Endothelin-1 and blood pressure in women

With more than one million polymorphisms (gene variant sites) in the human genome, the number of genetic association studies of various diseases and clinical outcomes is increasing rapidly. Unfortunately, conflicting results have been obtained in many cases, and only 16% of the genetic associations identified have been subsequently replicated with formal statistical significance [1]. This relates to the underpowering of studies (small sample size), misclassification bias from errors in case–control assignment, genotyping errors and confounding by ethnic origin, age, diet and use of drugs. In this issue of the journal, Funalot et al. [2] report that the genes encoding endothelin-converting enzyme-1 (ECE-1) and endothelin-1 (ET-1) interact to modulate blood pressure levels in women, but not in men. ET-1 (a 21-amino-acid peptide) is a potent vasoconstrictor, generated by ECE-1 from big-ET-1 (a 38-amino-acid peptide). Its effects are mediated via ETA and ETB receptors, and ET receptor blockade lowers blood pressure. Large clinical trials with ET receptor antagonists have not yet been conducted for hypertension [3]. ECE-1 exists in four isoforms (ECE-1a, ECE-1b, ECE-1c and ECE-1d), and one gene, encompassing four alternative promotors, is responsible for the synthesis of these isoforms [4,5]. The isoforms differ only by their N-terminal amino-acid tail, which determines their respective cellular localization. The catalytic domain of the isoforms, a, c and d, is orientated towards the outside of the cell, whereas isoform b is found intracellularly [6]. Funalot et al. [2] studied a polymorphism in the 5′-regulatory region of the ECE-1b gene (338 bp upstream from the translation start site), resulting in a binding site for the transcription factor E2F-2. This C>A substitution is associated with increased promoter activity, as demonstrated in promoter-reporter gene experiments [7]. In untreated hypertensive German women (n = 101), the A allele had already been found to display an intermediate (codominant) effect on both daytime and night-time systolic and diastolic blood pressure [7]. Using participants of the Etude du Veillisement Artériel (EVA) study, a large epidemiological study in Nantes, France, Funalot et al. [2] were able to confirm this finding. The strength of their study is the large sample size (n = 1198, of whom 698 were women). There are also some discrepancies versus the German study: blood pressure was higher in AA women only, suggesting a recessive effect of this gene variant in the French population. Furthermore, the French study included both normotensive and hypertensive women (whereas all women in this study were post-menopausal) and 25% of the French study population used antihypertensive medication. The latter may have affected the outcome because antihypertensive treatment (in particular with angiotensin-converting enzyme inhibitors) lowers plasma ET-1 levels [8], thereby interfering with the alterations in ET-1 levels that are expected to occur with respect to the presence of the A allele. The percentage of AA women taking antihypertensive medication in the EVA study (34%) tended to be higher (P = NS), but no information is provided by Funalot et al. [2] on either the type of medication or the consistency of the effect in the absence of medication. To explain the association between the ECE1 C-388A polymorphism and blood pressure, it is assumed that the A allele, by increasing the ECE-1b concentrations, results in a rise in the levels of ET-1. Evidence for this concept is currently lacking. Muller et al. [9] have recently proposed that ECE-1b, through heterodimerization with the other ECE-1 isoforms, regulates extracellular ECE-1 activity via translocation of the plasma membrane isoforms to intracellular compartments. Thus, an increase in ECE-1b might also lead to a decrease in extracellular ET-1. Moreover, feedback mechanisms could result in decreased big-ET-1 levels and/or an altered density of ETA and ETB receptors, thereby counteracting a rise in ET-1 and/or the consequences of such a rise. In a similar fashion, feedback mechanisms normalize the angiotensin II levels in subjects carrying the T allele of the angiotensinogen M235T polymorphism: their elevated angiotensinogen levels are accompanied by decreased renin concentrations, and thus the degree of angiotensin I generation in TT subjects resembles that in MT and MM subjects [10]. Furthermore, it should be noted that there are non-ECE-1 ET-1-generating enzymes (chymase and neutral endopeptidase) [6], and that the concentrations of the ET-1-degrading enzymes may vary. To complicate things even further, ECE-1 also hydrolyses other peptides (e.g. bradykinin, angiotensin I and substance P), although at lower efficacy [11,12]. Funalot et al. [2] have taken the above compensatory/counteracting mechanisms into consideration, at least in part, by looking for an interaction between the ECE1 C-338A polymorphism and a polymorphism of the ET-1 gene (EDN1 K198N). The latter polymorphism is located in the coding region of the ET-1 gene, and has already been associated with blood pressure levels in overweight individuals [13]. It did not associate with blood pressure in the present study. Interestingly, however, the effect of the ECE1 C-338A variant on blood pressure was apparant only in homozygous EDN1 KK women. Unfortunately, at this stage, the functionality of the EDN1 K198N polymorphism is unknown. It might be speculated that the KK genotype represents a variant that is unable to downregulate big-ET-1 synthesis in the face of increased ECE-1b levels. Future studies investigating ET-1 levels with respect to the various genotypes are required to clarify this issue. Finally, it is unclear why the ECE-1b effect is observed in women only. Funalot et al. [2] speculate on an interaction between sex hormones and the endothelin system. A comparable interaction exists between sex hormones and the renin–angiotensin system [14–16] because oestrogens and androgens control angiotensinogen and renin synthesis, respectively [17,18]. The androgen-stimulated ET-1 activity that is proposed by Funalot et al. [2] to explain the lack of effect of endothelin system genetic variants in men might thus be attributed to angiotensin II stimulating the production of ET-1 [19]. Additional studies on genetic variants of the endothelin system in premenopausal women may shed light on the possible contribution of oestrogens. In summary, the large study conducted by Funalot et al. [2] confirms and extends previous findings on the relationship between the ECE1 and EDN1 genes and blood pressure in women. Accordingly, it provides a firm basis for future studies aiming at delineating the exact mechanism underlying this relationship. Such studies might involve the measurement of ET-1 levels with respect to ECE1 and EDN1 genotype, age, gender, sex hormones and/or drug treatment.