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CARDIOVASCULAR

Protective Effect of Chronic Vitamin C Treatment on Endothelial Function of Apolipoprotein E-Deficient Mouse Carotid Artery

Departments of Anesthesiology and Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic and Foundation, Rochester, Minnesota

Received January 13, 2003; accepted March 14, 2003.

	Abstract

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Endothelium-dependent relaxations are impaired in carotid artery of apolipoprotein E-deficient (apoE^–/–) mice. This impairment seems to be due to increased formation of superoxide anions and inactivation of endothelial nitric oxide (NO). In the present study, we tested hypothesis that chronic treatment with vitamin C may prevent endothelial dysfunction by increasing release of NO from endothelial cells. C57BL/6 and apoE^–/– mice were treated for 26 weeks with Western-type fat diet with and without 1% vitamin C. Vasomotor function of isolated carotid arteries was studied by video dimension analyzer. Expression of endothelial NO synthase (eNOS) and platelet-endothelial cell adhesion molecule-1 (PECAM-1) protein were evaluated by Western blotting. Levels of cGMP and cAMP were measured by radioimmunoassay. In apoE^–/– mice, vitamin C significantly augmented relaxations to acetylcholine (10^–9–10^–5 mol/l), but did not affect relaxations to NO donor diethylammonium-(Z)-1-(N,N-diethylamino) diazen-1-1,2-diolate (DEA-NONOate; 10^–9–10^–5 mol/l). In contrast, vitamin C reduced relaxations to acetylcholine and DEA-NONOate in C57BL/6 mice. Interestingly, vitamin C significantly increased basal cGMP levels in C57BL/6 mice but did not affect cGMP formation in apoE^–/–. Vitamin C treatment did not affect expression of eNOS protein, whereas elevated expression of PECAM-1 protein in apoE^–/– mice was returned to normal level. Our findings demonstrate that chronic treatment with vitamin C prevents endothelial dysfunction of carotid artery induced by hypercholesterolemia. This effect seems to be mediated by preservation of NO bioavailability in endothelial cells.

In our previous study, we demonstrated that long-term treatment with vitamin C has a beneficial effect on endothelial function of apoE^–/– aorta (d'Uscio et al., 2003). However, it is well established that pharmacology of cerebrovascular tree is different from pharmacology of peripheral circulation (Rang et al., 2001). Therefore, the rationale for the present study was based on the fact that in vivo effect of chronic vitamin C treatment on endothelial function of carotid arteries has not been studied. Furthermore, understanding of mechanisms responsible for beneficial effects of vitamin C on vascular function in vivo is incomplete. We hypothesized that chronic treatment with an antioxidant, vitamin C, may protect endothelial function of carotid arteries by preserving normal bioavailability of nitric oxide. If correct, this hypothesis may also help to explain the mechanisms underlying decreased risk of stroke in humans with high plasma concentration of vitamin C (Simon et al., 1998).

	Materials and Methods

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Animal Groups. Male C57BL/6J (wild-type) mice and homozygous apoE-deficient mice (C57BL/6J-ApoE^–/–) were obtained at the age of 4 to 5 weeks from The Jackson Laboratory (Bar Harbor, ME). Housing facilities and all experimental protocols were approved by the Institutional Animal Care and Use Committee of Mayo Clinic (Rochester, MN). C57BL/6J mice and apoE-deficient mice were fed for 26 weeks a lipid-rich Western-type diet (0.15% cholesterol and 42% milk fat by weight, TD88137; Harlan Teklad, Madison, WI) without or with Vitamin C (1%/kg diet) (Plump et al., 1992).

Blood Sample and Body Weight. Body weight was measured with triple beam balance (Ohaus, Florham Park, NJ). Blood samples were obtained through puncture of the right ventricle. The blood was immediately transferred to a tube containing heparin (1000 U) and centrifuged at 4°C for 10 min. Plasma was separated immediately at 4°C and kept at –80°C until assayed. Plasma total cholesterol was determined using a colorimetric-based assay on a Cobas Mira system. Vitamin C was stabilized with 10% meta-phosphoric acid by removing protein and analyzed by UV absorbance after elution from a reversed-phase high-performance liquid chromatography with phosphate buffer (pH 2).

Analysis of Vascular Reactivity with Arteriography. Experiments were performed on 7-mm-long carotid rings from mice that had been anesthetized with pentobarbital (60 mg/kg i.p.) as detailed in our previous study (d'Uscio et al., 2003). Carotid arteries were carefully removed and placed immediately into cold (4°C) modified Krebs-Ringer bicarbonate solution (118.6 mmol/l NaCl, 4.7 mmol/l KCl, 2.5 mmol/l CaCl₂, 1.2 mmol/l MgSO₄, 1.2 mmol/l KH₂PO₄, 25.1 mmol/l NaHCO₃, 0.026 mmol/l calcium-ethylenediamine-tetraacetic acid, and 10.1 mmol/l glucose). Carotid arteries were dissected free from connective tissue in cold Krebs' solution and transferred to an arteriograph (Living Systems Instrumentation, Burlington, VT). At the beginning of each experiment, vessels were equilibrated for 45 min at 50 mm Hg and wall thickness and diameter were measured. Vessels were contracted with thromboxane analog 9,11-dideoxy-11,9-epoxymethano-prostaglandin F₂ (U46619 [GenBank] ; 3 x 10^–8 mol/l). When a steady tone was established, acetylcholine (10^–9–10^–5 mol/l) or DEA-NONOate (10^–9–10^–5 mol/l) were added in cumulative manner. Data are given as changes in diameter (micrometers) of the artery obtained during contraction with U46619 [GenBank] .

Drugs and Chemical Agents. Acetylcholine hydrochloride and 3-isobutyl-1-methylxathnine (IBMX) were from Sigma-Aldrich (St. Louis, MO). DEA-NONOate and U-46619 were from Cayman Chemical (Ann Arbor, MI). DEA-NONOate was prepared as stock solutions in 1.5 mol/l Tris buffer, pH 8.8. U46619 [GenBank] was dissolved in 1 part of 100% ethanol and then diluted with 9 parts of water. The remaining drugs were dissolved in distilled water. All drugs except IBMX were then diluted in Krebs' solution and concentrations are expressed as final molar concentration (moles per liter).

Measurement of the Level of cGMP and cAMP in the Carotid Artery. A radioimmunoassay technique was used to determine the levels of cGMP and cAMP, as reported previously (d'Uscio et al., 2001). Artery was initially incubated in minimal essential media with 10% albumin (Sigma-Aldrich) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA) in a 5% CO₂ incubator at 37°C for 30 min. After this 30-min period, tissue was incubated another 30 min in IBMX (10^–4 mol/l) to inhibit the degradation of cyclic nucleotides by phosphodiesterases. The carotid tissue was then removed from the media and quickly frozen in liquid nitrogen. After homogenization, cGMP and cAMP levels were measured using radioimmunoassay kits (Amersham Biosciences, Inc., Piscataway, NJ). Protein assay was conducted by DC protein assay kit (Bio-Rad, Hercules, CA). The results were expressed as picomoles per milligram of protein.

Western Blot Analysis. Western blot was done as reported previously (d'Uscio et al., 2001). Briefly, after collection and removal of connective tissue, carotid arteries were homogenized on ice in lysis buffer (pH 7.5) containing 50 mM Tris-HCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% SDS, 0.1% deoxycholate, 1% IGEPAL, and a 100-fold dilution of a mammalian protease inhibitor cocktail (all from Sigma-Aldrich). Equal amounts of protein (30 µg/lane) were separated by 7.5% SDS-PAGE and transferred to nitrocellulose membrane (Amersham Biosciences, Inc.). For eNOS protein analysis, monoclonal anti-eNOS (1:100; Transduction Laboratories, San Diego, CA) was used. For PECAM-1 protein analysis, polyclonal anti-PECAM-1 (1: 500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used. Actin (1:50,000; Sigma-Aldrich) was used as internal control. Bands were visualized by enhanced chemiluminescence using a commercially available kit (Amersham Biosciences, Inc.). Densitometry was carried out using NIH Image (Scion-Image; Scion Corporation, Frederick, MD), and the results were expressed in relative densitometry compared with actin.

Statistical Analysis. Results are expressed as mean ± S.E.M., for n animals used in each experimental protocol. One-way analysis of variance with multiple comparisons adjustment (Dunnett's method) determined the statistical significance of differences between the vitamin C, total cholesterol, cGMP, cAMP, and optical intensity values in the different experimental groups. Repeated measures analysis of variance with multiple comparisons adjustment (Bonferroni's method) determined the statistical significance of differences between contraction and relaxation levels in the different experimental groups. A value of P < 0.05 was considered statistically significant.

	Results

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Body Weight and Morphological Data. The body weights were 48.6 ± 0.7, 47.8 ± 0.5, 36.1 ± 1.5,* and 35.7 ± 1.7* g for C57BL/6J mice, C57BL/6J mice treated with vitamin C, apoE^–/– mice, and apoE^–/– mice treated with vitamin C, respectively (*P < 0.05 versus C57BL/BJ and C57BL/6J mice treated with vitamin C; n = 16–17). Diameters of the carotid arteries were 432.6 ± 8.38, 419.2 ± 4.68, 448.2 ± 9.02, and 423.9 ± 9.40 µm in C57BL/6J mice, C57BL/6J mice treated with vitamin C, apoE^–/– mice, and apoE^–/– mice treated with vitamin C, respectively (P = N.S.; n = 15–22). Wall thicknesses of the carotid arteries were 48.0 ± 1.65, 49.9 ± 1.52, 54.3 ± 1.72,* and 48.4 ± 1.53 µm in C57BL/6J mice, C57BL/6J mice treated with vitamin C, apoE^–/– mice, and apoE^–/– mice treated with vitamin C, respectively (*P < 0.05 versus C57BL/6J mice, C57BL/6J mice treated with vitamin C, and apoE^–/– mice treated with vitamin C; n = 15–22).

Plasma Vitamin C and Total Cholesterol. Plasma vitamin C levels were significantly lower in apoE^–/– mice compared with C57BL/6J mice (Fig. 1A). Chronic vitamin C treatment increased 2- to 3-fold in apoE^–/– and C57BL/6J mice treated with vitamin C compared with untreated animals (Fig. 1A). Plasma total cholesterol levels were significantly higher in apoE^–/– mice and apoE^–/– mice treated with vitamin C compared with C57BL/6J mice and C57BL/6J mice treated with vitamin C (Fig. 1B). Chronic vitamin C treatment had no effect on the cholesterol level in apoE^–/– mice.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Plasma level of vitamin C (A) and total cholesterol (B) in C57BL/6J mice and apoE–/– mice after 26 weeks on a Western-type or Western-type supplemented 1% vitamin C. A, plasma level of vitamin C was decreased in apoE–/– mice compared with C57BL/6J mice (*, P < 0.05; n = 4). Vitamin C supplementation increased plasma level of vitamin C in C57BL/6J mice and apoE–/– mice (, P < 0.05; n = 4). B, total cholesterol were significantly higher in apoE–/– mice compared with C57BL/6J mice (, P < 0.05; n = 4). Vitamin C did not affect the plasma level of total cholesterol (Ct, C57BL/6J mice; ApoE, apoE–/– mice; Vit C, vitamin C).

Vascular Reactivity. Concentration-response curves to U46619 [GenBank] were not significantly different among four groups of mice (Fig. 2). During submaximal contractions to U46619 [GenBank] , endothelium-dependent relaxations to acetylcholine were significantly impaired in the carotid artery of C57BL/6J mice treated with vitamin C (Fig. 3A). Endothelium-independent relaxations to the NO donor DEA-NONOate were also significantly impaired in the carotid artery of C57BL/6J mice treated with vitamin C (Fig. 3B). In contrast, endothelium-dependent relaxations to acetylcholine were normalized in the carotid artery of apoE^–/– mice treated with vitamin C (Fig. 4A). Endothelium-independent relaxations to the NO donor DEA-NONOate were not different between apoE^–/– mice and apoE^–/– mice treated with vitamin C (Fig. 4B).

View larger version (11K): [in this window] [in a new window]   Fig. 2. Contraction to U46619 [GenBank] in carotid arteries of C57BL/6J mice, C57BL/6J mice treated with vitamin C (A), apoE–/– mice, and apoE–/– mice treated with vitamin C (B). A, no differences were found between C57BL/6J mice and C57BL/6J mice treated with vitamin C [P = N.S.; analysis of variance (ANOVA) + Bonferroni's; n = 3]. B, no differences were found between apoE–/– mice and apoE–/– mice treated with vitamin C (P = N.S.; ANOVA + Bonferroni's; n = 3) (C57BL/6, C57BL/6J mice; ApoE, apoE–/– mice; Vit C, vitamin C).

View larger version (14K): [in this window] [in a new window]   Fig. 3. Endothelium-dependent relaxations to acetylcholine (A) and endothelium-independent relaxations to DEA-NONOate (B) in carotid arteries of C57BL/6J mice and C57BL/6J mice treated with vitamin C after 26 weeks on a Western-type or Western-type supplemented 1% vitamin C. A, relaxations to acetylcholine were impaired in C57BL/6J mice treated with vitamin C compared with C57BL/6J mice [P < 0.05; analysis of variance (ANOVA) + Bonferroni's; n = 12–13]. B, relaxations to DEA-NONOate were impaired in C57BL/6J mice treated with vitamin C compared with C57BL/6J mice (P < 0.05; ANOVA + Bonferroni's; n = 7) (C57BL/6, C57BL/6J mice; Vit C, vitamin C).

View larger version (14K): [in this window] [in a new window]   Fig. 4. Endothelium-dependent relaxations to acetylcholine (A) and endothelium-independent relaxations to DEA-NONOate (B) in carotid arteries of apoE–/– mice and apoE–/– mice treated with vitamin C after 26 weeks on a Western-type or Western-type supplemented 1% vitamin C. A, relaxations to acetylcholine were normalized in apoE–/– mice treated with vitamin C compared with apoE–/– mice [P < 0.05; analysis of variance (ANOVA) + Bonferroni's; n = 6]. B, no differences were found between apoE–/– mice and apoE–/– mice treated with vitamin C (P = N.S.; ANOVA + Bonferroni's; n = 6–7) (ApoE, apoE–/– mice; Vit C, vitamin C).

cGMP and cAMP Levels. Basal cGMP levels were significantly increased in carotid arteries of C57BL/6J mice treated with vitamin C compared with C57BL/6J, apoE^–/– mice, and apoE^–/– mice treated with vitamin C (Fig. 5A). In contrast, basal cAMP levels were not different among the four groups (Fig. 5B).

View larger version (12K): [in this window] [in a new window]   Fig. 5. Bar graphs showing basal cGMP (A) and cAMP (B) levels in carotid arteries of C57BL/6J, C57BL/6J treated with vitamin C, apoE–/– mice, and apoE–/– mice treated with vitamin C. The data are mean ± S.E.M. (n = 7–8), and a value of P < 0.05 indicated a significant difference (*, P < 0.05 versus C57BL/6J, apoE–/– mice, apoE–/– mice treated with vitamin C) (C57BL/6, C57BL/6J mice; ApoE, apoE–/– mice; Vit C, vitamin C).

eNOS Protein Expression. Western blot analysis detected similar eNOS protein expression in common carotid arteries of C57BL/6J, C57BL/6J treated with vitamin C, apoE^–/– mice, and apoE^–/– mice treated with vitamin C (Fig. 6).

View larger version (24K): [in this window] [in a new window]   Fig. 6. A, representative Western blot analysis of eNOS protein expression in carotid arteries of C57BL/6J, C57BL/6J treated with vitamin C, apoE–/– mice, and apoE–/– mice treated with vitamin C. B, relative densitometric analysis of eNOS protein normalized with actin. eNOS protein expression was similar among four groups. Results are mean ± S.E.M. (n = 3 experiments) (P = N.S.) (Ct, C57BL/6J mice; ApoE, apoE–/– mice; Vit C, vitamin C).

PECAM-1 Protein Expression. PECAM-1 expression was increased in apoE^–/– mice compared with C57BL/6J. Vitamin C treatment normalized the elevated expression of PECAM-1 protein in apoE^–/– mice (Fig. 7).

View larger version (26K): [in this window] [in a new window]   Fig. 7. A, representative Western blot analysis of PECAM-1 protein expression in carotid arteries of C57BL/6J, C57BL/6J treated with vitamin C, apoE–/– mice, and apoE–/– mice treated with vitamin C. B, relative densitometric analysis of PECAM-1 protein normalized with actin. PECAM-1 protein expression was significantly higher in apoE–/– mice among four groups. Results are mean ± S.E.M. (n = 3 experiments) (*, P < 0.05 versus C57BL/6J, C57BL/6J treated with vitamin C, and apoE–/– mice treated with vitamin C) (Ct, C57BL/6J mice; ApoE, apoE–/– mice; Vit C, vitamin C).

	Discussion

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This is the first study to examine the effect of chronic vitamin C treatment on vascular function of mouse carotid artery. In C57BL/6J mice, vitamin C significantly increased basal production of cGMP, most likely reflecting increased formation and release of NO. Indeed, chronic vitamin C treatment increases nitric-oxide synthase enzymatic activity in aortas of C57BL/6J mice (d'Uscio et al., 2003). Consistent with previous reports, increased local concentration of vascular NO caused reduced smooth muscle reactivity to both endogenous NO released by acetylcholine or exogenous NO generated by DEA-NONOate (Molina et al., 1987; Ohashi et al., 1998; d'Uscio et al., 2003). However, the most important finding of the present study is that chronic treatment with vitamin C has beneficial effect on endothelial function in hypercholesterolemic apoE^–/– mice carotid artery. These results are in agreement with our previous report demonstrating an important role of reactive oxygen species in pathogenesis of endothelial dysfunction in apoE^–/– carotid artery (d'Uscio et al., 2001). Unlike humans who depend on dietary intake to maintain normal circulating levels of vitamin C, mice are capable of synthesizing vitamin C (Bhattacharjee et al., 1985). Interestingly, mice and humans have comparable levels of plasma vitamin C (0.6–2.0 mg/dl; Levine et al., 1999). In apoE^–/– mice, we observed that plasma levels of vitamin C were significantly decreased compared with levels in C57BL/6J animals. Although the reason for this decrease is not apparent, it is most likely result of increased vitamin C catabolism due to hypercholesterolemia-induced oxidative stress (Muldoon et al., 1996). Low plasma vitamin C is associated with increased risk of stroke (Kurl et al., 2002). As expected, high cholesterol levels were detected in plasma of apoE^–/– mice and they were not affected by vitamin C treatment.

Vasodilatation in response to acetylcholine and DEANONOate was studied in arteries contracted with a thromboxane A₂ analog, U46619 [GenBank] . We did not detect any difference between C57BL/6J mice and apoE^–/– mice vascular reactivity to U46619 [GenBank] . However, it was surprising that vitamin C reduced relaxations to acetylcholine and DEA-NONOate in C57BL/6J mice. Measurements of cGMP levels in arterial wall indicated that vitamin C significantly increased concentration of nitric oxide second messenger (Chen et al., 2001). This effect was selective for cGMP because cAMP levels were not affected. It is unlikely that vitamin C increases cGMP levels by activation of guanylate cyclase because biochemical studies demonstrated that vitamin C does not directly activate the enzyme (Cherry and Wolin, 1989; Schrammel et al., 2000). It is also unlikely that vitamin C increased formation of reactive oxygen species, including superoxide anion and hydrogen peroxide. In that scenario, superoxide anions may account for impairment of relaxations mediated by nitric oxide, whereas hydrogen peroxide can activate guanylate cyclase and increase basal production of cyclic GMP (Cosentino and Katusic, 1995). In our recent study, we demonstrated that chronic treatment with vitamin C did not increase formation of superoxide anions in mouse aorta (d'Uscio et al., 2003). Although we did not measure NO, tissue levels of cGMP have traditionally served as a very good index of NO production (Lincoln et al., 2001). Indeed, in eNOS transgenic mice, basal levels of cGMP in arterial wall are high and relaxations to both acetylcholine and NO are reduced (Ohashi et al., 1998). Thus, it seems that in mouse carotid artery vitamin C may increase formation of NO, consistent with previously reported ability of vitamin C to stimulate NO biosynthesis in vitro and in vivo (Huang et al., 2000; Baker et al., 2001; Heller et al., 2001; d'Uscio et al., 2003).

The most striking finding of the present study is that vitamin C significantly improved endothelium-dependent relaxations to acetylcholine in carotid artery of apoE^–/– mice. The effect was most likely due to increased bioavailability of NO in endothelium because vitamin C did not affect relaxation of smooth muscle cells to DEA-NONOate. The exact mechanism underlying beneficial effect of vitamin C is not clear. Despite the fact that eNOS protein expression is not affected by vitamin C (Heller et al., 1999; Baker et al., 2001), we may have underestimated eNOS expression in ApoE^–/– mice due to increased carotid artery wall thickness. Therefore, up-regulation of eNOS expression in ApoE^–/– carotid arteries cannot be completely ruled out. Several other mechanisms may account for the observed improvement in endothelial function, including scavenging of superoxide anions (Jackson et al., 1998) and stabilization of eNOS cofactor tetrahydrobiopterin (Huang et al., 2000; Baker et al., 2001; Heller et al., 2001; d'Uscio et al., 2003). Our previous study provided evidence that long-term vitamin C treatment may protect tetrahydrobiopterin from oxidation in aortas of apoE^–/– carotid arteries (d'Uscio et al., 2003). In the present study, we did not measure tetrahydrobiopterin levels because of the technical difficulties caused by very small size of the mouse carotid arteries.

The results of the present study indicated that wall thickness is increased in apoE^–/– mice. Vitamin C treatment prevented this change in vascular structure. The exact mechanism underlying increased wall thickness of apoE^–/– carotid arteries and beneficial effect of vitamin C is unclear. However, our findings are consistent with the reported inverse relationship between vitamin C intake and carotid artery wall thickness in population included in Atherosclerotic Risk in Communities Study (Kritchevsky et al., 1995). Increase in carotid artery thickness is also consistent with reported increase in arterial blood pressure of apoE^–/– (Yang et al., 1999; Buzello et al., 2003).

PECAM-1 is a 130-kDa member of immunoglobulin superfamily that is expressed on the surface of circulating platelets, monocytes, neutrophils, selected T-cell subsets, and vascular endothelial cells (Davies et al., 1993). PECAM-1 participates in the adhesion cascade leading to extravasation of leukocytes to sites of inflammation (Vaporciyan et al., 1993). Pretreating monocytes or neutrophils with antibodies specific for PECAM-1 inhibits their emigration across vascular endothelial cells. We demonstrated that elevated expression of PECAM-1 in apoE^–/– mice was returned to normal level by vitamin C treatment. This may be an important mechanism underlying antiatherosclerotic effect of vitamin C (Lehr et al., 1994, 1995; Weber et al., 1996). Reduction of the leukocytes migration into the vascular wall can reduce local concentrations of superoxide anion (Weber et al., 1996) and contribute to restoration of NO biological activity.

The results of the present study demonstrate that chronic vitamin C treatment prevents endothelial dysfunction in carotid artery of apoE^–/– mice. This effect seems to be mediated by increased bioavailability of endothelial NO. We speculate that in humans dietary intake (or supplementation) of vitamin C could be an important factor in preservation of normal endothelial function of carotid artery and prevention of stroke.

	Acknowledgements

 
We thank Ana G. Rosales for help with statistical analysis and Janet Beckman for preparing the manuscript.

	Footnotes

 
This work was supported in part by National Institutes of Health Grants NS-37491 and HL-53524, the American Heart Association Bugher Foundation Award for the Investigation of Stroke, and Mayo Foundation. L.V.d'U. is the recipient of a Swiss National Science Foundation stipend, and an American Heart Association Northland Affiliate Fellowship.

DOI: 10.1124/jpet.103.049163.

ABBREVIATIONS: NO, nitric oxide; PECAM-1, platelet-endothelial cell adhesion molecule-1; apoE^–/–, apolipoprotein E-deficient; IBMX, 3-isobutyl-1-methylxathnine; eNOS, endothelial nitric oxide synthase; DEA-NONOate, diethylammonium-(Z)-1-(N,N-diethylamino) diazen-1-1,2-diolate.

Address correspondence to: Dr. Zvonimir S. Katusic, Department of Anesthesiology and Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905. E-mail: katusic.zvonimir{at}mayo.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Baker TA, Milstien S, and Katusic ZS (2001) Effect of vitamin C on the availability of tetrahydrobiopterin in human endothelial cells. J Cardiovasc Pharmacol 37: 333–338.[CrossRef][Medline]

Bhattacharjee J, Chakraborty AS, Sarkar NK, Basu A, and Mitra S (1985) Study of ascorbate status in murine and human leukaemias. J Comp Pathol 95: 87–91.[CrossRef][Medline]

Buzello M, Tornig J, Faulhaber J, Ehmke H, Ritz E, and Amann K (2003) The apolipoprotein E knockout mouse: a model documenting accelerated atherogenesis in uremia. J Am Soc Nephrol 14: 311–316.[Abstract/Free Full Text]

Cai H and Harrison DG (2000) Endothelial dysfunction in cardiovascular diseases. The role of oxidant stress. Circ Res 87: 840–844.[Abstract/Free Full Text]

Chen ZJ, Che D, and Chang CH (2001) Antioxidants, vitamin C and dithiothreitol, activate membrane-bound guanylate cyclase in PC 12 cells. J Pharm Pharmacol 53: 243–247.[CrossRef][Medline]

Cherry PD and Wolin MS (1989) Ascorbate activates soluble guanylate cyclase via H₂O₂-metabolism by catalase. Free Radic Biol Med 7: 485–490.[CrossRef][Medline]

Cosentino F and Katusic ZS (1995) Tetrahydrobiopterin and dysfunction of endothelial nitric oxide synthase in coronary arteries. Circulation 91: 139–144.[Abstract/Free Full Text]

Davies MJ, Gordon JL, Gearing AJ, Pigott R, Woolf N, Katz D, and Kyriakopoulous A (1993) The expression of the adhesion molecules ICAM-1, VCAM-1, PECAM and E-selectin in human atherosclerosis. J Pathol 171: 223–229.[CrossRef][Medline]

d'Uscio LV, Milstien S, Richardson D, Smith L, Katusic ZS (2003) Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity. Circ Res 92: 88–95.[Abstract/Free Full Text]

d'Uscio LV, Smith L, and Katusic ZS (2001) Hypercholesterolemia impairs endothelium-dependent relaxations in common carotid arteries of apolipoprotein E-deficient mice. Stroke 32: 2658–2664.[Abstract/Free Full Text]

Heller R, Munscher Paulig F, Grabner R, and Till U (1999) L-Ascorbic acid potentiates nitric oxide synthesis in endothelial cells. J Biol Chem 274: 8254–8260.[Abstract/Free Full Text]

Heller R, Unbehaun A, Schellenberg B, Mayer B, Werner-Felmayer G, and Werner ER (2001) L-Ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin. J Biol Chem 276: 40–47.[Abstract/Free Full Text]

Huang A, Vita JA, Venema RC, and Keaney JF Jr (2000) Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin. J Biol Chem 275: 17399–17406.[Abstract/Free Full Text]

Jackson TS, Xu A, Vita JA, and Keaney JF Jr (1998) Ascorbate prevents the interaction of superoxide and nitric oxide only at very high physiological concentrations. Circ Res 83: 916–922.[Abstract/Free Full Text]

Kritchevsky SB, Shimakawa T, Tell GS, Dennis B, Carpenter M, Eckfeldt JH, Peacher-Ryan H, and Heiss G (1995) Dietary antioxidants and carotid artery wall thickness. The ARIC Study. Atherosclerosis Risk in Communities Study. Circulation 92: 2142–2150.[Abstract/Free Full Text]

Kurl S, Tuomainen TP, Laukkanen JA, Nyyssonen K, Lakka T, Sivenius J, and Salonen JT (2002) Plasma vitamin C modifies the association between hypertension and risk of stroke. Stroke 33: 1568–1573.[Abstract/Free Full Text]

Lehr H, Frei B, and Arfors K (1994) Vitamin C prevents cigarette smoke-induced leukocyte aggregation and adhesion to endothelium in vivo. Proc Natl Acad Sci USA 91: 7688–7692.[Abstract/Free Full Text]

Lehr H, Frei B, Olofsson AM, Carew TE, and Arfors KE (1995) Protection from oxidized LDL-induced leukocyte adhesion to microvascular and macrovascular endothelium in vivo by vitamin C but not by vitamin E. Circulation 91: 1525–1532.[Abstract/Free Full Text]

Levine M, Rumsey SC, Daruwala R, Park JB, and Wang Y (1999) Criteria and recommendations for vitamin C intake. J Am Med Assoc 281: 1415–1423.[Abstract/Free Full Text]

Lincoln TM, Dey N, and Sellak H (2001) cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol 91: 1421–1430.[Abstract/Free Full Text]

Molina CR, Andresen JW, Rapoport RM, Waldman S, and Murad F (1987) Effect of in vivo nitroglycerin therapy on endothelium-dependent and independent vascular relaxation and cyclic GMP accumulation in rat aorta. J Cardiovasc Pharmacol 10: 371–378.[Medline]

Muldoon MF, Kritchevsky SB, Evans RW, and Kagan VE (1996) Serum total antioxidant activity in relative hypo- and hypercholesterolemia. Free Radic Res 25: 239–245.[Medline]

O'Brien KD, McDonald TO, Chait A, Allen MD, Alpers CE (1996) Neovascular expression of E-selectin, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content. Circulation 93: 672–682.[Abstract/Free Full Text]

Ohashi Y, Kawashima S, Hirata K, Yamashita T, Ishida T, Inoue N, Sakoda T, Kurihara H, Yazaki Y, and Yokoyama M (1998) Hypotension and reduced nitric oxide-elicited vasorelaxation in transgenic mice overexpressing endothelial nitric oxide synthase. J Clin Investig 102: 2061–2071.[Medline]

Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh A, Verstuyft JG, Rubin EM, and Breslow JL (1992) Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell 71: 343–353.[CrossRef][Medline]

Poston RN and Johnson-Tidey RR (1996) Localized adhesion of monocytes to human atherosclerotic plaques demonstrated in vitro: implications for atherogenesis. Am J Pathol 149: 73–80.[Abstract]

Rang HP, Dale MM, Ritter JM, and Gardner P (2001) The vascular system, in Pharmacology, 4th ed, pp 278–300, Churchill Livingstone, London.

Schrammel A, Koesling D, Schmidt K, and Mayer D (2000) Inhibition of purified soluble guanylyl cyclase by L-ascorbic acid. Cardiovasc Res 47: 602–608.[Abstract/Free Full Text]

Simon JA, Hudes ES, and Browner WS (1998) Serum ascorbic acid and cardiovascular disease prevalence in US adults. Epidemiology 9: 316–321.[CrossRef][Medline]

Vaporciyan AA, DeLisser HM, Yan HC, Mendiguren II, Thom SR, Jones ML, Ward PA, and Albelda SM (1993) Involvement of platelet-endothelial cell adhesion molecule-1 in neutrophil recruitment in vivo. Science (Wash DC) 262: 1580–1582.[Abstract/Free Full Text]

Weber C, Erl W, Weber K, and Weber PC (1996) Increased adhesiveness of isolated monocytes to endothelium is prevented by vitamin C intake in smokers. Circulation 93: 1488–1492.[Abstract/Free Full Text]

Yang R, Powell-Braxton L, Ogaoawara AK, Dybdal N, Bunting S, Ohneda O, and Jin H (1999) Hypertension and endothelial dysfunction in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol 19: 2762–2768.[Abstract/Free Full Text]

Zeiher AM, Drexler H, Wollshlager H, and Just H (1991) Modulation of coronary vasomotor tone in humans. Progressive endothelial dysfunction with different early stages of coronary atherosclerosis. Circulation 83: 391–401.[Abstract/Free Full Text]

Zhang SH, Reddick RL, Piedrahita JA, and Maeda N (1992) Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science (Wash DC) 258: 468–471.[Abstract/Free Full Text]

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