Shinozaki K. et al.: and Endothelial Function 1 I

Pteridines Vol. 17, 2006, pp. 11-15

Tetrahydrobiopterin and Endothelial Dysfunction in Cardiovascular Diseases

Kazuya Shinozaki', Atsunori Kashiwagi\ Masahiro Masada', Tomio Okamura'

'Department of Pharmacology and "Department of Medicine, Shiga University of Medical Science, Otsu, Japan. 'Laboratory of Biochemistry, Faeulty of Horticulture, Chiba University, Chiba, Japan

Abstract

Endothelial vasodilator dysfunction is a characteristic feature of patients at risk for coronary atheroscierosis. We have reported that insuHn rcsistance may be a pathogenic factor for endothehal dysfunction through impaircd endothelial synthase (eNOS) activity and increased oxidative breakdown of nitric oxide (NO) due to an enhanced fonnation of Superoxide anion, which arc caused by relative deficiency of tetrahydrobiopterin (BH4) in vascular endothelial cclls. Guanosine-triphosphate cyclohydrolase I, the rate-limiting cnzyme in the produc- tion of BH4, is decreased in the aorta of insulin-resistant rats and supplementation of BH4 restored the endothe- lial function and relieved oxidative tissue damage. The BH4 treatment may evoke these benefits not only by pro- viding eNOS with cofactor to enhance NO synthesis, but also by acting as an indirect and/or direct antioxidant to decrease Superoxide anion derived from the . A further understanding of the physiological and patho- logical roles and their regulation may lead to new therapeutic avenucs.

Key words: tetrahydrobiopterin, endothelial dysfunction, atheroscierosis, nitric oxide, insulin resistance

Introduction produce O2" due to uncoupled oxygen activation (6). In contrast, BH4 has been used to reverse vascular Endothelial damage is of considerable clinical dvsfunction m diabetes (7) and importancc because the endothelium is considcred to (gj^ suggesting that BH4 levels are insufficient in thesc play a central rolcm the pathogenesisofatherosclero- conditions. In this review, mechanisms by which SIS, and endothelial dysfunction has been imphcated m abnormal pter.dine metabolism affects endothelial prcceding to and hkely contributing to the develop- function and ultimately contnbutcs to ment of in these states (1.2). .^^ju be discussed Over the past several years, endothelium-derived relaxing factor, whose activity is accounted for nitric PterkUne Metabolism and eNOS Activity oxide (NO), has cmerged as a major regulator of Endothelial vasodilator dysfunction has been periphcral vascular tone (3). Subsequently, altered observed in patients with traditional coronary risk fac- endothelium-dependent vascular relaxation has been tors. even in the absence of evidence for atherosclerot- associated with enhanced degradation of NO by reac- ic lesions, which suggests that the endothelium is both tive oxygen species (ROS) in animal models of many a target and a mediator of atheroscierosis (9). Thcre is different diseases. These include , dia- a growing body of evidence demonstrating the coexis- betes, hypercholesterolemia, and insulin-resistant State tence of endothelial dysfunction in the insulin-resistant (4). states such as obesity, hypertension and type 2 diabetes Evidence indicales that (6R)-5,6,7,8-tetrahydro- (10.11). To investigate the underlying mechanisms for biopterin (BH4), which is an important allosteric effec- the relationship between insulin-resistant State and tor of (NOS) through stabiliza- impaired endothelial nitric oxide synthase (eNOS) tion of the dimeric active form of the enzyme (5), may function, we examined the endothelial function and play a key role in the control of endothelial NO and production of NO and O2" in isolated thoracic aortas Superoxide anion (O,") production in vivo. In fact, it obtained from rats. We used a fructose-induced has been demonstrated that blood vessels depleted of insulin-resistant model (fructose-fed rats), originally

Corrcspondence to: Tomio Okamura, MD, PhD, Department of Pharmacology, Shiga University of Mcdical Science, Tsukinowa-cho, Seta, Otsu, Shiga 520-2192, Japan, FAX 81-77-548-2183, Phone 81-77-548-2181 E-mail ol

Pteridines/VoL 17/No. 1 12 Shinozaki K. et al.: Tetrahydrobiopterin and Endothelial Function

described by Zavaroni et al (12). Organ Chamber duced during activation of purified NOS at suboptimal experiments showed that the maximal relaxation to concentrations of BH4 (20). Because eNOS mRNA both acetylcholine (a receptor-mediated agonist) and expression was not changed in fructose-fed rats (18), A23187 (a receptor-independent endothelium-depend- the impaired eNOS activity may be due to a decreased ent vasodilator) was significantly reduced, and the availability of the cofactor for endothelial synthesis of ED50 values were increased in the aortas from fmc- NO. To evaluate the possible role of BH4 for the mod- tose-fed rats as compared with those in the aortas from ulation of eNOS-dependent NO/O2" balance, we meas- normal diet-fed rats. Endothelium-dependent vasodila- ured both BH4 and BH2 Contents in aortic tissue with tion was restored by a scavenger of O2" (Cu,Zn-SOD) or without endothelium. We found that endothelial in fructose-fed rats. In addition, L-N-monometghyl BH4 content and GCH-I activity were significantly arginine (L-NMMA) not only inhibited the generation decreased, whereas the levels of 7,8-BH2 were of NO, but also suppressed the increased O2" produc- markedly increased in fructose-fed rats. In addition, tion in fructose-fed rats. On the other band, vasodilator with regard to the decreased DHPR activity, the responses to sodium nitroprusside were comparable marked decrement of BH4 relative to 7,8-BH2 between the groups. Thus, in the insulin-resistant State, (BH4/7,8-BH2 ratio) was observed in fructose-fed rats the reduced endothelium-dependent is (21). These findings indicate that abnormal pteridine explained by mechanisms of a reduced NO production metabolism in the vascular wall plays an important duc to decreased NOS activity as well as accelerated role in the regulation of endothelial NO production. degradation of NO by O2" within the vascular wall. An increasing body of evidence suggests that vas- Oxidized Biopterin and eNOS Uncoupling cular is involved in the pathogenesis of In agreement with these studies, previous studies many cardiovascular diseases, including hypercholes- have reported uncoupled eNOS contributes to O2" pro- terolcmia, diabetes, hypertension, and heart failure duction specifically within endothelial cells and is (13,14). Among many enzymatie systems that are associated with increased oxidation of BH4, forming capable of producing ROS, xanthine oxidase, BH2 and biopterin (Table I) (21-26). Since BH4 is rap- NAD(P)H oxidase, and uncoupled eNOS have been idly oxidized to 7,8-BH2, insufficient DHPR activity extensively studied in vascular cells. Of note, eNOS would lead to accumulation of 7,8-BH2, which has constitutivcly produccs both NO and O2", suggesting been shown to inhibit the stimulatory effects of BII4 on that the effective release of NO from the vascular NO synthase (18). Redox activity of the pteridine is endothelium depends on the relative concentrations of suggested by experiments showing that the oxidized these two radical species. derivative BHj binds with fairly high affinity to NOS Tetrahydrobiopterin is a critical cofactor for NOS without supporting catalytic activity (18). In isolated and appears to contribute to the enzymes to bind L- caninc cerebral arterics, auto-oxidation of cxogcnous arginine. It may acl as a both redox-active and BH4 causes endothelium-dependent contractions (27). ailosteric effector of NO synthase (15). In addition, To evaluate whether pteridine levels and the BH4/7,8- BH4 is found to prevent feedback Inhibition of NO BH2 ratio regulate the eNOS activity, we measured synthase by NO (16). The biosynthesis of intracellular eNOS activity in the presence and absence of pteridine BH4 is governed by two enzymes: guanosine-triphos- metabolites. The basal activity of recombinant eNOS phate cyclohydrolase I (GCH-I), which regulates its was maximally stimulated by addition of 10 mmol/1 rate of formation, and dihydropteridine reductase BH4 (28). Addition of 7,8-BH2 in the presence of 10 (DHPR), which regulates its rate of regeneration. |j.mol/l BH4 dose-dependently inhibited eNOS activity. Utilization of BH4 in the NO synthesis generates In the presence of Ca-~/calmodulin and NADPH, quinonoid dihydrobiopterin (qBH2) rather than BH^ eNOS generates O2", as assessed by lucigenin chemi- (15-17). Altematively, qBH2 may rearrange non-enzy- luminescence method. Addition of BH4 inhibited matically to BH2, which is no longer a substrate for eNOS-mediated Oj" generation and 1 mmol/1 BH4 DHPR (18). reduced the amount of O2" by 21%. In contrast, addi- It has been demonstrated that blood vessels deplet- tion of 7,8-BH2 in the presence of 1 0 |j,mol/l BH4 dose- ed of BH4 produce Oj" due to uncoupled oxygen acti- dependently stimulated eNOS-mediated O2" genera- vation (19). Since NO synthases have a number of fea- tion (28). The increment of 02~ production was inhib- tures in common with P-450 reductases and monooxy- ited to below the basal level after incubation with genases, the presence of the pterin is a key to the pro- either 100 U/ml Cu,Zn-SOD or 10 |xmol/l L-NMMA duction of NO in contrast to O2" or peroxides associat- (data not shown). These results demonstrate that high ed with P-450 reductase activity under certain condi- concentrations of 7,8-BH2 inhibit GCH-I and hence de tions. This concept is farther supported by biochemical novo synthesis of BH4 (18). Therefore, these results studies demonstrating that hydrogen peroxide is pro- suggest that insulin resistance induces vascular dys-

Pteridines/Vol. 17/No. 1 Shinozaki K. et al.\ Tetrahydrobiopterin and Endothelial Function 13

Table 1. BH4 and oxidized biopterin Contents in aortas from vascular disorders

7,8-BH2 BH4/7,8-BH2 Diseases BH4 Ref. I- biopterin + biopterin Hypertension DOCA-Salt (Rat) TT 4-i Landmesser, 2003 Prehypertensive SHR (Rat) N.D. Cosentino, 1998 N.D. Hypercholesterolemia Diet-induced (Rabbit) i N.D. Vasquez-Vivar, 2002 N.D. ApoE-KO (Mouse) —^ T T d 'Uscio, 2003 Diabetes Streptozotocin-induced (Rat) i TT TT Alp, 2003 Goto-Kakizaki (Rat) i TT TT Bitar, 2005 db/db (Mouse) T T Pannirselvam, 2003 Insulin Resistance Fructose-induced (Rat) T T Shinozaki, 1999 Zucker Fatty (Rat) T TT Shinozaki, 2005 N.D. = not determined function through alterations in the BH4/7,8-BH2 ratio. and markedly reduced the lipid peroxide content which were increased in fructose-fed rats. Furthermore, Therapeutic Strategies to Improve Endothelial increased binding activity of two redox-sensitive tran- Function scription factors, nuclear factor-kB (NF-kB) and acti- Bcncfical cffccts of BH4 stapplementation on vascu- vating protein-1 (AP-1), in fructose-fed rats was also lar endothelial function of experimental animals and prevented by the treatment with BH4 (32). We have humans have been reported by a number of laborato- recently reported that intramuscular overexpression of ries (22-26). Short-term administration of intraarterial pCAGGS-GCH-1 Plasmid DNA leads to persistent BH4 restored the impaired endothelium-dependent incrcasc in systcmic BH4 levels and preserved dilation in hypercholesterolemic patients (8,29). endothelium-dependent vascular relaxations in Zucker Recently, several reports have shown that acute admin- fatty rats (26). These results suggest that sufficient istration of BH4 improves endothelium-dependent supplementation with BH4 might help to prevent or forearm vasodilation in patients with hypertension and delay the occurrence of cardiovascular diseases. chronic haart failure (30), To further confirm the On the other hand, recent data suggest that oxida- hypothesis which abnormal pteridine metabolism tive degradation of BH4 by peroxynitrite (forming BHj induces uncoupling of cNOS, we investigated the and biopterin via the nonprotonated BH3 radical) is an effects of dietary treatment with BH4 on endothelium- explanation for reduced BH4 levels in dependent arterial relaxation and vascular oxidative and other vascular disease states (33). Experiments in stress in the aortas of insulin-resistant rats (31). Oral vitro and ex vivo indicate that peroxynitrite can oxi- supplementation of sapropterin hydrochloride (10 dize BH4 within minutes, at physiologically relevant mg/kg/day) for 8 weeks significantly increased the concentrations, and lead directly to eNOS uncoupling BH4 content and BH4/7,8-BH2 ratio in aortic tissues of and endothelial dysfunction (34). In vitro fmding fruetose-fed rats. Impairment of endothelium-depend- demonstrated that the ability of vitamin C to stimulate ent arterial relaxation in the aortic strips of the fruc- eNOS activity in cultured endothelial cells via chemi- tose-fed rats was reversed by BH4 treatment. The BH4 cal stabilizalion of BH4 (33). However, the importance treatment was associated with a 2-fold increase in of these observations in vivo remains to be confirmed. eNOS activity as well as a 70% reduction of endothe- Because BH4 treatment did not change the increased lial O2" production compared to those in fructose-fed oxidized biopterin levels in insulin-resistant State (31), rats. We also demonstrated that BH4 treatment of the we investigated the additional element to regulato vas- fructose-fed rats partially improved insulin sensitivity cular tone in the insulin-resistant State. In aortic

Pteridines/Vol. 17/No. 1 14 Shinozaki K. et al.: Tctrahydrobioptcrin and Endothelial Function

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