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(Circulation Research. 2003;92:88.)
© 2003 American Heart Association, Inc.

Molecular Medicine

Long-Term Vitamin C Treatment Increases Vascular Tetrahydrobiopterin Levels and Nitric Oxide Synthase Activity

Livius V. d’Uscio, Sheldon Milstien, Darcy Richardson, Leslie Smith, Zvonimir S. Katusic

From the Departments of Anesthesiology, and Molecular Pharmacology and Experimental Therapeutics (L.V.U., D.R., L.S., Z.S.K.), Mayo Clinic and Foundation, Rochester, Minn; and the National Institute of Mental Health (S.M.), NIH, Bethesda, Md.

Correspondence to Zvonimir S. Katusic, MD, PhD, Dept of Anesthesiology, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail katusic.zvonimir{at}mayo.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In cultured endothelial cells, the antioxidant, L-ascorbic acid (vitamin C), increases nitric oxide synthase (NOS) enzyme activity via chemical stabilization of tetrahydrobiopterin. Our objective was to determine the effect of vitamin C on NOS function and tetrahydrobiopterin metabolism in vivo. Twenty-six to twenty-eight weeks of diet supplementation with vitamin C (1%/kg chow) significantly increased circulating levels of vitamin C in wild-type (C57BL/6J) and apolipoprotein E (apoE)–deficient mice. Measurements of NOS enzymatic activity in aortas of apoE-deficient mice indicated a significant increase in total NOS activity. However, this increase was mainly due to high activity of inducible NOS, whereas eNOS activity was reduced. Significantly higher tetrahydrobiopterin levels were detected in aortas of apoE-deficient mice. Long-term treatment with vitamin C restored endothelial NOS activity in aortas of apoE-deficient mice, but did not affect activity of inducible NOS. In addition, 7,8-dihydrobiopterin levels, an oxidized form of tetrahydrobiopterin, were decreased and vascular endothelial function of aortas was significantly improved in apoE-deficient mice. Interestingly, vitamin C also increased tetrahydrobiopterin and NOS activity in aortas of C57BL/6J mice. In contrast, long-term treatment with vitamin E (2000 U/kg chow) did not affect vascular NOS activity or metabolism of tetrahydrobiopterin. In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of tetrahydrobiopterin and restoration of eNOS enzymatic activity.

Key Words: tetrahydrobiopterin • nitric oxide synthase • nitric oxide • antioxidants • superoxide anion

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Nitric oxide (NO) is a potent vasodilator and plays a key role in control of the cardiovascular system.¹ NO is mainly formed in endothelial cells from L-arginine by oxidation of its terminal guanidino-nitrogen,² requiring the cofactors NADPH, (6R)-5,6,7,8-tetrahydrobiopterin (BH₄), FAD, FMN, heme, and Zn²⁺.^3,4 The formation of NO occurs via endothelial NO-synthase (eNOS) which is expressed constitutively.^5,6 Relaxations in response to the abluminal release of endothelium-derived NO are associated with stimulation of soluble guanylyl cyclase (sGC) and in turn formation of cyclic guanosine 3',5'-monophosphate (cGMP) in vascular smooth muscle cells.⁷

Inducible NOS (iNOS) enzyme can be expressed in vascular smooth muscle cells, endothelium, and macrophages. This enzyme activity is Ca²⁺-independent and produces large amounts of NO; it is induced by cytokines such as interleukin 1ß and tumor necrosis factor- and hence is activated in atherosclerosis and inflammatory processes.^8–11 BH₄ is an essential cofactor required for activity of all NOS isoforms.^4,12 During activation of NOS, BH₄ is needed for allosteric and redox activation of its enzymatic activity.^4,13

Accumulating evidence suggests that alterations in the NO pathway, such as increased NO decomposition by superoxide anion (O₂^-) or altered NOS expressions, play a central role in endothelial dysfunction induced by hypercholesterolemia.¹⁴ This may be of major importance because NO can substantially inhibit several components of the atherogenic process, such as vascular smooth muscle cells contraction and proliferation, platelet aggregation, and monocyte adhesion.^15,16 It has been shown in several studies that antioxidants, vitamin C or vitamin E, reduced vascular oxidative stress^17–20 and increased NO-mediated endothelium-dependent relaxations.^21,22 In addition, vitamin C increased vasodilation of forearm resistance arteries in humans with hypercholesterolemia,²³ long-term smokers,²⁴ essential hypertension,²⁵ and coronary artery disease.^26,27 The molecular mechanisms underlying the in vivo antioxidant effects of vitamin C are not fully understood. More recent findings in cultured endothelial cells indicate that vitamin C may increase NOS enzymatic activity by chemical stabilization of BH₄.^28–30 Therefore, we hypothesized that the in vivo effect of vitamin C is mediated in part by its ability to protect BH₄ from oxidation and thereby increase enzymatic activity of eNOS. In this study, we compared the effects of vitamins C and E on BH₄ and NOS in wild-type and atherosclerotic mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Experimental Animals
Male C57BL/6J (wild-type) mice and homozygous apoE-deficient mice (4 to 5 weeks old) were obtained from Jackson Laboratory (Bar Harbor, Maine) and were fed a lipid rich Western-type diet (TD88137, Harlan Teklad)^31,32 without or with vitamin C (1%/kg diet) or vitamin E (2000 IU/kg diet) for 26 to 28 weeks. The dosages of vitamin C and vitamin E were based on previous studies.^18,33 Housing facilities and all experimental protocols were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic.

Plasma Vitamins C and E
A reverse-phase HPLC was used to determine plasma concentrations of vitamins C and E.

Lesion Assessment
Dissected aortas were opened longitudinally and fixed in 4% buffered paraformaldehyde for 2 hours and were stained in supersaturated Sudan IV solution for an additional 16 hours.³⁴

Vasomotor Reactivity
Isolated aortic rings were connected to a force transducer for recording of isometric force and placed in organ baths filled with 25 mL Krebs solution (37°C; 94% O₂/6% CO₂; pH 7.4).³⁵ Concentration-dependent response curves to acetylcholine (Ach), and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate) were cumulatively obtained during submaximal contractions to phenylephrine.

Quantification of Vascular O₂^- Production
Vascular O₂^- production was measured by lucigenin-enhanced chemiluminescence as described.³⁵

Measurement of Ca²⁺-Dependent NOS Enzyme Activity
Aortas were homogenized on ice in lysis buffer pH 7.5, and L-[¹⁴C]-Citrulline formation was measured as described previously.³⁵

Western Blot Analysis
Mouse monoclonal anti-eNOS (1:500), anti-iNOS (1:100; Transduction Labs), and anti-nitrotyrosine (0.5 µg/mL; Upstate Biotechnology) were used. As a loading control, blots were rehybridized with monoclonal anti-actin (Sigma).³⁵

Measurements of Tissue BH₄ and 7,8-BH₂/Biopterin
Biopterin levels were determined after differential oxidation in acid and base conditions by reverse-phase HPLC.^36–38

Measurements of Intracellular cGMP and cAMP
Radioimmunoassay kits (Amersham) were used to perform the measurements as described elsewhere.³⁷

Calculations and Statistical Analysis
Results are expressed as mean±SEM. Wild-type and apoE-deficient mice groups were compared separately by one-way ANOVA for multiple comparisons. For simple comparisons between two groups, an unpaired Student’s t test was used where appropriate. A value of P<0.05 was considered significant.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animal Characteristics
Plasma total cholesterol, LDL, and triglyceride concentrations were elevated while HDL levels were reduced in apoE-deficient mice as compared with wild-type mice (P<0.05; Table 1). Concomitant treatment with antioxidant vitamin C or E had no effect on the plasma lipid profile (Table 1).

View this table: [in this window] [in a new window]   Table 1. Characteristics of ApoE-Deficient and C57BL/6J Mice After 26 to 28 Weeks of Treatment

Plasma vitamin C levels were significantly reduced in apoE-deficient mice as compared with wild-type (P<0.05; n=5; Table 1). Conversely, plasma levels of vitamin E were increased in apoE-deficient mice (P<0.05; n=5; Table 1). Supplementation with vitamin C or E increased their concentrations 3- or 2-fold, respectively, in both wild-type and apoE-deficient mice (P<0.05; n=5; Table 1).

Morphology
Aortic lesion areas were significantly reduced by 51% after treatment of apoE-deficient mice with vitamin C (16.7±3.6%; P<0.05 versus apoE group: 34.0±2.7%; n=5). Vitamin E decreased lesion formation by 32% in apoE-deficient mice (data not shown).

Vascular Reactivity
Contractions to 80 mmol/L KCl and concentration-dependent contractions to phenylephrine were not statistically different between apoE-deficient and C57BL/6J mice groups (Table 1).

We have previously shown that in mice aortas, endothelium-dependent relaxation in response to Ach was L-NAME–sensitive.³⁵ Either vitamin C or E treatment significantly improved NO-mediated endothelium-dependent relaxations to Ach in aortas of apoE-deficient mice (83±2% or 71±3%, respectively; P<0.05 versus apoE group, maximal relaxation: 59±4%; Figure 1A, right). However, maximal relaxations to Ach were still impaired as compared with C57BL/6J mice (91±1%; P<0.05). In addition, maximal relaxations to Ach were significantly bigger in vitamin C–treated apoE-deficient mice as compared with mice treated with vitamin E (P<0.05; Figure 1A, right). In contrast, vitamin C significantly reduced endothelium-dependent relaxations to Ach in wild-type mice (78±3%; P<0.05), whereas vitamin E did not have any effect (Figure 1A, left).

View larger version (29K): [in this window] [in a new window]   Figure 1. Effects of antioxidants on endothelium-dependent relaxations to Ach and endothelium-independent relaxations to DEA-NONOate in the aorta of wild-type (C57BL/6J) and apoE-deficient mice after 26 to 28 weeks on a Western-type diet. A, Both vitamin C and E improved relaxations to Ach in apoE-deficient mice while, conversely, vitamin C impaired these relaxations (P<0.05; n=9 to 12; ANOVA+Bonferroni’s). Note that relaxations to Ach were still impaired in vitamin C–treated apoE-deficient mice as compared with C57BL/6J mice (P<0.05). B, Only vitamin C significantly improved relaxations to DEA-NONOate in apoE-deficient mice while, conversely, vitamin C impaired these relaxations in C57BL/6J mice (P<0.05; n=8 to 12; ANOVA+Bonferroni’s). Note that relaxations were still impaired in vitamin C–treated apoE-deficient mice as compared with wild-type mice (P<0.05; ANOVA+Bonferroni’s). Results are mean±SEM and expressed as percent relaxation of the submaximal contraction to phenylephrine (1 to 6x10-7 mol/L).

Endothelium-independent relaxations to the NO donor DEA-NONOate were reduced, and the concentration-response curve was shifted to right in apoE-deficient mice (pD₂: 7.4; P<0.05 versus wild-type mice: 8.5). Vitamin C, but not vitamin E, in part improved the sensitivity to DEA-NONOate in apoE-deficient mice (pD₂: 7.7; P<0.05 versus apoE mice; Figure 1B, right). In contrast, vitamin C reduced relaxations to the NO-donor in wild-type mice (pD₂: 8.2; P<0.05 versus wild-type group: 8.5; Figure 1B, left) without affecting maximal relaxations.

Ca²⁺-Dependent NOS Activity
In order to evaluate the mechanisms underlying effects of antioxidants on endothelium-dependent relaxations, we measured Ca²⁺-dependent NOS activity in aortas of apoE-deficient and wild-type mice as determined by conversion of L-[¹⁴C]arginine to L-[¹⁴C]citrulline in tissue homogenates. Vitamin C selectively increased Ca²⁺-dependent NOS activity in aortas from both wild-type and apoE-deficient mice (P<0.05; Figure 2A). Interestingly, vitamin C normalized enzyme activity in apoE-deficient mice to values similar to those found in aortas from wild-type mice. Conversely, vitamin C did not affect eNOS protein expression (Figure 2B; n=3), whereas vitamin E had no significant effects on eNOS protein expression or NOS activity in either apoE-deficient or wild-type mice (Figure 2).

View larger version (43K): [in this window] [in a new window]   Figure 2. A, Bar graphs showing Ca2+-dependent eNOS enzyme activity in the aorta of wild-type (C57BL/6J) and apoE-deficient mice after 26 to 28 weeks on a Western-type diet with or without antioxidants. L-[14C]citrulline formation was measured in aortic homogenates as described in Materials and Methods. Results are mean±SEM (n=7). *P<0.05 vs C57BL/6J mice (ANOVA+Bonferroni’s); P<0.05 vs C57BL/6J mice (unpaired t test); #P<0.05 vs apoE-deficient mice (ANOVA+Bonferroni’s). B, Representative Western blot analysis of eNOS protein expression in aortas of C57BL/6J and apoE-deficient mice. Bar graph indicates the results of relative densitometric analysis of eNOS expression as OD per mm2 aortic surface (n=3 to 4). Actin blots are shown as loading controls.

iNOS Enzyme Activity and Protein Expression
In the aortas of wild-type mice, Ca²⁺-independent NOS activity was very low as compared with Ca²⁺-dependent NOS activity (P<0.05; Figures 2A and 3A). iNOS activity was increased in apoE-deficient mice as compared with wild-type (P<0.05; Figure 3A). In addition, iNOS protein expression was also enhanced in apoE-deficient mice (P<0.05; Figure 3B). Antioxidant vitamins did not affect iNOS protein expression (Figure 3B). Interestingly, vitamin C selectively increased iNOS enzyme activity in wild-type (P<0.05; Figure 3A), whereas it had no effect in apoE-deficient mice.

View larger version (42K): [in this window] [in a new window]   Figure 3. A, Bar graphs showing Ca2+-independent NOS enzyme activity (iNOS) in the aorta of wild-type (C57BL/6J) and apoE-deficient mice after 26 to 28 weeks on a Western-type diet with or without antioxidants. L-[14C]citrulline formation was measured in aortic homogenates as described in Materials and Methods. Results are mean±SEM (n=7). *P<0.05 vs C57BL/6J mice; P<0.05 vs C57BL/6J with or without antioxidants (ANOVA+Bonferroni’s). B, Representative Western blot analysis of iNOS protein expression in aortas of C57BL/6J and apoE-deficient mice. Bar graphs indicate the results of the relative densitometry as compared with actin (n=3). *P<0.05 vs C57BL/6J with or without antioxidants (ANOVA+Bonferroni’s).

cGMP and cAMP Levels
Basal cGMP levels were reduced in aortas from apoE-deficient mice as compared with wild-type mice (P<0.05; Figure 4). Vitamin C treatment increased basal cGMP levels only in wild-type (P<0.05; Figure 4). Basal cAMP levels were not different between wild-type (30±6 pmol/mg) and apoE-deficient mice (25±2 pmol/mg) and after vitamin C treatment (34±4 and 26±3 pmol/mg) or after vitamin E treatment (33±4 and 27±4 pmol/mg), respectively.

View larger version (24K): [in this window] [in a new window]   Figure 4. Bar graphs showing basal cGMP levels in aortas of wild-type (C57BL/6J) and apoE-deficient mice. Results are mean±SEM (n=5 to 10). *P<0.05 vs C57BL/6J mice; P<0.05 vs C57BL/6J with or without antioxidants (ANOVA+Bonferroni’s).

Tetrahydrobiopterin Levels
Total aortic biopterin levels were increased in apoE-deficient mice as compared with wild-type mice (P<0.05). This increase was due to the elevation of BH₄ levels (P<0.05; Figure 5A). 7,8-BH₂/biopterin levels were not affected (NS; Figure 5B). The ratios of BH₄ to 7,8-BH₂/biopterin were not different between two groups of mice (Figure 5C).

View larger version (31K): [in this window] [in a new window]   Figure 5. Bar graphs showing BH4 levels (A), 7,8-BH2/biopterin levels (B), and BH4 to 7,8-BH2/biopterin ratio (C) in aortas of wild-type (C57BL/6J) and apoE-deficient mice after 26 to 28 weeks on a Western-type diet with or without antioxidants. Results are mean±SEM (n=4 to 7). *P<0.05 vs wild-type mice (ANOVA+Bonferroni’s); P<0.05 vs C57BL/6J mice (unpaired t test); #P<0.05 vs apoE-deficient mice (ANOVA+Bonferroni’s).

Treatment of apoE-deficient mice with vitamin C did not affect aortic BH₄ levels. In contrast, vitamin C significantly decreased 7,8-BH₂/biopterin levels in apoE-deficient mice (P<0.05; Figure 5B). Conversely, vitamin C significantly increased BH₄ levels without affecting on 7,8-BH₂/biopterin levels in wild-type mice (P<0.05; Figure 5), whereas vitamin E did not have any effect. Most importantly, vitamin C increased BH₄ to 7,8-BH₂/biopterin ratio in both apoE-deficient and wild-type mice (P<0.05; Figure 5C).

We also measured BH₄ and 7,8-BH₂/biopterin levels in the liver in order to determine whether vitamin C may affect BH₄ metabolism in tissues other than blood vessel. We found that in wild-type mice, 7,8-BH₂/biopterin was very low as compared with BH₄ (Table 2). On the other hand, 7,8-BH₂/biopterin levels were increased in apoE-deficient mice as compared with wild-type (P<0.05; Table 2). Consequently, BH₄ to 7,8-BH₂/biopterin ratio decreased in apoE mice (P<0.05). Vitamin C treatment did not have any effect on BH₄ and 7,8-BH₂/biopterin levels (NS; Table 2), whereas vitamin E slightly decreased BH₄ levels in apoE-deficient mice (P<0.05).

View this table: [in this window] [in a new window]   Table 2. Biopterin Levels in the Liver of ApoE-Deficient and C57BL/6J Mice After 26 to 28 Weeks of Treatment

Vascular O₂^- Production
Formation of O₂^- was increased 3-fold in apoE aortas (P<0.05 versus wild-type mice; Figure 6A). Both antioxidant vitamins significantly decreased O₂^- levels in apoE-deficient mice aortas (P<0.05 versus apoE group; Figure 6A), whereas they did not affect O₂^- production in wild-type mice.

View larger version (28K): [in this window] [in a new window]   Figure 6. A, Detection of superoxide anion in mouse aortas by lucigenin-enhanced chemiluminescence. Photon counts were averaged over 8 minutes and were expressed as counts per min per µg dry weight. Results are mean±SEM (n=7 to 9). *P<0.05 vs C57BL/6J mice; P<0.05 vs apoE-deficient mice (ANOVA+Bonferroni’s). B, Representative Western blot analysis of nitrotyrosine abundance in aortas of apoE-deficient mice. Lane 1 corresponds to 3 µmol/L nitrated bovine albumin as a positive control. Lanes 2 and 5 are apoE; lanes 3 and 6, apoE+vitamin C; and lanes 4 and 7, apoE+vitamin E groups. Lanes 5 to 7, Sodium dithionite (20 mmol/L) treatment (n=3 experiments). Actin blot is shown as loading control.

Detection of Nitrotyrosine
Western blot analysis showed an increased nitrotyrosine abundance in the aorta of apoE-deficient mice (n=4, Figure 6B), whereas in wild-type mice, nitrotyrosine could not be detected (data not shown). Both vitamin C and E reduced tissue nitrotyrosine abundance in apoE-deficient mice (Figure 6B). In order to confirm the specificity of the antibody, sodium dithionite was used to destroy the nitrotyrosine epitope (Figure 6B; lanes 5 to 7).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This is the first study to examine in vivo effects of long-term vitamin C treatment on NOS enzymatic activity and BH₄ metabolism in aortas of wild-type and apoE-deficient mice. We report a number of novel findings. First, vitamin C treatment increased total biopterin and BH₄ levels in aorta of wild-type mice. This increase was associated with increased enzymatic activity of eNOS, iNOS, and higher basal levels of cGMP, suggesting that vitamin C has a BH₄-dependent stimulatory effect on NO formation in normal arterial wall. Second, total biopterin, BH₄, and iNOS enzymatic activity were significantly higher in apoE-deficient mice as compared with wild-type mice. Third, supplementation with vitamin C improved endothelial dysfunction in apoE-deficient mice, reduced atherosclerotic lesions, and restored eNOS enzymatic activity. This is most likely due, in part, to the ability of vitamin C to protect BH₄ and to preserve biosynthesis of NO. Fourth, in contrast to vitamin C, vitamin E did not affect vascular NOS enzymatic activity or BH₄ metabolism. Thus, our results demonstrate that vitamin C (but not vitamin E) is an important regulator of BH₄ metabolism and NOS function in vivo.

BH₄ is an essential cofactor required for activity of NOS.⁶ Previous studies in cultured vascular endothelial cells demonstrated that vitamin C increases eNOS activity by increasing availability of BH₄.^28–30 Increased availability of BH₄ was not due to higher activity of GTP cyclohydrolase I, the rate-limiting enzyme in biosynthesis of BH₄. Rather, chemical stabilization of BH₄ by vitamin C may be the most likely explanation for previously reported observations. In the present study, we tested this concept in vivo by long-term dietary supplementation of vitamin C. Our findings support the idea that vitamin C may increase intracellular concentrations of BH₄ in the normal arterial wall. This, in turn, may activate NOS and increase formation of NO. Increased enzymatic activity of NOS and higher cGMP (but not cAMP) levels in arteries obtained from vitamin C–treated wild-type mice strongly suggest that formation of NO is selectively augmented by vitamin C treatment. It is interesting that iNOS is expressed in wild-type mouse arteries and its activity is very low as compared with Ca²⁺-dependent NOS activity. The fact that vitamin C did not affect expression of eNOS or iNOS protein, together with a significant increase in eNOS and iNOS enzymatic activity, suggest that availability of BH₄ may be a regulatory mechanism designed to control levels of NO production. It appears that in vivo intracellular concentration of BH₄ is subsaturating for vascular NOS isoforms.

Endothelium-dependent relaxations to Ach and endothelium-independent relaxations to DEA-NONOate were impaired in the aortas of vitamin C–treated wild-type mice. This finding is consistent with reported impairment of NO-induced relaxation in eNOS transgenic mice and arteries transduced with recombinant iNOS.^39,40 Vitamin C did not increase formation of O₂^- in normal arteries, ruling out chemical antagonism between O₂^- and NO as an explanation for impairment of relaxations mediated by NO. Downregulation of expression and function of soluble guanylate cyclase in vitamin C–treated aortas is the most likely reason behind reduced reactivity of vascular smooth muscle to NO.^39,41 Further studies are needed to determine the exact mechanism responsible for reduction of relaxations induced by endogenous or exogenous NO. Our results also call for further studies of BH₄ catabolism in normal arteries. Turnover of BH₄ in blood vessels appears to be very rapid. In isolated canine basilar arteries, incubation with a GTP cyclohydrolase I inhibitor for 6 hours resulted in 95% depletion of intracellular BH₄.⁴² The exact molecular mechanisms responsible for degradation of BH₄ that can be inhibited by vitamin C remain to be determined.

Proinflammatory cytokines, including tumor necrosis factor-, interferon-, and interleukin-1ß, stimulate BH₄ biosynthesis in cultured vascular endothelial cells.^43–46 This effect is due to upregulation of GTP cyclohydrolase I transcription, expression, and function.⁴⁶ Simmons and colleagues demonstrated that in cardiac microvascular endothelial cells cytokines cause coordinate induction of GTP cyclohydrolase I and iNOS.⁴⁵ Cytokines play a key role in pathogenesis of atherosclerosis, and therefore, it is not surprising that in the present study we detected 2-fold increases of BH₄ in aortas of apoE-deficient mice. This increase in BH₄ was associated with about 7-fold increase in iNOS enzymatic activity. Thus, the present in vivo findings are consistent with previously obtained results in cultured endothelial cells and support the hypothesis that biosynthesis of BH₄ is coordinated with induction and increased activity of iNOS. They are also consistent with reported increased plasma levels of neopterin, a by-product of BH₄ biosynthesis, in patients with atherosclerosis and coronary syndromes.^47,48

In apoE-deficient mice, vitamin C treatment did not affect aortic BH₄ levels, but did significantly reduce the BH₂ fraction, suggesting that vitamin C may protect BH₄ from oxidation. Catabolism of BH₄ has not been studied in apoE-deficient mice, and we can only speculate about molecular mechanisms underlying protection of BH₄. In a previous study, we demonstrated that peroxynitrite causes oxidation of BH₄.³⁸ This has been confirmed in two subsequent reports.^49,50 Whether endogenous peroxynitrite contributes to oxidation of BH₄ in vivo is unknown. Vitamin C could lessen redox cycling of BH₄ by decreasing intracellular O₂^- and peroxynitrite accumulation because BH₄ has been shown to undergo redox cycling with molecular oxygen, which results in the generation of O₂^-.⁵¹ However, because both vitamins C and E reduced production of O₂^- and nitrotyrosine, but only vitamin C had effects on BH₄ and NOS activity, it appears unlikely that O₂^-/peroxynitrite-mediated oxidation is responsible for oxidation of BH₄. Furthermore, vitamin C was very effective in increasing BH₄ levels in wild-type animals despite the absence of nitrotyrosine and very low O₂^- formation in their aortas. Studies in cultured vascular endothelial cells demonstrated that oxidation of BH₄ to quinonoid 6,7-[8H]-BH₂, rearrangement to 7,8-BH₂ and further oxidation to biopterin is most likely the main pathway of BH₄ degradation.³⁰ Based on the results obtained in cultured endothelium, the stabilizing effect of vitamin C may be due to a chemical reduction of quinonoid 6,7-(8)-BH₂ or BH₃ radical to BH₄.⁵² In addition, vitamin C could also increase the affinity of BH₄ for NOS enzyme by preserving thiols on NOS that are required for binding of the cofactor and, in turn, may stimulate NO-production.^28,30,53

Despite the fact that mice have ability to synthesize vitamin C (unlike humans), increased dietary intake of vitamin C stimulated NOS enzymatic activity in wild-type and apoE-deficient mice. It is possible that the high level of oxidative stress that was found in atherosclerotic apoE-deficient mice^35,49 may consume vitamin C. Indeed, plasma concentrations of vitamin C were significantly lower in apoE-deficient mice. This is consistent with results of epidemiological studies in humans demonstrating that plasma vitamin C concentrations are inversely related to increased risk for atherosclerosis.^54–57 Thus, supplementation of vitamin C may help to replace oxidized vitamin C in apoE-deficient mice. Why long-term treatment with vitamin C increases NOS activity in wild-type animals is unclear and remains to be determined.

Our study is the first to examine the effect of vitamin C on endothelial dysfunction and progression of atherosclerosis in apoE-deficient mice. As expected, vitamin C improved endothelial function, and reduced O₂^- and peroxynitrite formation. These effects could be independent of the effect of vitamin C on BH₄ metabolism. Endothelial cells can take up reduced or oxidized forms of ascorbic acid and accumulate concentrations up to 3 to 8 mmol/L.⁵⁸ This concentration of vitamin C can effectively scavenge O₂^- and protect NO from chemical inactivation.⁵⁹ With regard to the antiatherogenic effect of vitamin E, our results are in agreement with the previously reported ability of vitamin E to prevent development of atherosclerosis in apoE-deficient mice.¹⁸

The present study demonstrates that long-term treatment of C57BL/6J mice with vitamin C increases BH₄ levels in the vascular wall. This increase is coupled with increased eNOS enzymatic activity and high basal levels of cGMP. We also provide evidence that BH₄ metabolism may be an important component in pathogenesis of atherosclerosis. Coordinated upregulation of BH₄ availability and iNOS expression is probably designed to increase biosynthesis of NO in vascular wall exposed to proinflammatory cytokines. However, prolonged high activity of iNOS may be detrimental to vascular function due to "uncoupling" of the enzyme and subsequent increased formation of O₂^-.⁶⁰ Protection of BH₄ appears to be an important mechanism that may contribute to antiatherogenic effect of vitamin C.

	Acknowledgments

 
This work was supported by National Heart, Lung, and Blood Institute grants HL-53524, HL-58080, and HL-66958 and the Mayo Foundation. Livius V. d’Uscio is a recipient of a stipend from the Swiss National Sciences Foundation and a postdoctoral fellowship from the American Heart Association, Northland Affiliate. The authors would like to thank Karen Kloke for measurements of plasma vitamins and Janet Beckman for typing the manuscript.

Received July 30, 2002; revision received November 15, 2002; accepted November 15, 2002.

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