GCH1 haplotype determines vascular and plasma biopterin availability in coronary artery disease effects on vascular superoxide production and endothelial function

Abstract

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.

Figure 1. Effect of GCH1 Haplotype on Plasma/Vascular BH4

The presence of the haplotype (XO or XX) was associated with significantly lower levels of plasma tetrahydrobiopterin (BH4) (A) and total biopterins (tBio) (B). A similar effect was also observed in saphenous veins (SV) (C and D) and internal mammary arteries (IMA) (E and F).

Figure 2. Effect of GCH1 Haplotype on Vascular GTPCH mRNA Levels

The presence of the XX genotype was associated with significantly lower messenger ribonucleic acid (mRNA) levels of GTPCH compared with OO genotype. Shown are the data derived from 23 saphenous veins (SVs) and 17 internal mammary arteries (IMAs). *p < 0.05 versus OO.

Figure 3. Effect of GCH1 Haplotype on Vascular Superoxide, Oxidative Stress, and Endothelial Function

The presence of the X haplotype was associated with higher total superoxide (O ₂⁻) production (A) and greater L-NAME-inhibitable delta[O₂⁻] (B) in human saphenous veins. The X haplotype was also associated with significantly lower plasma BH4:tBio ratio (C) and higher plasma levels of oxidized low-density lipoprotein (ox-LDL) (D). In addition, the X haplotype was also associated with lower maximum vasorelaxations in response to acetylcholine (ACh) (E) in saphenous veins, although it had no impact on the vasomotor responses to sodium nitroprusside (SNP) (F).