Relationship:953

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Key Event Relationship Overview

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Description of Relationship

Upstream Event Downstream Event/Outcome
Tetrahydrobiopterin, Decrease NOS3, Uncoupling

AOPs Referencing Relationship

AOP Name Type of Relationship Weight of Evidence Quantitative Understanding
Oxidative Stress Leading to Arterial Stiffness Directly Leads to Strong Strong

Taxonomic Applicability

Name Scientific Name Evidence Links

How Does This Key Event Relationship Work

Oxidative stress leads to the excessive oxidation and depletion of BH4, resulting in endothelial nitric oxide synthase (eNOS) uncoupling where eNOS produces superoxide rather than nitric oxide (Förstermann and Münzel, 2006).

Weight of Evidence

Biological Plausibility

BH4 is an essential cofactor for eNOS and is required for its enzymatic activity in producing NO. The depletion of BH4 leading to eNOS uncoupling is well-studied in literature, thus there is strong biological plausibility for this link.

Two mouse studies showed limited BH4 induced eNOS uncoupling by reducing eNOS activity, leading to decreased nitric oxide and increased superoxide. In the mouse endothelial cell line sEnd.1, BH4 deficiency induced NOS3 uncoupling as determined by superoxide production and impaired vasodilation (Crabtree et al., 2009). In primary aortic endothelial cells of mice with GTPCH1 deletion, BH4 depletion significantly reduced eNOS activity, increased basal superoxide production and decreased NO bioactivity (Chuaiphichai et al., 2014). In rat hearts, BH4 content and eNOS activity were decreased in a time-dependent manner following myocardial ischemia with a marked decline after thirty minutes, while superoxide generation increased (Dumitrescu et al., 2007).

Bovine aortic endothelial cells (BAECs) undergoing hypoxia and reoxygenation had decreased BH4 and decreased NO production, which was partially restored bytreatment with xanthine oxidase inhibitor oxypurinol, N-acetyl-l-cysteine (NAC) and NAC+BH4 (De Pascali et al., 2014). Also in BAECs, inhibition of BH4 from treatment with 4-hydroxy-2-nonenal (4-HNE) decreased eNOS activity and NO production (Whitsett et al., 2007).

Many studies demonstrated that BH4 treatment reduced eNOS-mediated superoxide generation and increased NO formation in bovine, mouse, and rat endothelium (Chen et al., 2011; De Pascali et al., 2014; Landmesser et al., 2003; Ozaki et al., 2002; Shinozaki et al., 2000). Clinical studies reported improvement endothelial function in cardiovascular disease after treatment with BH4 (Wang et al., 2014).

Empirical Support for Linkage

Include consideration of temporal concordance here

Multiple studies demonstrated strong dependency between BH4 and eNOS uncoupling; decreased BH4 along with decreased eNOS activity, decreased NO production or increased superoxide generation were observed after stimulation with various perturbations.

Prolonged myocardial ischemia (>30 min) in isolated rat hearts caused large depletion of BH4 (95% depletion), which paralleled decreased eNOS activity (58% reduction) and increased superoxide generation (<0.01 RFU/mg to 0.3 RFU/mg)(Dumitrescu et al., 2007). Similarly, cardiac reperfusion patients exhibited decreased BH4 levels (100% to 68%), decreased eNOS activity (40% reduction) and increased superoxide production (37.83 to 65.02light unit/s/mg)(Jayaram et al., 2015).

In BAECs treated with selective GTPCH-1 inhibitor DAHP (10 mmol/L) or 4-HNE (25 μM) for 4 hours resulted in decreased BH4 (control: ~10 pmol/mg, DAHP: ~5 pmol/mg, 4-HNE: ~6 pmol/mg). At 24 hours, a reduction in NO (control: ~1700 pmol/mg, DAHP: ~1200 pmol/mg, 4-HNE: ~1100 pmol/mg) and increased superoxide formation (control: ~60 pmol/mg, DAHP: ~100 pmol/mg, 4-HNE: ~125 pmol/mg) were observed (Whitsett et al., 2007). Another study in BAECs using DAHP showed similar results for BH4 (control: ~20 pmol/mg, DAHP: ~12pmol/mg), superoxide (control: 100%, DAHP: 152%), and NO (control: 100%, DAHP: 61%) (Wang et al., 2008).

In BAECs undergoing hypoxia and reoxygenation (H/R), treatment with oxypurinol increased BH4 from 6.1 ± 0.9 pmol/mg (after H/R) to 11.9 ± 0.8 pmol/mg and increased NO from 34.2 ± 1.7% to 63.7 ± 3.0%, demonstrating that these key events are modulated together (De Pascali et al., 2014).

In a rat model of hypertension, BH4 depletion and eNOS uncoupling induced by aortic coarctation (AoCo) is reversed by treatment with clofibrate; eNOS activity increased from 7 ng L-cit/mg protein/30 min to 22.2 ng L-cit/mg protein/30 min, BH4 levels increased from 17.1 pmol to 32.5 pmol, and superoxide decreased from 32.22 FI to 24.6 FI) (Cervantes-Pérez et al., 2012).

Uncertainties or Inconsistencies

The uncoupling of eNOS may also occur through other mechanisms such as S-glutathionylation of eNOS and depletion of L-arginine (Zweier et al., 2011).

Quantitative Understanding of the Linkage

Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?

As BH4 is required for normal eNOS function, it would be possible that any change in BH4 may affect eNOS function. The studies above demonstrated that there are many modulators of the response-response relationships including cardiac reperfusion (Jayaram et al., 2015), DAHP (Wang et al., 2008; Whitsett et al., 2007), and 4-hydroxy-2-nonenal (Whitsett et al., 2007).

Evidence Supporting Taxonomic Applicability

References