**2. Prostanoids in endothelium**

Prostanoids are unstable lipid mediators that, after intracellular biosynthesis, are released outside the endothelial cell. It is believed that prostanoids work mostly locally in an autocrine or paracrine way. Prostanoids are ubiquitous compounds that coordinate a wide variety of physiological and pathological processes via membrane receptors on the surface of target cells (FitzGerald, 2002). Prostanoids include prostaglandins (such as prostaglandins D2, E2, F2, F2α, PGI2 –or prostacyclin-) and thromboxanes (such as TXA2) (Smith *et al.*, 2000; Linton & Fazio, 2002).

Prostanoids production is mainly regulated by the availability of free arachidonic acid and the activity of cyclooxygenases (COX). Release of arachidonate from cell membrane phospholipids is mediated through phospholipases, mainly phospholipase A2. Once arachidonate is released, it is sequentially converted to prostaglandins G2 and H2 by two COX isoenzymes (COX-1 and COX-2). The unstable prostaglandin H2 is then transformed into biologically active prostanoids by specific synthases in different cells. Prostaglandins interact with G-protein-coupled receptors, classified in function of their preferential affinity towards the five primary prostaglandins (Tsuboi *et al.*, 2002) (Figure 1).

COX isoenzymes (also known as prostaglandin endoperoxide synthases or prostaglandin H synthases) are the rate-limiting step in the formation of prostanoids from arachidonic acid and represent the main control mechanism for prostaglandin production (Smith *et al.*, 2000). Both enzymes have similar biochemical activity in converting arachidonic acid to PGH2 in tissue or in recombinant enzyme systems (Vane *et al.*, 1998).

COX-1 and COX-2 are encoded by two different genes derived from human chromosomes 9 and 1, respectively. In spite of there is a 60 - 65% homology between the isoforms within

A number of studies have demonstrated that estrogens exert significant benefits on the cardiovascular system, and at least a part of these benefits are attributed to the direct effect of estradiol on vascular endothelial cells (Mendelsohn & Karas, 1999; Rubanyi *et al.*, 2002; Sader & Celermajer, 2002). Estradiol is able to stimulate endothelial NO production through several mechanisms, including increased expression of NO synthases (mainly endothelial NO synthase), increased L-arginine availability, non-genomic activation of second messengers (Simoncini, 2009), translocation to intracellular sites, modulation of NO degrading sites (Tostes

Additionally, estradiol is able to exert antioxidant actions on endothelium (Shwaery *et al.*, 1998; Hermenegildo *et al.*, 2002), to modulate the renin-angiotensin system (Farhat *et al.*, 1996; Alvarez *et al.*, 2002), and to decrease endothelin-1 production (Mikkola *et al.*, 1995; Akishita *et al.*, 1998). Furthermore, estradiol regulates endothelial cell expression of adhesion

Estradiol has also been implicated on the regulation of prostanoids production in endothelial cells. Two main vascular prostanoids, prostacyclin and thromboxane A2, play an essential role in the maintenance of vascular homeostasis. Prostacyclin is a vasodilator and an inhibitor of platelet aggregation; in contrast, thromboxane A2 is a vasoconstrictor and a promoter of platelet aggregation. As a consequence of their opposing roles, an imbalance in prostacyclin or thromboxane production has been implicated in the physiopathology of many thrombotic and cardiovascular disorders. In this chapter, we will discuss clinical and experimental data that document the endothelial effects of estradiol on prostanoid

Prostanoids are unstable lipid mediators that, after intracellular biosynthesis, are released outside the endothelial cell. It is believed that prostanoids work mostly locally in an autocrine or paracrine way. Prostanoids are ubiquitous compounds that coordinate a wide variety of physiological and pathological processes via membrane receptors on the surface of target cells (FitzGerald, 2002). Prostanoids include prostaglandins (such as prostaglandins D2, E2, F2, F2α, PGI2 –or prostacyclin-) and thromboxanes (such as TXA2) (Smith *et al.*, 2000;

Prostanoids production is mainly regulated by the availability of free arachidonic acid and the activity of cyclooxygenases (COX). Release of arachidonate from cell membrane phospholipids is mediated through phospholipases, mainly phospholipase A2. Once arachidonate is released, it is sequentially converted to prostaglandins G2 and H2 by two COX isoenzymes (COX-1 and COX-2). The unstable prostaglandin H2 is then transformed into biologically active prostanoids by specific synthases in different cells. Prostaglandins interact with G-protein-coupled receptors, classified in function of their preferential affinity

COX isoenzymes (also known as prostaglandin endoperoxide synthases or prostaglandin H synthases) are the rate-limiting step in the formation of prostanoids from arachidonic acid and represent the main control mechanism for prostaglandin production (Smith *et al.*, 2000). Both enzymes have similar biochemical activity in converting arachidonic acid to PGH2 in

COX-1 and COX-2 are encoded by two different genes derived from human chromosomes 9 and 1, respectively. In spite of there is a 60 - 65% homology between the isoforms within

towards the five primary prostaglandins (Tsuboi *et al.*, 2002) (Figure 1).

tissue or in recombinant enzyme systems (Vane *et al.*, 1998).

*et al.*, 2003), and modulation of endogenous antagonist levels (Monsalve *et al.*, 2007).

molecules (Caulin-Glaser *et al.*, 1996; Abu-Taha *et al.*, 2009).

production and regulation, and their vascular consequences.

**2. Prostanoids in endothelium** 

Linton & Fazio, 2002).

species, the activity and the pattern of gene expression of these enzymes are differentially regulated (Davidge, 2001).

Fig. 1. Biosynthesis of prostaglandins and thromboxanes.

COX-1 has been considered to be the constitutively expressed protein, while COX-2 is induced at sites of inflammation. Following that hypothesis, COX-1 would generate prostaglandins for physiological, housekeeping functions like gastrointestinal mucosal integrity and regulation of renal blood flow, while COX-2 would form the prostaglandins responsible for inflammatory symptoms (Smith *et al.*, 2000; Parente & Perretti, 2003).

But this separation of functions is so not clear. For instance, COX-2 is constitutively expressed in some regions of the central nervous system, and in renal and uterus tissues, suggesting it may play a role under physiologic conditions (Kim *et al.*, 1999; FitzGerald, 2002; Cheng & Harris, 2004). In fact, both COX-1 and COX-2 are involved not only in physiological, but also in pathological processes. The importance of this topic has impelled many outstanding reviews (Vane *et al.*, 1998; Parente & Perretti, 2003; Cipollone *et al.*, 2008; Vanhoutte, 2009).

Regarding the vascular system, both isoenzymes are expressed in endothelium and smooth muscle cells. However, endothelial cells contain up to 20 times more COX than smooth muscle cells (DeWitt *et al.*, 1983). As mentioned before for other organs, COX-1 has usually been considered in endothelium as the constitutive isoform, while COX-2 is induced by a number of cardiovascular risk factors, such as cytokines, cholesterol, lipoproteins, and hypoxia. Actually, both COX isoenzymes share characteristics of constitutive and inducible enzymes in endothelium. Shear stress induces COX-1 gene expression in human umbilical vein endothelial cells (HUVEC) (Doroudi *et al.*, 2000), while clinical studies with a selective

Estradiol Regulation of Prostanoids Production in Endothelium 327

types of ER belong to the class of intracellular receptors classically defined as nuclear ligand-activated transcription factors. Activation of these receptors by the corresponding hormones affects gene expression by acting on specific sequences in the target genes, known

The contribution of both receptors to the regulation of vascular effects is still under study. There is controversy about whether estrogenic effects are mediated through ERα, ERβ or both. In the cardiovascular system, ERα and ERβ have been identified by different techniques in the endothelium, smooth muscle cells and adventitia and on adrenergic nerve endings of arteries from various territories and several species, including humans (Karas *et al.*, 1994; Kim-Schulze *et al.*, 1996; Venkov *et al.*, 1996; Register & Adams, 1998). Although it has been reported cultured endothelial cells do not express ERα (Toth *et al.*, 2008), other investigators have demonstrated the presence of both ERα and ERβ mRNA in endothelium (Wagner *et al.*, 2001) and data from our group demonstrate the protein expression of both

Although the relative significance of both ER subtypes in the vascular actions of estrogens is still under study, data from mice lacking either ERα (Pare *et al.*, 2002; Arnal *et al.*, 2010) or ERβ (Zhu *et al.*, 2002), and also from a non-functional mutation of ERα in a male patient (Sudhir *et al.*, 1997), reveal an impaired vascular function. Therefore, ERα is probably critical for the production of estrogen vascular protective actions (Favre *et al.*, 2010), but ERβ may have relevant functions at the vascular level too (Simoncini *et al.*, 2004). The changes in vascular cell gene and protein expression mediated by those ERα and/or ERβ produce the classic, and better known, longer-term effects of estrogen (Cano & Hermenegildo, 2000). ER act in the nucleus as ligand-activated transcription factors that regulate gene expression in response to hormone binding. ER can also regulate vascular cell gene expression in the absence of estrogen following activation by growth factor signalling pathways, a process referred to as ligand-independent transcriptional activation. In addition, ER are able to signal rapidly (from seconds to minutes), in an apparently non-nuclear manner, resulting in downstream activation of specific kinases and their effectors molecules (Mendelsohn &

The rapid effects of estrogens have been also explained by ER-independent mechanisms of action. For instance, the G protein-coupled receptor GPR30 has been proposed to be a third form of ER, although it is controversial whether GPR30 is a biologically-relevant ER or a collaborator in non-nuclear functions of the classical ER in certain contexts (Levin, 2009). Moreover, even though GPR30 is expressed in certain endothelial cells, there is currently a lack of clear evidence of a role for the receptor in estrogen action in

Rapid actions of estradiol include several endothelial pathways, being the activity of endothelial nitric oxide synthase the most studied (Chow *et al.*, 2010). Other signalling pathways acutely activated by estradiol in endothelium are several kinases, such as phosphotidylinositol 3-kinase (PI3K) (Razandi *et al.*, 2000), protein kinase C (PKC) (Akarasereenont *et al.*, 2000) and Rho-associated kinase (ROCK) (Simoncini *et al.*, 2006; Oviedo *et al.*, 2011). Rapid actions are responsible for the acute and potent vasorelaxation induced by estradiol both at physiological (Teoh *et al.*, 2000; Tep-areenan *et al.*, 2003) or supraphysiological (Naderali *et al.*, 2001; Salom *et al.*, 2002) concentrations, and it has been demonstrated in isolated vessels from animals such as rats (Tep-areenan *et al.*, 2003), pigs (Teoh *et al.*, 2000), rabbits (Salom *et al.*, 2002), guinea pigs (Naderali *et al.*, 2001), and even

as estrogen-response elements, and by modulating transcriptional events.

ERα and ERβ in HUVEC (Sobrino *et al.*, 2009; Sobrino *et al.*, 2010).

Karas, 2010).

endothelium (Wu *et al.*, 2011).

from human beings (Chester *et al.*, 1995).

inhibitor of COX-2 (celecoxib) have shown that this enzyme exerts control of most systemic prostacyclin production in healthy humans (McAdam *et al.*, 1999).

Although prostaglandins E2 and F2α can contribute to vascular phenotype, two main prostanoids play an essential role in vascular physiology: thromboxane A2 and prostacyclin. On the one hand, in the cardiovascular system, thromboxane A2 is predominantly derived from platelet COX-1, but can also be produced by other cell types including the endothelial cells. The stimulation of thromboxane receptors elicits not only platelet aggregation and smooth muscle contraction, but also the expression of adhesion molecules and the adhesion and infiltration of monocytes/macrophages (Nakahata, 2008). As platelets are anucleate, there can be no induction of COX-2 enzyme synthesis (Patrignani *et al.*, 1999). Thromboxane A2 can also be synthesized from endothelial cells (Sellers & Stallone, 2008). The third possibility is a transcellular formation of thromboxane A2 by platelets from prostaglandin H2 released by endothelial cells (Camacho & Vila, 2000). Thromboxane A2 promotes irreversible platelet aggregation, vasoconstriction, and smooth muscle proliferation and, consequently, plays an important role as a mediator of not only haemostasis, but also of acute vascular occlusive events and atherogenesis (FitzGerald, 2002; Weir *et al.*, 2003).

On the other hand, in most blood vessels prostacyclin is the main prostanoid produced by endothelial cells, and the endothelium is its major source (Moncada & Vane, 1978). Both COX-1 and COX-2 isoenzymes contribute to the production of endothelial prostacyclin. By stimulating its preferential receptor, prostacyclin is a potent inhibitor of platelet adhesion to the endothelial cell surface and of platelet aggregation, and generally acts as an endothelium-derived vasodilator and an inhibitor of vascular smooth muscle migration and proliferation (Moncada & Vane, 1978; Fetalvero *et al.*, 2007).

Under physiological conditions, endothelial prostacyclin is a counterregulatory influence to platelet-derived thromboxane, and eicosanoids produced by the COX pathways generally induce vasorelaxation. Nevertheless, in some pathologic circumstances, such as oxidative stress and dyslipidemia, there may be an imbalance where COX-dependent vasoconstrictors and platelet aggregation become more predominant. Reactive oxygen species, such as superoxide anion and hydrogen peroxide are derived from multiple sources within inflammatory leukocytes and vascular tissues including NADPH oxidase, uncoupled endothelial and inducible endothelial NO synthase, xanthine oxidase, cyclooxygenases, lipoxygenases, cytochrome P450 monooxygenases and excess substrate utilization by mitochondria. Additionally, NO reacts with superoxide anion to form the extremely potent oxidant, peroxynitrite. Low concentrations of peroxinitrite selectively nitrate and inactivate prostacyclin synthase, which not only eliminates the vasodilatory, growth-inhibiting, antithrombotic, and antiadhesive effects of prostacyclin, but also increases release of the potent vasoconstrictor, prothrombotic, growth- and adhesion-promoting agents, prostaglandin H2 and thromboxane A2 being in general deleterious to vascular function (Zou *et al.*, 2004; Schildknecht & Ullrich, 2009; Feletou *et al.*, 2010).

## **3. Estrogens actions on endothelium**

Estradiol is the most potent estrogen in humans, and exerts its actions mainly through binding and activation of estrogen receptors (ER). Two major subtypes of ER (ERα and ERβ) have been identified. These receptors vary not only in their tissue distributions, but also in their agonist/antagonist profile of several compounds (Cano & Hermenegildo, 2000). Both

inhibitor of COX-2 (celecoxib) have shown that this enzyme exerts control of most systemic

Although prostaglandins E2 and F2α can contribute to vascular phenotype, two main prostanoids play an essential role in vascular physiology: thromboxane A2 and prostacyclin. On the one hand, in the cardiovascular system, thromboxane A2 is predominantly derived from platelet COX-1, but can also be produced by other cell types including the endothelial cells. The stimulation of thromboxane receptors elicits not only platelet aggregation and smooth muscle contraction, but also the expression of adhesion molecules and the adhesion and infiltration of monocytes/macrophages (Nakahata, 2008). As platelets are anucleate, there can be no induction of COX-2 enzyme synthesis (Patrignani *et al.*, 1999). Thromboxane A2 can also be synthesized from endothelial cells (Sellers & Stallone, 2008). The third possibility is a transcellular formation of thromboxane A2 by platelets from prostaglandin H2 released by endothelial cells (Camacho & Vila, 2000). Thromboxane A2 promotes irreversible platelet aggregation, vasoconstriction, and smooth muscle proliferation and, consequently, plays an important role as a mediator of not only haemostasis, but also of acute vascular occlusive events and atherogenesis

On the other hand, in most blood vessels prostacyclin is the main prostanoid produced by endothelial cells, and the endothelium is its major source (Moncada & Vane, 1978). Both COX-1 and COX-2 isoenzymes contribute to the production of endothelial prostacyclin. By stimulating its preferential receptor, prostacyclin is a potent inhibitor of platelet adhesion to the endothelial cell surface and of platelet aggregation, and generally acts as an endothelium-derived vasodilator and an inhibitor of vascular smooth muscle migration and

Under physiological conditions, endothelial prostacyclin is a counterregulatory influence to platelet-derived thromboxane, and eicosanoids produced by the COX pathways generally induce vasorelaxation. Nevertheless, in some pathologic circumstances, such as oxidative stress and dyslipidemia, there may be an imbalance where COX-dependent vasoconstrictors and platelet aggregation become more predominant. Reactive oxygen species, such as superoxide anion and hydrogen peroxide are derived from multiple sources within inflammatory leukocytes and vascular tissues including NADPH oxidase, uncoupled endothelial and inducible endothelial NO synthase, xanthine oxidase, cyclooxygenases, lipoxygenases, cytochrome P450 monooxygenases and excess substrate utilization by mitochondria. Additionally, NO reacts with superoxide anion to form the extremely potent oxidant, peroxynitrite. Low concentrations of peroxinitrite selectively nitrate and inactivate prostacyclin synthase, which not only eliminates the vasodilatory, growth-inhibiting, antithrombotic, and antiadhesive effects of prostacyclin, but also increases release of the potent vasoconstrictor, prothrombotic, growth- and adhesion-promoting agents, prostaglandin H2 and thromboxane A2 being in general deleterious to vascular function

Estradiol is the most potent estrogen in humans, and exerts its actions mainly through binding and activation of estrogen receptors (ER). Two major subtypes of ER (ERα and ERβ) have been identified. These receptors vary not only in their tissue distributions, but also in their agonist/antagonist profile of several compounds (Cano & Hermenegildo, 2000). Both

prostacyclin production in healthy humans (McAdam *et al.*, 1999).

(FitzGerald, 2002; Weir *et al.*, 2003).

proliferation (Moncada & Vane, 1978; Fetalvero *et al.*, 2007).

(Zou *et al.*, 2004; Schildknecht & Ullrich, 2009; Feletou *et al.*, 2010).

**3. Estrogens actions on endothelium** 

types of ER belong to the class of intracellular receptors classically defined as nuclear ligand-activated transcription factors. Activation of these receptors by the corresponding hormones affects gene expression by acting on specific sequences in the target genes, known as estrogen-response elements, and by modulating transcriptional events.

The contribution of both receptors to the regulation of vascular effects is still under study. There is controversy about whether estrogenic effects are mediated through ERα, ERβ or both. In the cardiovascular system, ERα and ERβ have been identified by different techniques in the endothelium, smooth muscle cells and adventitia and on adrenergic nerve endings of arteries from various territories and several species, including humans (Karas *et al.*, 1994; Kim-Schulze *et al.*, 1996; Venkov *et al.*, 1996; Register & Adams, 1998). Although it has been reported cultured endothelial cells do not express ERα (Toth *et al.*, 2008), other investigators have demonstrated the presence of both ERα and ERβ mRNA in endothelium (Wagner *et al.*, 2001) and data from our group demonstrate the protein expression of both ERα and ERβ in HUVEC (Sobrino *et al.*, 2009; Sobrino *et al.*, 2010).

Although the relative significance of both ER subtypes in the vascular actions of estrogens is still under study, data from mice lacking either ERα (Pare *et al.*, 2002; Arnal *et al.*, 2010) or ERβ (Zhu *et al.*, 2002), and also from a non-functional mutation of ERα in a male patient (Sudhir *et al.*, 1997), reveal an impaired vascular function. Therefore, ERα is probably critical for the production of estrogen vascular protective actions (Favre *et al.*, 2010), but ERβ may have relevant functions at the vascular level too (Simoncini *et al.*, 2004). The changes in vascular cell gene and protein expression mediated by those ERα and/or ERβ produce the classic, and better known, longer-term effects of estrogen (Cano & Hermenegildo, 2000).

ER act in the nucleus as ligand-activated transcription factors that regulate gene expression in response to hormone binding. ER can also regulate vascular cell gene expression in the absence of estrogen following activation by growth factor signalling pathways, a process referred to as ligand-independent transcriptional activation. In addition, ER are able to signal rapidly (from seconds to minutes), in an apparently non-nuclear manner, resulting in downstream activation of specific kinases and their effectors molecules (Mendelsohn & Karas, 2010).

The rapid effects of estrogens have been also explained by ER-independent mechanisms of action. For instance, the G protein-coupled receptor GPR30 has been proposed to be a third form of ER, although it is controversial whether GPR30 is a biologically-relevant ER or a collaborator in non-nuclear functions of the classical ER in certain contexts (Levin, 2009). Moreover, even though GPR30 is expressed in certain endothelial cells, there is currently a lack of clear evidence of a role for the receptor in estrogen action in endothelium (Wu *et al.*, 2011).

Rapid actions of estradiol include several endothelial pathways, being the activity of endothelial nitric oxide synthase the most studied (Chow *et al.*, 2010). Other signalling pathways acutely activated by estradiol in endothelium are several kinases, such as phosphotidylinositol 3-kinase (PI3K) (Razandi *et al.*, 2000), protein kinase C (PKC) (Akarasereenont *et al.*, 2000) and Rho-associated kinase (ROCK) (Simoncini *et al.*, 2006; Oviedo *et al.*, 2011). Rapid actions are responsible for the acute and potent vasorelaxation induced by estradiol both at physiological (Teoh *et al.*, 2000; Tep-areenan *et al.*, 2003) or supraphysiological (Naderali *et al.*, 2001; Salom *et al.*, 2002) concentrations, and it has been demonstrated in isolated vessels from animals such as rats (Tep-areenan *et al.*, 2003), pigs (Teoh *et al.*, 2000), rabbits (Salom *et al.*, 2002), guinea pigs (Naderali *et al.*, 2001), and even from human beings (Chester *et al.*, 1995).

Estradiol Regulation of Prostanoids Production in Endothelium 329

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

**Prostacyclin** 

Ovine fetal PAEC 15 min 10 ↑ 52 % ↔ ↔ (Sherman *et al.*, 2002)

HUVEC 15 min 1 ↑ 57 % NA NA (Alvarez *et al.*, 2002)

HUVEC 30 min 1 ↑ 134 % NA NA (Alvarez *et al.*, 2002)

HUVEC 24 h 10 ↑ 26 % NA NA (Mikkola *et al.*, 1995)

HUVEC 24 h 0.1 ↑ 16 % NA NA (Mikkola *et al.*, 1996) HUVEC 24 h 1 ↑ 78 % <sup>↔</sup> <sup>↑</sup> (Akarasereenont *et al.*,

HUVEC 24 h 1 ↑ 30 % ↑ ↔ (Sobrino *et al.*, 2009)

HUVEC 24 h 10 ↑ 33 % ↑ ↔ (Sobrino *et al.*, 2010)

Human coronary EC 24 h 100 ↑ 45 % NA NA (Mueck *et al.*, 2002)

Bovine coronary EC 24 h 1 ↓ 83 % ↔ ↔ (Stewart *et al.*, 1999)

Ovine fetal PAEC 48 h 10 ↑ 64 % ↑ NA (Jun *et al.*, 1998)

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

**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

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

cells. HUVEC: human umbilical vein endothelial cells. EC: endothelial cells.

well as in isolated vascular preparations.

**production COX-1 COX-2 Reference** 

2000)

increased protein production (Su *et al.*, 2009).

**(nM)** 

**Cell type Time E2**

**Rapid effects** 

**Delayed effects** 

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