Molecular mechanisms of maternal vascular dysfunction in preeclampsia

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Review

Special Issue: Nurturing the Next Generation

Molecular mechanisms of maternal vascular dysfunction in preeclampsia Styliani Goulopoulou1,2 and Sandra T. Davidge3,4,5 1

Department of Integrative Physiology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX, USA Department of Obstetrics and Gynecology, University of North Texas Health Science Center, Fort Worth, TX, USA 3 Department of Obstetrics and Gynecology, University of Alberta, Edmonton, Canada 4 Department of Physiology, University of Alberta, Edmonton, Canada 5 Women and Children’s Health Research Institute, Edmonton, Canada 2

In preeclampsia, as a heterogeneous syndrome, multiple pathways have been proposed for both the causal as well as the perpetuating factors leading to maternal vascular dysfunction. Postulated mechanisms include imbalance in the bioavailability and activity of endothelium-derived contracting and relaxing factors and oxidative stress. Studies have shown that placenta-derived factors [antiangiogenic factors, microparticles (MPs), cell-free nucleic acids] are released into the maternal circulation and act on the vascular wall to modify the secretory capacity of endothelial cells and alter the responsiveness of vascular smooth muscle cells to constricting and relaxing stimuli. These molecules signal their deleterious effects on the maternal vascular wall via pathways that provide the molecular basis for novel and effective therapeutic interventions. Preeclampsia: a pregnancy-specific syndrome with gestational and postpartum vascular implications Preeclampsia is a pregnancy-specific syndrome and one of the leading causes of maternal and fetal mortality and morbidity. This syndrome is characterized by newly developed hypertension and proteinuria diagnosed after 20 weeks of gestation [1]. Common clinical manifestations include maternal vascular dysfunction, chronic immune system activation, renal dysfunction, and intrauterine growth restriction (IUGR). If left unmanaged, preeclampsia can lead to maternal seizures, stroke, multiorgan failure, and death [2]. While approximately 63 000 maternal and 500 000 infant deaths are attributed to preeclampsia annually [3], no early diagnosis criteria are established and there is no cure for this pregnancy syndrome. The exact cause of preeclampsia is currently unknown but there is a consensus that the placenta plays a cardinal role in the pathogenesis of preeclampsia because delivery of the placenta resolves the clinical symptoms. Changes in the oxygenation levels of the placenta due to failure of spiral artery transformation are thought to be responsible Corresponding author: Davidge, S.T. ([email protected]). Keywords: endothelial function; pregnancy; hypertension; preeclampsia. 1471-4914/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2014.11.009

for the underlying pathology of preeclampsia [4]. Recent discoveries of an imbalance in antiangiogenic factors and loss of cytoprotective mechanisms in preeclamptic placentas have provided new insights into the pathophysiology of this pregnancy syndrome and are promising prognostic and therapeutic targets [5–9]. Systemic maternal vascular dysfunction is a major phenotype of pregnancies with preeclampsia, contributing to

Glossary Endothelium-derived contracting factors (EDCFs): molecules produced in the vascular endothelium that signal smooth muscle cells in blood vessel walls to contract, constricting the blood vessel. EDCFs are released in response to various stimuli, such as Ang II, arachidonic acid, hypoxia, stretch, and the superoxide anion. Release of EDCFs is associated with pathological conditions. Endothelium-derived relaxing factors (EDRFs): molecules produced in the vascular endothelium that signal smooth muscle cells in blood vessel walls to relax, dilating the blood vessel. Hypertension: a chronic medical condition in which the blood pressure in the arteries is elevated. Intrauterine growth restriction (IUGR): attenuated growth of a fetus; the fetus does not reach its growth potential while in the mother’s womb. In cases of IUGR, the growing fetus weighs less than 90% of other babies at the same gestational age. Lectin-like oxidized low-density lipoprotein (oxLDL) receptor 1 (LOX-1): a lectin-like 52-kD receptor that mediates the recognition, internalization, and degradation of oxLDL by vascular endothelial cells. Microparticles (MPs): vesicles >100 nm in diameter that are released into the circulation via shedding from activated or apoptotic cells. Mitochondrial DNA (mtDNA): contains 37 genes and approximately 16 600 bp; organized as a circular, covalently closed, double-stranded DNA in most multicellular organisms. Nitric oxide (NO): free radical with cell signaling properties. Oxidative stress: a physiological state that describes an imbalance between the production of free radicals and antioxidant defense mechanisms. Postpartum: relating to or occurring in the period of time following the birth of a child. Prostacyclin (PGI2): a lipid molecule and member of the eicosanoid family that is released by healthy endothelium and induces smooth muscle relaxation. Proteinuria: the presence of an excess of serum proteins in the urine. Reactive oxygen species (ROS): chemically reactive molecules containing oxygen that play a role in cell signaling and homeostasis. Resistance vessels: small blood vessels that constitute the main part of total vascular resistance to blood flow. Spiral arteries: corkscrew-like arteries of the endometrium that are sensitive to hormonal and growth factor influences in the non-pregnant endometrium. Undergo remodeling and vascular smooth muscle disorganization during pregnancy due to the phenomenon of trophoblast invasion. Toll-like receptors (TLRs): single, membrane-spanning, noncatalytic receptors that recognize structurally conserved molecules derived from microbes (pathogen-associated molecular patterns) or dying cells (damage-associated molecular patterns). Trophoblast: specialized cells of the placenta. Trends in Molecular Medicine xx (2014) 1–10

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Review increased peripheral vascular resistance, maternal hypertension, and proteinuria [10]. The mechanisms of systemic vascular dysfunction in preeclamptic pregnancies involve an imbalance in the production of constrictors and dilators in vascular cells, hyper-responsiveness to constrictor stimuli, reduced endothelium-dependent dilation, and oxidative stress [1,10,11]. Many studies have shown that the interaction between circulating factors and the maternal endothelium significantly contribute to generalized vascular dysfunction [12–14]. These circulating factors are partly of placental origin, providing a link between placental and maternal vascular dysfunction [15]. The maternal response to these factors depends on the woman’s vascular health, which may be compromised due to pre-existing conditions such as obesity, diabetes, or poor nutrition, all of which are risk factors for developing preeclampsia [1]. The implications of preeclampsia extend beyond the course of gestation. Delivery of the placenta resolves the clinical symptoms; however, women with a history of preeclampsia have increased risk of developing cardiovascular disease later in life [16,17]. Women affected by preeclampsia have a three- or fourfold greater risk of developing hypertension [18], stroke, or heart disease 5–15 years after pregnancy [17] and a twofold greater risk of dying from cardiovascular or cerebrovascular disease compared with women who had a normal pregnancy [10]. The molecular mechanisms that link gestational complications with risk for cardiovascular disease after pregnancy are not well understood. Vascular dysfunction is an early marker of atherosclerosis and cardiovascular disease and a common feature of pregnancies with preeclampsia that persists many years after a pregnancy affected by this syndrome [19–21]. Thus, vascular dysfunction during gestation may account for an elevated risk of cardiovascular disease in women with a history of preeclampsia [10]. Preeclampsia is a heterogeneous syndrome and multiple pathways have been proposed for both the causal as well as the perpetuating factors leading to maternal vascular dysfunction. This review synthesizes knowledge from recent discoveries on the mechanisms of maternal vascular dysfunction in preeclampsia, elaborating specifically on the molecular changes in vascular endothelial and smooth muscle layers and on the mechanisms through which circulating factors affect vascular reactivity thus contributing to maternal vascular dysfunction. We propose that understanding the molecular mechanisms underlying preeclampsia-associated vascular dysfunction will provide insight into the clinical manifestations of this syndrome and also explain how pregnancy complications predispose the mother to future cardiovascular events. Endothelium-derived regulators of maternal vascular reactivity in preeclampsia Reduction of peripheral vascular resistance in early pregnancy followed by modest attenuation of mean arterial pressure in mid-gestation is one of the major hemodynamic adaptations to normal pregnancy [22]. In preeclampsia, however, the maternal vascular adaptations are aberrant and pregnant women with preeclampsia exhibit increased total vascular resistance and arterial blood pressure that persist until delivery [22]. Systemic endothelial dysfunction 2

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is central to the pathophysiology of preeclampsia and an underlying mechanism of increased vascular resistance and hypertension [1]. In response to mechanical and chemical stimuli, the endothelial cells release vasoactive molecules, which induce vascular smooth muscle relaxation [also known as endothelium-derived relaxing factors (EDRFs)] or contraction [also known as endothelium-derived contracting factors (EDCFs)], modulating vascular tone [23]. An imbalance between EDRF and EDCF bioavailability, and alterations in vascular smooth muscle responsiveness to these factors, may account for the hypertensive phenotype of preeclampsia, which is characterized by increased vasoconstriction and reduced vasodilation in the systemic maternal circulation. EDRFs Isolated arteries from women with preeclampsia exhibit reduced endothelium-dependent relaxation [24,25]. Experimental animal models of preeclampsia show signs of endothelial dysfunction in reproductive [26] and nonreproductive vessels [27,28]. The reduced endothelium-dependent relaxation observed in pregnancies with preeclampsia is attributed partly to reduced bioavailability of nitric oxide (NO) [29] and prostacyclin (PGI2) [30]. NO induces vascular smooth muscle relaxation via soluble guanylyl cyclase/cyclic guanosine monophosphate (sGC/cGMP)-dependent and independent mechanisms [31]. PGI2 is a product of arachidonic acid metabolism that induces vascular smooth muscle relaxation via receptor-mediated cyclic adenosine monophosphate (cAMP)-dependent mechanisms. Previous studies have shown increased endothelial NO synthase (eNOS) expression [32] in preeclampsia; however, increased arginase expression (a reciprocal regulator of NOS) [33], elevated levels of asymmetric dimethylarginine (ADMA) (a natural NOS inhibitor) [34], and increased peroxynitrite formation (formed through scavenging of NO by superoxide anions) [35] suggest that NO bioavailability is reduced (Figure 1). Urinary and plasma concentrations of PGI2 are also reduced in women with preeclampsia [36], favoring an imbalance between vasodilatory and vasoconstrictor prostanoids. Relaxation responses in some arteries cannot be fully explained by the contributions of NO and PGI2. Vascular smooth muscle hyperpolarization by endothelium-derived factors other than prostanoids and NO has been previously described [23]. The exact nature of these factors and mechanisms has not been defined, but myoendothelial gap junctions and hydrogen sulfate (H2S) are potential candidates [9,23]. Small myometrial arteries from women with preeclampsia showed reduced vasodilatory responses, which were attributed to attenuation in the contribution of endothelium-derived hyperpolarization (EDH) because of physical disruption of myoendothelial junctions [37] (Figure 1). Reduction in plasma H2S in pregnancies with preeclampsia has also been reported [9]. In addition, cystathionine-g-lyase (CSE), the primary H2S-synthesizing enzyme in the vasculature, is reduced in preeclampsia [9] (Figure 1). The EDH-mediated response involves the actions of small- (SKCa) and intermediate- (IKCa) but not large- (BKCa) conductance calcium-activated potassium channels. Chronic hypoxia inhibits pregnancy-induced

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↑ oxLDL

(6)

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ANGII K+

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ONOO– NO

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PGHS

TP

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Fe cGMP ↓ Relaxaon

↓ Vasodilaon ↑ Vasoconstricon

Constricon ↑ AT1-AA

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Figure 1. Simplified schematic representation of vascular processes affected by circulating and endothelium-derived factors in preeclampsia. (1) Increased arginase expression [reciprocally regulates nitric oxide (NO) synthase (NOS)], elevated levels of asymmetric dimethylarginine (ADMA), a natural NOS inhibitor, and increased peroxynitrite formation (formed through scavenging of NO by superoxide anions) contribute to a reduction in NO bioavailability, increased oxidative stress, and reduced endothelium-dependent relaxation. (2) Reduced levels of cyclic guanosine monophosphate (cGMP) due to enhanced phosphodiesterase (PDE) and soluble guanylyl cyclase (sGC) activity attenuate vascular smooth muscle relaxation. (3) Physical disruption of myoendothelial gap junctions (MEGJs), reduced production of hydrogen sulfate (H2S) due to reduced cystathionine-g-lyase (CSE) activity, and downregulation of small- (SKCa) and intermediate- (IKCa) conductance calcium-activated potassium channels lead to reduced endothelium-derived hyperpolarization (EDH), contributing to reduced vasodilation. (4) Increased production of endothelin (ET)-1 due to increased activity of matrix metalloproteinases (MMPs) and thromboxane A2 (TxA2) results in augmented vascular constriction via activation of ET receptor A (ETA) and the TxA2 receptor (TP), respectively. (5) Vasoconstrictor responses are potentiated by increased angiotensin (Ang) I receptor (AT1R) signaling. Circulating levels of AT1R autoantibodies (AT1-AAs) also enhance AT1R signaling, augmenting vasoconstriction. (6) Increased circulating oxidized low-density lipoprotein (oxLDL) binds and activates low-density lipoprotein receptor-1 (LOX-1) in endothelial cells causing the production of superoxide and peroxynitrite. (7) Overproduction of the antiangiogenic factors fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) antagonize the vasodilatory properties of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and transforming growth factor beta (TGF-b), respectively. Placenta-derived cell-free DNA (cfDNA), including mitochondrial and fetal DNA, and syncytiotrophoblast-derived microparticles (STBMs), interacts with endothelial cells, monocytes, and neutrophils to promote oxidative stress and inflammation. Abbreviations: AA, arachidonic acid; PGI2, prostacyclin; PGHS, prostaglandin endoperoxide synthase; bET-1, big ET-1; eNOS, endothelial NOS; ETB, ET receptor B.

upregulation of SKCa channel function in sheep uterine arteries [38]. The function of endothelial SKCa channels may be relevant to the vascular pathophysiology of preeclampsia since placental ischemia/hypoxia is considered a feature of this syndrome. The vascular smooth muscle responsiveness to EDRFs in pregnancies with preeclampsia has been understudied, but evidence suggests that it may contribute to maternal vascular dysfunction. cGMP is a second messenger downstream of NO/sGC signaling that facilitates vascular smooth muscle relaxation via activation of protein kinases or direct influences on potassium channels [39]. cGMP activity is regulated by the phosphodiesterase (PDE) enzyme family [31]. cGMP levels in the placental circulation are reduced and serum PDE activity is increased in pregnancies with

preeclampsia [31]. Reductions in endothelium-independent relaxation of the thoracic aorta with concomitant decreases in cGMP levels have been also found in animals with preeclampsia-like signs [40]. These findings were attributed to a reduction in sGC activity [40] (Figure 1). EDCFs In addition to EDRFs, the endothelium produces endothelin (ET)-1, prostanoids [e.g., thromboxane A2 (TxA2)], and angiotensin II (Ang II), which are all vasoconstrictors and have been implicated in the vascular dysfunction of preeclampsia and other hypertensive disorders. Elevated circulating levels of ET-1 have been reported in women with preeclampsia [36]. There is no evidence to suggest that increased ET-1 levels precede the manifestation of 3

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Review preeclampsia, suggesting that ET-1 may not play role in the genesis of the syndrome. This claim, however, warrants further investigation because circulating levels do not reflect tissue levels of the peptide. Nevertheless, many studies have confirmed the implication of ET-1 signaling in the excessive vasoconstriction seen in pregnancies with preeclampsia [41–44]. ET-1 transduces its effects via activation of ET receptor A (ETA) and ET receptor B (ETB), which are expressed in vascular smooth muscle and endothelial cells [44]. Activation of ETA in vascular smooth muscle cells induces vasoconstriction, whereas ETB activation in endothelial cells induces the release of NO and PGI2, promoting vasodilation, and facilitates ET-1 clearance [44]. In preeclampsia there is an imbalance between ET-1 production and receptor actions, promoting a vasoconstrictive state [43,44]. Enhanced ET-1-induced constriction was found in microvessels from the reduced uteroplacental perfusion (RUPP) rat model of preeclampsia [44] and this was associated with an increased intracellular calcium initial peak. Thus, it is possible that enhanced ET-1-induced vasoconstriction is due to a defect in ET-1-induced calcium influx involving voltage-gated and/or store- and receptor-operated channels. ETA antagonism ameliorated the hypertensive phenotype in various animal models of preeclampsia, suggesting that enhanced vasoconstrictor responses to ET-1 in preeclampsia may be ETA dependent [45]. Recently, the involvement of ETB in augmented ET-1-induced vasoconstrictor responses was also reported [44]. Expression of the ETB receptor and its ability to induce relaxation were attenuated in RUPP rats, suggesting that downregulation of the ETB receptor and its activity further contributes to enhanced ET-1 vasoconstrictive effects [44]. Abdalvand et al. [41] showed increased mesenteric artery responses to big ET-1 (bET-1) in the RUPP model of preeclampsia. These enhanced vasoconstrictor responses were attributed to alterations in the conversion of bET-1 to ET-1 within the endothelium due to increased activity of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9 [41] (Figure 1). Thus, derangements in the functionality of ET receptors, as well as alterations in ET-1 production, may occur in preeclampsia. TxA2 is an arachidonic acid metabolite derived from prostaglandin H synthase (PGHS) that is produced in platelets and endothelial cells. The constrictor effects of TxA2 on vascular smooth muscle are mediated by the TxA2 (TP) receptor, a member of the prostanoid family of heterotrimeric G protein-coupled receptors [46]. TxA2 is considered to play a role in preeclampsia-associated enhanced vasoconstriction [46] (Figure 1) and circulating levels of TxB2 (a TxA2 metabolite) are significantly increased in pregnancies with preeclampsia [30]. The vasoconstrictor effects of TxA2 in preeclampsia are amplified by its ability to potentiate the vasoconstrictor effects of Ang II [47] and ET-1 [48]. NO and PGI2 inhibit the actions of TxA2 via TP receptor desensitization [49]; however, this is not the case in pregnancies with preeclampsia, as NO and PGI2 production is impaired. Investigation of the global DNA methylation profiles of omental arteries from women with preeclampsia revealed that the thromboxane synthase gene (TBXAS1; encodes an enzyme that catalyzes the isomerization of prostaglandin 4

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H2 into thromboxane) was the most significantly hypomethylated gene in vessels from women with preeclampsia [50]. Reduced DNA methylation of the TBXAS1 promoter was associated with increased expression of thromboxane synthase in omental arteries of preeclamptic women [51]. Taken together, these data suggest that in preeclampsia, there is an imbalance in the production of vasoconstrictor (TxA2) and vasodilatory prostanoids (PGI2), which is modulated by epigenetic modifications. Women with preeclampsia also exhibit an augmented pressor response to Ang II and this response can be observed even before the manifestation of this syndrome [52]. Interestingly, circulating Ang II levels are not greater in women with preeclampsia compared with normal pregnant women [53]; however, Ang II signaling is augmented in preeclampsia, suggesting effects on Ang II receptor (AT1R) expression and/or cell signaling. Expression of AT1R is indeed increased in women with preeclampsia [54] (Figure 1). Furthermore, agonistic AT1R autoantibodies (AT1-AAs) have been found in the circulation of women with preeclampsia and these autoantibodies may be responsible for enhanced Ang II signaling [55,56] (Figure 1). AT1-AAs are able to activate the AT1R in a similar way to Ang II [56]. Signal cascades associated with binding of AT1R by AT1-AAs (or Ang II) involve phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/ 2), induction of NADPH oxidase, phosphorylation of nuclear factor kappa light chain enhancer of activated B cells (NF-kB), and promoter activation in the nucleus [55]. These intracellular cascades lead to upregulation of tissue factor, soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng), and ET-1 [57], all of which are notably upregulated in pregnancies with preeclampsia. Placenta-derived circulating factors as links between placental ischemia and maternal vascular dysfunction in preeclampsia Shallow trophoblast invasion of the spiral arteries during the early placentation process is a common feature of preeclampsia and contributes to inappropriate fluctuations of oxygen tension in the uteroplacental unit, leading to various cellular events that induce the release of antiangiogenic and proapoptotic factors [4,58]. Consequently, the placenta acts as a vector of these factors in the maternal systemic circulation. In support of the notion that placenta-derived factors contribute to maternal vascular dysfunction, addition of plasma or serum from pregnancies with preeclampsia to cultured endothelial cells increased endothelial cell platelet-derived growth factor (PDGF) mRNA and protein expression and the release of fibronectin, NO, and PGI2 [12,14]. In addition, incubation of isolated myometrial resistance vessels with plasma from women with preeclampsia reduced vascular relaxation responses [13]. The exact molecular pathways by which placenta-derived factors induce dysfunction of the maternal peripheral arteries are not fully understood. Evidence suggests that placenta-derived factors act on endothelial cells to induce secretion of proinflammatory cytokines and modify the production of vasoactive substances and reactive oxygen species (ROS), promoting vascular inflammation and vasoconstriction [59–61].

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Review Angiogenic factors Vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and sEng have angiogenic and vasoactive properties [10]. They play an important role in placental development and modulate the function of the placental and maternal systemic vasculatures [10]. In preeclampsia, however, changes in the production and signaling of these angiogenic factors account for the development of placental and maternal vascular dysfunction and the hypertensive phenotype [10]. VEGF transduces its biological effects through two high-affinity receptor tyrosine kinases, VEGF receptor-1 [also known as fetal liver tyrosine-like (Flt-1)] and VEGF receptor-2 [also known as kinase domain-related receptor (KDR) or Flk-1] [10]. Activation of KDR in endothelial cells induces NO and PGI2 release, promoting endothelium-dependent relaxation [62]. PlGF has 54% homology with VEGF, binds to Flt-1 but not to KDR, and potentiates the actions of VEGF [10]. The antiangiogenic protein soluble Flt-1 (sFlt-1) is the soluble form of the VEGF/PIGF receptor Flt-1 and is produced by alternative splicing of Flt-1 mRNA [10]. sFlt-1 antagonizes the binding of VEGF and PIGF with their receptors [63], inhibiting their vasodilatory effects (Figure 1). Circulating levels of VEGF and PIGF are reduced in women with preeclampsia [5] and experimental animal models with preeclampsia-like signs [64] and this may be attributed to increased levels of circulating sFlt-1 [5]. Infusion of sFlt-1 in pregnant rodents induces preeclampsia-like signs, increased expression of VEGF receptors, and reduced circulating levels of VEGF [65], suggesting a causal role of sFlt-1 in the pathogenesis of preeclampsia. In addition to attenuated endothelium-dependent relaxation, rodents infused with sFlt-1 exhibit a reduction in endothelium-independent relaxation [65]. The latter effect was abolished following incubation with a superoxide scavenger, suggesting that the detrimental effects of sFlt-1 on maternal vascular function are oxidative stress dependent. Circulating sFlt-1 in preeclampsia was considered to be of placental origin but additional sources of sFlt-1 have also been reported. Platelet–monocyte aggregates (PMAs) are able to produce sFlt-1 and PMA-specific sFlt-1 production is greater in women with preeclampsia [66]. Production of sFlt-1 in PMAs is regulated at the transcriptional level, through NF-kB-related mechanisms [66] that also contribute to the production of proinflammatory cytokines commonly found in the circulation of women with preeclampsia. Gene-expression profiling of placentas from women with preeclampsia revealed the involvement of sEng as another antiangiogenic factor in preeclampsia [67]. sEng is the soluble form of endoglin, a type I membrane glycoprotein located on cell surfaces and coreceptor for transforming growth factor beta (TGF-b) receptor complex. TGF-b1 signaling in vascular tissues induces endothelium-dependent relaxation via an eNOS-dependent mechanism. sEng inhibits binding of TGF-b1 to its receptor and consequently downregulates eNOS, leading to reduced NO formation and attenuated vasodilatory responses [8] (Figure 1). Circulating levels of sEng are elevated in women with preeclampsia [68]. It has been suggested that sFlt-1 and sEng

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act in concert to induce endothelial dysfunction, as mice injected with both molecules exhibited decreased cerebral perfusion, vascular thrombi, and endothelial swelling, but these effects were not seen in mice injected with either molecule alone [69]. MPs MPs are membrane vesicles (>100 nm) shed into the extracellular space under conditions of cell stress or injury [70]. MPs promote the production of oxidative stress in the vascular endothelium through processes that involve NADPH oxidase, PGHS, xanthine oxidase, and eNOS [70] (Figure 1). Further, MPs have vasoconstrictor effects by acting as a source of TxA2 for the vascular wall [71] and by increasing the expression of inducible NOS (iNOS) and PGHS-2 (inducible isoform) in smooth muscle cells [71]. Increased circulating levels of MPs have been found in several inflammatory and thrombotic diseases. Placentaderived syncytiotrophoblast MPs (STBMs) are found in the circulation by the third trimester of pregnancy and are increased in pregnancies with preeclampsia [72]. In vitro studies showed that artificially prepared STMBs reduced the viability of endothelial cells and affected their function [73,74]. Maternal omental arteries perfused with STBMs exhibited reduced endothelium-dependent relaxation [75]. By contrast, Van Wijk et al. [76] reported no direct vascular effect of STBMs on isolated mesometrial arteries from pregnant women. This dichotomy may be due to vascular bed differences. STBMs may also indirectly affect endothelial function via their interaction with other factors. Circulating STBMs come into contact not only with endothelial cells but also with maternal blood cells. Supernatants from cocultured human umbilical vein endothelial cells with STBMs activated isolated peripheral blood leukocytes and potentiated peripheral monocyte responses in vitro [60]. Messerli et al. [77] reported that STBMs shed by placental explants upregulated monocyte cell-surface expression of the adhesion molecule CD54 and induced the production of the proinflammatory cytokines IL-8, IL-6, and IL-1b. Given that STBM levels and activation of monocytes are elevated in preeclampsia, it is possible that STBM-induced monocytes produce soluble and cell-surface mediators of inflammation that transduce a proinflammatory signal in endothelial cells. Others have shown that STBMs activate neutrophils and induce the production of superoxide radicals, which may be the mediator of endothelial dysfunction in preeclampsia [78]. STBMs are potential contributors to preeclampsia-associated vascular dysfunction, but MPs of platelet and endothelial cell origin have also been found to be elevated in preeclampsia [79]. To date, there are no reports on the role of leukocyte- and vascular smooth muscle cell-derived MPs in pregnancies with preeclampsia. Given the diverse biological effects of MPs based on the vascular bed, the cell type of origin, and the nature of their interaction with the target cells, efforts should be made to identify the major cell contributor to preeclampsia-associated vascular dysfunction and the molecular mechanism by which MPs transduce their detrimental effects on the maternal vascular wall. 5

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Review Cell-free nucleic acids Cell-free fetal DNA is increased in pregnancies with preeclampsia and, most importantly, was increased in the circulation of women who were at high risk of developing preeclampsia before the onset of the symptoms [80]. Cellfree fetal DNA is almost exclusively of trophoblast origin and is not derived from the demise of circulating fetal cells [80,81]. Scharfe-Nugent et al. [82] demonstrated that fetal DNA is highly inflammatory, because fetal, but not adult, DNA activated human peripheral blood mononucleated cells (PBMCs), thereby inducing the release of the inflammatory cytokine IL-6. Treatment of mice with fetal DNA resulted in fetal resorption and systemic maternal inflammation and these effects were facilitated by activation of the immune receptor Toll-like receptor (TLR) 9 [82]. Circulating cell-free nucleic acids in the circulation of women with preeclampsia also include mitochondrial DNA (mtDNA), which is also increased in women with IUGR [83]. In a seminal paper, Zhang et al. [84] showed that mitochondrial fragments, including mtDNA, that are released into the extracellular space due to cell injury and death have proinflammatory and immunogenic properties via activation of pattern recognition receptors. mtDNA was able to specifically bind and activate TLR9 [84]. TLR9 is activated by hypomethylated CpG DNA, a common feature of fetal DNA and mtDNA sequences as well as bacterial DNA but not common in adult vertebrate DNA [81]. Therefore, bacterial infections, trophoblast shedding, and placental cell death (all characteristics of preeclampsia) may give rise to an immune response via a converging pathway related to TLR9 signaling (Figure 2). Interestingly, activation of other TLRs, such as TLR4 and the endolysosomic TLR3, TLR7, and TLR8, has been previously implicated in the development of preeclampsia [27,85,86]. Pregnant rats treated with synthetic ligands of TLR developed endothelial dysfunction, hypertension, systemic inflammation, and proteinuria [86], all characteristics of pregnancies with preeclampsia. Neutrophils have been suggested as a potential contributor to the increases in total cell-free DNA seen in pregnancies with preeclampsia [81]. Neutrophils are able to expel their genomic DNA into the extracellular environment in the form of neutrophil extracellular traps (NETs) [81]. NETs have been found in the intervillous space of placentas from women with preeclampsia and it is of particular interest that STBMs are able to induce NETosis and become trapped in the extruded NET structures [87]. These findings raise the possibility that neutrophils and the process of NETosis are links between placental shedding and injury with the maternal syndrome of preeclampsia [81]. Hypoxia-inducible factor 1 (HIF-1) HIF-1a is a subunit of the heterodimeric transcription factor HIF-1 that regulates the cellular response to low oxygen tension [88]. HIF-1a expression is increased in placentas from women with preeclampsia and overexpression of HIF-1a in pregnant mice upregulates sFLt-1, sEng, and ET-1, inducing a preeclamptic phenotype [89]. Taken together, these data suggest that HIF-1a is a possible mediator between the placenta and vasoactive molecules 6

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Placental apoptosis/necrosis

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Transcripon of proinflammatory genes and interferon-inducible genes Inflammaon and type I interferon response TRENDS in Molecular Medicine

Figure 2. Abbreviated schematic of Toll-like receptor (TLR) 9 signaling. Fetal and mitochondrial DNA (mtDNA) are increased in the circulation of women with preeclampsia [80,83]. Fetal DNA is released during placenta trophoblast shedding and mtDNA may be released from dying trophoblast cells. Fetal DNA and mtDNA contain unmethylated CpG DNA, which enters the intracellular space via class III phosphatidylinositol 3-kinase (PI3K)-mediated endocytosis to activate the pattern recognition receptor TLR9 located in endolysosomes. CpG DNA binding leads to recruitment of the adapter protein myeloid differentiation factor 88 (MyD88). The interaction of TLR9 with MyD88 activates signal transduction proteins such as members of the IL-1 receptor-associated kinase family (IRAK). Subsequently, IRAK proteins interact with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF-6) leading to its ubiquitination. These events result in nuclear localization of nuclear factor kappa light chain enhancer of activated B cells (NF-kB) and mitogenactivated protein kinase (MAPK) stimulation of the transcription factor activator protein 1 (AP-1), which trigger the production of proinflammatory cytokines. TLR9 also stimulates type I interferon production via activation of interferon regulatory factor 7 (IRF7).

that have deleterious effects on the maternal vascular wall in preeclampsia. Oxidative stress: a mediator of maternal vascular dysfunction in preeclampsia An imbalance between production of pro-oxidants and antioxidant scavenging mechanisms, namely oxidative stress, is considered a convergence point of various molecular pathways, proximal to endothelial dysfunction in preeclampsia [1,11]. The preeclamptic placenta shows signs of increased production of oxidants, lipid peroxides, and isoprostanes and reduced levels of antioxidant mechanisms [11]. Further, vessels from women with preeclampsia show increased eNOS in the presence of increased markers of the potent oxidant peroxynitrite and a decrease in the antioxidant superoxide dismutase [90]. Activation of the NADPH oxidase enzyme system plays a critical role in the production of superoxide [91] and has been proposed as a central mechanism in the induction of a pro-oxidant

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oxLDL/LOX-1, Nrf2, and HO-1 and multiple targets for novel therapies in preeclampsia. Concluding remarks and future perspectives Significant progress has been achieved in the past few years regarding the molecular mechanisms associated with the pathogenesis of preeclampsia. Specifically, the characterization of antiangiogenic factors (i.e., sFlt-1) [103] and endogenous cytoprotective pathways (i.e., HO-1/CO, CSE/H2S) [9,96,97] have provided scientific justification for recent therapeutic efforts such as sFlt-1 apheresis to reduce sFlt-1 in the maternal circulation [7] and statins to improve maternal vascular health [104]. In addition, work on the vasodilatory properties of relaxin [22], a hormone of placental origin, has led to the identification of relaxin as a potential therapy to improve maternal vascular reactivity in pregnancies with preeclampsia.

Environmental factors

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environment in the vascular endothelium of women with preeclampsia [1]. Superoxide induces eNOS uncoupling and reduces bioavailable NO, which also leads to increased peroxynitrite production. Subsequently, peroxynitrite promotes the production of EDCFs such as ET-1 while inhibiting the production of EDRFs such as PGI2 [1]. Formation of peroxynitrite and superoxide in the maternal vasculature in preeclampsia can be also induced by activation of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). Previous studies showed that LOX-1 activation in endothelial cells by plasma from women with preeclampsia increased the activity of NADPH oxidase, superoxide, and peroxynitrite production [35] (Figure 1). Further, peroxynitrite upregulated and maintained higher LOX-1 expression, suggesting a positive feedback loop where LOX-1 induces oxidative stress and, in turn, oxidative stress upregulates LOX-1 [35]. Recent evidence suggests a new regulatory mechanism for LOX-1, as activation of TLR4 signaling induced LOX-1 expression via a p38 mitogenactivated protein kinase (MAPK)/NF-kB pathway in mouse aorta [92]. Further, activation of LOX-1 downregulated endothelial IKCa3.1 channels [93], which are important electrical triggers in vasorelaxation that contribute significantly to endothelium-dependent dilatation. Oxidized low-density lipoprotein (oxLDL) is a ligand for LOX-1 and is increased in the circulation of women with preeclampsia [94]. In addition to oxLDL, other circulating factors that are increased in preeclampsia are able to activate LOX-1. These include anionic phospholipids, apoptotic cells, activated platelets, and bacteria [35]. The LOX-1 pathway may be one of the molecular links between circulating factors, oxidative stress, and maternal vascular dysfunction in preeclampsia. LOX-1 is expressed in endothelial cells and also in vascular smooth muscle cells [95]; however, the role of LOX-1 in vascular smooth muscle cells from pregnancies with preeclampsia has not been investigated. Heme oxygenase (HO)-1, the inducible form of HO, has been identified as a major placental protector from cellular damage during pregnancy [96]. HO-1 has antioxidant, antiapoptotic, and vasodilatory properties via the actions of its metabolites biliverdin, bilirubin, and carbon monoxide (CO) [97,98]. Expression of HO-1 was reduced in preeclamptic placentas and levels of CO were attenuated in the exhaled breath of women with preeclampsia, suggesting decreased HO-1 activity. HO-1 inhibition potentiated sFlt-1 and sEng production from placental villous explants [99] and in vivo induction of HO-1 reduced maternal hypertension, decreased placental sFlt-1 and oxidative stress, and increased circulating VEGF in an animal model of preeclampsia [100]. Thus, loss of HO-1 cytoprotective actions may be a significant contributor to the pathogenesis of preeclampsia [97]. The human HO-1 genes contain binding sites for multiple transcription factors, among which nuclear factor erythroid 2-related factor 2 (Nrf2) is considered the most important [101]. Interestingly, oxLDL is a regulator of Nrf2 but Nrf2 was less activated in placentas from preeclamptic pregnancies despite a high serum concentration of oxLDL [102]. Chigusa et al. [102] suggested that in preeclampsia internalization of oxLDL is reduced due to lower placental expression of LOX-1, leading to reduced Nrf2 and HO-1. These data suggest a common pathway for oxidative stress,

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Figure 3. Phenotypic characteristics of maternal vascular dysfunction and potential molecular targets. Oxidative stress, inflammation, and imbalance between constrictor and dilatory mechanisms are the main features of maternal vascular dysfunction during gestation and may predispose the mother to a high risk of cardiovascular disease later in life. This vascular phenotype is the result of the interaction between the actions of placenta-derived factors, other circulating factors, and the environment, as well as pre-existing conditions and genetic factors. Pharmacological manipulation of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), and Toll-like receptor (TLR) signaling may provide novel strategies to target maternal vascular dysfunction due to the role of these molecules/pathways in central inflammatory and vasoactive mechanisms. Furthermore, development of selective pharmacological agents that modulate the production and/or signaling of angiotensin receptor autoantibodies (AT1-AAs), microparticles (MPs), and cell-free nucleic acids (CpG DNA) will allow the investigation of their cellular mechanisms and will further our understanding regarding the vascular pathophysiology of preeclampsia. Finally, studies are needed to investigate the molecular mechanisms by which prostaglandin endoperoxide synthase 2 (PGHS-2), endothelin receptor B (ETB), and matrix metalloproteinases (MMPs) facilitate derangements of maternal vascular biology during preeclampsia.

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Review Box 1. Outstanding questions  What are the converging pathways in the occurrence of endothelial and vascular smooth muscle dysfunction in preeclampsia?  What are the contributions of placental debris (i.e., fetal and mtDNA) overproduction and deficiencies in clearance mechanisms (i.e., autophagy and mitophagy) to increased circulating cellfree DNA in preeclampsia?  What are the molecular links between maternal vascular dysfunction during a preeclamptic pregnancy and maternal cardiovascular risk following pregnancy?

Currently, the molecular links between preeclampsiaassociated maternal vascular dysfunction and maternal risk of cardiovascular disease later in life are unknown. The vascular pathophysiology of preeclampsia involves various circulating factors that act on the maternal vascular wall to disturb the balance between vasodilatory and vasoconstrictor mechanisms (Figure 1). It is unlikely that poor maternal vascular responses in preeclampsia are the result of the actions of a single factor/molecular pathway. The diversity in the nature of the potential instigators and their source, as well as the complex interactions between the proposed pathways, underscores the need for the discovery of common converging pathways that link placental dysfunction with systemic maternal vascular pathology. It is possible that these converging pathways are also links to maternal risk of cardiovascular disease following a pregnancy with preeclampsia. The pursuit of the identity of the molecular links between gestational vascular dysfunction and future cardiovascular risk should include investigation of the role of preeclampsia-related damage of the maternal vasculature versus the presence of pre-existing factors leading to cardiovascular disease. The studies summarized here describe the multiple and complex mechanisms associated with the development and perpetuating mechanisms of maternal vascular dysfunction. To the best of our knowledge, there are no data to support the importance of one pathway over the others in the end result of maternal vascular dysfunction in the heterogeneous syndrome of preeclampsia. We propose certain molecules (HO-1, Nrf2), biological processes (epigenetic modifications), and pathways (TLR signaling) as targets with potential therapeutic implications (Figure 3). Nevertheless, important basic questions remain to be answered (Box 1) before we can develop a comprehensive strategy targeting preeclampsia and maternal vascular pathophysiology. Although we have animal models to test hypotheses, we lack a robust animal model to address the complexity of preeclampsia. Thus, many of the molecular pathways reviewed here have been described and investigated in studies of other cardiovascular or chronic disease complications (i.e., hypertension, kidney disease, obesity, and diabetes). It is imperative, however, that preeclampsia be recognized as a unique syndrome, with the placenta serving as a source of activating factors and the maternal response being modulated by pregnancy. Acknowledgments S.T.D. is a Canada Research Chair in Maternal and Perinatal Cardiovascular Health. The Davidge laboratory receives funding from the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Canada, and the Women and Children’s Health Research 8

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Institute through the generous support of the Stollery Children’s Hospital Foundation and the Royal Alexandra Hospital Foundation. Research in the laboratory of S.G. is supported by the American Heart Association (13SDG17050056).

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