovascular events (Heitzer et al. 2001; Schachinger et al. 2000). Endothelial dysfunction is independently associated with cardiovascular events (Lerman and Zeiher 2005). The mechanisms for oxidative stress-induced endothelial dysfunction in cardiovascular diseases have been postulated. However, it is unclear whether these mechanisms work in humans. Renovascular hypertension is caused by renal artery stenosis, leading to stimulation of the renin-angiotensin system and increased production of its main active peptide, angiotensin II (Ang II), resulting in production of reactive oxygen species (ROS). Patients with renovascular hypertension are ideal models for determining how endothelium-dependent vasodilation is affected by excess Ang II and an Ang II-related increase in oxidative stress. In addition, it has been shown that although bilirubin at a high concentration acts as a cytotoxic metabolite, bilirubin, particularly unconjugated bilirubin, at a low concentration is a potent endogenous antioxidant (Kapitulnik 2004). Patients with Gilbert syndrome have mild unconjugated hyperbilirubinemia (Gilbert 1901). Therefore, Gilbert syndrome is an ideal model for determining how endothelium-dependent vasodilation is affected by bilirubin-related decrease in oxidative stress.
3
In this review, we take particular note of recent findings and the relationships between antioxidant property, oxidative stress, and endothelial function in humans. Is oxidative stress really involved in endothelial function in humans? In various pathophysiological states, activation of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase (XO), cyclooxygenase, and uncoupled endothelial nitric oxide synthase (eNOS) with tetrahydrobiopterin (BH4) oxidation and mitochondrial electron transport as well as inactivation of the antioxidant system, including superoxide dismutase, glutathione peroxidase and catalase, lead to an increase in ROS production and decrease in ROS degradation. (Cai and Harrison 2000; Rodriguez-Crespo and Gerber 1996; Kaufman 1993). An imbalance of reduced production of NO or increased production of ROS promotes endothelial dysfunction, leading to remodeling, platelet aggregation, loss of vasodilation, inflammation, and smooth muscle cell growth (Touyz 2004; Guzik et al. 2000; Sowers 2002). ROS directly inhibit NO activity (Cai and Harrison 2000). In addition, ROS activate the PI3K/ras/Akt/MAPK pathway, related to redox transcriptional factors, leading to redox gene expression, which results in inhibition of eNOS mRNA expression and eNOS activity. Indeed, several lines of evidence have shown, both in vitro and in vivo, the critical role of oxidative stress in endothelial function under the condition of excessive oxidative stress (Cai and Harrison 2000; Münzel et al. 2010; Virdis et al. 2013). However, the condition of excessive oxidative stress in experimental studies may have no application in humans, even in patients with cardiovascular disease. Patients with renovascular hypertension: NADPH oxidase NADPH oxidase is a multisubunit complex composed of cytosolic components, such as p47phox, p67phox and Rac 1, and membrane-spanning components, such as p22phox and gp91phox. NADPH oxidase should be the most important source of ROS in the vasculature (Dzau 2001). In an atherosclerotic state, Ang II-induced activation of NADPH oxidase is one of the major sources of ROS (Cai and Harrison 2000; Touyz 2004; Guzik et al. 2000; Dzau 2001). Upregulation of p22phox mRNA expression is a key component of Ang II-induced NADPH oxidase activation, and increased expression levels of other components also play an important role in NADPH oxidase under pathological conditions (Cai and Harrison 2000). Patients with renovascular hypertension have excess Ang II and Ang II-related increase in oxidative stress (Figure 1) (Higashi et al. 2002a). Renal angioplasty
4
decreased
plasma
renin
activity,
plasma
AngII
concentration,
and
serum
malondialdehyde-modified low-density lipoprotein (MDA-LDL) concentration and urinary
8-hydroxy-2’-deoxyguanosine
(8-OHdG)
excretion
in
patients
with
renovascular hypertension. After renal angioplasty, forearm blood flow response to acetylcholine was enhanced in these patients. Co-infusion of the antioxidant vitamin C augmented acetylcholine-induced vasodilation before angioplasty but not after angioplasty. The increase in maximal acetylcholine-induced vasodilation was associated with the decrease in urinary excretion of 8-OHdG and the decrease in serum concentration of MDA-LDL. These findings suggest that endothelial function is impaired in relation to the severity of oxidative stress in a clinical setting. Patients with Gilbert syndrome: bilirubin It is well known that ascorbic acid (vitamin C), urobilinogen, glutathione, tocopherol (vitamin E) ubiqunone (coenzyme Q10), -carotene, and lycopene are endogenous antioxidant properties in humans (Table 1). Unconjugated bilirubin is also a potent antioxidant (Stocker et al. 1987; Abraham and Kappas; 2008). Bilirubin is an end product of the heme oxygenase-1 (HO-1)/Bach1 pathway (Figure 2) (Muto et al. 2004; Sun et al. 2002). In brief, in the heme oxygenase-1 (HO-1) promoter region, there are two enhancer sequences called E1 and E2. In the basal state, HO-1 expression is turned off as Bach1 binds to these enhancer sequences, forming heterodimers with small Maf proteins. HO-1 activators including Nrf2 are not accessible to E1 and E2 because the Bach1 heterodimer has higher affinity to the sequence. In cases of cellular injury, including oxidative stress, it is thought that intracellular heme level is increased. These hemes strongly associate with Bach1, causing the Bach1 heterodimer to dissociate from DNA, which in turn allows transcriptonal activators to access E1 and E2, and thereby HO-1 expression is activated. Inducible HO-1 catalyzes the cleavage of heme to free iron, carbon monoxide and biliverdin. Biliverdin is rapidly converted to unconjugated bilirubin by biliverdin reductase. Unconjugated bilirubin is changed to the conjugated form of bilirubin through the uridine-diphosphate-glucuronosyltransferase isoform 1A (UGT1A). In patients with Gilbert syndrome, enzymatic activity of UGT1A1 is reduced by mutation of its gene, which is located on human chromosome 2 (Bosma et al. 1995). Therefore, conversion of unconjugated bilirubin to conjugated bilirubin is decreased, resulting in hyperbilirubinemia. Therefore, Gilbert syndrome is an ideal model for
5
determining how endothelium-dependent vasodilation is affected by bilirubin-related decrease in oxidative stress. The incidence of Gilbert syndrome in the general population is 5% to 10% (Owens and Evans 1975; Sieg et al. 1987). We evaluated the role of oxidative stress in endothelial function in patients with Gilbert syndrome under normal conditions without cardiovascular risk factors (Maruhashi et al. 2012). A total of 108 young men with Gilbert syndrome without cardiovascular risk factors and 108 age-matched healthy men (normal controls) were enrolled. Gilbert syndrome was diagnosed by the following criteria: mild unconjugated hyperbilirubinemia (1.2 to 6.0 mg/dL), normal values of hepatic biochemical tests, absence of liver disease or overt hemolysis, UGT1A activity of less than 35% of normal, and results of real-time PCR detection of UGT1A*28 mutation (Lin et al. 2006). In control subjects, the upper limit of the normal range for serum concentrations of bilirubin was 1.0 mg/dL. Serum concentration of bilirubin was significantly higher in patients with Gilbert syndrome than in control subjects. Serum concentration of MDALDL and urinary excretion of 8-OHdG were significantly lower in patients with Gilbert syndrome than in control subjects. There was no significant difference in other parameters between the two groups. Flow-mediated vasodilation (FMD) was significantly greater in patients with Gilbert syndrome than in normal control subjects. FMD correlated with serum concentrations of bilirubin and MDA-LDL and with urinary excretion of 8-OHdG in patients with Gilbert syndrome but not in control subjects. In addition, serum concentration of bilirubin correlated with MDA-LDL and urinary excretion of 8-OHdG in these patients. Patients with Gilbert syndrome had low levels of oxidative stress associated with hyperbilirubinemia and enhancement of endothelium-dependent vasodilation. These beneficial effects on the vasculature may contribute to reduced prevalence of vascular complications in atherosclerotic patients with Gilbert syndrome compared with that in atherosclerotic patients without Gilbert syndrome (Inoguchi et al. 2007). It is likely that superoxide plays an important role in vascular function even under a normal condition without cardiovascular risk factors. XO/uric acid In humans, the antioxidant uric acid circulates in greater concentrations than all other antioxidants (Table 1). Uric acid is an end product from xanthine through XO, which is one of the enzymatic sources of ROS, in higher mammals. Several investigators have shown that an XO inhibitor improves endothelial function in patients with
6
hyperuricemia, probably through a decrease in XO-induced ROS production (Yiginer et al. 2008; George et al. 2006). On the other hand, it has been reported that uric acid enhances the production of ROS by activation of the local renin-angiotensin system, particularly Ang II, in human endothelial vascular cells (Yu et al. 2010). Thus, uric acid is thought to mediate endothelial dysfunction by generating oxidative stress once absorbed into endothelial cells. Interestingly, although uric acid at a concentration of over 6 mg/dL induced generation of ROS and senescence and apoptosis of endothelial cells, uric acid at a concentration of 3 mg/dL had no effect on endothelial cells (Vítek et al. 2002). These findings suggest that XO is an exacerbating factor of endothelial dysfunction and that there may be a certain threshold for uric acid levels before proatherogenic properties become apparent. eNOS uncoupling/BH4 BH4 is an allosteric factor in the coupling of the oxidase and reductase domains of eNOS (Rodriguez-Crespo et al. 1996). While eNOS activation at optimal levels of BH4 leads to production of NO, under conditions of BH4 deficiency, electron flow from the reductase domain to the oxidase domain is diverted to molecular oxygen rather than to L-arginine, a substrate of NO, leading to eNOS uncoupling, which results in generation of ROS rather than NO (Kuzkaya et al. 2003). In addition, it has been reported that degradation of BH4 by ROS is associated with down-regulation of eNOS (Stoes et al. 1997). In clinical studies, supplementation of BH4 improved endothelial function in smokers, in patients with diabetes, hypertension, hypercholesterolemia, and chronic heart failure, and in elderly subjects (Setoguchi et al. 2003; Higashi et al. 2002; Higashi et al. 2006; Hayden and Tyagi 2003; Moat and McDowell 2005). Infusion of a NOS inhibitor abolished the BH4-induced enhancement of forearm vasorelaxation evoked by acetylcholine in patients with hypertension (Higashi et al. 2002b). These findings suggest that deficiency and/or oxidation of BH4 may contribute to endothelial dysfunction.
Clinical perspectives Oxidative stress plays a critical role in endothelial function and atherosclerosis in humans. Oxidative stress-related endothelial dysfunction should be a therapeutic target for atherosclerosis. Future studies are needed to evaluate the direct effects of exogenous and endogenous antioxidants on endothelial function and the prevention of
7
cardiovascular morbidity and mortality for long-term periods. Acknowledgments This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, Japan Heart Foundation Grant for Research on Hypertension and Metabolism, and a Grant for Research Foundation for Community Medicine.
Conflict of Interest None. Figure Legends Figure 1. Endothelial function and oxidative stress in patients with renovascular hypertension. (A) Circulating angiotensin II levels before and after angioplasty. (B) Circulating oxidative stress markers, urinary excretion of 8-hydroxy-2’-deoxyguanosine (8-OHdG) and serum malondialdehyde-modified low-density lipoprotein (MDA-LDL) concentration before and after angioplasty. (C) Effects of concomitant administration of the antioxidant vitamin C on forearm blood flow response to acetylcholine administration before and after angioplasty. (D) Correlations of the maximal response of forearm blood flow to administration of acetylcholine with urinary excretion of 8OHdG and serum MDA-LDL concentration before and after angioplasty. Modified from Ref. (Higashi et al. 2002a). Figure 2. Heme oxygenase-1 (HO-1)/Bach1/bilirubin pathway with and without oxidative stress. Unconjugated bilirubin is changed to the conjugated form of bilirubin through the uridine-diphosphate-glucuronosyltransferase isoform 1A (UGT1A). In patients with Gilbert syndrome, enzymatic activity of UGT1A1 is reduced by mutation of its gene.
8
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Table 1. Circulating Antioxidant in Humans Antioxidant properties Water-soluble Uric acid Ascorbic acid (vitamin C) Bilirubin (unconjugated) Urobilinogen Glutathione Lipid-soluble Tocopherol (vitamin E) Ubiqunone (coenzyme Q10) -carotene Lycopene
Concentration of plasma (mol/L)
Antioxidant proteins Enzyme Superoxide dismutase Catalase Glutathione peroxidase Non-enzyme Albumin Transferrin Iron-binding proteins/copper-binding proteins
300 50 15 10 2 30 2 0.5 0.5
C
A
Forearm b blood flow (mL/min/100 0 mL tissue)
Angiotensin III (pg/mL)
0
40 35 30 25 20 15 10 5
50 45
0
5
10
15
20
25
30
Before
After
P < 0.05
Baseline
15
30
Acetylcholine (µg/min)
7.5
P < 0.0001
Before angioplasty g p y Before angioplasty + vitamin C After angioplasty After angioplasty + vitamin C
P < 0.05
D
B
Serum MDA-L LDL (U/L) Max response tto acetylcholine (mL/min/100 0 mL tissue)
Figure 1
15
20
25
30
35
40 Urinary 8-OHdG excretion (ng/mg cr.)
0
0
30
10
10 0
r = - 0.51 P = 0.004
40
50
0
10
5
After
After angioplasty Before angioplasty
Before
5
10
15
20
25
30
20
0
Control
P < 0.05
20
30
40
50
0
30
60
90
120
150
180
P < 0.05 Urinary 8-OHdG G (µg/mg Cr.)
0
50
Control
200 Serum MDA-LDL (U/L)
150
250
r = - 0.39 P = 0.02
After angioplasty g p y Before angioplasty
After
P < 0.05
Before
100
P < 0.05
E2
small Maf
E2
Nrf2
Oxidative stress (yes)
small Maf
Bach1
Oxidative stress (no)
Figure 2
E1
E1
Heme
HO-1
HO-1
HO-1
Gilbert syndrome
Fe
CO
Biliverdin
Biliverdin reductase
Bilirubin (conjugated)
UGT1A
Bilirubin (unconjugated)
Antioxidant Anti-inflammation
