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Circulation. 2007;115:1789-1797
Published online before print March 26, 2007, doi: 10.1161/CIRCULATIONAHA.106.660134
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(Circulation. 2007;115:1789-1797.)
© 2007 American Heart Association, Inc.

Molecular Cardiology

Negative Regulation of Soluble Flt-1 and Soluble Endoglin Release by Heme Oxygenase-1

Melissa Cudmore, PhD; Shakil Ahmad, PhD; Bahjat Al-Ani, PhD; Takeshi Fujisawa, PhD; Heather Coxall, BSc; Kunal Chudasama, BSc; Luke R. Devey, MD; Stephen J. Wigmore, MD; Allyah Abbas; Peter W. Hewett, PhD; Asif Ahmed, PhD

From the Departments of Reproductive and Vascular Biology (M.C., S.A., B.A.-A., T.F., H.C., K.C., A. Abbas, P.W.H., A. Ahmed) and Liver Research Group (L.R.D., S.J.W.), Institute of Biomedical Research, The Medical School, University of Birmingham, and Birmingham Women’s Hospital (M.C., S.A., B.A.-A., T.F., H.C., K.C., A. Abbas, P.W.H., A. Ahmed), Edgbaston, Birmingham, UK.

Correspondence to Dr Asif Ahmed, Department of Reproductive and Vascular Biology, Medical School, University of Birmingham, Edgbaston, Birmingham, West Midlands, B15 2TT, UK. E-mail a.s.ahmed{at}bham.ac.uk

Received August 23, 2006; accepted January 19, 2007.


*    Abstract
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 *Abstract
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Background— Preeclampsia is characterized clinically by hypertension and proteinuria. Soluble Flt-1 (sFlt-1; also known as soluble vascular endothelial growth factor receptor-1 [VEGFR-1]) and soluble endoglin (sEng) are elevated in preeclampsia, and their administration to pregnant rats elicits preeclampsia-like symptoms. Heme oxygenase-1 (HO-1) and its metabolite carbon monoxide (CO) exert protective effects against oxidative stimuli. Thus, we hypothesized that HO-1 upregulation may offer protection against preeclampsia by inhibiting sFlt-1 and sEng release.

Methods and Results— Preeclamptic villous explants secreted high levels of sFlt-1 and sEng. Adenoviral overexpression of HO-1 in endothelial cells inhibited VEGF-mediated sFlt-1 release and interferon-{gamma}– and tumor necrosis factor-{alpha}–induced sEng release, whereas HO-1 inhibition potentiated sFlt-1 and sEng production from endothelial cells and placental villous explants. Consistent with these findings, mice lacking HO-1 produced higher levels of sFlt-1 and sEng compared with wild-type mice. Using selective ligands (VEGF-E and placental growth factor) and a receptor-specific inhibitor (SU-1498), we demonstrated that VEGF-induced sFlt-1 release was VEGFR-2 dependent. Furthermore, CO–releasing molecule-2 (CORM-2) or CO decreased sFlt-1 release and inhibited VEGFR-2 phosphorylation. Treatment of endothelial cells with statins upregulated HO-1 and inhibited the release of sFlt-1, whereas vitamins C and E had no effect.

Conclusions— The present study demonstrates that the HO-1/CO pathway inhibits sFlt-1 and sEng release, providing compelling evidence for a protective role of HO-1 in pregnancy, and identifies HO-1 as a novel target for the treatment of preeclampsia.


Key Words: endothelium • endothelium-derived factors • heme oxygenase-1 • preeclampsia • pregnancy • statins • angiogenesis


*    Introduction
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Cardiovascular disease and preeclampsia share some common risk factors, such as insulin resistance, obesity, diabetes mellitus, and inflammation.1,2 The disruption of endothelial homeostasis and inflammation are fundamental to the initiation and progression of atherosclerosis3 and preeclampsia.4 Preeclampsia is a maternal systemic endothelial disease defined clinically as hypertension and proteinuria after 20 weeks’ gestation that affects 3% to 8% of all pregnancies and women.5 Women with a history of preeclampsia and their offspring are at greater risk of developing cardiovascular disease later in life.6,7

Clinical Perspective p 1797

Preeclampsia involves dysregulated placental angiogenesis,8 resulting in the release of soluble antiangiogenic factors that induce systemic endothelial dysfunction.9 Two key antiangiogenic circulating factors that give the highest strength of association with preeclamptic outcome are soluble Flt-1 (sFlt-1) and soluble endoglin (sEng).10–12 Maternal serum levels of sFlt-1 are elevated 5 weeks before the clinical onset of preeclampsia.10,13–16 sEng, a placenta-derived 65-kDa cleaved form of endoglin (also known as CD105), a coreceptor for transforming growth factor-ß, is elevated in the serum of preeclamptic women 8 to 12 weeks before the clinical onset of the disease.12 In rats, administration of recombinant sFlt-1 or a vascular endothelial growth factor (VEGF)–neutralizing antibody results in glomerular endothelial cell damage and proteinuria,17 and adenoviral delivery of sFlt-1 to pregnant rats mimics the clinical manifestations of preeclampsia,18 suggesting that excess circulating sFlt-1 may play a role in the pathogenesis of preeclampsia. Indeed, we demonstrated that the conditioned medium from preeclamptic placenta exhibited reduced capillary tube–forming activity compared with that of normal placenta and that the removal of sFlt-1 from preeclamptic conditioned medium eliminates this suppressive activity. Like sFlt-1, sEng also inhibited capillary morphogenesis.19 Furthermore, sEng acts synergistically with sFlt-1 to induce endothelial dysfunction; simultaneous adenoviral administration of sFlt-1 and sEng induced severe preeclampsia-like symptoms in pregnant rats.19 However, the molecular mechanism(s) regulating the release of these antiangiogenic factors are unknown. Any intervention that would reduce the prevalence of these circulating factors not only may prolong the pregnancy but also protect the mother from permanent vascular damage.

It was proposed that the resolution of oxidative stress and inflammation associated with pregnancy may be controlled by vascular protective factors and that the lack of such compensatory systems leads to preeclampsia.20 Heme oxygenase-1 (HO-1) is an inducible, endoplasmic reticulum–bound enzyme that catalyzes the nicotinamide adenosine dinucleotide phosphate–cytochrome P450 reductase–dependent oxidation of heme to biliverdin in a 3-step process that liberates carbon monoxide (CO) and Fe2+.21 HO-1 is antiinflammatory and provides a defense against oxidant injury.22,23 Exogenous HO-1 is widely acknowledged to be protective against ischemia-reperfusion injury,24–27 and HO-1 is upregulated after reperfusion.28

HOs are also critical for the successful outcome of pregnancy.29 Administration of a HO-1 antagonist to pregnant rats resulted in complete fetal resorption,30 and adenoviral overexpression of HO-1 rescues pregnancy in abortion-prone mice.31 In women, HO-1 maintains uterine quiescence,32 which is further supported by lower end-tidal CO levels in women with uterine contractions, indicating reduced HO activity.33 Women with preeclampsia have significantly decreased CO concentrations in their exhaled breath compared with healthy pregnant women, indicating a decreased expression or activity of HO,34,35 and protein levels of HO-120 and HO-236–38 are decreased in preeclamptic placenta. In the present study, we report that adenoviral overexpression of HO-1 in endothelial cells results in a decreased production of sFlt-1 and sEng, whereas HO-1 small interfering RNA (siRNA) knockdown potentiated sFlt-1 and sEng release from endothelial cells, and pharmacological inhibition of HO activity in placental villous explants stimulated the release of sFlt-1 and sEng. The loss of HO activity may be central to the pathogenesis of preeclampsia.


*    Methods
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 *Methods
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Reagents
Recombinant growth factors were purchased from RELIATech (Brauschweig, Germany). Tin protoporphyrin-IX (SnPP) was obtained from Alexis Biochemicals (Nottingham, UK). The VEGF receptor-2 (VEGFR-2) tyrosine kinase inhibitor SU-1498 was purchased from Calbiochem (Nottingham, UK). Tricarbonyldichlororuthenium (II) dimer (CO-releasing molecule [CORM-2]), ruthenium (III) chloride hydrate (CORM-2 control), simvastatin, mevastatin, fluvastatin, farnesyl pyrophosphate, vitamin C, vitamin E, tumor necrosis factor (TNF-{alpha}), interferon-{gamma} (IFN-{gamma}), and all other cell culture reagents and chemicals were obtained from Sigma Aldrich (Poole, UK).

Cell Culture and Stimulations
Human umbilical vein endothelial cells (HUVECs) and porcine aortic endothelial cells expressing human VEGFR-2 (PAEVEGFR-2) were used as described previously.39 HUVECs were stimulated with VEGF-A (20 ng/mL), placental growth factor-1 (20 ng/mL), VEGF-E (20 ng/mL), TNF-{alpha} (10 ng/mL), or IFN-{gamma} (10 ng/mL), and conditioned media was collected and assayed for sFlt-1 or sEng by ELISA. For inhibitor studies, HUVECs were incubated with SU-1498 (10 µmol/L), SnPP (20 µmol/L), CORM-2 (50 µmol/L), inactivated CORM-2 (iCORM-2; 50 µmol/L; reconstituted CORM-2 exposed to air for 24 hours), statins (10 µmol/L), vitamin C (1 mmol/L), or vitamin E (1 mmol/L) for 30 minutes before the addition of VEGF-E.

Adenoviral Gene Transfer
The recombinant, replication-deficient adenovirus-encoding rat HO-1 (AdHO-1) was used as described previously.40 Optimal multiplicity of infection for AdHO-1 was determined to be 50 infective units/cell by Western blotting using a rabbit anti-HO-1 antibody (StressGen Biotechnologies Corp [now Nventa], Victoria, BC, Canada).

Quantitative Real-Time Polymerase Chain Reaction
Sample preparation and real-time polymerase chain reaction were performed as described previously.41 Briefly, mRNA was prepared with TRIzol and DNase-1 digestion/purification on RNAeasy columns (Qiagen, West Sussex, UK) and reverse transcribed with the cDNA Synthesis Kit (Promega, Madison, Wis). Triplicate cDNA samples and standards were amplified in SensiMix containing SYBR green (Quantace, London, UK) with primers specific for HO-1 (sense, 5'-GGG TGA TAG AAG AGG CCA AGA CT-3'; antisense, 5'-GCA GAA TCT TGC ACT TTG TTG CT-3')42 or ß-actin. The mean threshold cycle for each HO-1 was normalized to ß-actin and expressed relative to control.

siRNA Knockdown of HO-1
HUVEC were trypsinized, and {approx}1x106 cells were electroporated with {approx}3 µg HO-1 (sense, 5'-GGCAGAGGGUGAUAGAAGAUU-3'; antisense, 5'-UCUUCUAUCACCCUCUGCCUU-3')43 or control siRNA using the HUVEC kit II and Nucleofector (Amaxa GmbH, Cologne, Germany) as described previously.41

HO-1–Null Mice
The generation of the HO-1–/– mice has been previously described.44,45

Placental Tissue Collection and Preparation
Human placental tissue was obtained from normal pregnancies and gestationally matched pregnancies complicated by preeclampsia. Preeclampsia was defined as blood pressure >140/90 mm Hg on at least 2 consecutive measurements and proteinuria of at least 300 mg/24 h. Informed consent was obtained from the patients, and the study had the approval of the South Birmingham Ethical Committee (Birmingham, UK). Villous explants were prepared and exposed to hypoxia as described previously46,47 in the presence or absence of SnPP (20 µmol/L), VEGF (20 ng/mL), TNF-{alpha} (50 ng/mL), or simvastatin (10 µmol/L) for 24 hours, and conditioned media was assayed for sFlt-1 or sEng.

sFlt-1 and sEng ELISA
sFlt-1 levels were measured as previously described,47 and sEng was measured by ELISA according to the manufacturer’s instructions (R&D Systems, Abingdon, UK).

Immunoprecipitation and Western Blotting
After 48 hours of serum starvation, PAEVEGFR-2 were preincubated for 30 minutes with CORM-2 (50 µmol/L), iCORM-2 (50 µmol/L), CORM-2 control compound (100 µmol/L), or medium containing CO gas. Cells were stimulated with VEGF-E (20 ng/mL) for 10 minutes and lysed in radio immunoprecipitation assay buffer, and the lysate was subjected to overnight immunoprecipitation with rabbit anti–VEGFR-2 (C-1158) (Autogen Bioclear, Wiltshire, UK). Protein-A-agarose (Amersham-Pharmacia, Chalfont St. Giles, UK) –captured immunoprecipitates were separated on 6% SDS-PAGE, and phosphotyrosines were detected with mouse anti-PY99 (Autogen Bioclear, Wiltshire, UK). HUVECs were incubated for 24 hours with simvastatin (10 µmol/L), vitamin C (1 mmol/L), or vitamin E (1 mmol/L), and radio immunoprecipitation assay lysates were subjected to 15% SDS-PAGE and Western blotted with rabbit anti-HO-1 antibody (StressGen Biotechnologies Corp).

Statistical Analysis
All data are expressed as mean±SEM. Statistical comparisons were performed with 1-way ANOVA, followed by the Student-Newman-Keuls test as appropriate. Statistical significance was set at P<0.05.

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.


*    Results and Discussion
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HO-1 Is a Negative Regulator of VEGFR-2–Mediated sFlt-1 Release
Previous studies have shown that HO-1 protein is decreased20 and sFlt-1 release is increased in preeclamptic placenta.47,48 Circulating total VEGF18,49 is increased in preeclampsia, and VEGF is known to stimulate the release of sFlt-1 from endothelial cells47,50 and placental explants.47 Using selective ligands (VEGF-E and placental growth factor-1) and a receptor-specific inhibitor (SU-1498), we demonstrate that VEGF-induced sFlt-1 release is VEGF receptor-2 (VEGFR-2) dependent (see Figure I in the Data Supplement). To determine whether HO-1 affects sFlt-1 release, endothelial cells were infected with an AdHO-1. Overexpression of HO-1 inhibited VEGF-E–mediated release of sFlt-1 and reduced IFN-{gamma}–stimulated sFlt-1 production (Figure 1A). Consistent with these findings, the HO inhibitor SnPP (Figure 1B) and siRNA knockdown of HO-1 (Figure 1C) potentiated VEGF-E–induced sFlt-1 secretion, suggesting that HO-1 is a negative regulator of VEGFR-2–mediated sFlt-1 release in endothelial cells. In line with this, significantly higher levels of sFlt-1 were secreted from HO-1–null murine lung biopsy explants compared with wild-type litter mates (Figure 1D). The ability of HO-1 to suppress cytokine-induced damage20 and to inhibit sFlt-1 release strongly supports the concept that loss of HO activity may be central to the pathogenesis of preeclampsia.


Figure 1182458
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  		Figure 1. HO-1 negatively regulates sFlt-1 release. A, HUVECs were infected with 50 infective units/cell of AdHO-1, and HO-1 overexpression was confirmed by Western blotting (inset). After stimulation with VEGF-E (20 ng/mL) or IFN-{gamma} (10 ng/mL) for 24 hours, sFlt-1 levels in cell supernatants were assayed by ELISA. B, HUVECs were preincubated with SnPP (20 µmol/L) for 30 minutes and then stimulated with VEGF-E (20 ng/mL) for 24 hours, and sFlt-1 release was measured by ELISA. *P<0.01 vs VEGF-E. C, HUVECs were electroporated with HO-1 siRNA or control siRNA (MM), and HO-1 knockdown was confirmed by real-time polymerase chain reaction and Western blotting (see inset). ***P<0.01 vs MM siRNA. After stimulation with VEGF-E (20 ng/mL) for 24 hours, the conditioned media was assayed for sFlt-1. *P<0.05 vs control siRNA; **P<0.01 vs control siRNA+VEGF-E. D, Supernatants from lung biopsies of 8-week-old HO-1–/– (KO) and wild-type (WT) mice were collected after 24 hours and assayed for sFlt-1. *P<0.01 vs WT. Data are mean (±SEM) of ≥3 experiments performed in duplicate. The absence of HO-1 in the KO mice was confirmed by Western blotting (inset).

CO Inhibits VEGFR-2–Mediated sFlt-1 Release
To determine whether CO, the gaseous product of HO activity, was involved in the inhibitory effect on sFlt-1 release observed after HO-1 overexpression, HUVECs were treated with CORM-2.51 VEGF-E–stimulated sFlt-1 release was inhibited by CORM-2, a CO-releasing molecule, whereas inactivated CORM-2 (iCORM-2) had no effect (Figure 2A). This demonstrates that the CO produced by HO-1 is responsible, at least in part, for this inhibition. Women with preeclampsia have significantly decreased CO concentrations in their exhaled breath compared with healthy pregnant women, indicating decreased expression or activity of HO.34,35 Furthermore, women who smoke throughout their pregnancies are 33% less likely to develop preeclampsia29,52 and have reduced serum levels of sFlt-1.53 In contrast, women who use snuff, a form of smokeless tobacco, are at an increased risk of developing preeclampsia.54 It is likely that exposure to elevated concentrations of exogenous CO, one of the combustion products of cigarette smoke, offers protection against preeclampsia.55


Figure 2182458
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  		Figure 2. CO inhibits sFlt-1 release and VEGFR-2 phosphorylation. A, HUVECs were preincubated with 50 µmol/L CORM-2 or iCORM-2 for 30 minutes and stimulated with VEGF-E (20 ng/mL) for 24 hours, and sFlt-1 was measured in the supernatants by ELISA. Data are mean (±SEM) of ≥3 experiments performed in duplicate. PAEVEGFR-2 (PAER-2) cells were serum deprived for 48 hours and then preincubated with 50 µmol/L CORM-2, iCORM-2, or 100 µmol/L CORM-2 control for 30 minutes (B) or CO gas–saturated medium for 45 minutes and stimulated with VEGF-E (20 ng/mL) for 10 minutes (C). Cell lysates were immunoprecipitated with rabbit anti–VEGFR-2 and Western blotted with an anti-phosphotyrosine antibody (PY99). Western blots are representative of 4 different experiments.

Because sFlt-1 release was VEGFR-2 mediated (Figure I in the online Data Supplement), experiments were undertaken to ascertain whether CORM-2 could inhibit VEGFR-2 phosphorylation induced by VEGF-E. PAEVEGFR-2 cells were exposed to CORM-2 or CO-saturated medium before stimulation with VEGF-E for 10 minutes, and VEGFR-2 immunoprecipitates were Western blotted for phosphotyrosine residues. Both CORM-2 and CO inhibited VEGF-E–mediated tyrosine phosphorylation of VEGFR-2 (Figure 2B and 2C).

HO Inhibition Stimulates Release of sFlt-1 From Placental Explants
To determine whether inhibition of placental HO activity potentiates sFlt-1 release, normal placental explants were incubated with SnPP for 24 hours under tissue normoxia (5% O2; PO2=60 mm Hg), and sFlt-1 was assayed in the conditioned medium. This resulted in a >2-fold increase in sFlt-1 secretion (Figure 3A). HO activity depends on the availability of oxygen,21 and the activity of HO isolated from chorionic villi is reported to decrease under hypoxic conditions.56 Exposure to hypoxia (1% O2; PO2=16 mm Hg) resulted in elevated sFlt-1 release from normal placental villous explants (Figure 3A) as reported previously.47 Although not significant, the addition of SnPP resulted in a slight increase in sFlt-1 release under hypoxia (Figure 3A). Exposure of syncytiotrophoblast cultures to hypoxia resulted in a significant reduction in HO-1 expression.57 Under hypoxia, a decrease in but not a complete inhibition of HO activity occurs, which may account for the elevated sFlt-1 levels seen in preeclampsia. Interestingly, when preeclamptic placental explants were placed under atmospheric conditions, inhibition of HO led to an upregulation of sFlt-1 (Figure 3B), suggesting that HO is biologically active in the preeclamptic placenta but at a reduced level compared with normal. Preeclampsia is a heterogeneous disease, and it is likely that hypoxia is experienced only in certain areas, with the distribution and degree depending on the severity of the disease. Histological studies showed that preeclamptic placenta appears to be normal in certain areas but infarcted or damaged at other sites where decreased HO expression exists.37 If HO expression and activity were to be enhanced therapeutically, the enzyme would be active in areas that are sufficiently oxygenated and may offer protection.


Figure 3182458
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  		Figure 3. Inhibition of HO induces sFlt-1 release from placental explants. A, Placental villous explants incubated with SnPP (20 µmol/L) under hypoxia (1% O2) or tissue normoxia (5% O2) for 24 hours. B, Normal or preeclamptic placental villous explants were rested overnight and then incubated in the presence of SnPP (20 µmol/L) under Birmingham, UK, atmospheric conditions for 24 hours. Conditioned media was collected and assayed for sFlt-1 by ELISA. Data are mean (±SEM) of 3 separate experiments (n=9).

HO-1 Is a Negative Regulator of sEng Release
Maternal serum levels of sEng are elevated in preeclampsia 8 to 12 weeks before the clinical onset of the disease,12 and preeclamptic placental tissues express elevated endoglin.19 Here, we show that like sFlt-1,47 preeclamptic placental villous explants release significant amounts of sEng into the culture medium compared with normal gestationally matched explants (Figure 4A). Ischemia/reperfusion injury in certain areas of the placenta is associated with the progression of preeclampsia.58 Hepatic ischemia/reperfusion injury in HO-1–/+ mice results in increased inflammatory cell recruitment and induction of proinflammatory cytokines such as TNF-{alpha} and IFN-{gamma} compared with wild-type littermates.59 Exacerbated inflammation and elevated TNF-{alpha} and IFN-{gamma} levels also occur in preeclampsia.60–64 Interestingly, hypoxia induces IFN-{gamma} release,65 which has been shown to suppress HO-1 expression,66 whereas CO antagonizes this effect in macrophages.67 Similar to the inhibition of sFlt-1, adenoviral HO-1 overexpression inhibited basal and TNF-{alpha}– and IFN-{gamma}–mediated sEng release (Figure 4B). In addition, TNF-{alpha}–induced release of sEng from normal placental villous explants was potentiated by inhibition of HO activity with SnPP (Figure 4C). The hypothesis that HO-1 is a global protective factor is supported by our data showing that circulating sEng is elevated in serum from HO-1–/– mice compared with wild-type litter mates (Figure 4D).


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  		Figure 4. HO-1 negatively regulates sEng release. A, Levels of sEng released by normal or preeclamptic placental villous explants after 24 hours. *P<0.001 vs preeclamptic. Data are mean (±SEM) of 3 separate experiments (n=9). B, HUVECs infected with 50 infective units/cell of AdHO-1 were stimulated with 10 ng/mL TNF-{alpha} or IFN-{gamma} for 24 hours, and sEng was measured in the supernatants. Data are mean (±SEM) of 3 separate experiments (n=9). C, Placental villous explants were incubated in the presence of TNF-{alpha} (10 ng/mL) and/or SnPP (20 µmol/L) for 24 hours, and conditioned media was assayed for sEng by ELISA. *P<0.01 vs vehicle; **P<0.001 vs TNF-{alpha} (n=6). D, Circulating sEng levels measured in the plasma of 8-week-old HO-1–null (KO) and wild-type (WT) mice. *P<0.001 vs WT.

Vitamins C and E Have No Effect on Release of sFlt-1 or sEng
Increased oxidative stress is a causative factor of preeclampsia. This finding has led to several clinical trials of vitamin supplements in pregnant women at risk of developing preeclampsia. Disappointingly, the results of 2 recently published studies show that vitamins C and E do not prevent or reduce the severity of disease.68,69 In fact, the Vitamins in Preeclampsia study showed that vitamin supplementation has a detrimental effect on infant outcome.69 On the basis of our hypothesis, we assessed the effects of vitamins C and E on HO-1 expression and sFlt-1 and sEng release. These vitamins had no effect on HO-1 expression (Figure 5A) or on the release of sFlt-1 or sEng under basal or stimulated conditions (Figure 5B and 5C). It is possible that the lack of successful outcome of these trials may be due in part to the failure of these vitamins to modify the expression and/or secretion of these 2 key antiangiogenic proteins. Our data suggest that upregulation of endogenous protective factors like the HO pathway or other antioxidant systems such as the thioredoxin family of proteins may alleviate the symptoms of preeclampsia.


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  		Figure 5. Vitamins C and E do not affect HO-1 expression or release of sFlt-1 or sEng. A, HUVECs were incubated with 1 mmol/L vitamin C and/or vitamin E for 24 hours, and cell lysates were Western blotted for HO-1. B, HUVECs were incubated with 1 mmol/L vitamin C and/or vitamin E in the presence or absence of VEGF-E (20 ng/mL) for 24 hours, and the conditioned media was assayed for sFlt-1 (B) or sEng (C) by ELISA. Data are mean (±SEM) of ≥3 separate experiments performed in duplicate.

Statins Inhibit sFlt-1 Release
Agents that increase HO expression and reduce the release of antiangiogenic factors may be beneficial as therapeutic agents in preeclampsia. The antiinflammatory and antiproliferative effects of simvastatin occur in part through HO-1.70 Simvastatin was reported to upregulate HO-1 mRNA in HUVECs,71 and here we show that simvastatin upregulates HO-1 protein (Figure 6A). Although long-term treatment with atorvastatin after acute myocardial infarct increased circulating sFlt-1 levels,72 addition of simvastatin, fluvastatin, or mevastatin (Figure 6B) significantly decreased the basal production and VEGF-E–induced release of sFlt-1 from endothelial cells. This inhibition was specific to the cholesterol biosynthesis pathway because it could be rescued by supplementation with farnesyl pyrophosphate to bypass hydroxy-methyl-glutaryl–Coenzyme A reductase (Figure 6C). Furthermore, simvastatin decreased the VEGF-induced sFlt-1 release from normal-term placental villous explants (Figure 6D). Additionally, treatment with simvastatin led to a reduction in sEng release, although this was not statistically significant. Currently, statins are contraindicated in pregnancy because skeletal malformations in rat fetuses were reported with high-dose lovastatin (800 mg · kg–1 · d–1, 700 times the maximum recommended dose in humans) or active metabolites of statins.73 However, no increase in congenital abnormalities above that of the normal population has been reported when statin treatment was inadvertently continued throughout pregnancy in women with familial hypercholesterolemia.74–77 Once a predictive and accurate biomarker has been established for early-onset preeclampsia, pregnant women at risk of developing preeclampsia could be managed with statins. We postulate that statins may alleviate the symptoms of preeclampsia by upregulating HO-1 and inhibiting the 2 key antiangiogenic factors, which have been shown to underpin the clinical syndrome.


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  		Figure 6. Simvastatin upregulates HO-1 and inhibits sFlt-1 release. A, HUVECs were incubated with 10 µmol/L simvastatin (Sim) or vehicle (Veh) for 24 hours, and HO-1 was detected in cell lysates by Western blotting. B, HUVECs were pretreated with 10 µmol/L simvastatin, fluvastatin, or mevastatin for 30 minutes and stimulated with VEGF-E (20 ng/mL) for 24 hours, and sFlt-1 was measured in the supernatants. *P<0.002 vs vehicle control; #P<0.002 vs vehicle+VEGF-E. C, HUVECs were pretreated with simvastatin (10 µmol/L) for 30 minutes and stimulated with VEGF-E (20 ng/mL) and/or farnesyl pyrophosphate (Fpp; 50 µmol/L) for 24 hours, and sFlt-1 was assayed in the supernatants. D, sFlt-1 release from placental villous explants stimulated with VEGF-E (20 ng/mL) in the presence or absence of simvastatin (10 µmol/L) for 24 hours. Data are mean (±SEM) of ≥3 separate experiments performed in duplicate. *P<0.002.

The present report demonstrates that HO-1 inhibits sFlt-1 and sEng release from endothelial cells and placental explants and that the pathophysiology of preeclampsia may involve the loss of HO activity. We propose that the HO/CO pathway acts as a gatekeeper, preventing the onset of preeclampsia by inhibiting the production of antiangiogenic factors. Further support for this concept is that both HO-1 expression20 and HO activity78 are reduced in preeclamptic placenta. Our data provide the first evidence to support the concept that HO-1 acts as a negative regulator of sFlt-1 and sEng release to offer vascular protection against pregnancy-induced oxidative stress and exacerbated inflammation. This opens a new avenue of investigation for increasing our understanding of the cause of preeclampsia and provides a novel target for therapeutic intervention.


*    Acknowledgments
 
We thank Professor Augustine Choi for his kind gift of the AdHO-1 and Professor Anupam Agarwal for providing the HO-1–/– mice.

Sources of Funding

This work was supported by grants from the British Heart Foundation, Medical Research Council, and the European Vascular Genomics Network (www.evgn.org), a Network of Excellence supported by the European Community’s Sixth Framework Programme for Research Priority 1, Life Sciences, Genomics, and Biotechnology for Health (contract LSHM-CT-2003–503254).

Disclosures

None.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults and Discussion
 *References
 

  1. Seely EW, Solomon CG. Insulin resistance and its potential role in pregnancy-induced hypertension. J Clin Endocrinol Metab. 2003; 88: 2393–2398.[Abstract/Free Full Text]
  2. Rodie VA, Freeman DJ, Sattar N, Greer IA. Pre-eclampsia and cardiovascular disease: metabolic syndrome of pregnancy? Atherosclerosis. 2004; 175: 189–202.[CrossRef][Medline] [Order article via Infotrieve]
  3. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005; 352: 1685–1695.[Free Full Text]
  4. Redman CW, Sargent IL. Latest advances in understanding preeclampsia. Science. 2005; 308: 1592–1594.[Abstract/Free Full Text]
  5. Sibai B, Dekker G, Kupferminc M. Pre-eclampsia. Lancet. 2005; 365: 785–799.[Medline] [Order article via Infotrieve]
  6. Smith GC, Pell JP, Walsh D. Pregnancy complications and maternal risk of ischaemic heart disease: a retrospective cohort study of 129,290 births. Lancet. 2001; 357: 2002–2006.[CrossRef][Medline] [Order article via Infotrieve]
  7. Vatten LJ, Romundstad PR, Holmen TL, Hsieh CC, Trichopoulos D, Stuver SO. Intrauterine exposure to preeclampsia and adolescent blood pressure, body size, and age at menarche in female offspring. Obstet Gynecol. 2003; 101: 529–533.[Abstract/Free Full Text]
  8. Ahmed A. Heparin-binding angiogenic growth factors in pregnancy. Trophoblast Res. 1997; 10: 215–258.
  9. Yuan HT, Haig D, Ananth Karumanchi S. Angiogenic factors in the pathogenesis of preeclampsia. Curr Top Dev Biol. 2005; 71: 297–312.[Medline] [Order article via Infotrieve]
  10. Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, Schisterman EF, Thadhani R, Sachs BP, Epstein FH, Sibai BM, Sukhatme VP, Karumanchi SA. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med. 2004; 350: 672–683.[Abstract/Free Full Text]
  11. Levine RJ, Thadhani R, Qian C, Lam C, Lim KH, Yu KF, Blink AL, Sachs BP, Epstein FH, Sibai BM, Sukhatme VP, Karumanchi SA. Urinary placental growth factor and risk of preeclampsia. JAMA. 2005; 293: 77–85.[Abstract/Free Full Text]
  12. Levine RJ, Lam C, Qian C, Yu KF, Maynard SE, Sachs BP, Sibai BM, Epstein FH, Romero R, Thadhani R, Karumanchi SA. Soluble endoglin and other circulating antiangiogenic factors in preeclampsia. N Engl J Med. 2006; 355: 992–1005.[Abstract/Free Full Text]
  13. Chaiworapongsa T, Romero R, Kim YM, Kim GJ, Kim MR, Espinoza J, Bujold E, Goncalves L, Gomez R, Edwin S, Mazor M. Plasma soluble vascular endothelial growth factor receptor-1 concentration is elevated prior to the clinical diagnosis of pre-eclampsia. J Matern Fetal Neonatal Med. 2005; 17: 3–18.[Medline] [Order article via Infotrieve]
  14. Koga K, Osuga Y, Yoshino O, Hirota Y, Ruimeng X, Hirata T, Takeda S, Yano T, Tsutsumi O, Taketani Y. Elevated serum soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) levels in women with preeclampsia. J Clin Endocrinol Metab. 2003; 88: 2348–2351.[Abstract]
  15. Hertig A, Berkane N, Lefevre G, Toumi K, Marti HP, Capeau J, Uzan S, Rondeau E. Maternal serum sFlt1 concentration is an early and reliable predictive marker of preeclampsia. Clin Chem. 2004; 50: 1702–1703.[Free Full Text]
  16. Crispi F, Dominguez C, Llurba E, Martin-Gallan P, Cabero L, Gratacos E. Placental angiogenic growth factors and uterine artery Doppler findings for characterization of different subsets in preeclampsia and in isolated intrauterine growth restriction. Am J Obstet Gynecol. 2006; 195: 201–207.[CrossRef][Medline] [Order article via Infotrieve]
  17. Sugimoto H, Hamano Y, Charytan D, Cosgrove D, Kieran M, Sudhakar A, Kalluri R. Neutralization of circulating vascular endothelial growth factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces proteinuria. J Biol Chem. 2003; 278: 12605–12608.[Abstract/Free Full Text]
  18. Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE, Epstein FH, Sukhatme VP, Karumanchi SA. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest. 2003; 111: 649–658.[CrossRef][Medline] [Order article via Infotrieve]
  19. Venkatesha S, Toporsian M, Lam C, Hanai J, Mammoto T, Kim YM, Bdolah Y, Lim KH, Yuan HT, Libermann TA, Stillman IE, Roberts D, D’Amore PA, Epstein FH, Sellke FW, Romero R, Sukhatme VP, Letarte M, Karumanchi SA. Soluble endoglin contributes to the pathogenesis of preeclampsia. Nat Med. 2006; 12: 642–649.[CrossRef][Medline] [Order article via Infotrieve]
  20. Ahmed A, Rahman M, Zhang X, Acevedo CH, Nijjar S, Rushton I, Bussolati B, St John J. Induction of placental heme oxygenase-1 is protective against TNFalpha-induced cytotoxicity and promotes vessel relaxation. Mol Med. 2000; 6: 391–409.[Medline] [Order article via Infotrieve]
  21. Tenhunen R, Marver HS, Schmid R. Microsomal heme oxygenase: characterization of the enzyme. J Biol Chem. 1969; 244: 6388–6394.[Abstract/Free Full Text]
  22. Keyse SM, Tyrrell RM. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc Natl Acad Sci U S A. 1989; 86: 99–103.[Abstract/Free Full Text]
  23. Duckers HJ, Boehm M, True AL, Yet SF, San H, Park JL, Clinton Webb R, Lee ME, Nabel GJ, Nabel EG. Heme oxygenase-1 protects against vascular constriction and proliferation. Nat Med. 2001; 7: 693–698.[CrossRef][Medline] [Order article via Infotrieve]
  24. Melo LG, Agrawal R, Zhang L, Rezvani M, Mangi AA, Ehsan A, Griese DP, Dell’Acqua G, Mann MJ, Oyama J, Yet SF, Layne MD, Perrella MA, Dzau VJ. Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene. Circulation. 2002; 105: 602–607.[Abstract/Free Full Text]
  25. Katori M, Anselmo DM, Busuttil RW, Kupiec-Weglinski JW. A novel strategy against ischemia and reperfusion injury: cytoprotection with heme oxygenase system. Transpl Immunol. 2002; 9: 227–233.[CrossRef][Medline] [Order article via Infotrieve]
  26. Zhang X, Shan P, Jiang D, Noble PW, Abraham NG, Kappas A, Lee PJ. Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis. J Biol Chem. 2004; 279: 10677–10684.[Abstract/Free Full Text]
  27. Tsuchihashi S, Fondevila C, Kupiec-Weglinski JW. Heme oxygenase system in ischemia and reperfusion injury. Ann Transplant. 2004; 9: 84–87.[Medline] [Order article via Infotrieve]
  28. Maines MD, Mayer RD, Ewing JF, McCoubrey WK Jr. Induction of kidney heme oxygenase-1 (HSP32) mRNA and protein by ischemia/reperfusion: possible role of heme as both promotor of tissue damage and regulator of HSP32. J Pharmacol Exp Ther. 1993; 264: 457–462.[Abstract/Free Full Text]
  29. Bainbridge SA, Smith GN. HO in pregnancy. Free Radic Biol Med. 2005; 38: 979–988.[CrossRef][Medline] [Order article via Infotrieve]
  30. Alexandreanu IC, Lawson DM. Effects of chronic administration of a heme oxygenase substrate or inhibitor on progression of the estrous cycle, pregnancy and lactation of Sprague-Dawley rats. Life Sci. 2002; 72: 153–162.[CrossRef][Medline] [Order article via Infotrieve]
  31. Zenclussen AC, Fest S, Joachim R, Klapp BF, Arck PC. Introducing a mouse model for pre-eclampsia: adoptive transfer of activated Th1 cells leads to pre-eclampsia-like symptoms exclusively in pregnant mice. Eur J Immunol. 2004; 34: 377–387.[CrossRef][Medline] [Order article via Infotrieve]
  32. Acevedo CH, Ahmed A. Hemeoxygenase-1 inhibits human myometrial contractility via carbon monoxide and is upregulated by progesterone during pregnancy. J Clin Invest. 1998; 101: 949–955.[Medline] [Order article via Infotrieve]
  33. Hendler I, Baum M, Kreiser D, Schiff E, Druzin M, Stevenson DK, Seidman DS. End-tidal breath carbon monoxide measurements are lower in pregnant women with uterine contractions. J Perinatol. 2004; 24: 275–278.[CrossRef][Medline] [Order article via Infotrieve]
  34. Baum M, Schiff E, Kreiser D, Dennery PA, Stevenson DK, Rosenthal T, Seidman DS. End-tidal carbon monoxide measurements in women with pregnancy-induced hypertension and preeclampsia. Am J Obstet Gynecol. 2000; 183: 900–903.[CrossRef][Medline] [Order article via Infotrieve]
  35. Kreiser D, Baum M, Seidman DS, Fanaroff A, Shah D, Hendler I, Stevenson DK, Schiff E, Druzin ML. End tidal carbon monoxide levels are lower in women with gestational hypertension and pre-eclampsia. J Perinatol. 2004; 24: 213–217.[CrossRef][Medline] [Order article via Infotrieve]
  36. Barber A, Robson SC, Myatt L, Bulmer JN, Lyall F. Heme oxygenase expression in human placenta and placental bed: reduced expression of placenta endothelial HO-2 in preeclampsia and fetal growth restriction. FASEB J. 2001; 15: 1158–1168.[Abstract/Free Full Text]
  37. Lash GE, McLaughlin BE, MacDonald-Goodfellow SK, Smith GN, Brien JF, Marks GS, Nakatsu K, Graham CH. Relationship between tissue damage and heme oxygenase expression in chorionic villi of term human placenta. Am J Physiol Heart Circ Physiol. 2003; 284: H160–H167.[Abstract/Free Full Text]
  38. Zenclussen AC, Lim E, Knoeller S, Knackstedt M, Hertwig K, Hagen E, Klapp BF, Arck PC. Heme oxygenases in pregnancy II: HO-2 is downregulated in human pathologic pregnancies. Am J Reprod Immunol. 2003; 50: 66–76.[CrossRef][Medline] [Order article via Infotrieve]
  39. Bussolati B, Dunk C, Grohman M, Kontos CD, Mason J, Ahmed A. Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide. Am J Pathol. 2001; 159: 993–1008.[Abstract/Free Full Text]
  40. Otterbein LE, Kolls JK, Mantell LL, Cook JL, Alam J, Choi AM. Exogenous administration of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury. J Clin Invest. 1999; 103: 1047–1054.[Medline] [Order article via Infotrieve]
  41. Ahmad S, Hewett PW, Wang P, Al-Ani B, Cudmore M, Fujisawa T, Haigh JJ, le Noble F, Wang L, Mukhopadhyay D, Ahmed A. Direct evidence for endothelial vascular endothelial growth factor receptor-1 function in nitric oxide-mediated angiogenesis. Circ Res. 2006; 99: 715–722.[Abstract/Free Full Text]
  42. Smith G, Dawe RS, Clark C, Evans AT, Comrie MM, Wolf CR, Ferguson J, Ibbotson SH. Quantitative real-time reverse transcription-polymerase chain reaction analysis of drug metabolizing and cytoprotective genes in psoriasis and regulation by ultraviolet radiation. J Invest Dermatol. 2003; 121: 390–398.[CrossRef][Medline] [Order article via Infotrieve]
  43. Kweon MH, Adhami VM, Lee JS, Mukhtar H. Constitutive overexpression of Nrf2-dependent heme oxygenase-1 in A549 cells contributes to resistance to apoptosis induced by epigallocatechin 3-gallate. J Biol Chem. 2006; 281: 33761–33772.[Abstract/Free Full Text]
  44. Poss KD, Tonegawa S. Heme oxygenase 1 is required for mammalian iron reutilization. Proc Natl Acad Sci U S A. 1997; 94: 10919–10924.[Abstract/Free Full Text]
  45. Poss KD, Tonegawa S. Reduced stress defense in heme oxygenase 1-deficient cells. Proc Natl Acad Sci U S A. 1997; 94: 10925–10930.[Abstract/Free Full Text]
  46. Ahmed A, Dunk C, Ahmad S, Khaliq A. Regulation of placental vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) and soluble Flt-1 by oxygen: a review. Placenta. 2000; 21 (suppl A): S16–S24.[CrossRef][Medline] [Order article via Infotrieve]
  47. Ahmad S, Ahmed A. Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeclampsia. Circ Res. 2004; 95: 884–891.[Abstract/Free Full Text]
  48. Zhou Y, McMaster M, Woo K, Janatpour M, Perry J, Karpanen T, Alitalo K, Damsky C, Fisher SJ. Vascular endothelial growth factor ligands and receptors that regulate human cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome. Am J Pathol. 2002; 160: 1405–1423.[Abstract/Free Full Text]
  49. Baker PN, Krasnow J, Roberts JM, Yeo KT. Elevated serum levels of vascular endothelial growth factor in patients with preeclampsia. Obstet Gynecol. 1995; 86: 815–821.[Abstract]
  50. Hornig C, Barleon B, Ahmad S, Vuorela P, Ahmed A, Weich HA. Release and complex formation of soluble VEGFR-1 from endothelial cells and biological fluids. Lab Invest. 2000; 80: 443–454.[Medline] [Order article via Infotrieve]
  51. Motterlini R, Clark JE, Foresti R, Sarathchandra P, Mann BE, Green CJ. Carbon monoxide–releasing molecules: characterization of biochemical and vascular activities. Circ Res. 2002; 90: E17–E24.[CrossRef][Medline] [Order article via Infotrieve]
  52. Lain KY, Powers RW, Krohn MA, Ness RB, Crombleholme WR, Roberts JM. Urinary cotinine concentration confirms the reduced risk of preeclampsia with tobacco exposure. Am J Obstet Gynecol. 1999; 181 (pt 1): 1192–1196.[CrossRef][Medline] [Order article via Infotrieve]
  53. Powers RW, Roberts JM, Cooper KM, Gallaher MJ, Frank MP, Harger GF, Ness RB. Maternal serum soluble fms-like tyrosine kinase 1 concentrations are not increased in early pregnancy and decrease more slowly postpartum in women who develop preeclampsia. Am J Obstet Gynecol. 2005; 193: 185–191.[CrossRef][Medline] [Order article via Infotrieve]
  54. England LJ, Levine RJ, Mills JL, Klebanoff MA, Yu KF, Cnattingius S. Adverse pregnancy outcomes in snuff users. Am J Obstet Gynecol. 2003; 189: 939–943.[CrossRef][Medline] [Order article via Infotrieve]
  55. Bainbridge SA, Belkacemi L, Dickinson M, Graham CH, Smith GN. Carbon monoxide inhibits hypoxia/reoxygenation-induced apoptosis and secondary necrosis in syncytiotrophoblast. Am J Pathol. 2006; 169: 774–783.[Abstract/Free Full Text]
  56. Appleton SD, Marks GS, Nakatsu K, Brien JF, Smith GN, Graham CH. Heme oxygenase activity in placenta: direct dependence on oxygen availability. Am J Physiol Heart Circ Physiol. 2002; 282: H2055–H2059.[Abstract/Free Full Text]
  57. Newby D, Cousins F, Myatt L, Lyall F. Heme oxygenase expression in cultured human trophoblast cells during in vitro differentiation: effects of hypoxia. Placenta. 2005; 26: 201–209.[CrossRef][Medline] [Order article via Infotrieve]
  58. Hung TH, Charnock-Jones DS, Skepper JN, Burton GJ. Secretion of tumor necrosis factor-alpha from human placental tissues induced by hypoxia-reoxygenation causes endothelial cell activation in vitro: a potential mediator of the inflammatory response in preeclampsia. Am J Pathol. 2004; 164: 1049–1061.[Abstract/Free Full Text]
  59. Tsuchihashi S, Livhits M, Zhai Y, Busuttil RW, Araujo JA, Kupiec-Weglinski JW. Basal rather than induced heme oxygenase-1 levels are crucial in the antioxidant cytoprotection. J Immunol. 2006; 177: 4749–4757.[Abstract/Free Full Text]
  60. Yoneyama Y, Suzuki S, Sawa R, Yoneyama K, Power GG, Araki T. Relation between adenosine and T-helper 1/T-helper 2 imbalance in women with preeclampsia. Obstet Gynecol. 2002; 99: 641–646.[Abstract/Free Full Text]
  61. Wilczynski JR, Tchorzewski H, Banasik M, Glowacka E, Wieczorek A, Lewkowicz P, Malinowski A, Szpakowski M, Wilczynski J. Lymphocyte subset distribution and cytokine secretion in third trimester decidua in normal pregnancy and preeclampsia. Eur J Obstet Gynecol Reprod Biol. 2003; 109: 8–15.[CrossRef][Medline] [Order article via Infotrieve]
  62. Arriaga-Pizano L, Jimenez-Zamudio L, Vadillo-Ortega F, Martinez-Flores A, Herrerias-Canedo T, Hernandez-Guerrero C. The predominant Th1 cytokine profile in maternal plasma of preeclamptic women is not reflected in the choriodecidual and fetal compartments. J Soc Gynecol Investig. 2005; 12: 335–342.[Medline] [Order article via Infotrieve]
  63. Azizieh F, Raghupathy R, Makhseed M. Maternal cytokine production patterns in women with pre-eclampsia. Am J Reprod Immunol. 2005; 54: 30–37.[CrossRef][Medline] [Order article via Infotrieve]
  64. Banerjee S, Smallwood A, Moorhead J, Chambers AE, Papageorghiou A, Campbell S, Nicolaides K. Placental expression of interferon-gamma (IFN-gamma) and its receptor IFN-gamma R2 fail to switch from early hypoxic to late normotensive development in preeclampsia. J Clin Endocrinol Metab. 2005; 90: 944–952.[Abstract/Free Full Text]
  65. Naldini A, Carraro F, Silvestri S, Bocci V. Hypoxia affects cytokine production and proliferative responses by human peripheral mononuclear cells. J Cell Physiol. 1997; 173: 335–342.[CrossRef][Medline] [Order article via Infotrieve]
  66. Kitamuro T, Takahashi K, Ogawa K, Udono-Fujimori R, Takeda K, Furuyama K, Nakayama M, Sun J, Fujita H, Hida W, Hattori T, Shirato K, Igarashi K, Shibahara S. Bach1 functions as a hypoxia-inducible repressor for the heme oxygenase-1 gene in human cells. J Biol Chem. 2003; 278: 9125–9133.[Abstract/Free Full Text]
  67. Hegazi RA, Rao KN, Mayle A, Sepulveda AR, Otterbein LE, Plevy SE. Carbon monoxide ameliorates chronic murine colitis through a heme oxygenase 1-dependent pathway. J Exp Med. 2005; 202: 1703–1713.[Abstract/Free Full Text]
  68. Rumbold AR, Crowther CA, Haslam RR, Dekker GA, Robinson JS. Vitamins C and E and the risks of preeclampsia and perinatal complications. N Engl J Med. 2006; 354: 1796–1806.[Abstract/Free Full Text]
  69. Poston L, Briley AL, Seed PT, Kelly FJ, Shennan AH. Vitamin C and vitamin E in pregnant women at risk for pre-eclampsia (VIP trial): randomised placebo-controlled trial. Lancet. 2006; 367: 1145–1154.[CrossRef][Medline] [Order article via Infotrieve]
  70. Lee TS, Chang CC, Zhu Y, Shyy JY. Simvastatin induces heme oxygenase-1: a novel mechanism of vessel protection. Circulation. 2004; 110: 1296–1302.[Abstract/Free Full Text]
  71. Grosser N, Hemmerle A, Berndt G, Erdmann K, Hinkelmann U, Schurger S, Wijayanti N, Immenschuh S, Schroder H. The antioxidant defense protein heme oxygenase 1 is a novel target for statins in endothelial cells. Free Radic Biol Med. 2004; 37: 2064–2071.[CrossRef][Medline] [Order article via Infotrieve]
  72. Kodama Y, Kitta Y, Nakamura T, Takano H, Umetani K, Fujioka D, Saito Y, Kawabata K, Obata JE, Mende A, Kobayashi T, Kugiyama K. Atorvastatin increases plasma soluble Fms-like tyrosine kinase-1 and decreases vascular endothelial growth factor and placental growth factor in association with improvement of ventricular function in acute myocardial infarction. J Am Coll Cardiol. 2006; 48: 43–50.[Abstract/Free Full Text]
  73. Minsker DH, MacDonald JS, Robertson RT, Bokelman DL. Mevalonate supplementation in pregnant rats suppresses the teratogenicity of mevinolinic acid, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme a reductase. Teratology. 1983; 28: 449–456.[CrossRef][Medline] [Order article via Infotrieve]
  74. Freyssinges C, Ducrocq MB. Simvastatin and pregnancy [in French]. Therapie. 1996; 51: 537–542.[Medline] [Order article via Infotrieve]
  75. Gibb H, Scialli AR. Statin drugs and congenital anomalies. Am J Med Genet A. 2005; 135: 230–231.[Medline] [Order article via Infotrieve]
  76. Manson JM, Freyssinges C, Ducrocq MB, Stephenson WP. Postmarketing surveillance of lovastatin and simvastatin exposure during pregnancy. Reprod Toxicol. 1996; 10: 439–446.[CrossRef][Medline] [Order article via Infotrieve]
  77. Pollack PS, Shields KE, Burnett DM, Osborne MJ, Cunningham ML, Stepanavage ME. Pregnancy outcomes after maternal exposure to simvastatin and lovastatin. Birth Defects Res A Clin Mol Teratol. 2005; 73: 888–896.[CrossRef][Medline] [Order article via Infotrieve]
  78. Wang YP, Yu YH. Expression of endogenous heme oxygenase on surface of placental trophoblasts of pregnant women with intrauterine growth retardation of the fetus. Di Yi Jun Yi Da Xue Xue Bao. 2002; 22: 637–639.[Medline] [Order article via Infotrieve]
 

CLINICAL PERSPECTIVE

Preeclampsia is a maternal systemic disorder with a clinical presentation of hypertension and proteinuria after 20 weeks’ gestation that affects 3% to 8% of all pregnancies. Women with a history of preeclampsia are at an increased risk of developing cardiovascular disease later in life. Long-term risk for cardiovascular disease also is augmented in the offspring of preeclamptic women. Despite the many theories for preeclampsia, the delineation of the mechanism of action for potential therapeutic agents has been lacking. Because the only effective treatment for preeclampsia resolution is the delivery of the placenta, it is generally accepted that placental factors that inhibit the normal function of the endothelium are involved. Recent retrospective cross-sectional studies demonstrated that 2 anti-angiogenic factors, soluble Flt-1 and soluble endoglin, are elevated in the sera of preeclamptic women weeks before the clinical onset of the disease. In the present study, we have identified heme oxygenase-1, a cytoprotective enzyme, as the key component in suppressing the release of soluble Flt-1 and soluble endoglin from the placenta. We also show that treatment of endothelial cells with statins upregulated HO-1 and inhibited the release of soluble Flt-1, whereas vitamins C and E had no effect, providing a possible explanation for the failure of the recent antioxidant vitamin supplementation trials with vitamins C and E to prevent preeclampsia in women at risk. Our study opens a new avenue of investigation for increasing our understanding of the origin of preeclampsia and provides a novel therapeutic target in placental-based pregnancy complications.


*    Footnotes
 
The online-only Data Supplement, consisting of a figure, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.660134/DC1.




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