AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 290: H2466-H2471, 2006. First published January 20, 2006; doi:10.1152/ajpheart.00366.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/6/H2466    most recent
00366.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by d'Uscio, L. V.
Right arrow Articles by Katusic, Z. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by d'Uscio, L. V.
Right arrow Articles by Katusic, Z. S.

Increased vascular biosynthesis of tetrahydrobiopterin in apolipoprotein E-deficient mice

Livius V. d'Uscio and Zvonimir S. Katusic

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

Submitted 14 April 2005 ; accepted in final form 17 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies suggested that loss of tetrahydrobiopterin (BH4) may play an important role in the pathogenesis of vascular endothelial dysfunction induced by diabetes and hypertension. In contrast, controversial results have been reported regarding BH4 metabolism in experimental models of atherosclerosis. Therefore, the present study was designed to characterize the expression and activity of GTP-cyclohydrolase I, a rate-limiting enzyme in biosynthesis of BH4, during atherogenesis. BH4 levels were significantly increased in atherosclerotic aortas of apolipoprotein E (apoE)-deficient mice as compared with wild-type mice after 5 mo of Western diet treatment. This increase was further significantly enhanced in apoE-deficient mice fed for 9 and 14 mo. Removal of the endothelium almost eliminated BH4 in wild-type mice but not in apoE-deficient mice, suggesting that a major component of increased BH4 synthesis is localized in the vascular media of apoE-deficient mice. Oxidative products of BH4 were low and did not differ between wild-type and apoE-deficient mice over the course of this study. Increased protein expression and enzymatic activity of GTP-cyclohydrolase I were detected in aortas of apoE-deficient mice (P < 0.05), providing molecular mechanisms responsible for elevation of vascular BH4. In contrast to aortas, we did not detect any change in levels of BH4 and in GTP-cyclohydrolase I expression in the brain. Our results demonstrate selective increase of intracellular BH4 levels via elevation of GTP-cyclohydrolase I activity in vascular tissue of apoE-deficient mice.

guanosine 5'-triphosphate-cyclohydrolase I; atherosclerosis

ATHEROSCLEROSIS is a multifactorial vascular disease that can be triggered by a combination of genetic and risk factors such as diet, smoking, hypercholesterolemia, and hypertension, which cause endothelial dysfunction and thereby promote the development of atherosclerosis (29). Under these conditions, endothelium-dependent relaxations are reduced and the release of biologically active nitric oxide (NO) is impaired (8). The latter may be caused by breakdown of the endogenous NO by superoxide anion and/or decreased expression and activity of endothelial NO synthase (6, 20, 23, 24). Existing evidence suggests that, during activation of constitutive and inducible NO synthase isoforms, tetrahydrobiopterin (BH4) is needed for allosteric and redox activation of its enzymatic activity (27, 37). Biosynthesis of BH4 is initiated by activation of rate-limiting enzyme GTP-cyclohydrolase I (GTPCH I) (9).

In vitro studies demonstrated that suboptimal levels of BH4 can lead to uncoupling of NO synthase, which could become a major source of superoxide anion and thereby cause oxidative stress in vascular disease (41). Previous in vivo studies suggest that oxidation of BH4, as a consequence of increased oxidative stress in cardiovascular disease, may contribute to endothelial dysfunction in diabetic rats and DOCA-salt hypertensive mice (19, 33). Furthermore, alterations in BH4 levels have been reported in animal models of hypercholesterolemia and atherosclerosis (1, 7, 25, 40). However, expression and activity of GTPCH I, a rate-limiting enzyme in biosynthesis of BH4, during atherogenesis have not been determined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. Four- to five-week-old male wild-type (C57BL/6J) and homozygous apolipoprotein E (apoE)-deficient mice (C57BL/6J-ApoETm1Unc) were obtained from Jackson Laboratory (Bar Harbor, ME). Housing facilities and all experimental protocols were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic and comply with the "Guiding Principles in the Care and Use of Animals" of the American Physiological Society. Wild-type and apoE-deficient mice were fed a lipid-rich, Western-type diet (0.15% cholesterol and 42% of milk fat by weight; TD88137, Harlan Teklad, Madison, WI) for up to 14 mo to accelerate the development of atherosclerosis (26, 48). At 3, 5, 9, or 14 mo after treatments, the mice were anesthetized (60 mg/kg body wt ip pentobarbital), and blood samples were collected through right ventricle puncture. Blood was immediately transferred to a tube containing heparin and centrifuged at 4°C for 10 min. Plasma was stored at –80°C until assayed. Plasma lipid profile was determined using a colorimetric-based assay on a Cobas Mira system. Aortas were carefully removed and dissected free from connective tissue in cold (4°C) modified Krebs-Ringer bicarbonate solution (in mmol/l: 118.6 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.1 NaHCO3, 10.1 glucose, and 0.026 EDTA).

Lesion assessment. Aortas were opened longitudinally and fixed in 4% buffered paraformaldehyde for 2 h. Intraluminal lipid-rich lesions were stained in supersaturated Sudan IV solution in 38% isopropanol for an additional 16 h (32). After being stained, the aortas were washed in distilled water and kept in 4% formalin. Serial images of the submerged vessels were captured with a digital camera. Lesion analysis was performed with Image Pro Plus 3.0 software. The area of lesion formation in each aorta was measured and expressed as %lesion area per total area of the aorta.

Measurements of tissue BH4 and 7,8-BH2 + biopterin levels. Freshly isolated whole aortas and brains were homogenized in extraction buffer containing 50 mmol/l Tris (pH 7.4), 1 mmol/l dithiothreitol, and 1 mmol/l EDTA at 4°C and were centrifuged at 10,000 g (8 min at 4°C). One whole aorta is needed for n = 1 experiment. Biopterin levels were determined after differential oxidation in acid (which converts both BH4 and 7,8-BH2 to biopterin) and base (which converts only 7,8-BH2 to biopterin) conditions by reverse-phase HPLC (Beckman Coulter, Fullerton, CA) with a fluorescence detector (Jasco) as described previously (7). Data were collected and analyzed by 32 Karat chromatography software (Beckman Coulter) and normalized against tissue protein levels. BH4 content was calculated from the difference in biopterin levels after acid and base oxidations.

To evaluate the contribution of endothelium to BH4 synthesis, aortas with and without endothelium were studied in parallel. The dissected aorta was opened lengthwise and fixed on a siliconated dish. Endothelial surface was incubated with 0.1% collagenase in PBS for 5 min at room temperature (38). Endothelial cells were removed carefully and softly using a surgical blade; the aorta was washed twice with extraction buffer and was homogenized as described above. The removal of endothelium was confirmed by the absence of endothelial NO synthase protein expression.

Measurement of GTPCH I activity. Tissue supernatant homogenates, prepared as in the biopterin assay, were filtered using a Sephadex G25M column (Amersham, Piscataway, NJ) to remove endogenous neopterin, BH4, and phenylalanine. GTPCH I enzymatic activity was assayed using reverse-phase HPLC method (Beckman Coulter) by measurements of neopterin, which was derived from dihydroneopterin triphosphate after oxidation and phosphate treatment (39). Results were normalized against tissue protein levels.

Western blot analysis. Fresh aortas and brains were homogenized on ice in lysis buffer (pH 7.5) containing 50 mmol/l Tris·HCl, 0.1 mmol/l EDTA, 0.1 mmol/l EGTA, 0.1% SDS, 0.1% deoxycholate, 1% Igepal, and mammalian protease inhibitor cocktail (Sigma) and then centrifuged. Equal amounts of protein (100 µg/lane) from all groups were separated by SDS-PAGE and transferred to nitrocellulose membrane (Amersham) using a semidry electrophoretic transfer cell for Western analysis. Polyclonal anti-GTPCH-I was raised in rabbits against a peptide ERELPRPGASPPAEK of the mouse GTPCH-I (National Center for Biotechnology Information accession number NP032128; Invitrogen, Carlsbad, CA). The specificity of the GTPCH I antibody was verified by incubation of vascular smooth muscle cells in the presence or absence of LPS (1 µg/ml) for 6 h. GTPCH I is selectively increased in LPS-stimulated cells only (unpublished observations). Bands were visualized by enhanced chemiluminescence with the use of a commercially available kit (Amersham) and normalized on actin (Sigma, St. Louis, MO).

Calculations and statistical analysis. Results are expressed as means ± SE, and n indicates the number of animals from which tissues were harvested. Control and apoE-deficient mice groups were compared by two-way ANOVA for multiple comparisons. For simple comparisons between two age-matched groups of control and apoE-deficient mice, an unpaired Student's t-test was used where appropriate. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Mice characteristics. Plasma cholesterol and LDL and HDL levels were slightly increased in wild-type mice after 3, 5, 9, and 14 mo of Western-type diet (P < 0.05; Table 1). In contrast, Western diet treatment elevated plasma cholesterol levels in apoE-deficient mice (P < 0.05; Table 1). Of note, cholesterol and LDL levels were significantly elevated in apoE-deficient mice as compared with wild-type mice throughout the study (P < 0.05; Table 1). Interestingly, after 3 mo of treatment, plasma HDL levels were higher in apoE-deficient mice as compared with wild-type mice (P < 0.05; Table 1). After 5 mo of treatment onward, HDL levels declined.


View this table:
[in this window]
[in a new window]
  		Table 1. Characteristics of apoE-deficient and wild-type mice after 0–14 mo of treatment

 
Morphology. Before the beginning of Western-type diet treatment, there were no visible atherosclerotic plaques found in the aorta of 4- to 5-wk-old apoE-deficient mice. Only a few aortic lesion spots were observed after 3 mo of treatment (6.4 ± 0.3% of total area; P < 0.05 vs. 0 mo; n = 5). Five months of treatment markedly increased aortic plaque formation to 27.4 ± 2.9% in apoE-deficient mice (P < 0.05 vs. 0- and 3-mo treatment; n = 5). This increase was further enhanced after 9 and 14 mo on a Western diet (36.5 ± 0.4 and 44.3 ± 3.7% of total area, respectively; P < 0.05; n = 4). In wild-type mice, there was no lesion formation observed during the time course of the study.

Tissue BH4 levels. Time-dependent studies showed that BH4 levels were significantly increased in apoE-deficient mice aortas as compared with wild-type mice after 5 mo on Western diet (P < 0.05; Fig. 1B). This increase was further enhanced after 9 and 14 mo of treatment (P < 0.05). Oxidative products of BH4, 7,8-BH2 and biopterin, were slightly increased in both wild-type and apoE-deficient mice after 5 and 9 mo as compared with before treatment (P < 0.05). However, they were not different between wild-type and apoE-deficient mice aortas over the course of the study (Fig. 1C).


Figure 1
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 1. Original HPLC and fluorescence chromatograms of biopterin standard and of biopterin after acid and base oxidations are shown (A). Bar graphs show tetrahydrobiopterin (BH4) levels (B) and 7,8-dihydrobiopterin + biopterin levels (C) in aortas of wild-type (WT; C57BL/6J) and apolipoprotein E (apoE)-deficient mice on Western-type diet. Time-dependent studies showed that BH4 levels were increased in apoE-deficient mice aortas as compared with WT mice after 5 mo on diet (B). This increase was further enhanced after 9 and 14 mo. In contrast to BH4, the levels of its oxidative products 7,8-dihydrobiopterin and biopterin were not different between wild-type and apoE-deficient mice aortas over the course of this study (C). Results are means ± SE (n = 5–8). *P < 0.05 vs. age-matched C57BL/6J mice; {dagger}P < 0.05 vs. untreated mice (0 mo); #P < 0.05 vs. treated mice for 3 mo (two-way ANOVA).

 
To evaluate the site of BH4 synthesis, we determined BH4 levels in endothelial cells. We found that ~85% of total BH4 is present in endothelial cells of control wild-type mice, whereas only 15% is localized in the media (Fig. 2). Interestingly, BH4 levels were doubled in the endothelium of age-matched apoE-deficient mice on Western diet for 9 mo. However, removal of the endothelium did not eliminate all BH4; levels of BH4 were increased more than eight times in media of apoE-deficient mice (Fig. 2).


Figure 2
View larger version (9K):
[in this window]
[in a new window]
  		Fig. 2. Effect of endothelium removal (E–) on tetrahydrobiopterin (BH4) levels in aortas of WT ( C57BL/6J) and apoE-deficient mice on a Western-type diet for 9 mo is shown. Note that removal of the endothelium almost eliminated BH4 in aortas of WT mice but not in apoE-deficient mice. Results are means ± SE (n = 5). *P < 0.05 vs. aortas of same species with endothelium (E+); {dagger}P < 0.05 vs. C57BL/6J mice without endothelium (two-way ANOVA).

 
In contrast to the aorta, BH4 levels were unchanged in the brain of wild-type and apoE-deficient mice (Fig. 3A), suggesting that the increase in aortic BH4 levels is tissue specific and related to development of atherosclerosis (6, 20). In addition, there was no evidence of increased oxidation of BH4 in the brain of wild-type and apoE-deficient mice (Fig. 3B).


Figure 3
View larger version (13K):
[in this window]
[in a new window]
  		Fig. 3. Tetrahydrobiopterin levels (A) and levels of its oxidative products 7,8-dihydrobiopterin and biopterin (B) were unchanged in brains of WT (C57BL/6J) and apoE-deficient mice over the course of this study. Results are means ± SE (n = 5–8).

 
GTPCH I enzymatic activity and protein expression. To evaluate the mechanisms of increased BH4 levels in apoE-deficient mice, we determined GTPCH I enzymatic activity. GTPCH I activity was significantly increased in apoE-deficient mice aortas as compared with wild-type mice after 5 mo on a Western-type diet (Fig. 4). This increase was further enhanced after 9 and 14 mo (Fig. 4), indicating that the increase in BH4 levels is due to the increased de novo biosynthesis of BH4 via GTPCH I enzyme.


Figure 4
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 4. GTP-cyclohydrolase I (GTPCH I) activity was increased in apoE-deficient mice aortas as compared with WT (C57BL/6J) mice after 5 mo on a Western-type diet. This increase was further enhanced after 9 and 14 mo on diet. Results are means ± SE (n = 5–9). *P < 0.05 vs. age-matched C57BL/6J mice; {dagger}P < 0.05 vs. untreated mice (0 mo) (two-way ANOVA).

 
Western blot analysis revealed increased GTPCH I protein expression in aortas of apoE-deficient mice as compared with wild-type mice after 3 mo (P < 0.05; Fig. 5, A and B), which was further enhanced after 5 and 9 mo of treatment with a Western diet (P < 0.05; Fig. 5, A and B). In contrast, GTPCH I protein expressions were not different in the brain of wild-type and apoE-deficient mice during the time course of the study (Fig. 5C).


Figure 5
View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5. A: representative Western blot analysis of GTP-cyclohydrolase I (GTPCH I) protein expression in aortas of WT (C57BL/6J) and apoE-deficient mice. Upregulation of GTPCH I protein expression was noted in aortas of apoE-deficient mice as compared with WT mice. M, months. B: bar graphs indicate results of relative densitometry compared with actin. Results are means ± SE (n = 3 independent experiments). *P < 0.05 vs. age-matched C57BL/6J mice; {dagger}P < 0.05 vs. 3 mo (two-way ANOVA). C: representative Western blot analysis of GTPCH I protein expression in brains of WT (C57BL/6J) and apoE-deficient mice (n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first study to investigate vascular biopterin metabolism during progression of atherosclerosis in apoE-deficient mice. We report several novel findings. First, BH4 levels are selectively increased in the aorta of atherosclerotic apoE-deficient mice as compared with age-matched wild-type mice. Second, endothelium is a major source of BH4 in wild-type mice, whereas both endothelium and vascular media contribute to BH4 synthesis in apoE-deficient mice. Third, elevation of BH4 levels in atherosclerotic aorta is caused by the increased protein expression and enzymatic activity of GTPCH I, the rate-limiting enzyme required for de novo biosynthesis of BH4.

It is well established that lesions accommodate active sites of inflammation and immune responses, whereas cytokines appear to orchestrate the chronic development of atherosclerosis, leading to the formation of complex atherosclerotic plaques (47). In addition, we and others have shown that production of superoxide anion is increased in endothelial and vascular smooth muscle cells and atheromatous plaques, suggesting that oxidative stress is present in atherosclerotic arteries (6, 22). Furthermore, NO-mediated, endothelium-dependent relaxations to acetylcholine and Ca2+ ionophore are impaired in atherosclerotic aortas of apoE-deficient mice (1, 6, 20, 43, 46). Superoxide anion scavengers improve endothelial function, indicating that inactivation of NO by superoxide anion is responsible for endothelial dysfunction in atherosclerotic apoE-deficient mice (6, 20).

It has been proposed that BH4 is an important molecular target for oxidation by reactive oxygen species and that reduced bioavailability of vascular BH4 may result in impairment of NO-mediated endothelium-dependent vasodilatation. Indeed, there are several reports showing that in vivo administration of BH4 improves endothelium-dependent vasodilatation in hypercholesterolemia (34), cigarette smoking (10), diabetes (33), and atherosclerosis (36). Significant reduction of BH4 levels, caused by both increased oxidation and/or decreased biosynthesis of BH4, has been reported in blood vessels obtained from experimental animals with diabetes and hypertension (19, 33). However, in the present study, we provide evidence that BH4 levels are not different in aortas of wild-type and apoE-deficient mice after 3 mo of treatment with Western diet, despite increased cholesterol and LDL levels, suggesting that BH4 and BH2 are not affected during early stages of hypercholesterolemia. In contrast, after 5 mo of treatment onward, BH4 levels were significantly increased in atherosclerotic aorta of apoE-deficient mice, which is in line with the previous reports (1, 7). Interestingly, levels of 7,8-BH2 and biopterin, oxidative products of BH4, remained unchanged in the aorta of wild-type and apoE-deficient mice during the time course of the study, suggesting that oxidation of BH4 does not contribute to reported endothelial dysfunction induced by hypercholesterolemia (6, 20, 46). However, the findings are at variance with reported reduction of BH4 levels in apoE-deficient mice overexpressing endothelial NO synthase (25) or in hypercholesterolemic rabbits (40). The reason for discrepancy is not clear, but the most likely explanation is that studies by Ozaki et al. (25) and Vasquez-Vivar et al. (40) used atherosclerotic animals that had ~30 times higher circulating levels of cholesterol as compared with control animals. In contrast, we and others have observed that, in apoE-deficient mice, total cholesterol levels were increased only about three times (1, 13). Thus it appears that hypercholesterolemia tends to increase BH4 levels, whereas only severe increase in cholesterol levels can reduce availability of BH4. In contrast, in the brain, BH4 levels and GTPCH I protein expression were not different between wild-type and apoE-deficient mice, supporting our conclusion regarding selective increase in BH4 biosynthesis in the arterial wall.

To evaluate the mechanisms of increased BH4 levels in apoE-deficient mice, we determined vascular GTPCH I enzymatic activity. We provide evidence that GTPCH I activity is augmented in apoE-deficient mice aortas after 5 mo on a Western diet, indicating that the selective increase in BH4 levels was due to the increased de novo biosynthesis of BH4. These findings are consistent with reported high plasma levels of neopterin in patients with atherosclerosis and coronary syndromes (2, 31, 35, 44). Interestingly, we also detected upregulation of GTPCH I protein expression in the aorta of apoE-deficient mice after 5 and 9 mo, further suggesting that increased BH4 levels found in atherosclerotic aortas are caused by both increased enzymatic activity and protein expression of GTPCH I. The exact mechanism responsible for upregulation of GTPCH I and BH4 synthesis is unclear. However, atherosclerosis is an inflammatory disease, and the presence of inflammatory cytokines in atherosclerotic blood vessels has been well documented (29, 47). In addition, recent studies (4, 12, 16, 21, 42) confirmed the involvement of the cytokines and chemokines in the progression of atherosclerosis in apoE-deficient mice. Consistent with the findings of the present study, inflammatory cytokines such as tumor necrosis factor-{alpha}, interferon-{gamma}, and interleukin-1beta stimulate BH4 biosynthesis via upregulation of GTPCH I activity and protein expression in cultured vascular endothelial cells (11, 14, 28, 30, 45).

BH4 exerts its vascular protective effect by stimulation of NO production and/or protection of proteins from nitration by peroxynitrite or nitrogen dioxide (3, 18). Both pharmacologic and genetic elevation of BH4 in aortas of apoE-deficient mice has an antiatherogenic effect (1, 7). In addition, a recent study (15) showed that BH4 deficiency in the mutant hph-1 mouse accelerates atherosclerosis progression. On the basis of these observations, we speculate that elevation of BH4 biosynthesis during atherosclerosis development is an adaptive response designed to optimize NO production. Indeed, in our previous study, we demonstrated that in aortas obtained from apoE-deficient mice fed a Western diet for 6 mo, total enzymatic activity of NOS is three times higher as compared with control arteries (7). This phenomenon was caused by increased expression and activity of inducible NO synthase, which is localized for the most part in the vascular media of apoE-deficient mice (17). Thus coordinated upregulation of inducible NO synthase and GTPCH I in atherosclerotic vessels is most likely caused by inflammatory cytokines (5, 28). However, a detected increase of BH4 is apparently insufficient to prevent the uncoupling of endothelial NO synthase (1). Excessive demand of BH4 in atherosclerotic arteries expressing very high levels of inducible NO synthase could create relative deficiency of cofactor. This would explain the beneficial effects of BH4 supplementation on endothelial function of experimental animals and humans with atherosclerosis (1, 36).

In conclusion, our results demonstrate that BH4 (but not 7,8-BH2) levels are selectively increased in atherosclerotic apoE-deficient mouse aortas. Elevation of BH4 levels is most likely caused by the increased de novo biosynthesis via GTPCH I pathway. The exact role of arterial GTPCH I upregulation in pathogenesis of atherosclerosis is unclear and remains to be determined.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-53524, HL-58080, and HL-66958 and by the Mayo Foundation.


   ACKNOWLEDGMENTS
 
We thank Janet Beckman for secretarial assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. S. Katusic, Dept. of Anesthesiology, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905 (e-mail: katusic.zvonimir{at}mayo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alp NJ, McAteer MA, Khoo J, Choudhury RP, and Channon KM. Increased endothelial tetrahydrobiopterin synthesis by targeted transgenic GTP-cyclohydrolase I overexpression reduces endothelial dysfunction and atherosclerosis in ApoE-knockout mice. Arterioscler Thromb Vasc Biol 24: 445–450, 2004.[Abstract/Free Full Text]
  2. Auer J, Berent R, Labetanig E, and Eber B. Serum neopterin and activity of coronary artery disease. Heart Dis 3: 297–301, 2001.[CrossRef][Medline]
  3. Aulak KS, Miyagi M, Yan L, West KA, Massillon D, Crabb JW, and Stuehr DJ. Proteomic method identifies proteins nitrated in vivo during inflammatory challenge. Proc Natl Acad Sci USA 98: 12056–12061, 2001.[Abstract/Free Full Text]
  4. Branen L, Hovgaard L, Nitulescu M, Bengtsson E, Nilsson J, and Jovinge S. Inhibition of tumor necrosis factor-alpha reduces atherosclerosis in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol 24: 2137–2142, 2004.[Abstract/Free Full Text]
  5. Busse R and Mulsch A. Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells. FEBS Lett 275: 87–90, 1990.[CrossRef][ISI][Medline]
  6. D'Uscio LV, Baker TA, Mantilla CB, Smith L, Weiler D, Sieck GC, and Katusic ZS. Mechanism of endothelial dysfunction in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 21: 1017–1022, 2001.[Abstract/Free Full Text]
  7. D'Uscio LV, Milstien S, Richardson D, Smith L, and Katusic ZS. Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity. Circ Res 92: 88–95, 2003.[Abstract/Free Full Text]
  8. Harrison DG, Freiman PC, Armstrong ML, Marcus ML, and Heistad DD. Alterations of vascular reactivity in atherosclerosis. Circ Res 61: II74–II80, 1987.[Medline]
  9. Hatakeyama K, Harada T, Suzuki S, Watanabe Y, and Kagamiyama H. Purification and characterization of rat liver GTP cyclohydrolase I. Cooperative binding of GTP to the enzyme. J Biol Chem 264: 21660–21664, 1989.[Abstract/Free Full Text]
  10. Heitzer T, Brockhoff C, Mayer B, Warnholtz A, Mollnau H, Henne S, Meinertz T, and Münzel T. Tetrahydrobiopterin improves endothelium-dependent vasodilation in chronic smokers: evidence for a dysfunctional nitric oxide synthase. Circ Res 86: E36–E41, 2000.
  11. Huang A, Zhang Y, Chen K, Hatakeyama K, and Keaney JF Jr. Cytokine-stimulated GTP cyclohydrolase I expression in endothelial cells requires coordinated activation of nuclear factor-{kappa}B and Stat1/Stat3. Circ Res 96: 164–171, 2005.[Abstract/Free Full Text]
  12. Inoue S, Egashira K, Ni W, Kitamoto S, Usui M, Otani K, Ishibashi M, Hiasa K, Nishida K, and Takeshita A. Anti-monocyte chemoattractant protein-1 gene therapy limits progression and destabilization of established atherosclerosis in apolipoprotein E-knockout mice. Circulation 106: 2700–2706, 2002.[Abstract/Free Full Text]
  13. Katusic ZS and d'Uscio LV. Tetrahydrobiopterin: mediator of endothelial protection. Arterioscler Thromb Vasc Biol 24: 397–398, 2004.[Free Full Text]
  14. Katusic ZS, Stelter A, and Milstien S. Cytokines stimulate GTP cyclohydrolase I gene expression in cultured human umbilical vein endothelial cells. Arterioscler Thromb Vasc Biol 18: 27–32, 1998.[Abstract/Free Full Text]
  15. Khoo JP, Bursill C, Alp NJ, McAteer MA, and Channon KM. Endothelial tetrahydrobiopterin deficiency accelerates atherosclerotic progression by nitric oxide synthase uncoupling (Abstract). Circulation 112: II166, 2005.
  16. Kirii H, Niwa T, Yamada Y, Wada H, Saito K, Iwakura Y, Asano M, Moriwaki H, and Seishima M. Lack of interleukin-1beta decreases the severity of atherosclerosis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol 23: 656–660, 2003.[Abstract/Free Full Text]
  17. Kuhlencordt PJ, Chen J, Han F, Astern J, and Huang PL. Genetic deficiency of inducible nitric oxide synthase reduces atherosclerosis and lowers plasma lipid peroxides in apolipoprotein E-knockout mice. Circulation 103: 3099–3104, 2001.[Abstract/Free Full Text]
  18. Kuhn DM and Geddes TJ. Tetrahydrobiopterin prevents nitration of tyrosine hydroxylase by peroxynitrite and nitrogen dioxide. Mol Pharmacol 64: 946–953, 2003.[Abstract/Free Full Text]
  19. Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, and Harrison DG. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest 111: 1201–1209, 2003.[CrossRef][ISI][Medline]
  20. Laursen JB, Somers M, Kurz S, McCann L, Warnholtz A, Freeman BA, Tarpey M, Fukai T, and Harrison DG. Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation 103: 1282–1288, 2001.[Abstract/Free Full Text]
  21. Lee TS, Yen HC, Pan CC, and Chau LY. The role of interleukin 12 in the development of atherosclerosis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol 19: 734–742, 1999.[Abstract/Free Full Text]
  22. Miller FJ, Jr, Gutterman DD, Rios CD, Heistad DD, and Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res 82: 1298–1305, 1998.[Abstract/Free Full Text]
  23. Minor RL, Jr, Myers PR, Guerra R, Jr, Bates JN, and Harrison DG. Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta. J Clin Invest 86: 2109–2116, 1990.[ISI][Medline]
  24. Oemar BS, Tschudi MR, Godoy N, Brovkovich V, Malinski T, and Lüscher TF. Reduced endothelial nitric oxide synthase expression and production in human atherosclerosis. Circulation 97: 2494–2498, 1998.[Abstract/Free Full Text]
  25. Ozaki M, Kawashima S, Yamashita T, Hirase T, Namiki M, Inoue N, Hirata K, Yasui H, Sakurai H, Yoshida Y, Masada M, and Yokoyama M. Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deficient mice. J Clin Invest 110: 331–340, 2002.[CrossRef][ISI][Medline]
  26. Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh A, Verstuyft JG, Rubin EM, and Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell 71: 343–353, 1992.[CrossRef][ISI][Medline]
  27. Raman CS, Li H, Martasek P, Kral V, Masters BS, and Poulos TL. Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center. Cell 95: 939–950, 1998.[CrossRef][ISI][Medline]
  28. Rosenkranz-Weiss P, Sessa WC, Milstien S, Kaufman S, Watson CA, and Pober JS. Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells. Elevations in tetrahydrobiopterin levels enhance endothelial nitric oxide synthase specific activity. J Clin Invest 93: 2236–2243, 1994.[ISI][Medline]
  29. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362: 801–809, 1993.[CrossRef][Medline]
  30. Sakai N, Kaufman S, and Milstein S. Tetrahydrobiopterin is required for cytokine-induced nitric oxide production in a murine macrophage cell line (RAW 264). Mol Pharmacol 43: 6–10, 1993.[Abstract]
  31. Schumacher M, Eber B, Tatzber F, Kaufmann P, Esterbauer H, and Klein W. Neopterin levels in patients with coronary artery disease. Atherosclerosis 94: 87–88, 1992.[CrossRef][ISI][Medline]
  32. Sheehan DC and Hrapchak BB. Theory and Practice of Histotechnology. Columbus, OH: Battelle, 1987.
  33. Shinozaki K, Nishio Y, Okamura T, Yoshida Y, Maegawa H, Kojima H, Masada M, Toda N, Kikkawa R, and Kashiwagi A. Oral administration of tetrahydrobiopterin prevents endothelial dysfunction and vascular oxidative stress in the aortas of insulin-resistant rats. Circ Res 87: 566–573, 2000.[Abstract/Free Full Text]
  34. Stroes E, Kastelein J, Cosentino F, Erkelens W, Wever R, Koomans H, Lüscher T, and Rabelink T. Tetrahydrobiopterin restores endothelial function in hypercholesterolemia. J Clin Invest 99: 41–46, 1997.[ISI][Medline]
  35. Tatzber F, Rabl H, Koriska K, Erhart U, Puhl H, Waeg G, Krebs A, and Esterbauer H. Elevated serum neopterin levels in atherosclerosis. Atherosclerosis 89: 203–208, 1991.[CrossRef][ISI][Medline]
  36. Tiefenbacher CP, Bleeke T, Vahl C, Amann K, Vogt A, and Kubler W. Endothelial dysfunction of coronary resistance arteries is improved by tetrahydrobiopterin in atherosclerosis. Circulation 102: 2172–2179, 2000.[Abstract/Free Full Text]
  37. Tzeng E, Billiar TR, Robbins PD, Loftus M, and Stuehr DJ. Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer. Proc Natl Acad Sci USA 92: 11771–11775, 1995.[Abstract/Free Full Text]
  38. Van der Loo B, Labugger R, Skepper JN, Bachschmid M, Kilo J, Powell JM, Palacios-Callender M, Erusalimsky JD, Quaschning T, Malinski T, Gygi D, Ullrich V, and Luscher TF. Enhanced peroxynitrite formation is associated with vascular aging. J Exp Med 192: 1731–1744, 2000.[Abstract/Free Full Text]
  39. Vann LR, Twitty S, Spiegel S, and Milstien S. Divergence in regulation of nitric-oxide synthase and its cofactor tetrahydrobiopterin by tumor necrosis factor-{alpha}. Ceramide potentiates nitric oxide synthesis without affecting GTP cyclohydrolase I activity. J Biol Chem 275: 13275–13281, 2000.[Abstract/Free Full Text]
  40. Vasquez-Vivar J, Duquaine D, Whitsett J, Kalyanaraman B, and Rajagopalan S. Altered tetrahydrobiopterin metabolism in atherosclerosis: implications for use of oxidized tetrahydrobiopterin analogues and thiol antioxidants. Arterioscler Thromb Vasc Biol 22: 1655–1661, 2002.[Abstract/Free Full Text]
  41. Vasquez-Vivar J, Martasek P, Whitsett J, Joseph J, and Kalyanaraman B. The ratio between tetrahydrobiopterin and oxidized tetrahydrobiopterin analogues controls superoxide release from endothelial nitric oxide synthase: an EPR spin trapping study. Biochem J 362: 733–739, 2002.[CrossRef][ISI][Medline]
  42. Veillard NR, Kwak B, Pelli G, Mulhaupt F, James RW, Proudfoot AE, and Mach F. Antagonism of RANTES receptors reduces atherosclerotic plaque formation in mice. Circ Res 94: 253–261, 2004.[Abstract/Free Full Text]
  43. Wassmann S, Czech T, van Eickels M, Fleming I, Bohm M, and Nickenig G. Inhibition of diet-induced atherosclerosis and endothelial dysfunction in apolipoprotein E/angiotensin II type 1A receptor double-knockout mice. Circulation 110: 3062–3067, 2004.[Abstract/Free Full Text]
  44. Weiss G, Willeit J, Kiechl S, Fuchs D, Jarosch E, Oberhollenzer F, Reibnegger G, Tilz GP, Gerstenbrand F, and Wachter H. Increased concentrations of neopterin in carotid atherosclerosis. Atherosclerosis 106: 263–271, 1994.[CrossRef][ISI][Medline]
  45. Werner ER, Werner-Felmayer G, and Wachter H. Tetrahydrobiopterin and cytokines. Proc Soc Exp Biol Med 203: 1–12, 1993.[Abstract]
  46. Yaghoubi M, Oliver-Krasinski J, Cayatte AJ, and Cohen RA. Decreased sensitivity to nitric oxide in the aorta of severely hypercholesterolemic apolipoprotein E-deficient mice. J Cardiovasc Pharmacol 36: 751–757, 2000.[CrossRef][ISI][Medline]
  47. Young JL, Libby P, and Schonbeck U. Cytokines in the pathogenesis of atherosclerosis. Thromb Haemost 88: 554–567, 2002.[ISI][Medline]
  48. Zhang SH, Reddick RL, Piedrahita JA, and Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 258: 468–471, 1992.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 HypertensionHome page
L. V. d'Uscio and Z. S. Katusic
Erythropoietin Increases Endothelial Biosynthesis of Tetrahydrobiopterin by Activation of Protein Kinase B{alpha}/Akt1
Hypertension, July 1, 2008; 52(1): 93 - 99.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
N. Sasaki, T. Yamashita, T. Takaya, M. Shinohara, R. Shiraki, M. Takeda, N. Emoto, A. Fukatsu, T. Hayashi, K. Ikemoto, et al.
Augmentation of Vascular Remodeling by Uncoupled Endothelial Nitric Oxide Synthase in a Mouse Model of Diabetes Mellitus
Arterioscler. Thromb. Vasc. Biol., June 1, 2008; 28(6): 1068 - 1076.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
R. K. Upmacis, M. J. Crabtree, R. S. Deeb, H. Shen, P. B. Lane, L. E. S. Benguigui, N. Maeda, D. P. Hajjar, and S. S. Gross
Profound biopterin oxidation and protein tyrosine nitration in tissues of ApoE-null mice on an atherogenic diet: contribution of inducible nitric oxide synthase
Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2878 - H2887.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
A. L. Moens and D. A. Kass
Tetrahydrobiopterin and Cardiovascular Disease
Arterioscler. Thromb. Vasc. Biol., November 1, 2006; 26(11): 2439 - 2444.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/6/H2466    most recent
00366.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by d'Uscio, L. V.
Right arrow Articles by Katusic, Z. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by d'Uscio, L. V.
Right arrow Articles by Katusic, Z. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.