Review: Novel insights into the regulation of vascular tone by sphingosine 1-phosphate

Placenta 35, Supplement A, Trophoblast Research, Vol. 28 (2014) S86eS92

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Review: Novel insights into the regulation of vascular tone by sphingosine 1-phosphate D. Kerage a, b, D.N. Brindley c, D.G. Hemmings a, b, * a

Department of Obstetrics and Gynecology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada c Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada b

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 11 December 2013

Endothelial dysfunction leading to increased vascular tone is implicated in the pathogenesis of cardiovascular disease, hypertension and pregnancy-related complications like preeclampsia and intrauterine growth restriction. Vascular tone is regulated by a balance between vasoconstrictor and vasodilator signals. Some vascular mediators circulate in blood, whereas others are produced by the endothelium and are delivered to the underlying vascular smooth muscle cells (VSMCs). It is proposed that increased permeability of resistance arteries in preeclampsia allows access of circulating vasoactive factors to VSMCs leading to increased vascular tone. This review focuses on the role of sphingosine 1-phosphate (S1P). This sphingolipid enhances the endothelial barrier, but it can also disrupt the barrier under certain conditions. These S1P-mediated effects on the endothelial barrier have been demonstrated in cultured endothelial cells and in isolated venules. They depend on S1P concentrations, the S1P receptors expressed and the vascular bed. However, no studies have examined if vascular tone is regulated by S1P in resistance arteries through changes in endothelial permeability and the leakage of circulating vasoconstrictors. Our recent studies using the pressure myograph system show that access of infused vasoconstrictors to VSMCs is blocked under low S1P concentrations. Pathophysiological levels of infused S1P disrupt the barrier and maximally increase vascular tone by facilitating access of itself and a coinfused vasoconstrictor to the VSMCs. Interestingly, infusion of an intermediate physiological concentration of S1P showed a small increase in endothelial permeability with controlled leakage of a coinfused vasoconstrictor that led to sub-maximal vascular tone development. These and other studies delineate the important role of S1P in the regulation of vascular tone and emphasize how dysfunction of this regulation can lead to pregnancy-related disorders. Ó 2013 Published by IFPA and Elsevier Ltd.

Keywords: Endothelial permeability Pregnancy Uterine artery Vascular tone Resistance artery Gap junctions

1. Introduction Dramatic vascular adaptations in pregnancy compensate for the remarkable hemodynamic changes that occur to ensure adequate exchange of nutrients and oxygen at the maternalefetal interface [1]. All maternal vessels undergo adaptations but the most significant changes are observed in the uterine vasculature. In human beings and many animals, luminal diameters nearly double in size in arcuate, radial and main uterine arteries with little or no vascular wall thickening [2]. In addition to these structural changes, circulating vasodilator concentrations are increased and the sensitivity

* Corresponding author. Department of Obstetrics and Gynecology, 2-27 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. Tel.: þ1 780 492 2098; fax: þ1 780 492 1308. E-mail address: [email protected] (D.G. Hemmings). 0143-4004/$ e see front matter Ó 2013 Published by IFPA and Elsevier Ltd. http://dx.doi.org/10.1016/j.placenta.2013.12.006

of uterine arteries to vasodilators is enhanced while the response to vasoconstrictors is generally blunted. This review postulates a role for a circulating bioactive lipid, sphingosine 1-phosphate (S1P), in modulating these regulatory changes in vascular tone. Under normal physiological conditions, endothelial and nonendothelial-derived vasoactive agents impact vascular tone by acting on both endothelial cells and the underlying vascular smooth muscle cells (VSMCs) [3]. The balance of responses in VSMCs to vasodilator and vasoconstrictor signals regulates vascular tone. Adaptations in this normal balance along with appropriate structural remodeling are essential to maintain appropriate vascular tone during pregnancy [1]. In late pregnancy, there is increased sensitivity to and production of vasodilators including endothelium-derived hyperpolarization factors (EDHF), prostacyclin and nitric oxide (NO). This is accompanied by decreased sensitivity to vasoconstrictors such as endothelin-1, thromboxane A2 and angiotensin II. These changes decrease vascular resistance

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Abbreviations eNOS ICAM IL IUGR [Ca2þ]i NO PI3K PLC PTEN ROS S1P S1P1e5 SK TNF VCAM VEGF VSMC

endothelial nitric oxide synthase intercellular adhesion molecule interleukin intrauterine growth restriction intracellular free Ca2þ nitric oxide phosphatidylinositol 3-kinase phospholipase C phosphatase and tensin homolog reactive oxygen species sphingosine 1-phosphate sphingosine 1-phosphate receptors sphingosine kinase tumor necrosis factor vascular cell adhesion molecule vascular endothelial growth factor vascular smooth muscle cell

and contribute to the extensive increase in uteroplacental blood flow from baseline at 20e50 ml/min to 450e800 ml/min in late human pregnancy [2]. Numerous factors impact the production and release of vasoactive substances from the placenta and maternal endothelium including hormones and cytokines. An unanswered question is how circulating vasoactive factors pass across the endothelial barrier of arteries to reach the VSMCs and whether regulation of this process contributes to maintenance of normal vascular tone. We propose that the control of endothelial permeability will govern the access of circulating factors to VSMCs and regulate vascular tone. Moreover, disruption in this mechanism will be found in pregnancies complicated by vascular dysfunction. S1P is a vasoactive mediator and also controls the endothelial barrier [4e6]. This review will describe what is known about these dual roles of S1P in the vasculature and will also outline new findings on the regulation of vascular tone through the control of endothelial permeability by S1P. Potential therapeutics using S1P receptor agonists and antagonists are also discussed.

2. Endothelial barrier structure and function The endothelium regulates many biological processes including cardiovascular homeostasis, angiogenesis and inflammation. The endothelium also controls the transport of blood components to the surrounding tissues through paracellular and transcellular pathways. The transcellular pathway transports macromolecules across the endothelium through specialized vesicles. In paracellular transport, molecules move passively across the barrier between the endothelial cells [7]. Endothelial cells are connected to each other by adherens junctions, tight junctions and gap junctions and to the VSMCs through myoendothelial gap junctions. The adherens and tight junctions form cell to cell zipper-like adhesion complexes [7]. Vascular endothelial cadherin (VE-cadherin) is the major structural protein of adherens junctions [8]. VE-cadherin binds b-catenin, which in turn binds to a-catenin, an actin binding protein, which connects the adherens junctions to the actin cytoskeleton. Disruption of VEcadherin to b-catenin binding inhibits proper adherens junction assembly, resulting in decreased cellecell adhesion and barrier disruption [8]. Further stabilization of adherens junctions is mediated by the binding of a-catenin to a-actinin and vinculin. The important role of VE-cadherin in promoting endothelial integrity

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was demonstrated in a mouse model in which intravenous injection of anti-VE-cadherin antibodies increased pulmonary vascular permeability [8]. Tight junctions are also present between adjacent endothelial cells and they not only restrict the movement of molecules between endothelial cells to the sub-endothelial space, but they also prevent diffusion of plasma membrane proteins between the apical and basolateral compartments [7]. The binding of occludins, claudins, and junctional adhesion molecules with zona occludens proteins connects the tight junctions with the actin cytoskeleton, again promoting barrier integrity [9]. Gap junctions form conduits between adjacent cells allowing direct intercellular communication [10,11]. Six connexin subunits oligomerize to form a hemichannel in the plasma membrane, which can dock to another hemichannel in the plasma membrane of an adjacent cell, assembling a complete gap junction channel [11]. These gap junctions allow intercellular transport of small molecules including Ca2þ and cyclic nucleotides. Gap junctions formed by connexin 43 have been recently been linked to pregnancy adaptations involving [Ca2þ]i signaling [10]. Activation of phosphorylated eNOS and subsequent production of NO to induce vasodilation, an essential vascular adaptation in pregnancy, depends on increased [Ca2þ]i [12]. Both NO and prostacyclin increase cAMP/cGMP production, which in turn increases connexin 43 expression and gap junctions. Using freshly isolated uterine artery endothelial cells from a pregnant sheep, Bird et al. showed increased connexins 37 and 43 that correlated with increased eNOS expression [10]. Connexins, therefore, play an important role in regulating vascular tone through Ca2þ-mediated eNOS activation during pregnancy, but whether connexins are dysfunctional in complicated pregnancies such as preeclampsia, is currently under investigation [10]. However, Krupp et al. have recently shown in umbilical vein endothelium isolated from pregnancies complicated by preeclampsia that reduced NO was accompanied by a failure of sustained Ca2þ bursting [13]. In addition to the gap junctions between endothelial cells, myoendothelial gap junctions connect the endothelial cells to the underlying VSMCs [10]. The formation of these myoendothelial gap junctions is inversely correlated with arterial diameter and the number of VSMCs [14]. These junctions could, therefore, play important physiological roles in smaller resistance vessels. Myoendothelial gap junctions also play a role in vascular tone regulation and may be important for vascular adaptations in pregnancy [10,15]. 3. Regulation of endothelial barrier function Barrier integrity is regulated by both circulating and endothelialderived factors. The effectiveness of the barrier differs depending on the vascular bed; for example, it is greater in cerebral compared to mesenteric vasculature. One of the most studied signaling systems that maintains endothelial barrier integrity is angiopoietin-1, which signals through Tie2 to enhance endothelial barrier function by regulating the stress fiber formation [16]. In contrast, disruption of endothelial barrier function is common in vascular disorders mediated by inflammatory cytokines such as VEGF, thrombin, TNFa, IL-6, ICAM-1 and VCAM-1 [8,9]. Vascular inflammation is an important component of hypertension and cardiovascular disease. In pregnancy, inflammation contributes to preeclampsia and intrauterine growth restriction (IUGR) [17]. Detailed mechanisms of cytokine-induced disruption of the endothelial barrier have been described [7]. However, the contributions of changes in endothelial permeability to regulation of vascular tone in non-pregnant or pregnant conditions remain to be elucidated. Factors that can both enhance and decrease the endothelial barrier include S1P and NO. The opposing functions of S1P depend

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on its concentration and on the balance of signaling through the different S1P receptors. Activation of S1P1 receptor enhances the barrier [16], whereas activation of S1P2 or S1P3 receptors decreases it [5]. NO also regulates barrier function dependent on its concentration and likely on the signal that stimulates its production. For example, VEGF mediates permeability through the activation of eNOS [18], although VEGF pretreatment also impairs vasodilation by bradykinin [19] and reduces Ca2þ bursting activity of vessels from pregnant sheep to that found in vessels from non-pregnant sheep [20]. In contrast, S1P at physiological concentrations produces NO that protects against permeability [21,22]. Thus, along with its dual roles in vasodilation and vasoconstriction, S1P is a good candidate for controlling the leakiness of the barrier, which in turn regulates vascular tone. 4. S1P metabolism To understand the role of S1P, it is necessary to understand its metabolism. Sphingosine is the breakdown product of ceramide,

which can be formed de novo or by the sequential breakdown of sphingomyelin and ceramide (Fig. 1). Sphingosine is converted to S1P by sphingosine kinase-1 and -2 (SK-1 and -2) [23e25]. Intracellular S1P levels are tightly regulated because of its important role as a signaling mediator [24,26]. This is achieved by balancing S1P synthesis by SK-1 and -2 with its reversible degradation by S1P phosphatases-1 and -2 or irreversible degradation by S1P lyase [26,27]. S1P is secreted from endothelial cells through specific transporters including S1P transporter spinster homolog-2 [28] and possibly ABC transporters [29]. This extracellular S1P is then able to activate cell surface S1P receptors on endothelial cells or VSMCs. Extracellular S1P is dephosphorylated by cell surface lipid phosphate phosphatases to sphingosine, which decreases extracellular S1P levels and attenuates signaling through S1P receptors [30]. Extracellular sphingosine readily enters the cell to contribute to the pool of intracellular sphingosine. The major sources of circulating S1P are erythrocytes, platelets, leukocytes and the endothelium [31,32]. Plasma S1P concentrations, which directly contact the endothelium, are 0.4e1.1 mM. Approximately 50e70% of S1P in

Fig. 1. Intracellular sphingosine can be obtained directly by uptake of sphingosine from the circulation. Part of the extracellular sphingosine pool can be produced from S1P by the ecto-activities of lipid phosphate phosphatases, which are embedded in the plasma membrane. Alternatively, intracellular sphingosine is generated from ceramide, which is synthesized de novo, or it is derived from the degradation of sphingomyelin by various sphingomyelinases. Sphingosine is converted to S1P by sphingosine kinase-1 and -2. Intracellular S1P levels are also regulated reversibly by two S1P phosphatases-1 and -2 or through irreversible degradation by S1P lyase.

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plasma is carried by high-density lipoprotein, 30% by serum albumin, 5e10% by low-density lipoprotein and 2e5% by very lowdensity lipoprotein [33]. 5. S1P-mediated functions in biology There are five S1P receptors (S1P1e5), which are closely related. These receptors were originally named Edg for Endothelial differentiation gene (S1P1/Edg-1, S1P2/Edg-5, S1P3/Edg-3, S1P4/Edg-6 and S1P5/Edg-8). The receptors are coupled to heterotrimeric Gproteins. S1P1 is associated with Gi, (particularly subtypes Gia1 and Gia3); S1P2 with Gi, G12/13 and Gq; S1P3 with Gi, Gq or G12/13 subunits [34]. Various cell types express different combinations of these receptors. Endothelial cells primarily express S1P1 and S1P3 receptors, whereas S1P1, S1P2 and S1P3 receptors are expressed on VSMCs. S1P4 and S1P5 receptors are normally not detectable in the vascular system [35]. Activation of S1P receptors contributes to a broad range of biological functions including cardiovascular development, angiogenesis and dual effects on vascular tone and endothelial permeability [6,35e37]. At high concentrations, S1P is implicated in pathophysiological processes like apoptosis, osteoporosis, inflammation or disruption of the endothelial barrier [38]. 6. The role of S1P in regulation of endothelial permeability S1P-induced regulation of endothelial permeability depends on its concentration, the S1P receptors expressed and the vascular bed. Many studies demonstrate that signaling through S1P1 enhances endothelial barrier function and blocking S1P1 results in pulmonary vascular leakage [5]. S1P increases endothelial barrier function by reorganization of junctional proteins such as VE-cadherin and b-catenin at the cellecell contacts [5]. Silencing of S1P1 reduces VE-cadherin expression [39]. S1P also promotes the formation of endothelial tight junctions through S1P1 by redistributing zona occludens-1 to lamellipodia and cellecell junctions [40]. These events are mediated through S1P1 in a Gi/Rac/PI3K-dependent manner. Activation of S1P1 stimulates Gi-mediated activation of phospholipase C-b (PLC-b), increased [Ca2þ]i and activation of Rac1 and Cdc42. This induces actin reorganization, stabilization and restoration of the cellecell junctions [7,41]. In contrast, S1P2 and S1P3 weaken the endothelial barrier by disrupting adherens junctions and increasing paracellular permeability through Rho-A and PTEN activated pathways [5]. The disruption of cellular junctions through S1P2 or S1P3, occurs by Gq activation of PLC-b and increased [Ca2þ]i which is followed by a cascade of events that ultimately disassembles the junctions. Activation of G12/13 by S1P2 or S1P3 also stimulates Rho-A and promotes microtubule and actin cytoskeleton destabilization inducing disruption of cellular junctions [7,41]. S1P also impacts gap junctions in a cell-specific manner that may also operate in endothelial cells, although this has not yet been studied. S1P increases connexin 43 expression through p38-MAPK in skeletal muscle, stimulating differentiation [42]. S1P also inhibits gap junction communication in astrocytes through dephosphorylation of connexin 43 [43]. Alternatively, phosphorylation of connexin 43 in cardiomyocytes was found to protect against ischemia-reperfusion injury by decreasing gap junction function [44]. The majority of studies examining the role of S1P in endothelial permeability have been performed in cultured cells. In intact perfused rat mesenteric venules, S1P and an S1P1 receptor agonist, SEW2871, inhibited the induction of endothelial permeability by bradykinin and platelet activating factor [45,46]. Although physiological concentrations of S1P have barrier-enhancing properties in perfused venules [45e47], there are no studies on the regulation of endothelial barrier function by S1P in intact perfused resistance arteries.

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7. The role of S1P in regulation of vascular tone S1P-mediated regulation of vascular tone depends on whether the receptors on the endothelium are activated compared to those on the VSMCs [4]. S1P induces vasodilation through S1P1 or S1P3 receptors on the endothelium by activating eNOS, leading to NO production, whereas it promotes vasoconstriction through S1P2 and S1P3 receptors on the VSMCs [4,6]. Most studies investigating the vascular response to S1P in intact arteries were performed using a wire myograph system in which S1P could simultaneously access both VSMCs and endothelial cells. Under these conditions, S1P constricts renal, mesenteric, cerebral and basilar arteries from mammals and human placental arteries. Large conduit arteries, like aortas, exhibit vasodilator effects to S1P after preconstriction [6]. Although signals through both S1P2 and S1P3 induce vasoconstriction, their importance differs depending on the vascular bed. While activation of S1P3 induces constriction in cerebral arteries, S1P2 activation has no effect [48]. However, another group demonstrated that S1P2 stimulates S1P-mediated vasoconstriction in the pulmonary vasculature [49]. The signal transduction mechanisms involved in S1P-mediated vasodilation and vasoconstriction responses have been extensively reviewed elsewhere [4,6]. 8. A novel mechanism for regulation of arterial vascular tone by control of endothelial permeability The impacts of S1P on endothelial permeability or vascular tone have normally been studied independently. We propose that these two functions are linked and that S1P plays a crucial role in regulating vascular tone in resistance arteries by controlling endothelial permeability. Local S1P concentrations can be controlled rapidly through phosphorylation and dephosphorylation. Under normal physiological conditions, circulating levels of S1P maintain a relatively tight barrier with little leakage, which reduces access of circulating vasoconstrictors to the underlying VSMCs (Fig. 2). When a transient increase in vascular tone is required for normal responses to physiological challenges, increased signals through S1P3 on the endothelium could increase endothelial permeability allowing limited access of circulating vasoconstrictors to VSMCs. To investigate this, we developed a novel technique using the pressure myograph system where endothelial permeability and vascular tone are measured simultaneously after infusion of agonists and antagonists inside pressurized arteries compared to their addition to the bath. Infusion mimics the effects of factors circulating in the blood, whereas addition to the bath allows direct access to the VSMCs. We demonstrated differential effects on endothelial permeability and vascular tone depending on the S1P concentration infused inside uterine arteries from non-pregnant mice in the presence or absence of a thromboxane mimetic, U46619. Importantly, even though addition of U46619 to the bath generated the expected constriction response, infusion of the same concentration inside the artery had no effect. Infusion of a low physiological concentration of S1P (0.1 mM) maintained the endothelial barrier and no vasoconstriction occurred when U46619 was co-infused. Pathophysiological concentrations of S1P (10 mM) disrupted the endothelial barrier and increased permeability. This concentration of S1P also increased vascular tone even when infused alone. This demonstrates that S1P itself at high concentrations reaches the VSMCs through endothelial leakage. Interestingly, even though co-infusion of an intermediate concentration of S1P (1 mM) induced a small permeability effect, leakage of coinfused U46619 induced vasoconstriction. However, this vasoconstriction was less than that induced by adding the same concentration of U46619 directly to the bath. This shows that leakage through the endothelium need not be an all or nothing effect, but it

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Endothelium

Arterial lumen

Endothelium S1P3

SK

1 M

S1P

S1 S1P1

C

SK

0.1 M

S1P

Sub-endothelial space

VSMC Constriction

Disrupted endothelial barrier

ROK R ROK,, Ca2+

N D O vasoconstrictors

S1P S 1P3

S1P3 S1

S1

A

TP P

S1P2 S

N NO S1P1

B S1P, vasoconstrictors e.g. thromboxane A2

Dilation

Enhanced endothelial barrier

Fig. 2. Proposed mechanism for S1P-induced regulation of vascular tone. Circulating S1P acts on endothelial cells. At low concentrations of S1P (0.1 mM) the prevailing response is produced by S1P1 activation and this enhances the endothelial barrier, which blocks leakage of potential vasoconstrictors (A). Activation of S1P1 or S1P3 on the endothelium also produces NO, which induces dilation (B). At high concentrations (1 mM) S1P acts through S1P3 and counteracts the responses mediated through S1P1. This increases permeability (C) allowing leakage of vasoconstrictors (e.g. thromboxane A2 and S1P itself) to the sub-endothelial space, which increases the constriction of VSMCs through thromboxane A2/ prostaglandin (TP) receptors and S1P2 and S1P3 receptors (D). The figure depicts the extreme responses to low and high concentrations of S1P. We propose that there is a dynamic range between approximately 0.1 and 1 mM where S1P dynamically regulates barrier function as a means of controlling vascular tone.

could represent a regulatory continuum. In addition, other components such as S1P-induced NO production modulate not only the vasoconstriction response, but also barrier function [18,22]. It is also evident from our initial studies that different vascular beds differ in sensitivity to these S1P-mediated effects. Uterine arteries from non-pregnant mice had greater sensitivity than mesenteric arteries, strongly suggesting that this proposed mechanism for vascular tone regulation is important in pregnancy [21]. In pathophysiological conditions, inflammatory-induced changes in S1P receptor gene expression could lead to permeability-enhancing signals through S1P3 that override the barrier enhancing signals through S1P1. An increase in plasma or endothelial concentrations of S1P could also increase vascular permeability [5]. The pro-inflammatory cytokines, TNFa and IL1, and also cytomegalovirus infections increase the expression of sphingosine kinase-1 [23,50,51], likely leading to overproduction of S1P with adverse effects on endothelial barrier function. 9. Could targeting S1P actions be therapeutically useful in pregnancy-related disorders? Maladaptations of the maternal cardiovascular system to the challenges of pregnancy can result in gestational-associated pathologies including preeclampsia and IUGR. These pregnancies are complicated by perinatal morbidity and mortality, and increased long-term health risks for both mother and child [52]. Endothelial dysfunction and decreased uteroplacental perfusion occur in both preeclampsia and IUGR, but many gaps remain in understanding the mechanisms leading to the pathogenesis of these disorders [53,54]. Many maternal circulating inflammatory and vasoactive factors, which can negatively impact vascular function, originate from the placenta and contribute to these pregnancy complications [10,53,54]. At present, removal of the placenta remains the only effective treatment for preeclampsia.

Endothelial dysfunction involving decreased NO-mediated vasodilation and increased vascular tone [55] plays a role in both preeclampsia and IUGR [54]. Inflammatory agents such as reactive oxygen species (ROS), which quench NO and reduce its bioavailability, are increased under conditions of oxidative stress and are commonly identified in complicated pregnancies [17]. However, the concept that reduced NO activity alone is the primary cause of these complications has been challenged by studies showing that dietary supplementation with L-arginine, a substrate of eNOS, does not reverse the vascular problems [55]. These results support the existence of alternative pathways leading to increased vascular tone in addition to those that decrease vasodilation. Increased placental hypoxia and oxidative stress contribute to the production of inflammatory factors with barrier disruption effects (e.g. VEGF, TNFa, ROS). Increased vascular permeability through impaired barrier function is identified in preeclampsia [56]. Disruption of the endothelial barrier by any permeability factor could lead to leakage of circulating vasoconstrictors, increased vascular tone, maternal hypertension and restricted fetal growth. It is possible that prolonged exposure of the endothelium to ROS or cytokines will ultimately exhaust the endogenous antiinflammatory protective systems, leading to increased paracellular permeability [57]. This could then increase the access of potent vasoconstrictors like angiotensin II and endothelin-1, which typically are elevated in the blood of women with preeclampsia, to the underlying VSMCs. Key discoveries in the past decade have revealed the therapeutic potential of S1P-based treatments for endothelial pathologies [58e 61]. The most promising results have come from studying FTY720, a sphingosine mimetic. FTY720 is an oral pro-drug, which is endogenously phosphorylated to FTY720-P (an S1P mimetic) [61] and appears to mimic the barrier-enhancing capabilities of S1P [59] along with stimulating vasodilation [60]. However, the mechanisms for these vascular effects are not clearly understood even though the relatively well-known mechanism of action by

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FTY720-P on inhibition of lymphocyte egress is its activation of S1P1 followed by S1P1 internalization and degradation. This leads to long-term desensitization [62]. FTY720 is best known for this immunosuppressive effect and this forms the basis for its efficacious treatment of multiple sclerosis. The Federal Drug Administration recently approved the first oral drug therapy for this disease developed by Novartis Pharmaceuticals [63]. It is intriguing to consider using S1P or its mimetics for treating vascular dysfunction in pregnancy complications given our understanding of the importance of S1P to the regulation of both endothelial permeability and vascular tone. Although FTY720-P appears to be effective for treating endothelial-based disorders [59e61], there are concerns about its immunosuppressive effects. Also, the mechanisms underlying the therapeutic effects on the endothelium are not fully understood. A better option may be to target S1P1specific agonists alone or in combination with S1P3 antagonists. This should enhance the endothelial barrier by activating S1P1 and simultaneously inhibiting the disruption of the barrier by antagonizing S1P3. This could normalize cellecell communication by reforming the cellecell junctions and limit endothelial leakage of circulating vasoconstrictors to the underlying VSMC. Given the reversible nature of adherens junctions and tight junction assembly and disassembly, these treatments should be effective both as a protective measure before loss of function and as a rescue measure after disruption has already occurred. Although no S1P3-specific antagonists are currently being tested, S1P1-specific agonists currently under investigation have shown promise in preventing allograft rejection (AUY954) [64], autoimmune disease (RG3477) and acute kidney injury (SEW2871) [65].

10. Conclusions The mechanisms and factors that regulate endothelial permeability in cell culture models and in the venous system appear to be relatively well characterized. Control of endothelial barrier function in resistance arteries is emerging as an additional mechanism for regulation of vascular tone. However, the exact mechanisms through which increased endothelial permeability contributes to disease, particularly during pregnancy, remain to be elucidated. S1P regulates endothelial permeability and vascular tone. We propose that endothelial permeability contributes to disease through increased vascular tone mediated by leakage of circulating vasoconstrictors which then activate VSMCs. This mechanism along with the loss of functional gap junctions important for production of NO and prostacyclin may contribute to the pathophysiology of vascular complications in pregnancy. Future studies targeting S1P turnover and signaling may prove to be important therapeutically in pregnancy-related disorders such as preeclampsia or IUGR.

Conflict of interest statement We confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Acknowledgments This work was funded by grants from the Canadian Institutes of Health Research (CIHR), Natural Sciences and Engineering Research Council of Canada (NSERC) and Women and Children’s Health Research Institute (WCHRI) at the University of Alberta.

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