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TRANSCRIPT
PROTON PUMP INHIBITORS DECREASE SFLT-1 AND SOLUBLE
ENDOGLIN SECRETION, DECREASE HYPERTENSION AND RESCUE
ENDOTHELIAL DYSFUNCTION
Authors
Kenji Onda1,2* PhD, Stephen Tong1* PhD, Sally Beard1 BSc, Natalie Binder1 PhD, Masanaga
Muto3, Sevvandi N Senadheera4 PhD, Laura Parry4 PhD, Mark Dilworth5,6 PhD, Lewis
Renshall5,6 BSc, Fiona Brownfoot1 MBBS, Roxanne Hastie1 BSc, Laura Tuohey1 BSc, Kirsten
Palmer1 PhD, Toshihiko Hirano2, Masahito Ikawa3 PhD, Tu'uhevaha Kaitu'u-Lino1 PhD, and
Natalie J Hannan1 PhD.
1Translational Obstetrics Group, Department of Obstetrics and Gynaecology, Mercy Hospital
for Women, University of Melbourne, Heidelberg, Victoria, Australia, 3084; 2Department of
Clinical Pharmacology, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo,
Japan; 3Research Institute for Microbial Diseases, Osaka University, Japan; 4School of
Biosciences, University of Melbourne, Parkville, Victoria, Australia. 5Maternal and Fetal
Health Research Centre, Institute of Human Development, University of Manchester, 6 St
Mary's Hospital, Central Manchester University Hospitals NHS Trust, Manchester Academic
Health Science Centre, Manchester, United Kingdom.
* These authors contributed equally to this work.
Short title: Proton pump inhibitors to treat preeclampsia.
Word count: 6993
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Corresponding author: Professor Stephen Tong, Translational Obstetrics Group, Department
of Obstetrics and Gynaecology, University of Melbourne; Mercy Hospital for Women,
Heidelberg, 3084 Victoria, Australia. Ph: +61-3-8458 4377 Fax: +31-3-8458 4380. Email:
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Abstract
Preeclampsia is a severe complication of pregnancy. Anti-angiogenic factors soluble fms-like
tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG) are secreted in excess from the
placenta, causing hypertension, endothelial dysfunction, and multi-organ injury. Oxidative
stress and vascular inflammation exacerbate the endothelial injury. A drug that can block these
pathophysiological steps would be an attractive treatment option. Proton pump inhibitors
(PPIs) are safe in pregnancy where they are prescribed for gastric reflux. We performed
functional studies on primary human tissues and animal models to examine the effects of PPIs
on sFlt-1 and sENG secretion, vessel dilatation, blood pressure and endothelial dysfunction.
PPIs decreased sFlt-1 and sENG secretion from trophoblast, placental explants from
preeclamptic pregnancies, and endothelial cells. They also mitigated tumor necrosis factor-α
induced endothelial dysfunction: PPIs blocked endothelial Vascular Cell Adhesion Molecule-1
expression, leukocyte adhesion to endothelium and disruption of endothelial tube formation.
PPIs decreased Endothelin-1 secretion and enhanced endothelial cell migration. Interestingly,
the PPI esomeprazole vasodilated maternal blood vessels from normal pregnancies and cases
of preterm preeclampsia, but its vasodilatory effects were lost when the vessels were denuded
of their endothelium. Esomeprazole decreased blood pressure in a transgenic mouse model
where human sFlt-1 was over-expressed in placenta. PPIs up-regulated endogenous anti-
oxidant defenses, and decreased cytokine secretion from placental tissue and endothelial cells.
We have found PPIs decrease sFlt-1 and sENG secretion and endothelial dysfunction, dilate
blood vessels, decrease blood pressure, and have anti-oxidant and anti-inflammatory
properties. They have therapeutic potential for preeclampsia and other diseases where
endothelial dysfunction is involved.
Keywords: Preeclampsia, treatment, proton pump inhibitors, endothelial dysfunction,
hypertension, pregnancy
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INTRODUCTION
Preeclampsia affects 3-8% of pregnancies and is a leading cause of mortality during
pregnancy. It is globally responsible for 60,000 deaths annually, and far greater rates of fetal
losses1. Placental factors are released in excess into the maternal circulation, causing
hypertension and endothelial dysfunction. This leads to multi-system organ injury to the
maternal kidneys, liver, haematological system, and the brain (causing eclamptic seizures)1.
The only treatment for preeclampsia is delivery of the placenta as this removes the source of
placental derived factors responsible for the maternal organ injury1, 2. In preterm preeclampsia,
clinicians are often forced to deliver the fetus preterm to save the mother. However, this
inflicts iatrogenic prematurity, which can lead to cerebral palsy, chronic disability or death. A
treatment that quenches the severity of the maternal disease could allow these pregnancies to
safely advance to a gestation where neonatal outcomes are significantly improved. Severe
preeclampsia at any gestation can progress rapidly over hours and become life-threatening.
Therefore, a treatment that reduces the severity of preeclampsia at any gestation could
improve clinical outcomes.
Factors released in excess from the placenta in preeclampsia thought to play an important role
in causing endothelial dysfunction (and subsequent maternal organ injury) are the anti-
angiogenic factors soluble fms-like tyrosine kinase 1 (sFlt-1)3, 4 and soluble endoglin (sENG)1,
4-6. sFlt-1 is a splice variant of vascular endothelial growth factor (VEGF) receptor 1 and
comprises only the extracellular domain3, 4. sFlt-1 binds and sequesters circulating VEGF,
decreasing VEGF signaling and its ability to promote vascular homeostasis5. Furthermore,
recent evidence suggests sFlt-1 may induce hypertension by impairing endothelial nitric oxide
synthase (eNOS) phosphorylation and increasing angiotensin II sensitivity 7. sENG shares
sequence homology with the extracellular domain of full-length membrane bound endoglin, a
co-receptor which facilitates transforming growth factor receptor-β1, and transforming growth
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factor receptor-β3 signaling in endothelial cells. Like sFlt-1, sENG also antagonizes
endothelial receptor signaling by competing with full-length endoglin, decreasing its ability to
bind with its co-receptors to maintain vessel homeostasis.
The evidence linking sFlt-1 and sENG with the pathophysiology of preeclampsia is strong3-5.
Circulating levels are significantly elevated in women with preeclampsia and levels correlate
with disease severity 8. Administering sFlt-1 to rats causes hypertension and proteinuria,
hallmarks of preeclampsia 3. Adenoviral co-administration of both sFlt-1 and sENG to rats
recapitulate the full spectrum of multi organ injury seen in preeclampsia, including severe
proteinuria, thrombocytopenia, elevated liver enzymes and fetal growth restriction9.
Preeclampsia is also associated with placental and systemic oxidative stress 2, 10 and
intravascular inflammation2, which are thought to exacerbate the endothelial injury.
Therefore, an attractive candidate therapeutic for preeclampsia may be a drug that is
considered safe in pregnancy and can do the following: 1) decrease sFlt-1 and sENG secretion
2) decrease blood pressure 3) decrease endothelial dysfunction 4) up-regulate endogenous
anti-oxidant defenses and 5) decrease secretion of inflammatory cytokines. As far as we are
aware, no such drug has been reported.
Proton pump inhibitors (PPIs) are widely prescribed during pregnancy for symptomatic
gastric reflux. Large epidemiological studies have shown that they are safe in pregnancy, even
when administered during the first trimester11, 12.
It was reported that heme-oxygenase-1 (HO-1) may negatively regulate sFlt-1 secretion13.
Noting that it has also been reported that the PPI lansoprazole up-regulates HO-1 in gastric
mucosa 14, we hypothesized that PPIs might up-regulate HO-1, which should then decrease
sFlt-1 and sENG secretion. In this study, we report functional experiments on primary human
tissues and mouse models that show PPIs have diverse actions that may mitigate the
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pathophysiology underlying preeclampsia. Our data identifies PPIs as candidate therapeutics
for preeclampsia.
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METHODS
For detailed methods, please see supplementary methods
Methods overview
We performed functional experiments where we administered proton pump inhibitors to
primary human tissues or cells, and assessed their effects on sFlt-1 and sENG secretion,
endothelial dysfunction (using in vitro assays), whole vessel dilatation, and secretion of
cytokines. To perform these experiments, we examined primary tissues from placenta
(isolated cytotrophoblast cells or placental explant tissue from women diagnosed with
preeclampsia), endothelial cells (Human Umbilical Vein Endothelial Cells (HUVECs) and
uterine microvascular cells) and whole vessels isolated from the omentum (maternal fatty
apron tissue). All experiments were run with technical triplicates and each experiment
repeated a minimum of three times (using samples from different patients). We also
performed in vivo experiments where we administered esomeprazole to a mouse model of
preeclampsia (where sFlt-1 is overexpressed specifically in the placenta), and pregnant eNOS
-/- mice.
We obtained ethical approval to perform these studies. All women provided written informed
consent.
Statistical analysis. All in vitro experiments were performed with technical triplicates and all
experiments were repeated a minimum of three times. Data was tested for normal distribution
and statistically tested as appropriate. When three or more groups were compared a One-way
ANOVA (for parametric data) or Kruskal-Wallis test (for non-parametric data) followed by
multiple comparison with Dunnet or Bonferroni ad-hoc test where appropriate or post-hoc
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analysis using either the Tukey (parametric) or Dunn’s test (non-parametric). When two
groups were analysed, either an unpaired t-test (parametric) or a Mann-Whitney test (non-
parametric) was used. Data is expressed as mean fold change from control ±SEM.
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RESULTS
Proton pump inhibitors decrease sFlt-1 expression and secretion from placenta and
endothelium
In preeclampsia, excess secretion of sFlt-1 is thought to cause hypertension, endothelial
dysfunction and subsequent maternal organ injury. Administering PPIs (lansoprazole,
rabeprazole and esomeprazole) to primary trophoblast cells dose-dependently reduced sFlt-1
secretion (Fig. 1A, this ELISA detects all sFlt-1 variants) and reduced mRNA expression of
the two major sFlt-1 variants present in placenta; sFlt-1 e15a and sFlt-1 i13 (Supplementary
Fig. S1A,B). They also reduced sFlt-1 secretion from placental explants obtained from normal
(Supplementary Figure S1C) and preeclamptic pregnancies (Figure 1B, see Supplementary
Table 1 for clinical information). PPIs also decreased sFlt-1 e15a protein secretion from
preeclamptic placental explants (Figure 1C). sFlt-1 e15a (or sFlt-1 v14) is the main sFlt-1
variant in placenta15 that has only been described quite recently15, 16.
Endothelium is another major tissue source of sFlt-1. When administered to human umbilical
vein endothelial cells (HUVECs), PPIs dose-dependently reduced sFlt-1 secretion (Figure 1D)
mRNA expression (Supplementary Figure S1D, E) of the two sFlt-1 variants, sFlt-1 e15a and
sFlt-1 i13. When we compared five PPIs, we found esomeprazole and rabeprazole were the
most potent in reducing sFlt-1 secretion from HUVECs (Figure 1E). PPIs also decreased sFlt-
1 secretion from primary uterine microvascular cells that, unlike HUVECs, are a mix of
venous and arterial endothelium (Supplementary Figure S1F). Placental hypoxia increases
sFlt-1 secretion, and there is evidence its release may be regulated by Hypoxia Inducible
factor-1α (HIF-1α)17, 18. PPIs markedly decreased HIF-1α protein expression (Figure 1F),
raising the possibility their ability to decrease sFlt-1 secretion may be mediated through HIF-
1α.
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Proton pump inhibitors decrease sENG expression and secretion from placenta and
endothelium
Soluble endoglin (sENG) is the second major anti-angiogenic factor released in excess from
the placenta in preeclampsia, and may play a role in severe disease5, 8, 9. PPIs reduced sENG
secretion from primary trophoblast (Figure 2A), HUVECs (Figure 2B), uterine microvascular
cells (Supplementary Figure S2A) and placental explants from cases of preterm preeclampsia
(Figure 2C). When we compared the ability of five PPIs to reduce sENG secretion from
HUVECs, we identified rabeprazole and esomeprazole were the most potent (Figure 2D).
Interestingly, PPIs also induced pro-angiogenic vegfa mRNA expression in primary
trophoblast (Figure 2E) and HUVECs (Supplementary Figure S2B), but did not up-regulate
its family member, placental growth factor (not shown).
At the same doses used in our PPI experiments (up to 100 M) pravastatin (proposed as a
treatment for preeclampsia19, 20) did not significantly affect sFlt-1 or sENG secretion from
HUVECs (Supplementary Figure S2C-D). We conclude PPIs reduce secretion of sFlt-1 and
sENG from primary placental and endothelial cells, and increase both placental and
endothelial vegfa mRNA expression.
The decrease in sFlt-1 and sENG secretion observed with proton pump inhibitors is not
explained by inhibition of V-ATPase.
We next set out to examine possible molecular pathways to explain how PPIs may be
inhibiting sFlt-1 and sENG secretion. As a medication for gastric reflux, PPIs were
specifically designed to inhibit H+/K+ ATPase V-ATPase (a proton pump in the gastric
epithelium) to decrease gastric acidity. Of note, the V-ATPase enzyme is also present in
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placenta 21-23. To examine whether the reduction in sFlt-1 and sENG secretion observed with
PPI treatment is mediated through V-ATPase inhibition, we examined the effects of blocking
V-ATPase by administering bafilomycin, a potent V-ATPase inhibitor 24. Indeed, bafilomycin
dose-dependently decreased sFlt-1 secretion by human trophoblast (Supplementary Figure
S3A). However, bafilomycin did not decrease intracellular levels of sFlt-1 protein, or the
mRNA expression of the sFlt-1 variants (sFlt-1 i13 and sFlt-1 e15a; Supplementary Figure
S3B-D). In contrast, we confirmed esomeprazole (added as a positive control in the same
experiment) significantly decreased sFlt-1 and mRNA expression of the two sFlt-1 variants.
Furthermore, bafilomycin did not decrease sFlt-1 secretion from HUVECs, and in fact
significantly increased sENG secretion from HUVECs (Supplementary Figure S3E-F). Given
blocking V-ATPase with bafilomycin did not decrease intracellular sFlt-1 protein or mRNA
expression, or consistently reduce sFlt-1 and sEng secretion, it is unlikely that PPIs decrease
the production of these anti-angiogenic factors through V-ATPase.
Reduced sFlt-1 and sENG secretion with proton pump inhibitors is not explained by
altered expression of cargo export proteins.
ADP-ribosylation factor 1 (ARF1) and Ras-related Protein 11 (RAB11) are proteins involved
in the secretion and transport of proteins from cells. Given Jung et al 25 previously concluded
these proteins facilitate sFlt-1 secretion from cells, we examined whether PPIs may decrease
sFlt-1 secretion by inhibiting the expression of these proteins. Treating trophoblast with
esomeprazole in fact increased ARF1 expression (Supplementary Figure S4A-B) and did not
affect RAB11 expression (Supplementary Figure 4 SC-D). Thus, it seems unlikely that these
molecules are involved in the decrease in sFlt-1 secretion induced by PPIs.
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Proton pump inhibitors vasodilate whole human vessels ex vivo and decrease blood
pressure in an animal model of preeclampsia.
Vasoconstriction and hypertension are hallmarks of preeclampsia. We next examined whether
PPIs have vasoactive properties. We isolated maternal omental arteries obtained at caesarean
section from normal and preeclamptic women and performed pressure myography ex vivo. We
first induced vasoconstriction by incubating arteries in U46619 (thromboxane agonist -
thromboxane is elevated in preeclampsia26), then added cumulative doses of esomeprazole.
Esomeprazole caused potent dilation of arteries from both preeclamptic and normal
pregnancies (Figure 3A-B, see Supplementary Table 2 for clinical characteristics of the
preeclampsia cohort). The vasodilatory effects of esomeprazole appeared to be mediated
through the endothelium, since vessels denuded of the endothelium did not dilate in response
to esomeprazole (Figure 3C). To obtain in vivo evidence that PPIs are vasoactive, we added
esomeprazole to a transgenic preeclampsia mouse model where human sFlt-1 is
overexpressed specifically in the placenta27. As expected, a significant increase in blood
pressure (both systolic and diastolic) was observed in untreated mice from E16.527. However,
daily administration of 150ug of esomeprazole (which approximately equates to a 30 mg dose
in humans) from E8.5 completely abrogated this increase in blood pressure (Figure 3D).
There was a non-significant trend towards decreased maternal proteinuria in mice treated with
esomeprazole compared to controls, and there were no differences in measurements of various
fetal or placental parameters (Supplementary Figure S5A-E). There was also a non-significant
trend towards a decrease in the blood pressure of non-pregnant female mice administered
esomeprazole compared to mice receiving vehicle (Supplementary Figure S5F). We
confirmed the presence of human sFlt-1 in the mouse serum obtained at the time of sacrifice
(Supplementary Figure S5G). The fact that was no difference in sFlt-1 levels in this animal
model given sFlt-1 is expected given it is autonomously expressed by the lentiviral plasmid.
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Proton pump inhibitors may be promoting vasodilation by modulating eNOS and
endothelin-1 expression
Endothelial nitric oxide synthase (eNOS) is an enzyme in the endothelium that produces
Nitric Oxide (NO), a potent vasodilator that signals to the underlying vascular smooth muscle.
Furthermore, it has recently been shown that sFlt-1 may be directly inducing hypertension in
preeclampsia by impairing eNOS phosphorylation7. We examined whether PPIs may be
inducing vasorelaxation via the eNOS pathway. When we treated HUVECs with
esomeprazole (Figure 4A) there was a significant increase in phosphorylated eNOS (p-eNOS,
the active form). Co-treatment of Tumour Necrosis Factor-α (TNF-α; a pro-inflammatory
cytokine that circulates in excess in preeclampsia28 and likely contributor to endothelial
dysfunction29) with esomeprazole and rabeprazole at different doses all induced a non-
significant increase in p-eNOS protein (Supplementary Figure S6A-B). This data suggests the
PPIs up-regulate p-eNOS expression. To obtain further evidence for this, we next
administered esomeprazole to pregnant mice lacking eNOS (eNOS-/-). These mice are
chronically hypertensive, which persists while they are pregnant30. In contrast to the striking
decrease in blood pressure seen in wild-type mice where sFlt-1 was over-expressed in
placenta (Figure 3D), there was only a modest and non-significant decrease in blood pressure
with esomeprazole treatment when the eNOS gene is absent (Figure 4B). Our data suggests
esomeprazole has vasodilatory properties and can decrease blood pressure in vivo (animal
model) and ex vivo (whole maternal vessels from women with preeclampsia). Furthermore,
this may be mediated through the endothelium, where p-eNOS may play a role.
Endothelin-1 is potent vasoconstrictor released from the endothelium into the circulation and
is increased in preeclampsia31. Administering PPIs to HUVECs reduced endothelin-1 mRNA
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expression (Figure 4C) and protein secretion (Figure 4D). PPIs also decreased endothelin-1
mRNA expression in uterine microvascular endothelial cells (Supplementary Figure S6C).
Proton pump inhibitors rescue endothelial dysfunction in vitro
Given the importance of endothelial dysfunction in preeclampsia 2, 5, another therapeutic
strategy may be to identify drugs that act at the level of the endothelium to reduce
dysfunction. PPIs potently blocked TNF-α-induced up-regulation of vascular cell adhesion
molecule-1 (VCAM-1) in HUVECs (Figure 5A, Supplementary Figure S7A) and primary
uterine microvascular cells (Supplementary Figure S7B). PPIs also decreased VCAM-1
expression in HUVECs when preeclamptic serum was used instead of TNF-α to induce
endothelial dysfunction (Supplementary Figure S7C).
Given VCAM-1 promotes leukocyte adhesion to the endothelium, we performed a monocyte
adhesion assay. PPIs dose-dependently decreased TNF-α-induced leukocyte adhesion to
HUVECs (Figure 5B). We next performed endothelial tube forming experiments and found
esomeprazole rescued TNF-α-induced tube disruption (Figure 5C). Additionally, we examined
endothelial cell migration using the xCELLigence assay, which allows cell migration to be
continuously monitored in real time. Treating HUVECs with sFlt-1 reduced cell migration but
this was rescued with the addition of either esomeprazole or lansoprazole (Figure 5D).
Therefore, we found PPIs rescued endothelial dysfunction in multiple in vitro assays.
Proton pump inhibitors up-regulate expression of endogenous anti-oxidant proteins
Excessive oxidative stress and inflammation is likely to exacerbate the placental and
endothelial dysfunction that occurs in preeclampsia10. Therefore, reducing oxidative stress and
inflammation may help decrease disease severity. Administering PPIs to primary trophoblast
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cells induced nuclear translocation of the transcription factor, nuclear factor (erythroid-
derived 2)-like 2 (Nrf2), to the nucleus (Figure 6A). One of the targets of Nrf2 is heme
oxygenase-1 (HO-1), which has anti-oxidant and cytoprotective actions32. PPIs potently
induced HO-1 expression in primary trophoblast (Figure 6B, Supplementary Figure S8A),
placental explants from women with preterm preeclampsia (Figure 6C), HUVECs (Figure 6D,
Supplementary Figure S8B) and uterine microvascular cells (Supplementary Figure S8C).
They also up-regulate NAD(P)H dehydrogenase Quinone 1 (NQO1) and thioredoxin (TXN)
in HUVECs and trophoblasts, both anti-oxidant molecules that are Nrf2 targets
(Supplementary Figure S8D-E). These data demonstrate PPIs up-regulate endogenous anti-
oxidant molecules in placenta and endothelium.
Proton pump inhibitors decrease secretion of cytokines from placenta and endothelium
Adding PPIs to placental explants from preterm preeclamptic pregnancies reduced secretion
of cytokines, including Interleukin (IL) IL1-β, IL-6, IL-10, and C-C Motif Chemokine Ligand
(CCL) CCL2 and CCL7 (Supplementary Figure S9A). Furthermore, PPIs reduced secretion
of cytokines from endothelial cells treated with either TNF-α or preeclamptic serum to induce
endothelial dysfunction (Supplementary Figure S9B-C). These data suggest PPIs can decrease
cytokine secretion from placenta and endothelium.
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DISCUSSION
In this study, we have identified PPIs as candidate treatments for preeclampsia. They appear
to have diverse biological actions that could be useful to block important pathophysiological
processes thought to occur in preeclampsia2, 5. PPIs decrease secretion of sFlt-1 and sENG
from primary trophoblast, placental explants from normal and preeclamptic pregnancies, and
from two types of primary endothelial cells. The PPI esomeprazole vasodilates human vessels
from normal and preeclamptic pregnancies, and reduces blood pressure in a transgenic mouse
model where human sFlt-1 is overexpressed in placenta.
The ability of esomeprazole to vasodilate vessels ex vivo and reduce blood pressure in vivo
may be mediated through the endothelium, perhaps by increasing p-eNOS expression. Indeed,
for our preeclampsia animal model we note that sFlt-1 is autonomously expressed in the
placenta meaning it is unlikely PPIs can directly decrease placental sFlt-1 secretion in that
experiment. Furthermore, PPIs appear to block endothelial dysfunction in a number of in vitro
assays. Finally, PPIs up-regulate endogenous anti-oxidant molecules and decrease secretion of
cytokines from placental tissues and endothelial cells.
We believe our study has several strengths. We performed a significant body of functional
experiments on seven types of primary human tissues where we administered a number of
PPIs at various doses. Importantly, we included vessels and placenta from cases of preterm
preeclampsia and biological replicates were performed on samples from different patients.
Hence we believe the functional evidence we have generated is strong.
While we have generated evidence suggesting PPIs may alter molecular signaling involved in
vessel dilation, we have not uncovered a unifying molecular mechanism to explain how PPIs
have such diverse biological effects (ranging from vessel dilatation, decreasing sFlt-1 and
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sENG secretion to decreasing endothelial dysfunction). It is unclear whether PPIs are exerting
these effects by acting on one, or multiple molecular targets.
We have explored relevant pathways to determine the molecular mechanism by which PPIs
decreased sFlt-1 and sENG. Initially we embarked on these studies with the hypothesis that
PPIs may up-regulate HO-114, and may reduce sFlt-1 and sENG secretion given it had
previously been proposed that HO-1 was central to preeclampsia and that HO-1 regulates
sFlt-1 and sENG production13. However, we have since published data demonstrating that
HO-1 is not decreased in preeclampsia and does not regulate placental sFlt-1 or sENG
secretion. As such, we do not believe the effects of sFlt-1 and sENG are mediated by the
ability of PPIs to up-regulate HO-1. Instead, we examined V-ATPase enzyme (given PPIs
were designed to inhibit this proton pump) and the ARF1 and RAB11 cargo proteins (given a
previous report suggesting they shuttle sFlt-1 out of cells). We were unable to show they
mediate the decrease in sFlt-1 and sENG secretion induced by PPIs. We have ongoing studies
to try to determine the molecular mechanisms behind the actions of PPIs, specifically the
decrease in anti-angiogenic factors, given such a discovery might yield new insights into new
drug targets.
Ghebremariam et al33 reported that the PPI omeprazole reduced eNOS and phospho-eNOS
(active eNOS) expression and reduced nitric oxide release from isolated veins. They presented
a qualitative blot (without densitometric analysis) to suggest omeprazole decreased eNOS
protein expression, and experiments demonstrating omeprazole impaired vascular reactivity.
Furthermore, they showed administrating lansoprazole to non-pregnant mice increased
circulating asymmetrical dimethylarginine (antagonist of eNOS), although blood pressure was
not reported. In contrast, we generated a body of data to show the opposite – that PPIs
upregulate p-eNOS and induce vasodilation both ex vivo and in vivo. We administered
different doses of esomeprazole or rabeprazole with, or without TNFα stimulation and in all
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cases there was a significant increase, or a trend toward an increase in phosphorylated eNOS
protein, (quantitative densitometric analysis). Importantly, we also found esomeprazole
potently vasodilated whole maternal blood vessels, and reduced blood pressure in an animal
model of preeclampsia. Finally, we observed a possible trend towards a decrease in blood
pressure when we administered esomeprazole to non-pregnant mice, but there was certainly
no evidence that the blood pressure increased. It is difficult to reconcile our findings with
Ghebremariam et al33. Despite these conflicting findings, we suggest clinical studies to
explore the potential of PPIs to treat preeclampsia are well justified given our preclinical data
suggests PPIs appear to have other biological effects (besides vasorelaxation) that may also be
beneficial in treating preeclampsia and there is strong epidemiological evidence to suggest
PPIs are safe when administered during pregnancy (as discussed below).
We note it is difficult to know how much drug is seen locally at target tissues. However, we
would make some observations. Firstly, the surface area of the target tissues (vessel
endothelium and placenta) is vast and easily accessible to circulating drugs. Secondly, we
would anticipate that some decrease in sFlt-1 and sENG may be sufficient to affect the disease
progression given these factors are present in normal pregnancies. It may not be necessary to
block their secretion entirely. Thirdly, we treated our mouse model (where sFlt-1 was
overexpressed in the placenta) with esomeprazole at a dose that equates to approximately 30
mg daily dosing in humans (using a formula published by Regan Shaw et al 34) and this
completely abrogated an increase in blood pressure. Lastly, we performed multiple dose-
response studies and multiple PPIs to show the various biological responses are present at a
range of doses. Importantly, the doses we used are in the micromolar range, these doses were
chosen since the PPIs are found in the circulation at these concentrations35-37, thus we
anticipate this will be the range that is clinically effective. It may be worthwhile undertaking
further in vitro studies using drug doses within the nanomolar range as these might shed
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further clues as to the mechanisms by which PPIs are exerting the diverse biological effects
we have observed.
Recent data has emerged combining microarray or ultrasound findings with histopathology38
defining further subtypes of preeclampsia, highlighting the heterogeneity of the disease.
Leavey et al39 found specific correlations with microarray data (from large cohorts of
preeclamptic placentas) with clinical findings and propose subclasses of preeclampsia distinct
to the ‘canonical’ disease (which is driven by anti-angiogenic factors). They identified a
cluster of patients they coined ‘immunological preeclampsia’ where the main drivers of the
disease may be immunological 39. It is therefore possible that PPIs might be less effective for
subtypes of preeclampsia where anti-angiogenic factors play less of a role. As the field of
preeclampsia subtyping matures, it may be feasible to stratify response to PPIs treatment in
future clinical trials with preeclampsia subtyping.
There have been very few orally available treatment options identified for preeclampsia that
have reached clinical trials. In regards to treating preeclampsia, the field has perhaps been
most focused on the potential use of pravastatin, a drug designed to inhibit cholesterol
synthesis40. There is preclinical evidence that pravastatin may have been useful to treat
preeclampsia20, 27, 41. A recent clinical trial of pravastatin to treat preterm preeclampsia has
ceased recruitment and the results have yet to be published (ISRCTN23410175). A
pharmacokinetic study of 20 participants has just been reported (NCT01717586) 42. However,
a potential drawback in using pravastatin is that it has been assigned category X classification
by the Food and Drug Administration. While we agree there are valid arguments to support
the use of statins to treat pregnancies complicated by preterm preeclampsia, a drug that is
thought to be safe in pregnancy will be more clinically acceptable.
In contrast to pravastatin, large epidemiological studies have demonstrated PPIs are safe in
pregnancy, where they are widely prescribed to relieve symptomatic gastric reflux in
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pregnancy11, 12. In 2010, Pasternak et al11 examined 1st trimester exposure, a time in pregnancy
where there is significant organogenesis and the fetus is most vulnerable to teratogens.
Examining 5082 pregnancies exposed to PPIs and 840,968 that were not exposed, they
concluded that 1st trimester exposure to PPIs was not associated with an increased risk of
major birth defects11. A systematic review published in 2009 (1530 pregnancies exposed to
PPIs and 133,410 non-exposed controls) did not identify an increase in congenital
abnormalities43. A subsequent large study examining 112,022 pregnancies (of which 1186
were exposed to PPIs) confirmed no increase in congenital anomalies, fetal growth restriction
or adverse neonatal outcomes (including preterm birth or low Apgar scores)12. Thus, very
large cohort studies indicate PPIs are safe during pregnancy.
Given our preclinical data, and the reassuring epidemiological data suggesting PPIs are safe, it
would seem justifiable to proceed to clinical trials to examine whether PPIs could be used to
either prevent preeclampsia, or to treat women diagnosed with preeclampsia. As such, we are
currently recruiting women with preterm preeclampsia to a phase II randomized clinical trial,
The Preeclampsia Intervention with esomeprazole (PIE Trial)44. We will examine whether
esomeprazole can safely prolong gestation in women diagnosed with preterm preeclampsia as
well as improve fetal, maternal and neonatal outcomes.
Perspectives
We have shown in preclinical studies that PPIs potently decrease placental release of sFlt-1
and sENG, decrease endothelial dysfunction, are vasodilatory and have anti-oxidant and anti-
inflammatory properties. They may represent a potential therapeutic option for preeclampsia
and perhaps other diseases where there is significant endothelial dysfunction. If PPIs are
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found to be useful to treat or prevent preeclampsia, they may significantly reduce maternal
and perinatal mortality globally.
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Acknowledgements: We thank the women who participated in this study and kindly donated
tissues. We also thank the research midwives Gabrielle Pell, Debra Jinks, Rachel Murdoch
and Genevieve Christophers and the Obstetrics/midwifery staff at the Mercy Hospital for
Women for collecting tissue and serum samples.
Sources of Funding: This work is supported by the National Health and Medical Research
Council (NHMRC) of Australia (ST, NJH, TKL, KP) and the Austin Medical Research
Foundation. ST and TKL are supported by NHMRC Fellowships. NJH is supported by a
University of Melbourne CR Roper Fellowship.
Disclosures: None
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Novelty and significance
What is new?
Proton pump inhibitors decrease placental and endothelial secretion of of soluble fms-like
tyrosine kinase-1 secretion and soluble endoglin.
Proton pump inhibitors vasodilate whole vessels and rescues hypertension in a model
model of preeclampsia
Proton pump inhibitors rescue endothelial dysfunction, up-regulates anti-oxidant genes
and decreases cytokine secretion from endothelial cells.
What is relevant?
Preeclampsia is a serious complication of pregnancy characterized by hypertension,
endothelial dysfunction and multi-organ injury.
Inflammation and oxidative stress exacerbate the endothelial injury.
A drug that is safe in pregnancy that can block these processes could be used to treat or
prevent preeclampsia.
Summary
We propose proton pump inhibitors are candidate drugs to prevent or treat preeclampsia, but
clinical trials are needed.
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Figure 1. Proton pump inhibitors decrease sFlt-1 expression and secretion in placental
and endothelial tissues, and HIF-1α expression. Relative sFlt-1 levels in the media
following 24 h treatment with PPIs administered to (A) primary trophoblast and (B)
preeclamptic explants. (C) Relative levels of sFlt-1 e15a (placental specific sFlt-1 variant)
in the media from preeclamptic explants following PPI treatment. (D) Relative sFlt-1 levels
in HUVECs after PPI treatment. (E) IC30 analysis (concentration of drug required to inhibit
sFlt-1 secretion from baseline by 30%) for five PPIs administered to HUVECs, which
demonstrates Rab and Eso are the most potent at reducing sFlt-1 secretion. (F) Western blot
and densitometric analysis of HIF-1α expression in HUVECs treated with PPIs at 100 μM
for 24 h. Lansoprazole (Lans), rabeprazole (Rab), esomeprazole (Eso), omeprazole (Ome),
pantoprazole (Pant). PPIs were administered at 5-100 μM concentrations, or as indicated.
Data are mean fold change from control ± SEM (*p<0.05, **p<0.01, ***p<0.001,
****p<0.0001). (n ≥3)
Figure 2. Proton pump inhibitors decrease sENG secretion from placental and
endothelial tissues, and increase VEGF expression. Relative sENG levels in the media
following PPI treatment for 24 h in (A) primary trophoblasts (B) HUVECs and (C)
preeclamptic explants. (D) IC30 (concentration of drug required to decrease sENG secretion
from baseline by 30%) were calculated for five PPIs, which demonstrates Eso and Rab are the
most potent at inhibiting sENG secretion. (E) VEGFA mRNA expression in primary
trophoblast treated with PPIs for 24 h. Lansoprazole (Lans), rabeprazole (Rab), esomeprazole
(Eso), omeprazole (Ome), pantoprazole (Pant). sENG and VEGFA mRNA expression were
measured in the same samples as the experiments shown in figure 1.
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Figure 3. Proton pump inhibitors vasodilate human vessels ex vivo from women with
preterm preeclampsia, and decrease blood pressure in vivo. (A-C) Pressure myography
measurements of human whole arterial vessels isolated from the omentum obtained at
caesarean section. Vessels were first bathed with U44619 (thromboxane agonist) to induce
vasoconstriction, then treated with up to 100 μM of esomeprazole. (A) Representative
vasoresponse recordings from blood vessels obtained from a vessel from a healthy
normotensive pregnancy (eso nor, and vehicle), a women with severe preterm preeclampsia
(HELLP syndrome (Haemolysis, elevated liver enzymes and low platelets) who required
delivery at 26 weeks’ gestation (eso PE). (B) Vasorelaxation in response to esomeprazole (100
μM) where whole vessels were taken from 11 normal pregnancies (normotensive) and 6 cases
of preterm preeclampsia (PE). *p<0.05. (C) Vasorelaxation in response to esomeprazole (1
nM-300 μM) where the omental artery was either left intact or denuded of the endothelium.
(D) Blood pressure in transgenic mice where sFlt-1 is overexpressed in the placenta, and
treated with either vehicle (n=8) or esomeprazole (n=9). ## p<0.01 comparing blood
pressures to levels seen at E10.5 within the respective groups; ***p<0.001 comparing systolic
blood pressures between the groups at E16.5 (systolic vs systolic), *p<0.05 comparing
systolic and diastolic blood pressures between the groups at E18.5.
Figure 4. Proton pump inhibitors upregulate phosphorylated eNOS, do not change Bp in
eNOS-/- mice and decrease ET-1 secretion. (A) Western blot analysis of endothelial nitric
oxide synthase (eNOS) and phosphorylated-eNOS (p-eNOS) in HUVECs. (B) Mean (systolic
and diastolic) blood pressure of eNOS-/- mice treated with either vehicle (n=7) or
esomeprazole (n=9). NP – Non pregnant. ^p =0.07. (C) Relative endothelin-1 (ET-1) mRNA
expression and (D) protein secretion from HUVECs treated with TNF-α (10ng/ml) for 24 h
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and either Rab or Eso at 5-100 μM concentrations. Data are mean ± SEM. Lansoprazole
(Lans), rabeprazole (Rab), esomeprazole (Eso). *p<0.05, ** p<0.01.(n ≥ 3)
Figure 5. PPIs decrease endothelial dysfunction in in vitro assays (A) Vascular cell
adhesion molecule-1 (VCAM) expression in HUVECs with TNF-α treatment and increasing
doses of rabeprazole (Rab) and esomeprazole (Eso). (B) Monocyte adhesion to HUVECs
following TNF-α treatment and increasing doses of lansoprazole (Lans) rabeprazole (Rab)
and esomeprazole (Eso). (C) Endothelial tube formation assay. TNF-α disrupts HUVECs tube
formation and this was rescued with eso (100 M). (D) HUVEC migration (xCELLigence)
assay; sFlt-1 (125 ng/ml) reduced migration, but this was rescued by administering either Eso
or Lans. PPIs were administered at 5-100 μM concentrations, or as indicated. Data are mean ±
SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (n ≥ 3)
Figure 6. Proton pump inhibitors up-regulate anti-oxidant factors in placental and
endothelial tissues. (A) Immunocytochemistry demonstrating nuclear translocation of
nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein (in green) in primary trophoblast
after treatment with PPIs at 100 μM. Note nuclear localization of Nrf2 (demonstrated by co-
staining with DAPI (blue; nuclei stain) with PPI treatment. The high powered inset in Eso
panel and arrowheads highlight cells with Nrf2 nuclear translocation, indicated by green/blue
nuclear fluorescence. (B) Increased heme oxygenase-1 (HO-1) protein (green) in primary
trophoblast after treatment with PPI (100 μM). Note increase cytoplasmic expression with
treatment. (C) HO-1 mRNA expression in preeclamptic placental explants treated with PPIs
(100 μM of Lans, 100 μM of Rab or 5-100 μM of Eso) (D) Western blot of HO-1 levels in
HUVECs treated with PPIs (5-100 μM). Lansoprazole (Lans), rabeprazole (Rab),
esomeprazole (Eso). Cobalt protoporphyrin (CoPP) (10 μM) is a positive control that up-
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regulates HO-1. Data are mean ± SEM. **** p<0.0001.
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