**4. Estrogens actions on prostanoids vascular regulation**

The actions of estrogens on prostanoids vascular production will be presented in two sections: first, those experimental data that are rapid in onset and short in duration (within minutes; "non-genomic" effects); second, those experimental data that are delayed in onset and prolonged in duration (require from hours to days to occur; "genomic" effects).

#### **4.1 Rapid effects of estrogens on prostanoids vascular production**

Physiological concentrations of estradiol cause a rapid stimulation of prostacyclin synthesis (within 15 min) in two types of cultured endothelial cells, ovine fetal pulmonary artery endothelial cells (PAEC) (Sherman *et al.*, 2002) and HUVEC (Alvarez *et al.*, 2002). In both cases, the use of the ER-antagonist ICI182780 reveals that the increased prostacyclin production is mediated through ER activation. In ovine PAEC, it seems mediated through ERβ, since prostacyclin stimulation with estradiol was fully blocked by the ERβ- antagonist RR-tetrahydrochrysene. Moreover, in ovine PAEC, the increased prostacyclin production is not related to changes in COX-1 or COX-2 protein abundance, thus reinforcing the implication of non-genomic mechanisms (Table 1) (Sherman *et al.*, 2002).

COX-pathway activity is involved in the acute relaxation afforded by estradiol. In rat mesenteric vessels and aorta, COX inhibition with indomethacin enhances the vasodilator potency of estradiol, suggesting an endogenous release of vasoconstrictor prostanoids (Tepareenan *et al.*, 2003). But estradiol also indirectly affects vasoreactivity to contracting and relaxing substances. In epicardial porcine coronary arteries activation of ERα, but not of ERβ, reduces thromboxane A2 vasoconstriction, indicating another indirect and vasodilator function of ERα (Traupe *et al.*, 2007). Other data suggest that the estrogen-dependent constrictor prostanoid mechanism in the vascular wall may play an important role in the regulation of vascular tone in the female, in both normal and pathophysiological states. In that way, endogenous estrogen acts as an important regulator of constrictor prostanoid function in female rat aorta, involving the potentiation of COX-2 and thromboxane synthase expression in vascular wall (Li *et al.*, 2008).

Data from studies performed on postmenopausal women also supports the importance of COX-pathway in acute estrogen-induced vasodilation. Estradiol induces a rapid increase of cholinergic cutaneous vasodilation, which is eliminated in women treated for 6 weeks with celecoxib, suggesting that the COX-2 pathway plays a specific role in the rapid estradiolinduced vasodilation in postmenopausal women (Calkin *et al.*, 2002).

Some studies have been performed at times that do not clarify whether the observed effects are genomic or non-genomic actions. Estrogen stimulates rat aortic endothelial cell release of prostacyclin after 1 hour of incubation without affecting COX-1 levels (Myers *et al.*, 1996). That stimulation could be mediated by NO, since endogenous NO increased production of prostacyclin and thromboxane A2 in bovine coronary microvascular endothelial cells within 1 hour, without affecting COX-1 or COX-2 expressions (Davidge *et al.*, 1995). At molecular level, exposure of HUVEC for 40 min to estradiol significantly increases the expression of roughly 250 genes, measured by DNA microarray analysis. Among them, the COX-2 gene was the strongest up-regulated by estradiol. In fact, estradiol induced COX-2 gene

In addition, steroid hormone genomic and non-genomic effects may occur simultaneously and may act at different levels, revealing the complexity of estrogen regulation of vascular

The actions of estrogens on prostanoids vascular production will be presented in two sections: first, those experimental data that are rapid in onset and short in duration (within minutes; "non-genomic" effects); second, those experimental data that are delayed in onset

Physiological concentrations of estradiol cause a rapid stimulation of prostacyclin synthesis (within 15 min) in two types of cultured endothelial cells, ovine fetal pulmonary artery endothelial cells (PAEC) (Sherman *et al.*, 2002) and HUVEC (Alvarez *et al.*, 2002). In both cases, the use of the ER-antagonist ICI182780 reveals that the increased prostacyclin production is mediated through ER activation. In ovine PAEC, it seems mediated through ERβ, since prostacyclin stimulation with estradiol was fully blocked by the ERβ- antagonist RR-tetrahydrochrysene. Moreover, in ovine PAEC, the increased prostacyclin production is not related to changes in COX-1 or COX-2 protein abundance, thus reinforcing the

COX-pathway activity is involved in the acute relaxation afforded by estradiol. In rat mesenteric vessels and aorta, COX inhibition with indomethacin enhances the vasodilator potency of estradiol, suggesting an endogenous release of vasoconstrictor prostanoids (Tepareenan *et al.*, 2003). But estradiol also indirectly affects vasoreactivity to contracting and relaxing substances. In epicardial porcine coronary arteries activation of ERα, but not of ERβ, reduces thromboxane A2 vasoconstriction, indicating another indirect and vasodilator function of ERα (Traupe *et al.*, 2007). Other data suggest that the estrogen-dependent constrictor prostanoid mechanism in the vascular wall may play an important role in the regulation of vascular tone in the female, in both normal and pathophysiological states. In that way, endogenous estrogen acts as an important regulator of constrictor prostanoid function in female rat aorta, involving the potentiation of COX-2 and thromboxane synthase

Data from studies performed on postmenopausal women also supports the importance of COX-pathway in acute estrogen-induced vasodilation. Estradiol induces a rapid increase of cholinergic cutaneous vasodilation, which is eliminated in women treated for 6 weeks with celecoxib, suggesting that the COX-2 pathway plays a specific role in the rapid estradiol-

Some studies have been performed at times that do not clarify whether the observed effects are genomic or non-genomic actions. Estrogen stimulates rat aortic endothelial cell release of prostacyclin after 1 hour of incubation without affecting COX-1 levels (Myers *et al.*, 1996). That stimulation could be mediated by NO, since endogenous NO increased production of prostacyclin and thromboxane A2 in bovine coronary microvascular endothelial cells within 1 hour, without affecting COX-1 or COX-2 expressions (Davidge *et al.*, 1995). At molecular level, exposure of HUVEC for 40 min to estradiol significantly increases the expression of roughly 250 genes, measured by DNA microarray analysis. Among them, the COX-2 gene was the strongest up-regulated by estradiol. In fact, estradiol induced COX-2 gene

and prolonged in duration (require from hours to days to occur; "genomic" effects).

**4. Estrogens actions on prostanoids vascular regulation** 

**4.1 Rapid effects of estrogens on prostanoids vascular production** 

implication of non-genomic mechanisms (Table 1) (Sherman *et al.*, 2002).

induced vasodilation in postmenopausal women (Calkin *et al.*, 2002).

expression in vascular wall (Li *et al.*, 2008).

function (Tostes *et al.*, 2003).

expression and new COX-2 protein synthesis by 40 and 60 min, respectively, and quickly stimulated the secretion of prostacyclin and prostaglandin E2 in an ER-dependent manner (Pedram *et al.*, 2002). A similar, significant induction of COX-2 mRNA levels is obtained in human placental villous endothelial cells exposed to estradiol up to 1 hour, without an increased protein production (Su *et al.*, 2009).


Table 1. Summary of estradiol effects on cultured cell production of prostacyclin. E2: concentration of estradiol in nM. Prostacyclin production is expressed as increased (↑) or decreased (↓) percentage of control values. COX-1 and COX-2 expression: increased (↑), decreased (↓), unchanged (↔) or not available (NA). PAEC: pulmonary artery endothelial cells. HUVEC: human umbilical vein endothelial cells. EC: endothelial cells.

#### **4.2 Delayed (genomic) effects of estrogens on prostanoids vascular production**

Estrogen also exerts vascular delayed effects on the metabolism of prostaglandins and the activity of COX, as has been demonstrated in studies performed both in cultured cells, as well as in isolated vascular preparations.

A time-course analysis performed in HUVEC, demonstrates that estradiol effects on prostacyclin production were evident only after 8 or 24 hours (10 and 1 nM estradiol, respectively), suggesting an ER-mediated genomic effect (Sobrino *et al.*, 2010). Moreover, physiological concentrations of estradiol stimulate the production of prostaglandins, mainly

Estradiol Regulation of Prostanoids Production in Endothelium 331

to decrease COX-2 expression in mesenteric arteries from ovariectomized aged rats

However, similar experiments have occasionally produced contradictory results. Exposure of culture bovine coronary microvascular endothelial cells to physiological concentrations of estradiol for 24 hours reduced both prostacyclin and thromboxane A2 production (Stewart *et al.*, 1999). In the case of whole tissue experiments, arteries respond in a different way to estradiol. This is the case of artery segments from different vascular beds (thoracic aorta, pulmonary artery, ear artery and coronary arteries) from adult male rabbits in which estradiol may cause both vasorelaxation and vasoconstriction (Saetrum *et al.*, 2002). Moreover, in thoracic aorta the contractile effects of estrogen are mediated through the

These discrepancies could indicate a difference in the effects of estrogen depending on its previous serum concentration, the reproductive condition of the animal and/or the vascular

**AA**

**Membrane phospholipids**

↑**COX-1**

**PLA2**

**COX-2**

↑**PGI2**

↑**PGIS**

Fig. 2. Estradiol-induced modifications on prostanoid pathway through ERα. Estradiol (E2), acting on ERα, increased cyclooxygenase-1 (COX-1) and prostacyclin synthase (PGIS) expression, resulting in an increased prostacyclin (PGI2) production. Phospholipase A2, cyclooxygenase-2 (COX-2) and thromboxane synthase (TXAS) expressions, along with thromboxane A2 production (TXA2), remained unchanged after exposure to estradiol.

The regulatory actions of estradiol on the prostanoids biosynthesis pathway are mediated through ER activation. The use of ICI182780, a non-selective ER-antagonists, completely blocks estradiol-stimulated prostacyclin production in ovine fetal PAEC (Jun *et al.*, 1998), HUVEC (Sobrino *et al.*, 2009), and tamoxifen blocked it in HUVEC (Mikkola *et al.*, 1995;

**PGH2**

TXA2

**TXAS**

bed being studied (Armstrong *et al.*, 2002; Saetrum *et al.*, 2002; Tostes *et al.*, 2003).

release of vasoconstrictor prostaglandins (Saetrum *et al.*, 2002).

**ER**α

**E2**

Mikkola *et al.*, 1996).

(Armstrong *et al.*, 2002).

prostacyclin, in a variety of cultured endothelial cells, such as ovine fetal PAEC (Jun *et al.*, 1998), HUVEC (Mikkola *et al.*, 1995; Mikkola *et al.*, 1996; Akarasereenont *et al.*, 2000; Sobrino *et al.*, 2010), and human coronary endothelial cell (Mueck *et al.*, 2002). In the vast majority of experiments, cells were exposed to estradiol during 24-48 hours, and the increase of prostacyclin above control values ranged from 16 % to 78 % (Table 1).

In addition, it has been shown that estradiol stimulates production of prostaglandins in a variety of preparations of arteries, including ovine uterine arteries (Vagnoni & Magness, 1998; Janowiak *et al.*, 1998; Habermehl *et al.*, 2000), mesenteric arteries from ovariectomized rats (Davidge & Zhang, 1998; Armstrong *et al.*, 2002), rat cerebral blood vessels (Ospina *et al.*, 2002; Ospina *et al.*, 2003), and aorta from ovariectomized monkeys (O'Sullivan *et al.*, 2001). The increase of prostacyclin production is of a similar magnitude to that obtained with cultured endothelial cells.

Concerning endothelial production of thromboxane A2 under estradiol, the literature indirectly suggests that estrogen would have a beneficial effect by decreasing the production of thrombogenic compounds. In men, there is a decrease in the formation of thromboxane A2 after the use of high dosage intra-muscular estrogen therapy (Henriksson *et al.*, 1996). Moreover, the ratio of *in vivo* prostacyclin to thromboxane A2 formation increases 2-fold during estrogen replacement therapy (Mueck *et al.*, 2001). Nevertheless, it has also been documented that estrogen increases platelet activation with the liberation of thromboxane A2 in women treated with hormone replacement therapy (Oliveira *et al.*, 2005) and that estrogen enhances the constrictor prostanoid function in female rat aorta (Li *et al.*, 2008). However, in cultured endothelial cells, thromboxane A2 production remains unchanged when measured after exposure to estradiol (Sobrino *et al.*, 2010).

The regulatory role of estradiol on the biosynthesis pathway of prostanoids in endothelium (Figure 1) has been scarcely studied, with the only exception of COX-1 and COX-2 expressions. Concerning the first enzyme, soluble phospholipase A2 is highly expressed in HUVEC and umbilical smooth muscle cells (Ost *et al.*, 1998), but it does not appear to be regulated by estradiol. Phospholipase A2 expression remains unchanged after exposure to estradiol in HUVEC (Sobrino *et al.*, 2010) or in human myometrial cells prepared from second trimester pregnant women (Korita *et al.*, 2004).

The roles of COX-1 and/or COX-2 isoenzymes in endothelial cells exposed to estradiol have been studied in some cases, and both enzymes have been implicated in estradiol effects. In ovine fetal PAEC after exposure to estradiol, both COX-1 mRNA and protein expressions were up-regulated (Jun *et al.*, 1998), whereas in HUVEC estradiol only induces COX-2 expression protein (Akarasereenont *et al.*, 2000). Genome-wide analysis performed in HUVEC exposed to estradiol for 24 hours demonstrated that COX-1 was among the 5 % of proteins which expression was changed more than 1.5 fold-times compared to controls, data that was verified by western blot analysis (Sobrino *et al.*, 2009).

Prostacyclin increased levels after vessel stimulation with estradiol have been mainly associated with enhanced expression of COX-1. In this way, COX-1 protein content is increased in rat (Ospina *et al.*, 2002) and mice (Geary *et al.*, 2001) cerebral blood vessels, and in ovine uterine arteries in response to treatment with estrogen (Rupnow *et al.*, 2002). Indeed, COX-1 (but not COX-2) expression is increased in the endothelium of the ovine uterine artery during the follicular phase of the ovarian cycle and during pregnancy, where estrogen levels are highest (Janowiak *et al.*, 1998; Habermehl *et al.*, 2000). In spite of the classic consideration of COX-2 as an inducible enzyme, it seems that COX-2 does not contribute to the estradiol-increased prostacyclin production. Estrogen has even been found

prostacyclin, in a variety of cultured endothelial cells, such as ovine fetal PAEC (Jun *et al.*, 1998), HUVEC (Mikkola *et al.*, 1995; Mikkola *et al.*, 1996; Akarasereenont *et al.*, 2000; Sobrino *et al.*, 2010), and human coronary endothelial cell (Mueck *et al.*, 2002). In the vast majority of experiments, cells were exposed to estradiol during 24-48 hours, and the increase of

In addition, it has been shown that estradiol stimulates production of prostaglandins in a variety of preparations of arteries, including ovine uterine arteries (Vagnoni & Magness, 1998; Janowiak *et al.*, 1998; Habermehl *et al.*, 2000), mesenteric arteries from ovariectomized rats (Davidge & Zhang, 1998; Armstrong *et al.*, 2002), rat cerebral blood vessels (Ospina *et al.*, 2002; Ospina *et al.*, 2003), and aorta from ovariectomized monkeys (O'Sullivan *et al.*, 2001). The increase of prostacyclin production is of a similar magnitude to that obtained with

Concerning endothelial production of thromboxane A2 under estradiol, the literature indirectly suggests that estrogen would have a beneficial effect by decreasing the production of thrombogenic compounds. In men, there is a decrease in the formation of thromboxane A2 after the use of high dosage intra-muscular estrogen therapy (Henriksson *et al.*, 1996). Moreover, the ratio of *in vivo* prostacyclin to thromboxane A2 formation increases 2-fold during estrogen replacement therapy (Mueck *et al.*, 2001). Nevertheless, it has also been documented that estrogen increases platelet activation with the liberation of thromboxane A2 in women treated with hormone replacement therapy (Oliveira *et al.*, 2005) and that estrogen enhances the constrictor prostanoid function in female rat aorta (Li *et al.*, 2008). However, in cultured endothelial cells, thromboxane A2 production remains unchanged

The regulatory role of estradiol on the biosynthesis pathway of prostanoids in endothelium (Figure 1) has been scarcely studied, with the only exception of COX-1 and COX-2 expressions. Concerning the first enzyme, soluble phospholipase A2 is highly expressed in HUVEC and umbilical smooth muscle cells (Ost *et al.*, 1998), but it does not appear to be regulated by estradiol. Phospholipase A2 expression remains unchanged after exposure to estradiol in HUVEC (Sobrino *et al.*, 2010) or in human myometrial cells prepared from

The roles of COX-1 and/or COX-2 isoenzymes in endothelial cells exposed to estradiol have been studied in some cases, and both enzymes have been implicated in estradiol effects. In ovine fetal PAEC after exposure to estradiol, both COX-1 mRNA and protein expressions were up-regulated (Jun *et al.*, 1998), whereas in HUVEC estradiol only induces COX-2 expression protein (Akarasereenont *et al.*, 2000). Genome-wide analysis performed in HUVEC exposed to estradiol for 24 hours demonstrated that COX-1 was among the 5 % of proteins which expression was changed more than 1.5 fold-times compared to controls, data

Prostacyclin increased levels after vessel stimulation with estradiol have been mainly associated with enhanced expression of COX-1. In this way, COX-1 protein content is increased in rat (Ospina *et al.*, 2002) and mice (Geary *et al.*, 2001) cerebral blood vessels, and in ovine uterine arteries in response to treatment with estrogen (Rupnow *et al.*, 2002). Indeed, COX-1 (but not COX-2) expression is increased in the endothelium of the ovine uterine artery during the follicular phase of the ovarian cycle and during pregnancy, where estrogen levels are highest (Janowiak *et al.*, 1998; Habermehl *et al.*, 2000). In spite of the classic consideration of COX-2 as an inducible enzyme, it seems that COX-2 does not contribute to the estradiol-increased prostacyclin production. Estrogen has even been found

prostacyclin above control values ranged from 16 % to 78 % (Table 1).

when measured after exposure to estradiol (Sobrino *et al.*, 2010).

second trimester pregnant women (Korita *et al.*, 2004).

that was verified by western blot analysis (Sobrino *et al.*, 2009).

cultured endothelial cells.

to decrease COX-2 expression in mesenteric arteries from ovariectomized aged rats (Armstrong *et al.*, 2002).

However, similar experiments have occasionally produced contradictory results. Exposure of culture bovine coronary microvascular endothelial cells to physiological concentrations of estradiol for 24 hours reduced both prostacyclin and thromboxane A2 production (Stewart *et al.*, 1999). In the case of whole tissue experiments, arteries respond in a different way to estradiol. This is the case of artery segments from different vascular beds (thoracic aorta, pulmonary artery, ear artery and coronary arteries) from adult male rabbits in which estradiol may cause both vasorelaxation and vasoconstriction (Saetrum *et al.*, 2002). Moreover, in thoracic aorta the contractile effects of estrogen are mediated through the release of vasoconstrictor prostaglandins (Saetrum *et al.*, 2002).

These discrepancies could indicate a difference in the effects of estrogen depending on its previous serum concentration, the reproductive condition of the animal and/or the vascular bed being studied (Armstrong *et al.*, 2002; Saetrum *et al.*, 2002; Tostes *et al.*, 2003).

Fig. 2. Estradiol-induced modifications on prostanoid pathway through ERα. Estradiol (E2), acting on ERα, increased cyclooxygenase-1 (COX-1) and prostacyclin synthase (PGIS) expression, resulting in an increased prostacyclin (PGI2) production. Phospholipase A2, cyclooxygenase-2 (COX-2) and thromboxane synthase (TXAS) expressions, along with thromboxane A2 production (TXA2), remained unchanged after exposure to estradiol.

The regulatory actions of estradiol on the prostanoids biosynthesis pathway are mediated through ER activation. The use of ICI182780, a non-selective ER-antagonists, completely blocks estradiol-stimulated prostacyclin production in ovine fetal PAEC (Jun *et al.*, 1998), HUVEC (Sobrino *et al.*, 2009), and tamoxifen blocked it in HUVEC (Mikkola *et al.*, 1995; Mikkola *et al.*, 1996).

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Moreover, data support a specific role for ERα on estradiol-stimulated prostacyclin production. Recently, selective ERα agonist 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1h-pyrazole (PPT), selective ERβ agonist (diarylpropionitril; DPN), and selective ERα antagonist, methylpiperidino-pyrazole (MPP), have become available (Krom *et al.*, 2007). With their use, it has been demonstrated that estradiol increased prostacyclin production, and COX-1 and prostacyclin synthase protein and gene expressions through ERα, whereas COX-2, phospholipases and thromboxane synthase expression remained unaltered (Sobrino *et al.*, 2010). The regulatory role of estradiol through ERα is supported by other studies. Estradiol increases levels of COX-1 in cerebral blood vessels from wild-type mice but was ineffective in ERα knockout mice (Geary *et al.*, 2001) and also increases COX-1 expression through ERα in ovine endothelial cells transfected with the human COX-1 promoter (Gibson *et al.*, 2005).
