Induction of vascular GTP-cyclohydrolase I and endogenous tetrahydrobiopterin synthesis protect against inflammation-induced endothelial dysfunction in human atherosclerosis

Abstract

Background: The endothelial nitric oxide synthase cofactor tetrahydrobiopterin (BH4) is essential for maintenance of enzymatic function. We hypothesized that induction of BH4 synthesis might be an endothelial defense mechanism against inflammation in vascular disease states.

Methods and results: In Study 1, 20 healthy individuals were randomized to receive Salmonella typhi vaccine (a model of acute inflammation) or placebo in a double-blind study. Vaccination increased circulating BH4 and interleukin 6 and induced endothelial dysfunction (as evaluated by brachial artery flow-mediated dilation) after 8 hours. In Study 2, a functional haplotype (X haplotype) in the GCH1 gene, encoding GTP-cyclohydrolase I, the rate-limiting enzyme in biopterin biosynthesis, was associated with endothelial dysfunction in the presence of high-sensitivity C-reactive protein in 440 coronary artery disease patients. In Study 3, 10 patients with coronary artery disease homozygotes for the GCH1 X haplotype (XX) and 40 without the haplotype (OO) underwent S Typhi vaccination. XX patients were unable to increase plasma BH4 and had a greater reduction of flow-mediated dilation than OO patients. In Study 4, vessel segments from 19 patients undergoing coronary bypass surgery were incubated with or without cytokines (interleukin-6/tumor necrosis factor-α/lipopolysaccharide) for 24 hours. Cytokine stimulation upregulated GCH1 expression, increased vascular BH4, and improved vasorelaxation in response to acetylcholine, which was inhibited by the GTP-cyclohydrolase inhibitor 2,4-diamino-6-hydroxypyrimidine.

Conclusions: The ability to increase vascular GCH1 expression and BH4 synthesis in response to inflammation preserves endothelial function in inflammatory states. These novel findings identify BH4 as a vascular defense mechanism against inflammation-induced endothelial dysfunction.

Figure 1

Vaccination with Salmonella typhi vaccine (n=10) induced a significant elevation of interleukin-6 (IL-6) at 8 and 12 hours, whereas IL-6 levels returned to baseline after 24 hours (A). Conversely, high-sensitivity C-reactive protein (CRP) levels were significantly increased only 24 hours after vaccination (B). Flow-mediated dilation (FMD) followed a similar pattern to that of IL-6, with a significant reduction at 8 and 12 hours after vaccination and a return to baseline at 24 hours (C). Infusion of placebo (n=10) had no significant effect on IL-6, CRP, or FMD over 24 hours. Values are expressed as mean±SEM. Two-way repeated-measures ANOVA revealed a significant time-by-group interaction for IL-6 (P<0.01), CRP (P<0.05), and FMD (P<0.01). *P<0.05, **P<0.01 vs baseline by paired t test followed by Bonferroni post hoc correction for 3 tests.

Figure 2

Vaccination with Salmonella typhi vaccine (n=10) induced a significant elevation of tetrahydrobiopterin (BH4; A) and total biopterins (B), with their peak elevation between 8 and 12 hours. Infusion of placebo (n=10) had no significant effect on interleukin-6, C-reactive protein, or flow-mediated dilation over 24 hours. Postintervention values are presented as percent of baseline (each value indexed to their 0-hour value) and are expressed as mean±SEM. Two-way repeated-measures ANOVA revealed a significant time-by-group interaction for both BH4 and total biopterins (P<0.01 for both). *P<0.01 vs baseline by paired t test followed by Bonferroni post hoc correction for 3 tests.

Figure 3

In patients with coronary artery disease, the GCH1 haplotype (40 OO and 10 XX) had no significant effect on flow-mediated dilation (FMD; A) or endothelium-independent vasodilation in response to nitrates (GTN; B). Values are expressed as mean±SEM.

Figure 4

When the population of patients with coronary artery disease was divided according to tertiles of plasma high-sensitivity C-reactive protein (hsCRP, as a marker of their background inflammatory status), carriage of the X haplotype was associated with significantly lower flow-mediated dilation (FMD; A), higher plasma tetrahydrobiopterin (BH4; B), and higher plasma total biopterins (tBio; C) than O homozygotes only in those patients at the highest tertile of plasma hsCRP. Values are expressed as mean±SEM. Two-way ANOVA for independent samples revealed significant hsCRP tertile-by-genotype interactions for FMD (P<0.05), plasma BH4 (P<0.01), and total biopterins (P<0.05). *P<0.05 vs OO in individual comparisons using unpaired t test after Bonferroni correction for 3 tests.

Figure 5

When patients with coronary artery disease were recruited by GCH1 genotype (40 OO and 10 XX) to undergo Salmonella typhi vaccination, we observed that vaccination induced a similar elevation of interleukin-6 (IL-6) at 8 hours in both genotypes (A), whereas C-reactive protein (hsCRP) levels remained unchanged at 8 hours in both genotypes (B). Values are expressed as mean±SEM. Two-way repeated-measures ANOVA revealed no significant time-by-genotype interaction. Within-groups comparisons (8 vs 0 hours) were performed with an unpaired t test. *P<0.01 vs Oh.

Figure 6

When patients with coronary artery disease were recruited by GCH1 genotype (40 OO and 10 XX) to undergo Salmonella typhi vaccination, we observed that vaccination induced a significant elevation of tetrahydrobiopterin (BH4; A) and total biopterins (tBiopterins; B) at 8 hours only in OO but not in XX genotypes. However, flow-mediated dilation (FMD) was reduced significantly only in the presence of XX haplotype (C). Values are expressed as mean±SEM. Two-way repeated-measures ANOVA revealed significant time-by-genotype interaction for BH4, total biopterins, and FMD (P<0.05 for all). *P<0.05, **P<0.01 vs 0 hours by unpaired t test.

Figure 7

Incubation of saphenous vein (SV; A) and internal mammary artery (IMA; B) segments for 24 hours with a combination of cytokines (tumor necrosis factor-α 4 ng/mL plus interleukin-6 10 nmol/L) and lipopolysaccharide (80 ng/mL) induced a significant elevation of vascular tetrahydrobiopterin (BH4), an effect that was prevented by the GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP; 1 mmol/L). Similarly, cytokine stimulation for 24 hours upregulated GCH1 gene expression in both SV (C) and IMA (D) segments, effects that were not modified by DAHP. The same combination of cytokines also increased vasorelaxation of SV segments in response to acetylcholine (ACh), an effect that was prevented by DAHP (E), whereas it had no effect on vasorelaxation in response to sodium nitroprusside (SNP; F). There were no significant differences in phenylephrine precontractions between the groups in either the ACh curve (E; Control: 1.54±0.72g [79.6±7.1% of KCl contraction] ; DAHP: 1.84±0.64g [64.0±8.6% of KCl contraction]; cytokines: 0.85±0.31g [86.6±3.7% of KCl contraction]; cytokines+DAHP: 1.02±0.26g[85.7±6.3% of KCl contraction]) or the SNP curve (F; Control: 1.14±0.14g [79.5±6.1% of KCl contraction]; DAHP: 2.21±0.66 g [62.4±10.5% of KCl contraction]; cytokines: 1.1±0.68g [66.9±10.5% of KCl contraction]; cytokines+DAHP: 1.17±0.42g [78.9±15.6% of KCl contraction]). *P<0.01 vs control (no cytokines, no DAHP).