Does endothelial tetrahydrobiopterin control the endothelial NO synthase coupling state in arterial resistance arteries?

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This article is a Commentary on Chuaiphichai S, Crabtree MJ, McNeill E, Hale AB, Trelfa L, Channon KM et al. (2017). A key role for tetrahydrobiopterin‐dependent endothelial NOS regulation in resistance arteries: studies in endothelial cell tetrahydrobiopterin‐deficient mice. Br J Pharmacol 174: 657–671. https://doi.org/10.1111/bph.13728

Abbreviations

BH4

tetrahydrobiopterin

eNOS

endothelial NOS

Gch1^fl/flTie2cre

endothelial‐specific GTPCH knockout

GTPCH

GTP‐cyclohydrolase‐1

Chuaiphichai et al. (2017) have investigated whether a genetic endothelial‐specific GTP‐cyclohydrolase‐1 (GTPCH) deficiency in Gch1^fl/flTie2cre mice will lead to endothelial NOS (eNOS) uncoupling and an impaired endothelium‐dependent relaxation in arterial resistance arteries. As expected, genetic GTPCH deletion resulted in a significant loss of GTPCH protein in mesenteric arteries accompanied by an almost complete absence of tetrahydrobiopterin (BH4) in the endothelial cell layer. Despite unchanged eNOS protein expression and phosphorylation levels at Ser¹¹⁷⁷ and Thr⁴⁹⁵, eNOS‐derived NO formation was virtually absent. Instead, increased endothelial formation of ROS was evident in vessels of Gch1^fl/flTie2cre mice, supporting the presence of uncoupled eNOS. The mesenteric arteries of Gch1^fl/flTie2cre mice developed a super‐sensitivity to vasoconstrictors such as phenylephrine and the thromboxane A₂ agonist U46619 and a shift of vascular tone homeostasis towards prostacyclin‐ and hydrogen peroxide‐dependent vasodilatation. The endothelial dysfunction observed in the vessels of Gch1^fl/flTie2cre mice was reversed by exogenous delivery of sepiapterin, a BH4 precursor. These findings mirror the results obtained with cultured endothelial cells (sEND.1 mouse endothelial cell line) upon genetic deletion of GTPCH by treatment with specific siRNA. In these studies, BH4 levels were substantially reduced and, as another indication of eNOS uncoupling (Zou et al., 2002), the eNOS dimer/monomer ratio was decreased by approximately 50% despite unchanged total eNOS protein levels. In addition, the observations made in resistance arteries strikingly resemble those obtained in arterial conductance vessels (aorta) of endothelial GTPCH‐deficient mice (Chuaiphichai et al., 2014).

A controversial discussion has continued for many years concerning the relevance of BH4 levels for the eNOS coupling state and implications for the development of endothelial dysfunction. BH4 levels have been shown to be up‐ rather than down‐regulated in whole aortic tissue and plasma despite obvious endothelial dysfunction (Antoniades et al., 2007; Kossmann et al., 2014). The inherent problem is that BH4 synthesis is also strongly linked to inflammation in vascular tissue and, thus, BH4 levels are typically increased along with an increase in the expression of the inducible NOS (iNOS) since BH4 is required for iNOS activity (Kossmann et al., 2014).

GTPCH is constitutively active in macrophages and is an enzyme that can be induced by pro‐inflammatory cytokines, suggesting that vascular BH4 levels, upon infiltration of macrophages, will not simply reflect endothelial BH4 content. Indeed, using a model of angiotensin II‐induced hypertension, our group recently established an essential role of vascular inflammation for the underlying pathogenesis (Wenzel et al., 2011). In a subsequent study, increased vascular oxidative stress, eNOS uncoupling, impaired aortic NO formation and endothelial dysfunction were observed despite a significant up‐regulation of aortic GTPCH protein and accordingly higher vascular levels of BH4 (Kossmann et al., 2014). The eNOS uncoupling, despite more vascular BH4, was demonstrated by two independent methods, L‐NAME‐inhibitable endothelial ROS formation and increased eNOS S‐glutathionylation respectively. The latter parameter can be viewed as an established marker for eNOS uncoupling (Chen et al., 2010).

Channon and co‐workers have stressed for a long time the concept that endothelial GTPCH and BH4 levels are the essential determinants of eNOS functionality and coupling state. Indeed, as shown previously in a clinical study, vascular but not plasma BH4 is an important determinant of eNOS coupling state, endothelium‐dependent vasodilatation and superoxide production in human vessels, whereas plasma BH4 instead reflects a systemic inflammatory response (Antoniades et al., 2007). With the present studies, Chuaiphichai et al. again provide molecular proof of their postulate and clearly demonstrate that an endothelial deficiency of BH4 is sufficient to cause endothelial dysfunction and eNOS uncoupling in arterial resistance arteries as measured by endothelium‐specific, L‐NAME‐inhibitable ROS formation and diminished eNOS dimer/monomer ratio.

Besides BH4, many other ‘redox switches’ within the enzyme eNOS were identified and discussed with respect to their relevance to render the enzyme inactive or even uncoupled [among them zinc–sulfur complex oxidation (Zou et al., 2002), S‐glutathionylation (Chen et al., 2010), phosphorylation at Tyr⁶⁵⁷ and Thr⁴⁹⁵ and asymmetric dimethylarginine (ADMA) synthesis and degradation, reviewed in Daiber et al. (2017)], challenging the unique role of BH4 deficiency in this process, especially in the presence of different cardiovascular risk factors. As a potential link between these different ‘redox switches’ in eNOS, a close interaction between BH4 availability and eNOS S‐glutathionylation was reported previously (Crabtree et al., 2013). However, this link was not confirmed in the present work nor was a correlation found between BH4 levels and activating/inactivating phosphorylations (Ser¹¹⁷⁷, Thr⁴⁹⁵) of eNOS.

The results by Chuaiphichai et al. substantially contribute to our understanding of the mechanism underlying the development of endothelial dysfunction in resistance arteries, which can be regarded as an early correlate of atherosclerosis and a major trigger for the development of future cardiovascular disease (Panza et al., 1990; Vita et al., 1990) and prognosis (Schachinger et al., 2000; Gokce et al., 2002) (for a review, see Daiber et al., 2017). Their results may also explain our previous clinical data that BH4 infusion corrected endothelial dysfunction in chronic smokers and therefore reversed eNOS uncoupling, whereas the structural analogue tetrahydroneopterin (NH4), which cannot bind to eNOS, had no effect on endothelial dysfunction (Heitzer et al., 2000).

Arterial hypertension is the most important cardiovascular risk factor in industrialized countries because of its very high prevalence (Lim et al., 2012; Murray et al., 2012). In 1991, Nakazono and co‐workers demonstrated that the administration of heparin‐binding superoxide dismutase was able to significantly lower arterial blood pressure in spontaneously hypertensive rats, suggesting that increased production of ROS at least in part mediates arterial hypertension (Nakazono et al., 1991). Further research established the vascular NADPH oxidase as a significant superoxide source (Rajagopalan et al., 1996). In 2002, Mollnau et al. were the first to demonstrate the presence of an uncoupled eNOS in the setting of angiotensin II‐induced hypertension (Mollnau et al., 2002). It is known that in the setting of increased oxidative stress within endothelial cells (e.g. induced by the NADPH oxidase), BH4 may be oxidized to the ^•BH3 radical (Kuzkaya et al., 2003), all of which can result in eNOS uncoupling (Forstermann and Munzel, 2006). The important implication is that a combination of antioxidant therapy and BH4 supplementation may be required in order to successfully treat cardiovascular diseases (Munzel et al., 2010).

This pathophysiology may be one of the reasons why, in large‐scale clinical trials, non‐selective antioxidant drugs have failed to show any health benefits for the treatment of cardiovascular disease (Munzel et al., 2010).

Conflict of interest

The authors declare no conflicts of interest.

Münzel, T. , and Daiber, A. (2017) Does endothelial tetrahydrobiopterin control the endothelial NO synthase coupling state in arterial resistance arteries?. British Journal of Pharmacology, 174: 2422–2424. doi: 10.1111/bph.13827.