A possible protective role of Nrf2 in preeclampsia

Accepted Manuscript Title: A Possible Protective Role of Nrf2 in Preeclampsia Author: Nisreen Kweider Berthold Huppertz Mamed Kadyrov Werner Rath Thomas Pufe Christoph Jan Wruck PII: DOI: Reference:

S0940-9602(14)00036-3 http://dx.doi.org/doi:10.1016/j.aanat.2014.04.002 AANAT 50863

To appear in: Received date: Revised date: Accepted date:

3-2-2014 15-4-2014 16-4-2014

Please cite this article as: Kweider, N., Huppertz, B., Kadyrov, M., Rath, W., Pufe, T., Wruck, C.J.,A Possible Protective Role of Nrf2 in Preeclampsia, Annals of Anatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.04.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A Possible Protective Role of Nrf2 in Preeclampsia Nisreen Kweider, Berthold Huppertz, Mamed Kadyrov, Werner Rath, Thomas Pufe, Christoph Jan Wruck Nisreen Kweider, M.D. Department of Anatomy and Cell Biology; Medical Faculty; RWTH Aachen

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University; Aachen; Germany. Wendlingweg 2, 52074 Aachen, Germany; [email protected] Berthold Huppertz, Prof. Institute of Cell Biology, Histology and Embryology, Center for Molecular

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Medicine, Medical University of Graz, Harrachgasse 21/7, 8010 Graz, Austria

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[email protected]

Mamed Kadyrov, PD. M.D. Department of Anatomy and Cell Biology; Medical Faculty; RWTH Aachen University; Aachen; Germany. Wendlingweg 2, 52074 Aachen, Germany;

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MEDIAN Kliniken, Baden-Württemberg, Germany [email protected]

Werner Rath, Prof. Obstetrics and Gynecology; Medical Faculty, University Hospital of the RWTH,

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Aachen, Germany. Wendlingweg 2, 52074 Aachen, Germany; [email protected] Thomas Pufe, Prof. Department of Anatomy and Cell Biology; Medical Faculty; RWTH Aachen

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University; Aachen; Germany. Wendlingweg 2, 52074 Aachen, Germany; [email protected]

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Christoph Jan Wruck, PD. PhD. Department of Anatomy and Cell Biology; Medical Faculty; RWTH Aachen University; Aachen; Germany. Wendlingweg 2, 52074 Aachen, Germany; [email protected]

Correspondence Author: Nisreen Kweider M.D., Department of Anatomy and Cell Biology; Medical Faculty; RWTH Aachen University; Aachen; Germany. Wendlingweg 2, 52074 Aachen Tel: +49 241 80 89 550, Fax +49 241 80 82 431, Email: [email protected]

Running title: Nrf2 in preeclampsia

Summary 1 Page 1 of 24

Excess release of reactive oxygen species (ROS) is a major cause of oxidative stress. This disturbance has been implicated as a cause of preeclampsia, a pregnancy-related disorder characterized by hypertension and proteinuria. Increased oxidative stress leads to trophoblast apoptosis/necrosis and alters the balance between pro- and anti-angiogenic factors, resulting in generalized maternal endothelial dysfunction. Trials using antioxidants have significantly failed to improve the condition of, or in any way protect, the mother from the life-threatening complications of this syndrome. Nuclear

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factor-erythroid 2-related factor 2 (Nrf2) is a potent transcription activator that regulates the expression of a multitude of genes that encode detoxification enzymes and anti-oxidative proteins. Recent discussion on evidence of a link between Nrf2 and vascular angiogenic balance has focussed

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on the downstream target protein, heme oxygenase-1 (HO-1). HO-1 metabolizes heme to biliverdin, iron and carbon monoxide (CO). HO-1/CO protects against hypertensive cardiovascular disease and

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contributes to the sustained health of the vascular system. In one animal model, sFlt-1 (soluble fmslike tyrosine kinase-1) has induced blood pressure elevation, but the induction of HO-1 attenuated the hypertensive response in the pregnant animals. The special conditions under which Nrf2 participates in

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the pathogenesis of preeclampsia are still unclear, as is whether Nrf2 attenuates or stimulates the processes involved in this syndrome. In this review, we summarize recent theories about how Nrf2 is

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involved in the pathogenesis of preeclampsia and present the reasons for considering Nrf2 as a

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therapeutic target for the treatment of preeclampsia.

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Key words: Nrf2, preeclampsia, heme oxygenase-1, CO

Introduction Preeclampsia is a pregnancy-related disorder that affects about 2-5 % of all pregnancies. Worldwide, preeclampsia constitutes a major cause of maternal and foetal morbidity and mortality (Duley, 2009;

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Steegers et al., 2010). In developing countries, preeclampsia is a leading cause of maternal mortality, with estimates exceeding 60,000 maternal deaths per year (Hogberg, 2005; Steegers et al., 2010). Preeclampsia is generally defined as new hypertension (diastolic blood pressure of ≥ 90 mm Hg and/or systolic blood pressure of ≥ 140 mm Hg) and/or proteinuria (≥ 0.3 g/day) after 20 weeks of gestation (ACOG, 2013). The clinical symptoms of the advanced stages include seizures, renal failure, intrauterine growth restriction (IUGR), and Hemolysis, Elevated Liver enzymes and Low Platelets

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(HELLP) syndrome. Any of the aforementioned symptoms is potentially life-threatening. Preeclampsia impacts the health of both the mother and the foetus, and risks to the mother persist beyond her reproductive years. Women diagnosed with preeclampsia are estimated to be twice as

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likely to suffer future cardiovascular or cerebrovascular events as unaffected women (Brown et al., 2013). Although the estimated 10-year risk of cardiovascular disease after delivery is low (less than

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5%), the risk of cardiovascular disease is expected to increase rapidly with increasing age of the mother (van Rijn et al., 2013; Yinon et al., 2010).

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Despite a burst of research on preeclampsia, the disease has remained the subject of much theorizing over the past decade (Huppertz, 2008; Schlembach, 2003; Sibai et al., 2005; Telang et al., 2013), and to date none of these authors’ hypotheses has become clearly established (Genest et al., 2012). One

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hypothesis has highlighted ‘excessive oxidative stress’ as the cause of clinical symptoms of preeclampsia (Burton and Jauniaux, 2011; Redman, 2011); indeed, all the hypotheses generally

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concede a dramatic increase in oxidative stress within the placenta (Miehe et al., 2005; Murray, 2012; Roberts and Bell, 2013). However, the aetiology of this oxidative stress remains unknown today, with

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each theory suggesting a different explanation.

The loss of balance between reactive oxygen species (ROS) production and antioxidant scavenging capacity exaggerates placental oxidative and, especially, endoplasmic reticulum stress (Burton and Yung, 2011; Redman, 2011). Critical elements of the intrinsic defence mechanism against oxidative stress are the upregulation of direct ROS scavenging enzymes, phase II detoxification proteins and other antioxidants that carry antioxidant response elements (AREs) in their promoter regions. One main regulator of AREs is nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor. Under normal physiological conditions in the placenta, The balance between the elements of the intrinsic oxidative stress defence mechanism is maintained mainly through the Nrf2-ARE signalling pathway (Chapple et al., 2012; Howden, 2013; Lee et al., 2005; Wruck et al., 2009). Recent evidence suggests that impairment of Nrf2 signalling may be involved in the pathogenesis of preeclampsia (Kweider et al., 2011; Kweider et al., 2012; Loset et al., 2011). Evidence is accumulating for the beneficial effects of Nrf2 activation in cardiovascular and hypertensive disorders (Li et al., 2011), and further research is warranted to determine how Nrf2 and hypertensive factors interact in pregnancy, as their interaction may profoundly affect the 3 Page 3 of 24

pathophysiology of preeclampsia. To this end, we have reviewed recent evidence for the role of Nrf2 in the pathogenesis of preeclampsia with a focus on evaluating any therapeutic implications.

Nrf2 exerts cytoprotective effects against multiple disorders Nrf2 confers multi-organ protection (Lee et al., 2005) by mediating a broad-based set of adaptive responses to intrinsic and extrinsic cellular stresses (Kensler et al., 2007; Wakabayashi et al., 2010).

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Several studies have reported that Nrf2 protects cells against a wide variety of toxic insults (carcinogens, electrophiles, ROS, inflammation, calcium disturbance, ultra violet light, and cigarette smoke) (Lee et al., 2005).

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Nrf2’s importance in protecting against several disorders has been demonstrated through analysis of Nrf2 knockout (KO) mice: Lee et al. have reported that targeted deletion of Nrf2 in mice was followed

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by development of regenerative immune-mediated hemolytic anemia in the animals (Lee et al., 2004); activation of Nrf2 signalling by synthetic triterpenoid CDDO-imidazolide conferred protection against

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acute kidney injury from ischemia-reperfusion (Liu et al., 2013); disruption of Nrf2 induced a state of dysregulation of the innate immune response during sepsis, interfering with proinflammatory signalling (Fragoulis et al., 2012; Thimmulappa et al., 2006; Wruck et al., 2011a); and quantitative

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analysis of oesophageal cornification in knockout mice has revealed that they exhibit decreased stratification, which has been linked to the regulation of epithelial cornification and, hence, to callus

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formation and wound healing in skin (Wruck et al., 2011b).

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Normally, most Nrf2 is degraded by proteasome. Upon exposure to electrophiles or ROS, however, Nrf2 is stabilized and accumulates in the nucleus, which leads to the activation of Nrf2 target genes. An understanding of the general mechanisms that underlie Nrf2 degradation and stabilization in response to oxidative stress should therefore raise our capability of interacting therapeutically with this defence system.

Regulation of Nrf2 activity

Nrf2 is a potent transcriptional activator that plays an important role in the inducible expression of many cytoprotective genes in response to oxidative and electrophilic stress (Kensler et al., 2007; Lee et al., 2005; Mitsuishi et al., 2012). It is a basic leucine zipper transcription factor and a member of the Cap’n’Collar transcription factor family. It has a conserved basic region-leucine zipper domain that binds to the antioxidant response element (ARE) [ (T/C) TGCTGA (C/G) TCA (T/C) ]. ARE is a cisacting regulatory element found in promoter regions of several genes that encode phase II detoxification enzymes (e.g. glutathione S transferases: GSTs) and antioxidant proteins (e.g. NADPH quinone oxidoreductase: NQO1 and heme oxygenases: HO) (Favreau and Pickett, 1993; Hine and

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Mitchell, 2012; Wakabayashi et al., 2010; Wild et al., 1999). Under normal conditions, Nrf2 is bound to a negative regulator, Keap1 (Kelch-like ECH-associating protein 1), which negatively regulates Nrf2 activity because of its rapid proteasomal degradation (Itoh et al., 1999). In the presence of electrophiles or of ROS, Nrf2 degradation ceases, whereupon stabilized Nrf2 accumulates in the nucleus. There, it heterodimerizes with small Maf proteins and activates target genes for cytoprotection by interacting with the ARE sequences in those genes (Itoh et

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al., 2004) (Figure 1A). The general structure of the Keap1-Nrf2 complex under normal conditions has been emerged from biochemical and structural analyses. Keap1 is a cysteine-rich protein, most of

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which can be modified in vitro by different oxidants and electrophiles. It exists as a homo-dimer (Zipper and Mulcahy, 2002), each one of which contains the N-terminal BTB-(broad complex-

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tramtrack-bric-a-brac)-domain and serves as an adapter for the Cullin3/Ring Box 1 (Cul3/Rbx1) E3 ubiquitin ligase complex (Zhang et al., 2004). This finally leads to ubiquitination of Nrf2 and its subsequent degradation by a proteasome. How cysteine modifications in Keap1 can lead to Nrf2

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activation is still unclear; however, two mechanisms have been described that potentially apply to this process. One is the “hinge and latch” mechanism, which involves a conformational change in the Keap1 homo-dimer that dissociates the DLG motif from the Keap1 DC domain and suppresses the

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ubiquitination of Nrf2. The other is Cul3 dissociation (Kansanen et al., 2013; Taguchi et al., 2011). In both models, E3 ligase activity subsequently declines, and the result is inefficient Nrf2 ubiquitination

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genes.

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and its translocation into the nucleus, where it binds to the AREs and drives expression of its target

At the C-terminal reside globular domains called DC domains that are located apart from each other (Mitsuishi et al., 2012; Ogura et al., 2010) (Figure 1B, Keap 1). Molecular dissection of Nrf2 identified six Nrf2-ECH (Neh) domains (Figure. 1B, Nrf2). Analysis of the structural properties of Nrf2 has indicated that one Neh2 molecule interacts with two molecules of Keap1 at two binding sites, the stronger-binding ETGE motif and the weaker-binding DLG motif (Kobayashi et al., 2002; Tong et al., 2006). Once reactive thiols of Keap1 are modified at residues C151, C273 and C288 by electrophiles or by ROS (Kansanen et al., 2013), Cul3-dependent ubiquitination of Nrf2 is readily inhibited. This leads to nuclear translocation of the protein, whereby newly-synthesized Nrf2 escapes proteasomal degradation and can accumulate in the nucleus (Itoh et al., 1999; Kobayashi et al., 2002; Suzuki et al., 2013). Electrophiles may directly interact with cysteine residues present in Keap1 and thereby stimulate Nrf2 dissociation from Keap1; hence, they may stimulate Nrf2 stabilization. Induction of many cytoprotective genes is the result (Dinkova-Kostova et al., 2005; Itoh et al., 2010; Osburn and Kensler, 2008; Satoh et al., 2013). Modification of thiols of Keap1 is only one of the pathways that disrupt proteasomal degradation of Nrf2; proteins such as p21 and p62 can bind to either Nrf2 or Keap1 and thereby disrupt the interaction between the two (Kansanen et al., 2013; Taguchi et

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al., 2011). Another mechanism that regulates Nrf2 activation is phosphorylation of Nrf2 by various protein kinases (PKC, PI3K/Akt, and JNK). Protein kinase C has been shown to phosphorylate Nrf2 in its Neh2 domain, breaking the association between Nrf2 and Keap1 and thus promoting translocation of Nrf2 into the nucleus (Huang et al., 2002). Recent studies have described a participation by JNK (cjun N-terminal kinase 1/2) and ERK (extracellular signal-regulated kinase) in Nrf2 activation (Bryan

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et al., 2013; Wruck et al., 2007), and pERK kinase has been shown to phosphorylate Nrf2 and promote cell survival (Cullinan et al., 2003) as well. Vascular endothelial growth factor (VEGF) has also been

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reported to increase phosphorylation of ERK1/2 and thus to activate Nrf2, which in turn is released from Keap1 and translocated into the nucleus under the control of ERK signalling pathways (Kweider

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et al., 2011). However, the exact site of phosphorylation has yet to be identified.

One recent study discusses the impact of hypoxia and of the hypoxia-regulated protein Siah2 on the

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Nrf2 regulatory pathway. Baba et al. have shown that the hypoxia-activated E3 ubiquitin ligase Siah2 regulates both non-phosphorylated Nrf2 and Nrf2 phosphorylated by the stress-related kinase PKC. PKC is activated by hypoxia and may be involved in regulating hypoxia-induced ROS (Baba et al.,

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2013). If hypoxia (or rather, mainly hypoxia-reperfusion) would be correlated with certain characteristics of preeclampsia to be discussed below, this would be consistent with a possible

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involvement of Nrf2 in this syndrome.

Nrf2 and Preeclampsia

Preeclampsia and oxidative stress

Pregnancy brings about a state of oxidative stress arising from high ROS production and lipid peroxidation, both of which increase towards the end of normal pregnancy in any case (Belo et al., 2004; Chamy et al., 2006; Sies, 1991). Throughout a normal pregnancy, antioxidant capacity (mainly) in the placenta rises to counteract this increased oxidative stress (Toescu et al., 2002). Moreover, pregnancy normally brings a general increase in inflammatory response, especially towards the end of the third trimester (Redman et al., 1999), with activation of mono-, granulo- and lymphocytes, all of which produce ROS (Burton and Jauniaux, 2011; Redman and Sargent, 2003; Silva et al., 2013). Substantial evidence implicates placental oxidative stress in the pathophysiology of the clinical symptoms of preeclampsia. Placental stress seems to be highest in early-onset type preeclampsia and may stimulate the release of circulating factors that further worsen the maternal syndrome (Redman, 2011). The hypertension and proteinuria manifested by this syndrome seem secondary to a diffuse maternal endothelial dysfunction (Roberts et al., 1989). Oxidative stress occurs when ROS production

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overwhelms the endogenous anti-oxidant defences. A wide range of cellular responses may be induced at this juncture depending upon the severity of the insult and on the compartment in which ROS are generated (Burton and Jauniaux, 2011; Roberts and Hubel, 2009). Oxidative stress is indubitably evident in the placenta in preeclampsia, with increased concentrations of protein carbonyls, lipid peroxides, nitrotryosine residues and DNA oxidation (Burton and Jauniaux, 2011; Myatt and Cui, 2004).

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As mentioned above, the aetiology of preeclampsia is still a mystery, with different hypotheses pointing toward a rise in oxidative stress in the placenta. Whether the aetiology of preeclampsia conforms to the hypotheses around malinvasion of the extravillous trophoblast (Redman, 2011)

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(Figure 2 B, D) or to those around dysregulation of the villous trophoblast as early as the first trimester (Huppertz, 2008) (Figure 2 C), in any case, preeclampsia culminates in abnormal shedding of

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trophoblast material into maternal blood (Huppertz, 2010). Oxidative stress within the placenta is directly associated with this trophoblast dysregulation (Huppertz and Peeters, 2005; Huppertz et al., 2013; Lyall et al., 2013). Free radical generation in the placenta is normally controlled by appropriate

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antioxidant levels (Burton and Jauniaux, 2011); in preeclampsia, however, the levels of several detoxifying enzymes are significantly reduced (Hubel et al., 1999; Loverro et al., 1996) (George and

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Granger, 2013; Miranda Guisado et al., 2012).

In early-onset cases of preeclampsia, altered blood flow due to malinvasion of extravillous trophoblast

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may compound problems in the villous trophoblast. In such cases, oxidative stress and physical disruption to the placental villous architecture cause the additional problems, rather than hypoxia or

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reduced flow (Burton et al., 2009). This hypothesis has been supported by a variety of studies. According to Burton, 2009 the impaired trophoblast invasion, with defective spiral artery remodelling followed by high-resistance vessels, will not reduce placental perfusion but rather increase velocity of blood flowing into the placenta. Consequently, damage to the villous surface and reduced maternalfetal oxygen exchange by turbulent blood flow induces oxidative stress within the placenta (Burton, 2009). Another study, by Goswami et al., has shown that, in early-onset cases, only those patients suffering from both IUGR and preeclampsia showed abnormal release of trophoblast material (Figure 2 D), while those suffering from pure early onset IUGR did not (Figure 2 B) (Goswami et al., 2006). Since most of the antioxidants and detoxifying enzymes are regulated via the Nrf2/ARE system, dysregulation of Nrf2 may contribute to the pathophysiology of preeclampsia.

The role of Nrf2 in preeclampsia Wruck et al. have provided the first experimental data showing that Nrf2 is active exclusively within the villous cytotrophoblast of preeclamptic placentas, which strongly suggests that these cells suffer from oxidative stress caused by ROS (Wruck et al., 2009). In general, the nuclei of the

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syncytiotrophoblast steadily drop their transcriptional activity, while old nuclei are shed via syncytial knot formation (Huppertz et al., 1998). In Wruck et al.’s study, higher numbers of syncytial knots are detected in placentas from preeclamptic women. The authors suggest that oxidative stress during preeclampsia necessitates increased expression and transfer of antioxidative enzymes from cytotrophoblast into syncytiotrophoblast via enhanced syncytial fusion, and that the increased syncytial knot formation they detect results from this enhanced enzyme transfer.

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In vitro cell models have served researchers well in their quest for a better understanding of the molecular mechanisms by which Nrf2 supports and regulates trophoblast function and, hence, normal placentation. Nrf2 has been investigated in several different cell models related to pregnancy and

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placental development, including human umbilical vein endothelial cells (HUVECs) (Heiss et al., 2009) and BeWo cells (as a model for syncytiotrophoblast formation) (Kweider et al., 2011). The

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upregulation of HO-1 via Nrf2 activation has been shown to have properties that are effective in preventing chemically induced oxidative damage in BeWo cells (Kweider et al., 2011). This study

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finds the compound sulforaphane, which is present at high levels in broccoli and induces Nrf2, to be involved both in the induction of cytoprotective Nrf2-driven genes and in the activation VEGF secretion into supernatant of these cells. Preserving placental levels of VEGF and PlGF may be crucial

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for normal placentation and hence for healthy pregnancies. Recently, these findings have received further support, as a disruption in Nrf2 signalling has been found to impair the angiogenic capacity of endothelial cells (Valcarcel-Ares et al., 2012).

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Analysis of genes and gene networks in the human placenta has suggested that Nrf2-mediated

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responses in the placenta are impaired in patients with preeclampsia. Loset et al. analyses the single genes, canonical pathways, and gene-gene networks that are likely to be important in the pathogenesis of preeclampsia. They report that Nrf2-mediated oxidative stress response was overrepresented in the decidua of patients with preeclampsia (Loset et al., 2011), which supports the findings of Wruck el al. A controversial recent study claims to have demonstrated that placentas from preeclamptic patients show reduced nuclear accumulation of Nrf2 associated with decreased HO-1 mRNA (Chigusa et al., 2012). The authors provide evidence for Nrf2 signalling that is disrupted such that it fails to increase the expression of anti-oxidative genes in the placenta. As a consequence, both mother and foetus are potentially affected by oxidative stress.

Genetic profiling of extravillous and villous cytotrophoblast has revealed that lower HO-1 expression is associated with lower cell motility and reduced trophoblast invasion (Bilban et al., 2009). Recent data have shown that Nrf2 contributes to cell invasion and migration (Florczyk et al., 2013; Heiss et al., 2009). As discussed above, invasive extravillous trophoblast, which is associated with cases of early-onset IUGR and with preeclampsia, shows impaired Nrf2 signalling as compared to control cases (Kweider et al., 2012). All in all, despite the importance of Nrf2, one should note that very few studies to date have reported on any role Nrf2 that may have in pregnancy-related disorders, to say 8 Page 8 of 24

nothing of preeclampsia specifically. Studies have also been performed on plasma or placental levels of multiple ARE gene-driven proteins such as superoxide dismutase (SOD), glutathione peroxidase and HO-1 in preeclampsia. As compared to healthy pregnancies, the levels of mRNA and of circulating SOD-proteins and HO-1-proteins were differentially expressed in complicated pregnancies including those characterized by preeclampsia. Nakamura et al. report lower mRNA concentrations of HMOX-1, SOD-1, and catalase in the cellular

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component of blood from Japanese preeclamptic patients (Nakamura et al., 2009). The activities of SOD and catalase were reduced in platelets from patients with preeclampsia, which correlated with

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increased protein carbonylation (Pimentel et al., 2013). At the same time, Martinez-Fierro et al. described under- or overexpression of the oxidative stress-related genes HMOX-1 and SOD-1 in

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maternal peripheral blood mononuclear cells (Martinez-Fierro et al., 2013).

Although the data in the literature do not always line up well across studies, the few current studies at

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our disposal agree that placental Nrf2 signalling is disturbed in preeclampsia patients. These studies also clearly indicate that dysregulation of Nrf2 is involved in the pathogenesis of preeclampsia.

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Nrf2 activation and hypertension

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Potential mechanisms involved in the beneficial impact of Nrf2 activation in preeclampsia

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Hypertensive patients exhibit impaired endogenous and exogenous antioxidant defence mechanisms in the endothelium (Russo et al., 1998). Preeclamptic patients typically possess low total antioxidant capacity and increased placental oxidative potential. It is therefore a strong possibility that Nrf2 may be a potent factor either in prevention or attenuation of the symptoms of preeclampsia. ROS-mediated, cytokine-induced trophoblast apoptosis (Smith et al., 1999) and oxygen differentially regulate apoptosis events (Myatt and Cui, 2004). Furthermore, ROS-mediated cytokines impair contractile and vasodilator responses, impair vascular remodelling and alter the vascular mechanics of hypertension (Mam et al., 2010). Several Nrf2 target genes such as NADPH oxidase, xanthine oxidase, superoxide dismutase (SOD 3), catalase, glutathione peroxidase (GPx 1), thioredoxin and HO-1 have been reported to protect against hypertension associated with multiple disorders including preeclampsia (Houston, 2013a; Mansego et al., 2011; Voelkel et al., 2013) (George et al., 2013; Monu et al., 2013). The ROS species that damaged tissues predominately produce neutralize nitric oxide (NO) and lead to downstream production of other ROS, such as hydrogen peroxide, hydrogen radicals, and perioxynitrite (Houston, 2013b). NO is the primary vasodilator, anti-hypertensive, and anti-atherosclerosis mediator in the endothelium (Houston, 2013b). In an animal model of deoxycorticosterone dismutase DOCA-salt-induced hypertension, activation of Nrf2 via epicatechin prevented an increase in systolic blood pressure as well as the proteinuria induced by DOCA-salt treatment (Gomez-Guzman et al., 9 Page 9 of 24

2012). The hypotensive effect of Nrf2-inducers has been clinically tested: patients with type 2 diabetes mellitus complicated by chronic renal disease were treated with bardoxolone in a randomized clinical trial. Baroxolone is radiation mitigator; i.e., an agent that protects cells from radiation-induced damage, and it operates on both Nrf2-dependent and Nrf2-independent pathways (Kim SB, 2011). Treatment with bardoxolone improved renal function and preserved blood pressure in the patients studied (Rojas-Rivera et al., 2012).

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All these data suggest that Nrf2 may be an important regulator of blood pressure. However, it must be emphasized that no difference in blood pressure between Nrf2 knockout and wild-type mice could be

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identified (Li et al., 2011), even as the same study showed that ang II-induced hypertrophic signalling was inhibited by forced activation of Nrf2.

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The molecular mechanisms by which Nrf2 regulates blood pressure still need to be dissected further and the environmental settings in which Nrf2 lowers blood pressure must still be investigated in more

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detail. Even so, the present state of knowledge about Nrf2 and its role in preeclampsia already offers some hope for new perspectives in designing and developing a novel class of Nrf2 activators to treat

Heme oxygenase-1

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preeclampsia.

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HO-1 is an antioxidant enzyme that catalyses the degradation of heme. Degradation of heme produces biliverdin, carbon monoxide (CO) and free iron (Fe2+) (Barbagallo et al., 2013; Li Volti et al., 2008;

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Maines, 1988; Tenhunen et al., 1968). Biliverdin is then converted by biliverdin reductase into bilirubin. These metabolites have conferred cellular and tissue protection in multiple models of injury and disease including oxidative or inflammatory injury, ischemia/reperfusion injuries, and vascular injury/disease (Levytska et al., 2013; Ndisang and Mishra, 2013). The human HO-1 genes contain multiple binding sites for multiple transcription factors, among which are Nrf2, Sp 1, NFκB, HNF-1 and others (Ryter et al., 2006). The most important factor seems to be Nrf2 (Alam et al., 1999). HO-1 upregulates VEGF in endothelial cells, keratinocytes, macrophages and tumour cells (Dulak et al., 2008). The poor vascularity of wounded skin in HO-1 knockout mice confirms that HO-1 is an important mediator of the pro-angiogenic activity of VEGF. In placental development, a reduction of HO-1 is associated with several gestational complications: recurrent miscarriage, spontaneous abortion and preeclampsia (Bainbridge and Smith, 2005). The physiological importance of HO-1 in pregnancy has also been found in partial HO-1 knock-out mice: these mice showed morphological changes in the placenta and elevated maternal diastolic blood pressure and plasma sFlt-1 levels (Zhao et al., 2009). The vasodilator function of CO is very important in controlling maternal vascular tone and in maintaining sufficient maternal-foetal circulation (Zhao et al., 2009). In vitro experiments on 10 Page 10 of 24

endothelial cells have demonstrated that induction of HO-1 and its metabolite CO inhibits the release of sFlt-1 and sEng from these cells (Cudmore et al., 2007; Cudmore et al., 2006). These antiangiogenic factors are significantly increased in women with early-onset preeclampsia, and they may contribute to endothelial dysfunction in these patients (Fasshauer et al., 2008; Stepan, 2009; Stepan et al., 2009; Verlohren et al., 2012). In a rat model of sFlt-1-induced hypertension, the activation of HO1 attenuated the induced blood pressure increase and enhanced the level of unbound VEGF in pregnant

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hypertensive rats (George et al., 2011). In this model, improved endothelial function was measurable by reduction in vascular expression of preproendothelin mRNA (George et al., 2011). Moreover, the inhibition of HO-1 activity induces hypertension in pregnant rats (George et al., 2013). Yet another

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mechanism of HO-1 induction described by Kang and colleagues may exert hypotensive effects: they show that HO-1/CO can ameliorate the effect of angiotensin II and subsequently reduce its

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hypertensive effects (Kang et al., 2011). Potentially, HO-1 induction also has prophylactic effects on atherosclerosis by virtue of its inhibiting neointimal formation through activation of phosphatidyl

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inositol 3-kinase (Kim et al., 2010).

These findings may justify the hypothesis that pharmacological interventions to increase HO-1 activity in both the placenta and the maternal endothelium may be a useful treatment option for preeclampsia,

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in that it might restore disturbed maternal cardiovascular functions. HO-1 expression can be induced by many compounds, some of which have therapeutic properties. These include statins (Grosser et al.,

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2004), probucol and probucol analogues (Deng et al., 2004), natural Nrf2 activator (kavalactones methysticin) (Wruck et al., 2008), sulforaphane (Wu and Juurlink, 2001), andrographolide (Guan et

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al., 2013) and others. Unfortunately, statins have been identified as teratogenic, and the risks associated with probucol are not well studied and remain to be elucidated. The potential of natural Nrf2 inducers for treating preeclamptic mothers, however, remains largely unexplored, with present findings certainly justifying further research into the possibility. Additional details about the potential clinical impact of Nrf2 activation in preeclampsia are summarized in Table 1.

Conclusion

The role of Nrf2 in the pathogenesis of preeclampsia is a new and promising channel for future research. Evidence is accumulating that Nrf2 plays a key role in many pathways of villous and extravillous trophoblast and that it preserves endothelial function and restores the balance between pro- and anti-angiogenic factors (Figure 3). Clinical trials with antioxidants have so far yielded poor results in preventing preeclampsia. Present studies, in the hope that Nrf2 may have greater therapeutic potential than that shown by antioxidant vitamin therapies, have focussed increasingly on the efficiencies of endogenous activation of antioxidant enzyme activities. As consideration of the present literature has shown, inducing Nrf2 activity might be a promising alternative approach to enhancing the endogenous protective system against oxidative stress. As such, innovations involving

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supplementation by Nrf2 inducers in therapeutic settings deserve consideration. They would seem to be a worthwhile objective for intensive research in future clinical trials.

Acknowledgments

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The authors would like to thank Wolfgang Graulich for producing the illustration in Figure (2).

References

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Verlohren, S., Herraiz, I., Lapaire, O., Schlembach, D., Moertl, M., Zeisler, H., Calda, P., Holzgreve, W., Galindo, A., Engels, T., Denk, B., Stepan, H., 2012. The sFlt-1/PlGF ratio in different types of hypertensive pregnancy disorders and its prognostic potential in preeclamptic patients. Am J Obstet Gynecol 206, 58 e51-58.

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Voelkel, N.F., Bogaard, H.J., Al Husseini, A., Farkas, L., Gomez-Arroyo, J., Natarajan, R., 2013. Antioxidants for the treatment of patients with severe angioproliferative pulmonary hypertension? Antioxidants & redox signaling 18, 1810-1817.

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Wakabayashi, N., Slocum, S.L., Skoko, J.J., Shin, S., Kensler, T.W., 2010. When NRF2 talks, who's listening? Antioxidants & redox signaling 13, 1649-1663. Wild, A.C., Moinova, H.R., Mulcahy, R.T., 1999. Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2. The Journal of biological chemistry 274, 33627-33636.

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Wruck, C.J., Claussen, M., Fuhrmann, G., Romer, L., Schulz, A., Pufe, T., Waetzig, V., Peipp, M., Herdegen, T., Gotz, M.E., 2007. Luteolin protects rat PC12 and C6 cells against MPP+ induced toxicity via an ERK dependent Keap1-Nrf2-ARE pathway. Journal of neural transmission. Supplementum, 57-67.

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Wruck, C.J., Fragoulis, A., Gurzynski, A., Brandenburg, L.O., Kan, Y.W., Chan, K., Hassenpflug, J., Freitag-Wolf, S., Varoga, D., Lippross, S., Pufe, T., 2011a. Role of oxidative stress in rheumatoid arthritis: insights from the Nrf2-knockout mice. Annals of the rheumatic diseases 70, 844-850. Wruck, C.J., Gotz, M.E., Herdegen, T., Varoga, D., Brandenburg, L.O., Pufe, T., 2008. Kavalactones protect neural cells against amyloid beta peptide-induced neurotoxicity via extracellular signal-regulated kinase 1/2-dependent nuclear factor erythroid 2-related factor 2 activation. Molecular pharmacology 73, 1785-1795. Wruck, C.J., Huppertz, B., Bose, P., Brandenburg, L.O., Pufe, T., Kadyrov, M., 2009. Role of a fetal defence mechanism against oxidative stress in the aetiology of preeclampsia. Histopathology 55, 102-106. Wruck, C.J., Wruck, A., Brandenburg, L.O., Kadyrov, M., Tohidnezhad, M., Pufe, T., 2011b. Impact of Nrf2 on esophagus epithelium cornification. International journal of dermatology 50, 1362-1365. Wu, L., Juurlink, B.H., 2001. The impaired glutathione system and its up-regulation by sulforaphane in vascular smooth muscle cells from spontaneously hypertensive rats. J Hypertens 19, 1819-1825. Yinon, Y., Kingdom, J.C., Odutayo, A., Moineddin, R., Drewlo, S., Lai, V., Cherney, D.Z., Hladunewich, M.A., 2010. Vascular dysfunction in women with a history of preeclampsia and intrauterine growth restriction: insights into future vascular risk. Circulation 122, 1846-1853. Zhang, D.D., Lo, S.C., Cross, J.V., Templeton, D.J., Hannink, M., 2004. Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex. Molecular and cellular biology 24, 10941-10953. Zhao, H., Wong, R.J., Kalish, F.S., Nayak, N.R., Stevenson, D.K., 2009. Effect of heme oxygenase-1 deficiency on placental development. Placenta 30, 861-868. Zipper, L.M., Mulcahy, R.T., 2002. The Keap1 BTB/POZ dimerization function is required to sequester Nrf2 in cytoplasm. The Journal of biological chemistry 277, 36544-36552.

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Figure legends

Figure 1. A: Schematic presentation of the Nrf2-ARE system. Under physiologic conditions, Nrf2 binds to the cytoskeletal protein Keap1, resulting in the polyubiquitination of Nrf2 by a cullin 3 ubiquitin

ligase

complex.

Nrf2

is

thus

targeted

for

degradation

by

proteasome.

Upon exposure to reactive oxygen species (ROS), electrophiles or vascular endothelial growth factor (VEGF), specific cysteine residues on Keap1 are modified to induce Nrf2 activity (“stabilization”). The effect is that Nrf2 translocates to the nucleus, where it heterodimerizes to small Maf protein (another transcription factor) and binds to ARE sequences in the promoter regions of multiple detoxifying and antioxidant genes. B: Domain structures of Keap1 and Nrf2. Abbreviations: N-terminal region (N-term), broad complex19 Page 19 of 24

tramtrack-bric-a-brac (BTB), and intervening region (IVR). DGR domains consist of kelch-repeat domains. The DC domain comprises a C-terminal region (C-term) and the DGR domains. Nrf2 identifies six Neh domains. The Neh2 molecule interacts with two molecules of Keap1 via two binding sites, the stronger-binding ETGE motif and the weaker-binding DLG motif. Figure 2. Trophoblast turnover and spiral artery remodelling in normal and pathological pregnancies. (A) During normal pregnancy the cytotrophoblasts (CTB) continuously fuse with the overlying

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syncytiotrophoblast (STB). Within this multinucleated layer, differentiation and, subsequently, late apoptosis occur. As a final point of trophoblast turnover, late apoptotic material is packed into

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syncytial knots, which are released into the maternal circulation as apoptotic corpuscles. CTB from anchoring villi change into an invasive phenotype called extravillous trophoblast (EVTs). The EVTs

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invade the uterine decidua and myometrium. A subset of the EVTs in the decidua and myometruim migrates towards the uterine spiral arteries, replaces their endothelial cells and transforms them into large conduit vessels of low resistance. (B) Intra uterine growth restriction (IUGR) is characterized by

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shallow invasion of EVTs into decidua and myometrium, resulting in incomplete transformation of the spiral arteries. Differentiation of the STB remains normal.

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(C) Preeclampsia: The villous CTB and EVT compartments seem to be unaffected, without any changes in proliferation, fusion or invasion rates. However, the STB no longer differentiates appropriately and releases increased amounts of syncytial material by means of necrosis, aponecrosis

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and apoptosis. (D) IUGR and preeclampsia: Both cell types are affected. The EVT fails to invade

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properly into decidua and myometrium, and the STB no longer differentiates normally and shows different modes of release (preeclampsia!). Modified from Huppertz B, Preg. Hyper.: An Int. J. Women’s Card. Health (2010), doi:10.1016/j.preghy.2010.10.003 Figure 3. Schematic diagram illustrating the possible mechanisms conferred by Nrf2 to protect against preeclampsia.

Table 1: Potential clinical importance of Nrf2 activation

Effect

Antioxidant involved

Mechanism of action

References

Inhibition of vasoconstriction

The stress-inducible modulator; (SQSTM1) Sequestosome1.

regulates the redox-sensitive voltage-gated potassium (Kv) channels, hence modulate the migration and proliferation of arterial smooth muscle cells.

Ishii et al., 2013

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Ashino et al., 2013, Lee et al., 2012

CO

inhibits the production of endothelin

Liu et al., 2010

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NQO1 and HO-1

attenuates of PDGF-dependent ROS production, thereby regulating VSMC migration and vascular remodelling

Alteration susceptibility to atherosclerosis and attenuation vascular remodeling

HO-1/Fe²+

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HO-1/CO

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Inhibition of the production of inflammatory cytokines

inhibits the increased levels of TNF-α, IL-1β, IL-6, ICAM-1, and their mediator, Rangasamy et al., 2005 Jin et al., 2008 NF-κB, thereby attenuating the inflammatory response after traumatic injury.

Kim et al., 2010

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inhibits the neointimal formation via activation of phosphatidyl inositol 3-kinase

unknown

leads to the survival and differentiation of hematopoietic stem progenitor cells (HSPC)

Merchant et al., 2011

HO-1/CO

leads to inflammatory revascularization due to elevated production of angiogenic factors

Florczyk et al., 2013 Kweider et al., 2011

ameliorates of the effects of Ang II

Kang et al., 2011

Proangiogenic effect

Reduction of hypertension

HO-1/CO

Phase II detoxifiying enzymes

preserves renal dopamine receptors (D1R) and inhibits hypertension

Banday and Lokhanwala 2013

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Ryter et al., 2002

SOD1 and HO1

proteasome inhibitor-mediated protection of vascular cells against oxidative stress.

Dreger et al., 2010

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activates NFkB, upregulates the antiapoptotic proteins A1, 20, c-lAP2, Bcl-2

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Enhancement of endothelial cells survival and prevents their apoptosis

CO

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