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CLINICAL RESEARCH: VASCULAR DISEASE

GCH1 Haplotype Determines Vascular and Plasma Biopterin Availability in Coronary Artery Disease

Effects on Vascular Superoxide Production and Endothelial Function

Charalambos Antoniades, MD, PhD^_*, Cheerag Shirodaria, MRCP^_*, Tim Van Assche, PhD^_*, Colin Cunnington, MRCP^_*, Irmgard Tegeder, MD, PhD^,, Jörn Lötsch, MD, PhD, Tomasz J. Guzik, MD, PhD^_*,, Paul Leeson, MRCP, PhD^_*, Jonathan Diesch, HNC^_*, Dimitris Tousoulis, MD, PhD, FACC^¶, Christodoulos Stefanadis, MD, FACC, FESC^¶, Michael Costigan, PhD, Clifford J. Woolf, MD, PhD, Nicholas J. Alp, MD, PhD, MRCP^_* and Keith M. Channon, MD, FRCP^_*,_*

^_* Department of Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom
Neural Plasticity Research Group, Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Pharmazentrum Frankfurt/ZAFES, Institute for Clinical Pharmacology, Johann Wolfgang Goethe University, Frankfurt am Main, Germany
Departments of Medicine and Pharmacology, Jagiellonian University, Cracow, Poland
^¶ Athens University Medical School, 1st Cardiology Department, Hippokration Hospital, Athens, Greece.

Manuscript received November 2, 2007; accepted December 12, 2007.

^_* Reprint requests and correspondence: Dr. Keith M. Channon, Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom. (Email: keith.channon{at}cardiov.ox.ac.uk).

Objectives: This study sought to determine the effects of endogenous tetrahydrobiopterin (BH4) bioavailability on endothelial nitric oxide synthase (eNOS) coupling, nitric oxide (NO) bioavailability, and vascular superoxide production in patients with coronary artery disease (CAD).

Background: GTP-cyclohydrolase I, encoded by the GCH1 gene, is the rate-limiting enzyme in the biosynthesis of BH4, an eNOS cofactor important for maintaining enzymatic coupling. We examined the associations between haplotypes of the GCH1 gene, GCH1 expression and biopterin levels, and the effects on endothelial function and vascular superoxide production.

Methods: Blood samples and segments of internal mammary arteries and saphenous veins were obtained from patients with CAD undergoing coronary artery bypass grafting (n = 347). The GCH1 haplotypes were defined by 3 polymorphisms: rs8007267G<A, rs3783641A<T, and rs10483639C<G (X haplotype: A, T, G; O haplotype: any other combination). Vascular superoxide (± the eNOS inhibitor N^G-nitro-L-arginine methyl ester [L-NAME]) was measured by lucigenin-enhanced chemiluminescence, whereas the vasorelaxations of saphenous veins to acetylcholine were evaluated ex vivo.

Results: Haplotype frequencies were OO 70.6%, XO 27.4%, and XX 2.0%. The X haplotype was associated with significantly lower vascular GCH1 messenger ribonucleic acid expression and substantial reductions in both plasma and vascular BH4 levels. In X haplotype carriers both vascular superoxide and L-NAME–inhibitable superoxide were significantly increased, and were associated with reduced vasorelaxations to acetylcholine.

Conclusions: GCH1 gene expression, modulated by a particular GCH1 haplotype, is a major determinant of BH4 bioavailability both in plasma and in the vascular wall in patients with CAD. Genetic variation in GCH1 underlies important differences in endogenous BH4 availability and is a determinant of eNOS coupling, vascular redox state, and endothelial function in human vascular disease.

Key Words: tetrahydrobiopterin • endothelial nitric oxide synthase • GTP-cyclohydrolase I • superoxide • haplotype • atherosclerosis

Abbreviations and Acronyms   ACh = acetylcholine   BH4 = tetrahydrobiopterin   BH2 = dihydrobiopterin   B = biopterin   CABG = coronary artery bypass grafting   CAD = coronary artery disease   CRP = C-reactive protein   eNOS = endothelial nitric oxide synthase   GTPCH = GTP-cyclohydrolase I   IMA = internal mammary artery   L-NAME = NG-nitro-L-arginine methyl ester   NO = nitric oxide   O2 – = superoxide radical   ONOO– = peroxynitrite   Ox-LDL = oxidized low-density lipoprotein   RT-qPCR = real-time quantitative polymerase chain reaction   SNP = sodium nitroprusside   SV = saphenous vein

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