Endothelin, sex, and pregnancy: unique considerations for blood pressure control in females

Abstract

Endothelin-1 (ET-1) is a potent vasoconstrictor, and dysregulation of the endothelin (ET) system has been implicated in the development of hypertension. Sex differences in the ET system have been identified in ET receptor expression and activation, levels of ET-1, and downstream mediators of the ET system. More specifically, males have greater ET-1/ET_A receptor activation, whereas females exhibit greater ET_B receptor activation. These differences have been suggested to contribute to the sex differences observed in blood pressure control, with greater ET_B receptor activation in females potentially acting as an important pathway contributing to the lower prevalence of hypertension in young females compared with age-matched males. This hypothesis is further supported by studies in pregnancy; the role of the ET system is enhanced during pregnancy, with dysregulation of the ET system resulting in preeclampsia. Further research is necessary to elucidate the relative roles of the ET system in blood pressure control in both sexes and to further explore the potential benefits of pharmacological ET blockade in women.

hypertension is a complex and multifaceted disease, and there are well-established sex differences in many aspects of blood pressure (BP) control (34, 66). Sex differences in BP have been recognized since the 1940s (7), where college-aged men were found to have higher BP than college-aged women. It is now well established that males have higher BP relative to age-matched females until the sixth decade of life, although sex differences in BP extend beyond absolute BP readings alone. There are also sex differences in the relationship between elevations in BP with the impact of hypertension on end-organ damage (6, 19), which may be particularly relevant given the recent early findings of the Systolic Blood Pressure Intervention Trial (SPRINT) trial indicating that reductions in BP below the commonly recommended target result in improved cardiovascular outcomes in adults 50 yr and older with hypertension (64). There is also an ever-expanding basic science literature documenting sex differences in the molecular mechanisms regulating BP. The goal of this short review is to highlight recent work examining sex differences in the contribution of the endothelin (ET) system to BP control.

Endothelin-1 (ET-1) has been described as the most potent vasoconstrictor substance identified to date, and overactivation or dysfunction of the ET system has been implicated in the development and progression of hypertension in both clinical and experimental studies (5, 27, 28, 46, 53). ET-1 acts via two G protein-coupled receptors, ET_A and ET_B. ET_A receptors are found predominately on vascular smooth muscle cells, and activation of ET_A receptors induces vasoconstriction and promotes increases in inflammation and oxidative stress. ET_A receptors are also found in the renal proximal tubule and collecting duct and likely contribute to ET-1-induced sodium retention, all of which will act in a prohypertensive fashion to increase BP. Indeed, ET_A receptor antagonism decreases BP in patients with resistant hypertension (4, 63). In contrast, ET_B receptors are predominately found on the vascular endothelium and renal collecting duct cells, and activation leads to vasodilation through enhanced nitric oxide (NO) production, natriuresis, and diuresis resulting in decreases in BP. Consistent with these data, inhibition of the ET_B receptor, either via pharmacological blockade or genetic modification, results in salt-sensitive hypertension (47, 52).

Sex Differences in the ET System

There are numerous sex differences in the ET system and these differences have been suggested to contribute to sex differences in BP control (5, 26, 53, 61). The major papers focused on sex differences in the endothelin system are summarized in Table 1. Plasma ET-1 levels are increased in hypertension, and males have higher ET-1 levels than females, both in humans and in experimental animal models (11, 37, 45). Although the mechanism(s) responsible for sex differences in the ET system are still being investigated, sex hormones have been implicated. Female sex hormones suppress ET-1 levels by inhibiting ET-1 mRNA expression and endothelin-converting enzyme activity and decrease ET_B receptor expression (61). Recent studies have further shown that estrogen replacement following ovariectomy of female Sprague-Dawley rats decreases ET_A receptor expression in the lung and ET_B expression in the renal inner medulla (17). In contrast, testosterone has been suggested to increase ET-1 (61), although a recent paper reported that orchidectomy increases ET-1, ET_A, and ET_B receptors in the rat portal vein, suggesting that testosterone may also suppress the ET system (49). Regardless, these data suggest tissue-specific effects of sex hormones on the ET system and underscore the importance of further defining the impact of both male and female sex hormones on the ET system.

Table 1.

Summary of papers demonstrating a role for ET-1 in sex differences in hypertension

Topic	Reference
Males have higher ET-1 levels than females	Polderman et al. (45)
Ovarian hormones regulate endothelin receptor expression	Gohar et al. (17)
Orchidectomy increases ET-1 and ET receptor expression in the portal vein	Rossignoli et al. (49)
Males have a higher expression of ETA and ETA:ETB ratio compared to females	Ergul et al. (12)
Renal ETB receptor function is preserved in females during ANG II hypertension	Kittikulsuth et al. (25)
Male SHRs exhibit greater vasoconstriction in response to ET-1	Fortes et al. (13)
Men exhibit greater ETA-mediated vasoconstriction than age-matched women	Stauffer et al. (55)
ETB receptor mediates vasoconstriction in males, but vasodilation in females	Kellogg et al. (24)
Intramedullary ET-1 infusion increases diuretic and natriuretic responses in female rats, but not males	Nakano et al. (40)
ETB receptor limits ET-1 induced increases in oxidative stress in females	Sullivan et al. (56)
ETB deficiency results in a greater decrease in renal NOS activity in females	Taylor et al. (57)
ET-1 results in a greater increase of calcium release from inner medullary collecting duct cells in males compared to females	Jin et al. (22)

ET, endothlin; ET-1, endothelin-1; NOS, nitric oxide synthase.

In addition to sex difference in the levels of expression of the ET system, males are also more sensitive to ET-1-induced vasoconstriction than females, and this has been linked to sex differences in ET receptor expression in the renal medulla and saphenous veins; males have greater ET_A receptor expression compared with females (12, 25, 61). As a result, males have greater ET_A-mediated vasoconstriction than females in both clinical and basic science studies (12, 13, 55, 60). In contrast, females exhibit enhanced ET_B-mediated dilation that antagonizes ET_A-mediated increases in vascular tone (55). In additional studies, treatment of men and women with an ET_B receptor antagonist to determine the contribution of the ET_B receptor to resting vascular tone found that in men, ET_B receptors mediate vasoconstriction, whereas, in women, ET_B receptors mediate vasodilation (24). The authors concluded that this response was most likely mediated by greater ET_B receptor expression in vascular smooth muscle cells in cutaneous vessels of men to induce constriction, whereas in women there was greater ET_B receptor expression in the endothelium mediating dilation. These data further suggest a more prominent role for ET_B receptor function in females compared with males, which may contribute to more advantageous cardiovascular outcomes in females relative to males.

There are also sex differences in the impact of ET receptor activation on renal function. ET-1 is critical in the regulation of salt and water excretion by the kidney with ET_B receptor activation inducing diuretic and natriuretic responses via a NO synthase (NOS) 1, cGMP, and protein kinase G pathway in male Sprague-Dawley rats (41). Followup studies by the same group expanded this work to determine the impact of sex of the animal on medullary ET_B function and found that renal medullary infusion of the ET_B receptor antagonist S6c increases urine flow rate and sodium excretion in both sexes; however, intramedullary infusion of ET-1 caused significant diuretic and natriuretic responses in female but not male rats (40). Additional studies found that female rats exhibit an ET_A-dependent diuresis and natriuresis that are dependent on activation of NOS1, whereas male rats exhibit an ET_A-mediated reduction in medullary blood flow not observed in females. NOS1 has also been shown to contribute to ET_B-dependent shear stress-mediated NO production in the renal collecting duct (20), although if there are sex differences in ET_B-NOS1, mediated NO production in isolated collecting duct cells has not been directly examined. Separate studies following chronic ANG II infusion in male and female rats found that renal medullary ET_B receptor function remains intact in female rats but is lost in male rats after ANG II infusion (25). Male rats exhibit a decrease in ET_B receptor expression after ANG II infusion, and as a result, medullary infusion of S6c has no effect on water or sodium excretion. In contrast, female rats maintain ET_B expression and ET_B agonist-induced diuresis and natriuresis with ANG II hypertension, although urine flow rate and sodium excretion decrease in hypertensive females compared with normotensive controls. Together, these studies suggest 1) sex-specific actions of ET_A receptor activation in the renal medulla, and 2) sex differences in the contribution of ET_B receptor activation to modulate renal function. Moreover, both of these findings remain consistent with females having less ET-mediated cardiovascular and renal injury relative to their male counterparts.

Sex differences in the ET system further extend to vasoactive mediators downstream of receptor activation. ET-1 stimulates the generation of oxidative stress, modulates NO levels, and induces inflammation. Moreover, each of these individual pathways have been shown to contribute to the development of hypertension, and all exhibit sex differences (66). The mechanism(s) responsible for causing sex differences in oxidative stress, NO, and inflammation are still under investigation; however, the ET system may be a prime candidate. There is indirect evidence supporting the hypothesis that ET_B receptor activation is critical in the ability of female rats to limit ET-1-induced increases in oxidative stress relative to males based on BP measurements (56); however, males may be more dependent on the ET_B receptor to limit increases in vascular oxidative stress in response to deoxycorticosterone acetate (DOCA)-salt treatment (23). Female rats also have greater ET_B receptor expression than males, and female rats lacking ET_B receptors exhibit a greater decrease in renal NOS activity than males (57). In addition as discussed above, female rats exhibit a NOS1-ET_A-dependent diuresis and natriuresis not observed in males (40), suggesting that females exhibit greater ET_A- and ET_B-mediated increases in NO compared with males. Chronic ET-1 infusion also increases immune cell infiltration in male experimental animals (51). Although the impact of ET-1 on inflammation has not been examined in females, it is tempting to speculate that greater ET_B activation in females contributes to the more anti-inflammatory immune profile seen in female rats (58).

Finally, recent studies indicate a sex difference in the ability of ET-1 to increase calcium release in freshly isolated inner medullary collecting duct cells; ET-1 results in greater increases in calcium release in cells from males compared with females (22). Taken together, greater ET-1/ET_A receptor activation in males may result in greater increases in oxidative stress and a proinflammatory immune profile which contributes to impaired renal and vascular function contributing to increases in BP. In contrast, enhanced or maintained ET_B function in females offers renal and cardiovascular protection, in part by maintaining NO production, which may act to limit increases in BP.

The endothelin system is arguably more idiosyncratic than most BP control systems and many of these subtleties could provide important mechanistic insights to BP control (for a complete review on the role of ET system see Ref. 27), yet they remain understudied with regard to sex. For example, ET_B receptors are important for the clearance of circulating ET-1, and the rise in BP following antagonism of ET_B receptors in vivo likely reflects increased ET_A activation due to increased circulating levels of ET-1 as opposed to a loss of ET_B-mediated vasodilation. The ET system has also been suggested to contribute to sex-specific alterations in vascular function with age (30, 32, 55). More studies are needed to better understand the impact of sex on the local regulation of the ET system across the lifespan to provide greater insight as to the regulation of cardiovascular and renal health at all ages.

Sex Differences in ET Control of BP

Despite numerous reported sex differences in the ET system as outlined above, the vast majority of the studies examining the impact of ET on BP have been conducted exclusively using male experimental animals. Based on the potential of greater ET_B receptor activation to be an important mechanism regulating cardiovascular heath in females and the fact that approximately half of the hypertensive population is female, understanding the role of ET in hypertensive females is an important, although understudied, area of research. Moreover, additional studies are needed to establish if sex differences in the ET system are physiologically relevant.

Studies of ET_B receptor-deficient rats have been used as a tool to explore the role of the ET system in BP control as well as sex differences in BP; we are unaware of any studies that have directly compared BP responses to chronic ET-1 infusion. Male rats lacking functional ET_B receptors have a higher BP compared with females when maintained on a normal-salt (NS) diet (57), despite comparable ET-1 levels between the sexes. In response to a high-salt (HS) diet, BP increases in both sexes, although female ET_B-deficient rats exhibit a greater increase in BP than males (56, 57). More specifically, systolic BP increased from ∼130 to ∼165 mmHg in male ET_B-deficient rats, and from ∼125 to ∼175 mmHg in females; resulting in a 15-mmHg greater increase in BP in females. Since even modest decreases in BP result in significant cardiovascular protection, this would suggest a physiologically relevant difference in the impact of the ET system on overall cardiovascular health between the sexes. Moreover, the exaggerated BP response to HS in female rats is attenuated by treatment with the antioxidant Tempol, suggesting that females are more sensitive to loss of ET_B receptor function and that the ET_B system typically acts in the female to limit ET-mediated increases in oxidative stress, and in turn, BP (56). However, a recent study potentially challenges this conclusion. BP in both of the above studies was measured by tail-cuff analysis. When BP is instead measured by telemetry in male and female ET_B receptor-deficient rats on NS and HS, there are no apparent sex differences in BP with mean arterial pressure in both sexes increasing from ∼130 mmHg at the beginning of HS treatment to ∼160 mmHg after 4 wk (52). The authors then examined acute BP responses to air-jet stress in male and female ET_B receptor-deficient rats and found that females maintained on both normal-salt (NS) and HS diets were more sensitive to acute stress-induced increases in BP versus males, which could explain the greater increase in BP reported when BP was measured by tail-cuff. The total mean arterial pressure response to acute air-jet stress, expressed as area under the curve, was ∼35 mmHg × 3 min in males versus ∼60 mmHg × 3 min in females following 3 wk on HS. The greater acute increase in BP was not mediated by increased vascular reactivity to ET−1, since male ET_B receptor deficient rats exhibited a greater increase in BP with intravenous infusion of ET−1 compared with females on NS, and both sexes exhibited comparable increases in BP with infusion of an ET_B agonist. HS exacerbated the acute increase in BP with ET-1 infusion, but there were no apparent sex differences. The authors concluded that female rats have reduced ET_A receptor activity when on a HS diet compared with males, and may still support the hypothesis that females in particular are dependent on ET_B receptor activation to limit increases in BP in response to stressful stimuli.

Additional support for this hypothesis is found in DOCA-salt hypertension. A bolus injection of ET-1 results in a transient decrease in BP followed by a prolonged increase in BP in both sexes; however, female DOCA-salt rats exhibit a greater decrease in BP compared with males (59). Injection of an ET_B receptor agonist also results in a greater depressor response in females (∼7 mmHg decrease in males vs. ∼18 mmHg in females), although females also exhibit an attenuated increase in BP following the transient dip compared with males (∼14 mmHg increase in males vs. ∼6 mmHg in females). These data suggest greater ET_B function in DOCA-salt females and greater ET_A receptor function in males, a conclusion that was further supported by additional studies showing that male DOCA rats exhibit a greater decrease in basal BP in response to an ET_A receptor blocker versus females (∼43 mmHg decrease in males vs. ∼14 mmHg decrease in females; 58). Separate studies directly assessed the role of the ET_B receptor in BP control in DOCA-salt hypertension using wild-type and ET_B receptor-deficient rats (23). Of interest, the sex difference in BP typically exhibited in wild-type rats with DOCA-salt, where males have greater increases in BP than females (16), was abolished in ET_B receptor-deficient rats despite the finding that ET_B-deficient rats of both sexes display significantly greater increases in BP with DOCA-salt compared with wild-type rats. These findings indicate that ET_B receptor activation typically functions to limit DOCA-salt-induced increases in BP in the female and further support a critical role for the ET-1/ET_B system in regulating BP in females in response to a stressor. Consistent with these results, blockade of ET_B receptors has been reported to result in greater increases in BP to angiotensin II infusion in female rats versus males (53).

ET System in Normal Pregnancy

Additional support for a prominent role for the ET_B receptor in the regulation of BP in females is found in pregnancy. During normal pregnancy, there is a significant peripheral and renal vasodilation to accommodate the plasma volume expansion required for optimal pregnancy outcomes. Previous studies have demonstrated a critical role of adaptations in the ET system in mediating the vascular and renal hemodynamic response to healthy pregnancy. Pharmacological inhibition of the ET_B receptor prevents the renal vasodilation and renal hyperfiltration that occurs in normal pregnancy (9), and ET_B antagonism increases BP and reduces fetal growth during pregnancy providing further evidence that activation of the ET_B receptor is necessary for optimal pregnancy outcomes in the rat (33). Recent work by Khalil and colleagues (35) indicates an increase in ET_B receptor expression during a normotensive, healthy pregnancy, with corresponding increases in ET_B receptor-mediated vasorelaxation and microvascular activity. Activation of the ET/ET_B pathway in healthy pregnancy is thought to be stimulated by the pregnancy hormone relaxin (10, 42), and other factors including estrogen, vascular endothelial growth factor, and placental growth factor may play a role in this response as well (31, 43, 44). In addition, ET-1 contributes to the contractile tone of the uteroplacental vasculature, and this tone diminishes near term (2). These studies demonstrate that activation of the ET system is critical in maintaining a normal cardiovascular response to pregnancy.

ET System in Preeclampsia

Dysregulation of the ET system during pregnancy has been implicated in the pathogenesis of preeclampsia, a syndrome of new onset hypertension and proteinuria during pregnancy that is associated with significant morbidity and mortality of the mother and fetus. Many groups have demonstrated increases in circulating ET-1 in the plasma of preeclamptic women compared with normotensive pregnant controls (15). Increases in ET-1 levels positively correlate with increases in soluble Fms-like tyrosine kinase-1 (sFLT-1) levels (62) in preeclamptic women, and the ratio of sFlt-1 to PLGF (placental growth factor) is a strong predictor of disease in women with preeclampsia (1), suggesting that increases in ET-1 may also serve as a predictor of disease progression in these women. These data also suggest that the antiangiogenic factors released by the placenta stimulate the production of ET-1, which further contributes to the maternal syndrome of preeclampsia. A summary of select papers that focus on the role of the endothelin system in preeclampsia highlighted in this review are included in Table 2 (for more complete reviews on the role of the ET system in preeclampsia please see the following recent reviews 15, 18, and 50). Based on the central role played by the ET system in normal pregnancy combined with the above data supporting a critical role for ET_B receptor activation in females in response to stressors, it is not surprising that ET receptor activation has been shown to impact BP in preeclampsia.

Table 2.

Select papers from this review demonstrating a role for ET-1 in the pathogenesis of preeclampsia

Topic	Reference
Elevated ET-1 positively correlates with sFlt-1 in preeclamptic women compared to normotensive controls	Verdonk et al. (62)
Circulating ET-1 is elevated in the RUPP model of placental ischemia	Roberts et al. (48)
AT1-AA infusion increases circulating ET-1 in a rodent model of preeclampsia	Brewer et al. (8)
Circulating ET-1 is elevated in an animal model of HELLP syndrome	Morris et al. (38)
Infusion of sFlt-1 in a pregnant rat increases circulating ET-1	Murphy et al. (39)
Pharmacological blockade of the ETA receptor attenuates BP in the RUPP	Alexander et al. (3)
Amelioration of maternal syndrome of preeclampsia by TNF-α blockade significantly reduces plasma ET-1 levels	LaMarca et al. (29)
Mice injected with IgG from preeclamptic women have elevated plasma ET-1 and develop the maternal syndrome of preeclampsia	Zhou et al. (65)
ETB receptor is downregulated in the RUPP rat, with a significant decrease in ETB-mediated nitric oxide production	Mazzuca et al. (36)
ETB deficient rats exhibit elevations in BP during late pregnancy	Spradley (54)

RUPP, reduced uteroplacental perfusion pressure; HELLP, hemolysis, elevated liver enzymes, low platelet count; AT1-AA, angiotensin II type I receptor autoantibody; TNF-α, tumor necrosis factor-α; BΠ, blood pressure.

To specifically examine the role of the ET system in the pathogenesis of preeclampsia, several investigators have explored this pathway in rodent models of the disease. Plasma ET-1 levels are elevated in the reduced uteroplacental perfusion pressure (RUPP) rat model, which induces placental ischemia to mimic a preeclamptic phenotype (48), and blockade of the ET_A receptor attenuates the BP response to RUPP in the pregnant rat (3). Further studies in the RUPP rat indicated that ET-1 may serve as a common pathway for various vasoactive factors that are increased during preeclampsia. Tumor necrosis factor-α (TNF-α) and the angiotensin II type 1 receptor autoantibody (AT₁-AA) are elevated during late pregnancy in the RUPP model (29, 48). Pharmacological blockade of TNF-α ameliorates the maternal syndrome, including significantly reducing the placental and renal levels of ET-1 (29). Similarly, blocking the AT₁ receptor significantly reduces the production of ET-1 in human umbilical venous endothelial cells exposed to RUPP sera. ET-1 production also increases in pregnant rats infused with factors linked to the development of preeclampsia including sFlt-1, either alone or in combination with s-endoglin (38, 39) or the AT1-AA (8). In all of these models, blockade of the ET_A receptor attenuates the rise in BP. Injection of IgG from women with preeclampsia also induces ET-1 expression and increases BP in mice, and the BP response is again attenuated by ET_A receptor blockade (65). Together, these studies suggest that ET-1 and activation of the ET_A receptor may be serving as a common target for multiple vasoactive factors in the pathogenesis of preeclampsia.

In addition to increased activation of the ET_A receptor during preeclampsia, recent studies suggest that dysregulation of the ET_B receptor in pregnancy may also contribute to the development of pregnancy-induced hypertension. Support for this notion is found in the RUPP rat model, expression of the ET_B receptor decreases in the aorta and the mesenteric microvessels compared with normal pregnant controls (35). Additionally, this study also found that the RUPP rats had significantly decreased ET_B receptor-mediated NO production compared with normotensive pregnant controls (36). The role of the ET_B receptor in preeclampsia has been directly assessed using the RUPP model in ET_B-deficient rats. The typical decrease in renal vascular myogenic tone that is observed during normal pregnancy is absent in ET_B receptor-deficient rats (21), and recent work has demonstrated that these rats have a higher BP during late pregnancy compared with wild-type counterparts (54). Not only does the RUPP procedure result in an exacerbated increase in BP in ET_B-deficient rats compared with the transgenic control RUPP rats, fetal outcomes are also affected in the ET_B-deficient RUPP rats (54). The pups of the ET_B-deficient rats are significantly smaller than the transgenic controls following the RUPP procedure, and there is a significant decrease in placental sufficiency, a marker for placental function.

Clinical Considerations

ET-receptor antagonists are not approved to treat hypertension based, in large part, on the failure of clinical trials to reach the primary endpoint as well as the availability of safe alternative BP-lowering agents (28). Although the ET_A receptor antagonist darusentan significantly reduced ambulatory BP in patients with resistant hypertension, the primary end point of the trial, clinic BP, was not significantly different from placebo on the final day of the trial. As a result, the development of the drug was discontinued (4, 63). However, the possibility exists that women in particular may benefit from pharmacological targeting of the ET pathway, specifically agents that would increase ET_B receptor activation, to lower BP. Indeed, ET receptor antagonists are used in the treatment of pulmonary arterial hypertension (PAH), and women with PAH exhibit a greater increase in 6-min walk distance over 12 wk than men (14), suggesting a sex difference in functional responses to ET receptor antagonists. Currently, ET_A receptor antagonists are contraindicated during pregnancy due to teratogenic effects during the first trimester, although, these antagonists may be useful in the third trimester when the dangers of preeclampsia are the greatest. The development of newer agents that do not cross the placental barrier or delayed treatment in pregnancy could provide systemic benefit to the mother to improve the symptoms of preeclampsia and prolong pregnancy to improve fetal outcomes.