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Key Event Relationship Overview

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Description of Relationship

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

AOPs Referencing Relationship

AOP Name Type of Relationship Weight of Evidence Quantitative Understanding
Oxidative Stress Leading to Hypertension 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 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 to produce NO. The depletion of BH4 leading to eNOS uncoupling is well-studied, thus there is strong biological plausibility for this link.

Two mouse studies showed limited BH4 availability 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 eNOS 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).

BAECs undergoing hypoxia and reoxygenation had decreased BH4 and decreased NO production, which was partially restored bytreatment with the xanthine oxidase inhibitor oxypurinol, N-acetyl-l-cysteine (NAC) and NAC+BH4 (De Pascali et al., 2014). Inhibition of BH4 due to treatment with 4-HNE decreased eNOS activity and NO production in BAECs (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). Oral treatment with BH4 in hypertensive patients significantly decreased blood pressure.

Empirical Support for Linkage

Include consideration of temporal concordance here

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

Prolonged myocardial ischemia (>30 min) in isolated rat hearts caused extensive BH4 depletion (95% depletion), which was paralleled by decreased eNOS activity (58% reduction) and increased superoxide generation from <0.01 relative fluorescence unit/mg to 0.3 measured using a fluorescence detector-based assay (Dumitrescu et al., 2007). Similarly, cardiac reperfusion patients exhibited a reduction in BH4 levels by 32%, decreased eNOS activity by 40% and increased superoxide production from 37.83 to 65.02 light unit/s/mg as detected using lucigenin-enhanced chemiluminescence (Jayaram et al., 2015).

In BAECs treated with 10 mmol/L DAHP or 25 μM 4-HNE for 4 hours, BH4 levels decreased (control: 10 pmol/mg, DAHP: 4.8 pmol/mg, 4-HNE: 6.1 pmol/mg). At 24 hours, a reduction in NO (control: 1678 pmol/mg, DAHP: 1274 pmol/mg, 4-HNE: 1106 pmol/mg) and increased superoxide formation (control: 59 pmol/mg, DAHP: 97 pmol/mg, 4-HNE: 122 pmol/mg) were observed (Whitsett et al., 2007). Another study in BAECs using DAHP showed similar results for BH4 (control: 20.5 pmol/mg, DAHP: 11.8 pmol/mg), superoxide (control: 100%, DAHP: 257%), and NO (control: 96%, DAHP: 60%) (Wang et al., 2008).

In BAECs undergoing hypoxia and reoxygenation (H/R), treatment with oxypurinol increased BH4 levels 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 6.927±3.475 ng L-citrulline/mg protein/30 min to 23.2 ± 9.034 ng L-citrulline/mg protein/30 min, BH4 levels increased from 17.144±2.251 pmol to 32.534±5.809 pmol, and superoxide decreased from relative fluorescence intensity of 32.22±2.903 to 24.59±1.124 (Cervantes-Pérez et al., 2012). Note that normal eNOS activity converts L-arginine to L-citrulline and produces NO; thus L-citrulline is an indirect measure of eNOS activity.

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 could 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-HNE (Whitsett et al., 2007).

Evidence Supporting Taxonomic Applicability

The relationship between BH4 depletion and eNOS uncoupling was observed in humans (Jayaram et al., 2015), cows (Wang et al., 2008; Whitsett et al., 2007), mice (Chuaiphichai et al., 2014; Crabtree et al., 2009) and rats (Cervantes-Pérez et al., 2012; Dumitrescu et al., 2007).


Cervantes-Pérez, L.G., Ibarra-Lara, M. de la L., Escalante, B., Del Valle-Mondragón, L., Vargas-Robles, H., Pérez-Severiano, F., Pastelín, G., and Sánchez-Mendoza, M.A. (2012). Endothelial nitric oxide synthase impairment is restored by clofibrate treatment in an animal model of hypertension. Eur. J. Pharmacol. 685, 108–115.

Chen, C.-A., Lin, C.-H., Druhan, L.J., Wang, T.-Y., Chen, Y.-R., and Zweier, J.L. (2011). Superoxide induces endothelial nitric-oxide synthase protein thiyl radical formation, a novel mechanism regulating eNOS function and coupling. J. Biol. Chem. 286, 29098–29107.

Chuaiphichai, S., McNeill, E., Douglas, G., Crabtree, M.J., Bendall, J.K., Hale, A.B., Alp, N.J., and Channon, K.M. (2014). Cell-autonomous role of endothelial GTP cyclohydrolase 1 and tetrahydrobiopterin in blood pressure regulation. Hypertension 64, 530–540.

Crabtree, M.J., Tatham, A.L., Al-Wakeel, Y., Warrick, N., Hale, A.B., Cai, S., Channon, K.M., and Alp, N.J. (2009). Quantitative regulation of intracellular endothelial nitric-oxide synthase (eNOS) coupling by both tetrahydrobiopterin-eNOS stoichiometry and biopterin redox status: insights from cells with tet-regulated GTP cyclohydrolase I expression. J. Biol. Chem. 284, 1136–1144.

De Pascali, F., Hemann, C., Samons, K., Chen, C.-A., and Zweier, J.L. (2014). Hypoxia and reoxygenation induce endothelial nitric oxide synthase uncoupling in endothelial cells through tetrahydrobiopterin depletion and S-glutathionylation. Biochemistry (Mosc.) 53, 3679–3688.

Dumitrescu, C., Biondi, R., Xia, Y., Cardounel, A.J., Druhan, L.J., Ambrosio, G., and Zweier, J.L. (2007). Myocardial ischemia results in tetrahydrobiopterin (BH4) oxidation with impaired endothelial function ameliorated by BH4. Proc. Natl. Acad. Sci. U. S. A. 104, 15081–15086.

Förstermann, U., and Münzel, T. (2006). Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation 113, 1708–1714.

Jayaram, R., Goodfellow, N., Zhang, M.H., Reilly, S., Crabtree, M., De Silva, R., Sayeed, R., and Casadei, B. (2015). Molecular mechanisms of myocardial nitroso-redox imbalance during on-pump cardiac surgery. Lancet Lond. Engl. 385 Suppl 1, S49.

Landmesser, U., Dikalov, S., Price, S.R., McCann, L., Fukai, T., Holland, S.M., Mitch, W.E., and Harrison, D.G. (2003). Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J. Clin. Invest. 111, 1201–1209.

Ozaki, M., Kawashima, S., Yamashita, T., Hirase, T., Namiki, M., Inoue, N., Hirata, K., Yasui, H., Sakurai, H., Yoshida, Y., et al. (2002). Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deficient mice. J. Clin. Invest. 110, 331–340.

Shinozaki, K., Nishio, Y., Okamura, T., Yoshida, Y., Maegawa, H., Kojima, H., Masada, M., Toda, N., Kikkawa, R., and Kashiwagi, A. (2000). Oral administration of tetrahydrobiopterin prevents endothelial dysfunction and vascular oxidative stress in the aortas of insulin-resistant rats. Circ. Res. 87, 566–573.

Wang, Q., Yang, M., Xu, H., and Yu, J. (2014). Tetrahydrobiopterin improves endothelial function in cardiovascular disease: a systematic review. Evid.-Based Complement. Altern. Med. ECAM 2014, 850312.

Wang, S., Xu, J., Song, P., Wu, Y., Zhang, J., Chul Choi, H., and Zou, M.-H. (2008). Acute inhibition of guanosine triphosphate cyclohydrolase 1 uncouples endothelial nitric oxide synthase and elevates blood pressure. Hypertension 52, 484–490.

Whitsett, J., Picklo, M.J., and Vasquez-Vivar, J. (2007). 4-Hydroxy-2-nonenal increases superoxide anion radical in endothelial cells via stimulated GTP cyclohydrolase proteasomal degradation. Arterioscler. Thromb. Vasc. Biol. 27, 2340–2347.