Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:719-721

This Article
Right arrow Extract Freely available
Right arrow Full Text (PDF)
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
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van Hinsbergh, V. W. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van Hinsbergh, V. W. M.
Related Collections
Right arrow Pathophysiology
Right arrow Cell signalling/signal transduction
Right arrow Genetically altered mice
Right arrow Endothelium/vascular type/nitric oxide
(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:719.)
© 2001 American Heart Association, Inc.

Editorials

NO or H2O2 for Endothelium-Dependent Vasorelaxation

Tetrahydrobiopterin Makes the Difference

Victor W. M. van Hinsbergh

From the Gaubius Laboratory, Leiden, and the Department of Physiology, Institute for Cardiovascular Research, Vrije Universiteit Medical Center, Amsterdam, the Netherlands.

Correspondence to Dr Victor W.M. van Hinsbergh, Gaubius Laboratory TNO-PG, Zernikedreef 9, 2301 CE Leiden, Netherlands. E-mail vwm.vanhinsbergh{at}pg.tno.nl


Key Words: nitric oxide synthase • hydrogen peroxide • superoxide anion • catalase GTP cyclohydrolase I

Rapid adaptation of the diameter of blood vessels is an important regulatory mechanism for regulating blood pressure and for limiting tissue damage resulting from wounding or ischemia. Endothelial cells contribute to vasoregulation by the production of potent vasodilators. Loss or reduction of endothelium-dependent vasorelaxation is one of the reflections of endothelial dysfunction, an early hallmark in the development of atherosclerosis. Studies in patients have pointed to an impairment of endothelium-dependent vasodilation in a number of conditions that also increase the risk of developing cardiovascular complications, including hypercholesterolemia, hypertension, and diabetes.1 2 3 4 This impairment is due to a reduced availability of NO, a major endothelium-dependent vasorelaxing factor.5 6 Several studies have indicated that supplementation of tetrahydrobiopterin (H4B), a cofactor of the endothelial NO synthase, can improve endothelium-dependent arterial vasoregulation in atherosclerotic and diabetic patients.7 8 9 10 In the April issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Cosentino et al11 use transgenic mice to achieve a 60% reduction of arterial H4B, and they demonstrate that this reduction is accompanied by a marked decrease of NO production while H2O2 production is increased.

Endothelial cells produce NO from L-arginine by NO synthase-3 (NOS-3), also called constitutive or endothelial NOS. The enzyme is constitutively present in endothelial cells and becomes active by interaction with the Ca2+/calmodulin complex, which is transiently generated after exposure of the endothelial cell to vasoactive agents, or by phosphorylation at serine 1117 by the serine/threonine protein kinase Akt.12 13 Arterial shear forces and estrogen are among the factors that can activate NOS-3 via Akt. NOS-3 consists of 2 enzymatic domains, a flavin-containing reductase domain and a heme-containing oxygenase domain. These domains are connected by a regulatory calmodulin-binding domain. Binding of the Ca2+/calmodulin complex orients the other domains in such a position that NADPH-derived electrons generated on the reductase domain flow to the oxygenase domain.12 Phosphorylation at position Ser1117 probably causes a comparable effect.13 The oxygenase domain of NOS-3 contains an iron ion (Fe3+) that binds oxygen on reduction (Fe2+), and this complex finally causes the conversion of L-arginine to NO and L-citrulline. This sequence of events occurs adequately only if the cofactor H4B is bound to the NOS-3 protein.14 15 On the basis of structural analysis, it has been concluded that H4B does not directly participate in the oxygen activation reactions that are critical for NO formation but that it probably has a function in the stabilization of the enzyme.16 In the absence of H4B, the NOS-3 enzyme is "uncoupled" and produces mainly superoxide instead of NO, as became clear from electron spin resonance studies.17 18 In this case, the intermediate Fe2+-O2 complex dissociates and forms superoxide (O2-) and the original Fe3+ group of the NOS-3 protein.

Endothelium-dependent vasodilation can also be induced by other mediators. Prostacyclin and prostaglandin E2 induce vasodilation in specific regions of the circulatory system. Furthermore, endothelium-derived hyperpolarizing factors (EDHFs) play a major role in the vasoregulation of intramyocardial arteries/arterioles. In endothelial NO synthase–deficient mice, endothelium-dependent vasoregulation is largely taken over by EDHFs.19 Products generated by cytochrome P-450 monooxygenase activity, in particular, epoxyeicosatrienoic acids, have been identified as EDHFs.20 The cytochrome P-450 subtype CYP9 was identified as a major contributor to EDHF generation.21 Two recent studies have indicated that hydrogen peroxide (H2O2) is also an EDHF.11 22 Matoba et al22 showed that H2O2 elicited hyperpolarization of smooth muscle cells and vasorelaxation of small mesenteric arteries in the mouse. Cosentino et al11 now provide evidence that under conditions of H4B deficiency, H2O2 instead of NO acts as an endothelium-derived relaxing factor in the mouse aorta and that NOS-3 is an important source of this EDHF. Although previous studies have shown that H2O2 is able to induce vasorelaxation by activating soluble guanylate cyclase in smooth muscle cells,23 24 the present studies provide first evidence that it indeed acts as such in a physiological setting.

The discovery that H2O2 generated by uncoupled NOS-3 is active in endothelium-dependent vasorelaxation comes from an elegant series of experiments in which Cosentino et al11 used H4B-deficient mice to alter the activity of NOS-3. Given the importance of the H4B for proper functioning of NOS-3, these authors considered using a mouse model of hyperphenylalaninemia, the hph-1 mouse. This mouse displays a 90% deficiency in the activity of GTP cyclohydrolase I, a key enzyme in the de novo H4B synthesis. As a consequence, these animals have a 60% reduction of H4B in their aortas. The residual H4B is probably generated by the so-called salvage pathway, in which sepiapterin is the substrate for H4B synthesis. The hph-1 mice exhibited a normal blood pressure and a normal vasoconstrictive response to norepinephrine. Unexpectedly, the endothelium-dependent vasorelaxation on acetylcholine stimulation of preconstricted aortas was normal. This response was not due to NO production, which was markedly impaired by the H4B deficiency, but to H2O2. The endothelium-derived vasorelaxation induced by acetylcholine in the aortas of hph-1 mice was reduced by catalase, the enzyme that scavenges H2O2. This is in contrast to the situation in wild-type animals, which mainly produced NO as an endothelium-derived vasorelaxant. The vasodilation in hph-1 mice was completely inhibited by NG-monomethyl-L-arginine, which directly couples the generation of H2O2 to the production of reactive oxygen species, in particular, superoxide anion by NOS-3. Conversion of superoxide anions in H2O2 by an exogenous supply of superoxide dismutase increased vasodilation in the hph-1 mice. The study of Cosentino et al leads to 2 important conclusions. First, it adds firm experimental evidence to the suggestion that H4B deficiency impairs NO production by NOS-3. An adequate NO production is not only important for vasorelaxation; NO is also considered to be a protective factor that counteracts atherosclerosis. Furthermore, NO is involved in the prevention of platelet activation, counteracting inflammatory functions of the endothelium, including the expression of leukocyte adhesion molecules and chemokines, the regulation of the endothelial barrier function, and angiogenesis.25 26 27 28 Second, the study shows that in healthy aortas of young hph-1 mice, acetylcholine-dependent vasorelaxation can be caused by H2O2; thus, it recognizes H2O2 as a potential and pathophysiologically relevant endothelium-derived mediator of vasorelaxation.

The study of Cosentino et al11 indicates that fewer superoxide anions can be detected in the hph-1 mouse aorta than in the wild-type mouse aorta. One might have anticipated that because of the uncoupling of NOS-3, superoxide would increase, but the data show the reverse. The authors suggest that superoxide is converted to hydrogen peroxide. The mechanism by which this conversion occurs is not yet clear. Apparently, H4B deficiency induces the increased conversion. However, whether this is directly related to the altered mode of action of NOS-3 or to a complex formation between NOS-3 and superoxide dismutase or whether enzymatic activities are induced in the endothelium of hph-1 mice because of the increased demand of H2O2 generation remains to be clarified. Heme oxygenase (HO)-2 may be a candidate to play a role in such a process, because both HO-2 and NOS-3 are constitutively present in endothelial cells, and HO-2 activity is inhibited by NO.29 30 HO activity induces manganese superoxide dismutase in astrocytes.31 Therefore, it would be interesting to know whether HO activity could also induce superoxide dismutase activity in endothelial cells.

The recognition of H4B as a crucial cofactor for proper NOS-3 activity provides an interesting clinical perspective. Several studies have shown that supplementation of H4B or 5-methyltetrahydrofolate, an active folate form that is thought to be involved in H4B regeneration, can indeed improve endothelium-dependent vasodilation in patients with atherosclerosis, coronary artery disease, and type II diabetes7 8 9 10 32 Furthermore, vitamin C stabilizes H4B and thus may improve the endothelial H4B status and NO production.33 Because H4B and folate can be given orally as a nutrient, these data reveal an important new potential target for preventive strategies for counteracting cardiovascular complications.

Several additional questions come from the study of Cosentino et al.11 In the mouse study, supplementation of H4B improved NO production, but because of the compensatory action of H2O2, vasoregulation was not altered. In patient studies, H4B administration improved vasoregulation. The production of H2O2 may vary considerably between different types of small and large arteries. If H2O2 is also generated in human arteries in which a partial H4B deficiency exists, one wonders to what extent it acts on vasorelaxation in atherosclerotic arteries. The increased dimensions of the arteries in humans and the presence of multiple cell layers in the thickened intima, which are not involved in vasodilation but certainly are able to interact with and modify reactive oxygen species, make it difficult to predict its efficacy. Another issue to be resolved is the effect of enhanced endothelial H2O2 generation on the long-term function of the vascular endothelium. The investigators used young animals (aged 8 weeks) for their studies. Because H2O2 generates the highly reactive hydroxyl radical in the Fenton reaction, which is generally considered as very damaging, one wonders whether the increased production of H2O2 by NOS-3 in H4B-deficient mice may cause vascular damage at advanced age. If this were the case, partial H4B deficiency, such as is seen in various groups of patients prone to cardiovascular disease, would not only cause reduced efficacy of endothelium-dependent vasoregulation but would also increase endothelial damage in due course. Even if defense mechanisms are appropriate in the healthy vessel, an increased H2O2 production in dysfunctioning endothelial cells may accelerate the progression of vascular disease. If such a mechanism plays a role, improvement of the H4B status of the endothelium would give long-term benefits for the vessel wall by improving NO generation and by reducing H2O2-mediated damage in the endothelium. A systematic long-term evaluation of dietary supplementations that improve the vascular H4B status in various types of patients at risk for developing vascular complications is urgently required.

References

  1. Flavahan NA. Atherosclerosis or lipoprotein-induced endothelial dysfunction: potential mechanisms underlying reduction in EDRF/nitric oxide activity. Circulation. 1992;85:1927–1938.[Free Full Text]
  2. Linder L, Kiowski W, Buhler FR, Luscher TF. Indirect evidence for release of endothelium-derived relaxing factor in human forearm circulation in vivo: blunted response in essential hypertension. Circulation. 1990;81:1762–1767.[Abstract/Free Full Text]
  3. King GL, Shiba T, Oliver J, Inoguchi T, Bursell SE. Cellular and molecular abnormalities in the vascular endothelium of diabetes mellitus. Annu Rev Med. 1994;45:179–188.[Medline] [Order article via Infotrieve]
  4. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DG, Miller OI, Sullivan ID, Lloyd JK, Deanfield JE. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet. 1992;340:1111–1115.[Medline] [Order article via Infotrieve]
  5. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288:373–376.[Medline] [Order article via Infotrieve]
  6. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987;327:524–526.[Medline] [Order article via Infotrieve]
  7. Stroes E, Kastelein J, Cosentino F, Erkelens W, Wever R, Koomans H, Luscher T, Rabelink T. Tetrahydrobiopterin restores endothelial function in hypercholesterolemia. J Clin Invest. 1997;99:41–46.[Medline] [Order article via Infotrieve]
  8. Maier W, Cosentino F, Lutolf RB, Fleisch M, Seiler C, Hess OM, Meier B, Luscher TF. Tetrahydrobiopterin improves endothelial function in patients with coronary artery disease. J Cardiovasc Pharmacol. 2000;35:173–178.[Medline] [Order article via Infotrieve]
  9. Tiefenbacher CP, Bleeke T, Vahl C, Amann K, Vogt A, Kubler W. Endothelial dysfunction of coronary resistance arteries is improved by tetrahydrobiopterin in atherosclerosis. Circulation. 2000;102:2172–2179.[Abstract/Free Full Text]
  10. Heitzer T, Krohn K, Albers S, Meinertz T. Tetrahydrobiopterin improves endothelium-dependent vasodilation by increasing nitric oxide activity in patients with type II diabetes mellitus. Diabetologia. 2000;43:1435–1438.[Medline] [Order article via Infotrieve]
  11. Cosentino F, Barker JE, Brand MP, Heales SJ, Werner ER, Tippins JR, West N, Channon KM, Volpe M, Lüscher TF. Reactive oxygen species mediate endothelium-dependent relaxations in tetrahydrobiopterin-deficient mice. Arterioscler Thromb Vasc Biol. 2001;21:496–502.[Abstract/Free Full Text]
  12. Abu-Soud HM, Stuehr DJ. Nitric oxide synthases reveal a role for calmodulin in controlling electron transfer. Proc Natl Acad Sci U S A. 1993;90:10769–10772.[Abstract/Free Full Text]
  13. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999;399:601–605.[Medline] [Order article via Infotrieve]
  14. Schmidt HH, Hofmann H, Schindler U, Shutenko ZS, Cunningham DD, Feelisch M. No .NO from NO synthase. Proc Natl Acad Sci U S A. 1996;93:14492–14497.[Abstract/Free Full Text]
  15. Wever RM, van Dam T, van Rijn HJ, de Groot F, Rabelink TJ. Tetrahydrobiopterin regulates superoxide and nitric oxide generation by recombinant endothelial nitric oxide synthase. Biochem Biophys Res Commun. 1997;237:340–344.[Medline] [Order article via Infotrieve]
  16. Raman CS, Li H, Martasek P, Kral V, Masters BS, Poulos TL. Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center. Cell. 1998;95:939–950.[Medline] [Order article via Infotrieve]
  17. Stroes E, Hijmering M, van Zandvoort M, Wever R, Rabelink TJ, van Faassen EE. Origin of superoxide production by endothelial nitric oxide synthase. FEBS Lett. 1998;438:161–164.[Medline] [Order article via Infotrieve]
  18. Vásquez-Vivar J, Kalyanaraman B, Martasek P, Hogg N, Masters BS, Karoui H, Tordo P, Pritchard KA. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U S A. 1998;95:9220–9225.[Abstract/Free Full Text]
  19. Brandes RP, Schmitz-Winnenthal FH, Feletou M, Godecke A, Huang PL, Vanhoutte PM, Fleming I, Busse R. An endothelium-derived hyperpolarizing factor distinct from NO and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial NO synthase knockout mice. Proc Natl Acad Sci U S A. 2000;97:9747–9752.[Abstract/Free Full Text]
  20. Rosolowsky M, Campbell WB. Role of PGI2 and epoxyeicosatrienoic acids in relaxation of bovine coronary arteries to arachidonic acid. Am J Physiol. 1993;264:H327–H335.[Abstract/Free Full Text]
  21. Fleming I, Michaelis UR, Bredenkotter D, Fisslthaler B, Dehghani F, Brandes RP, Busse R. Endothelium-derived hyperpolarizing factor synthase (cytochrome P450 2C9) is a functionally significant source of reactive oxygen species in coronary arteries. Circ Res. 2001;88:44–51.[Abstract/Free Full Text]
  22. Matoba T, Shimokawa H, Nakashima M, Hirakawa Y, Mukai Y, Hirano K, Kanaide H, Takeshita A. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice. J Clin Invest. 2000;106:1521–1530.[Medline] [Order article via Infotrieve]
  23. Burke TM, Wolin MS. Hydrogen peroxide elicits pulmonary arterial relaxation and guanylate cyclase activation. Am J Physiol. 1987;252:H721–H732.[Abstract/Free Full Text]
  24. Barlow RS, White RE. Hydrogen peroxide relaxes porcine coronary arteries by stimulating BKCa channel activity. Am J Physiol. 1998;275:H1283–H1289.
  25. Zeiher AM, Fisslthaler B, Schray-Utz B, Busse R. Nitric oxide modulates the expression of monocyte chemoattractant protein 1 in cultured human endothelial cells. Circ Res. 1995;76:980–986.[Abstract/Free Full Text]
  26. De Caterina R, Libby P, Peng HB, Thannickal VJ, Rajavashisth TB, Gimbrone MA, Shin WS, Liao JK. Nitric oxide decreases cytokine-induced endothelial activation: nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest. 1995;96:60–68.
  27. van Hinsbergh VWM. Endothelial permeability for macromolecules: mechanistic aspects of pathophysiological modulation. Arterioscler Thromb Vasc Biol. 1997;17:1018–1023.[Free Full Text]
  28. Ziche M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, Geppetti P, Ledda F. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest. 1994;94:2036–2044.
  29. Zakhary R, Gaine SP, Dinerman JL, Ruat M, Flavahan NA, Snyder SH. Heme oxygenase 2: endothelial and neuronal localization and role in endothelium-dependent relaxation. Proc Natl Acad Sci U S A. 1996;93:795–798.[Abstract/Free Full Text]
  30. Ding Y, McCoubrey WK, Maines MD. Interaction of heme oxygenase-2 with nitric oxide donors: is the oxygenase an intracellular "sink" for NO? Eur J Biochem. 1999;264:854–861.[Medline] [Order article via Infotrieve]
  31. Frankel D, Mehindate K, Schipper HM. Role of heme oxygenase-1 in the regulation of manganese superoxide dismutase gene expression in oxidatively-challenged astroglia. J Cell Physiol. 2000;185:80–86.[Medline] [Order article via Infotrieve]
  32. Verhaar MC, Wever RM, Kastelein JJ, van Dam T, Koomans HA, Rabelink TJ. 5-Methyltetrahydrofolate, the active form of folic acid, restores endothelial function in familial hypercholesterolemia. Circulation. 1998;97:237–241.[Abstract/Free Full Text]
  33. Heller R, Unbehaun A, Schellenberg B, Mayer B, Werner-Felmayer G, Werner ER. L-Ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin. J Biol Chem. 2001;276:40–47.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
H. Cai
Hydrogen peroxide regulation of endothelial function: Origins, mechanisms, and consequences
Cardiovasc Res, October 1, 2005; 68(1): 26 - 36.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Extract Freely available
Right arrow Full Text (PDF)
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
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van Hinsbergh, V. W. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van Hinsbergh, V. W. M.
Related Collections
Right arrow Pathophysiology
Right arrow Cell signalling/signal transduction
Right arrow Genetically altered mice
Right arrow Endothelium/vascular type/nitric oxide

ATVB Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 2001 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.