which all activate the PI3K/Akt pathway, inhibited the release of sEng from endothelial cells. Inhibition of the PI3K/Akt
pathway, by overexpression of phosphatase and tensin homolog (PTEN) or a dominant-negative isoform of Akt
(Aktdn) induced sEng release from endothelial cells and prevented the inhibitory effect of VEGF-A. Conversely, over-
expression of a constitutively active Akt (Aktmyr) inhibited PTEN and cytokine-induced sEng release. Systemic deliv-
ery of Aktmyrto mice significantly reduced circulating sEng, whereas Aktdnpromoted sEng release. Phosphorylation of
Akt was reduced in preeclamptic placenta and this correlated with the elevated level of circulating sEng. Knock-down
of Akt using siRNA prevented HO-1-mediated inhibition of sEng release and reduced HO-1 expression. Further-
more, HO-1 null mice have reduced phosphorylated Akt in their organs and overexpression of Aktmyrfailed to sup-
press the elevated levels of sEng detected in HO-1 null mice, indicating that HO-1 is required for the Akt-mediated
inhibition of sEng.
The loss of PI3K/Akt and/or HO-1 activity promotes sEng release and positive manipulation of these pathways offers
a strategy to circumvent endothelial dysfunction.
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Endothelium † Soluble endoglin † HO-1 † PI3K/Akt † HO-1 † Preeclampsia
Loss of Akt activity increases circulating
soluble endoglin release in preeclampsia:
identification of inter-dependency between Akt-1
and heme oxygenase-1
Melissa J. Cudmore1, Shakil Ahmad1, Samir Sissaoui2, Wenda Ramma1, Bin Ma1,2,
Takeshi Fujisawa1, Bahjat Al-Ani2, Keqing Wang1, Meng Cai1, Fatima Crispi3,
Peter W. Hewett2, Eduard Grataco ´s3, Stuart Egginton2, and Asif Ahmed1,2*
1University / BHF Centre for Cardiovascular Science, Queen’s Medical Research Institute, College of Medicine and Veterinary Medicine, University of Edinburgh, 47 Little France
Crescent, Edinburgh, EH16 4TJ, UK;2Department of Reproductive and Vascular Biology, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, B15 2TT, UK;
and3Department of Maternal-Fetal Medicine (Institut Clı ´nic de Ginecologia, Obstetricia i Neonatologia), Hospital Clinic-IDIBAPS, University of Barcelona and Centro de
Investigacion Biomedica en Red en Enfermedades Raras, Barcelona, Spain
Received 3 December 2010; revised 21 January 2011; accepted 31 January 2011; online publish-ahead-of-print 16 March 2011
Endothelial dysfunction is a hallmark of preeclampsia. Desensitization of the phosphoinositide 3-kinase (PI3K)/Akt
pathway underlies endothelial dysfunction and haeme oxygenase-1 (HO-1) is decreased in preeclampsia. To identify
therapeutic targets, we sought to assess whether these two regulators act to suppress soluble endoglin (sEng), an
antagonist of transforming growth factor-b (TGF-b) signalling, which is known to be elevated in preeclampsia.
Vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), angiopoietin-1 (Ang-1), and insulin,
Neutralization of transforming growth factor (TGF)-b leads to
endothelial dysfunction characterized by impaired endothelium-
mediated vasodilatation and elevated expression of surface
adhesion molecules, resulting in increased leucocyte adhesion.1
Endoglin (CD105), a transmembrane co-receptor for TGF-b1
and TGF-b3, is predominantly expressed by activated, proliferating
* Corresponding author. Tel: +44 131 242 9217, Fax: +44 131 242 6779, Email: firstname.lastname@example.org.
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for non-commercial purposes provided that the original authorship is properly and fully attributed; the Journal, Learned Society and Oxford University Press are attributed as the
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European Heart Journal (2012) 33, 1150–1158
endothelium during angiogenesis2,3and regulates the activity of
endothelial nitric oxide synthase (eNOS).4,5Proteolytic cleavage
of the extracellular domain of endoglin gives rise to soluble endo-
glin (sEng), which functions to neutralize TGF-b signalling.6
It has been shown that an increase in circulating sEng has direct,
significant, negative effects on endothelial health in vivo.1Soluble
endoglin was also shown to abrogate in vitro tube formation,
prevent TGF-b1 induction of eNOS phosphorylation, and
abolish activation of TGF-b1-mediated Smad 2/3-dependent luci-
ferase reporter activity.6In addition, sEng was shown to enhance
lung and liver microvascular permeability, cause focal endotheliosis
in kidney glomeruli, and block TGF-b-induced rat arterial vasodila-
tion.6High levels of plasma sEng have been associated with vascu-
lar disorders, such as systemic sclerosis,7atherosclerosis,8familial
hypertension,7malaria,9and most notably preeclampsia;10a
novel risk factor for cardiovascular disease in women.11Recently,
sEng was implicated as a likely cause of the reduced number of
regulatory T cells observed in the systemic circulation of pree-
clamptic women.12In addition, sEng was shown to act synergisti-
cally with soluble Flt-1 (sFlt-1), the natural antagonist of vascular
endothelial growth factor (VEGF), to induce maternal endothelial
dysfunction and severe preeclampsia in animal studies.6
Haeme oxygenase-1 (HO-1) is an inducible, cytoprotective, and
anti-inflammatory enzyme. It is widely acknowledged to provide a
defence against oxidant damage13,14and to be protective against
ischaemia-reperfusion injury.15–18Haeme oxygenase-1 null mice
have systemic endothelial damage and have greatly elevated circu-
lating sEng.19Haeme oxygenase-1 inhibits sEng release, from the
placenta and the endothelium, mediated by proinflammatory cyto-
kines, such as, tumour necrosis factor (TNF-a) and interferon-g
(IFN-g).19Significantly, a recent publication showed that the angio-
tensin receptor agonistic auto-antibody stimulates sEng, in vivo, by
upregulation of TNF-a and this upregulation can be prevented by
induction of HO-1 using haemin20confirming our earlier study.
Cellular systems employ a number of endogenous protective
mechanisms to defend against cell damage and death. The phos-
phoinositide 3-kinase (PI3K)/Akt and HO pathways are two impor-
tant examples. Activation of the PI3K/Akt pathway is crucial for
endothelial cell homeostasis and survival after vascular injury.21
Numerous growth factors, including VEGF-A,22basic fibroblast
growth factor (FGF-2),23angiopoietin-1 (Ang-1),24and insulin,25
exert their protective effect via activation of the PI3K/Akt pathway.
To date, the mechanism responsible for sEng release has not
been addressed. In this study, we sought to understand the
mechanistic regulation of sEng release and investigated the involve-
ment of two central regulators of vascular homeostasis; the PI3K/
Akt and inducible HO-1 pathways.
Reagents and antibodies
Recombinant VEGF and FGF-2 were purchased from RELIATech
(Brauschweig, Germany). Angiopoietin-1 was purchased from R&D
Systems (Abingdon, UK). Monoclonal antibody, anti-PTEN (A2B1)
and polyclonal antibodies, anti-Endoglin (C-term), anti-Endoglin
(N-term) were from Autogen Bioclear Ltd (Wiltshire, UK). Mono-
clonal antibody, anti-HO-1 was purchased from Abcam (UK).
anti-phospho-Akt (ser 473) antibodies were purchased from New
England Biolabs Ltd (Hertfordshire, UK). Polyclonal rabbit anti-HO-1
antibody was purchased from StressGen Biotechnologies Corporation
(Canada). Human TNF-a and IFN-g, monoclonal anti-b-actin, insulin,
and all other cell culture reagents and chemicals purchased from
Sigma-Aldrich Company Ltd (Dorset, UK).
Soluble endoglin ELISA
Soluble endoglin was measured in culture supernatants using the com-
mercial ELISA kits according to manufacturer’s instructions (R&D
Human umbilical vein endothelial cells (HUVECs) were isolated,
characterized, and cultured as previously described.26Experiments
were performed on third or fourth passage cells. Human umbilical
vein endothelial cells were stimulated with VEGF (20 ng/mL), TNF-a
(10 ng/mL), or IFN-g (10 ng/mL) and media collected and assayed
for sEng by ELISA.
Adenoviral gene transfer
expression of wild-type (WT) human PTEN (AdPTEN), catalytically
inactive human PTEN (AdPTENdn), dominant-negative Akt (Thr308
to Ala and Ser473 to Ala, AdAktdn), and constitutively active, myristoy-
lated Akt (AdAktmyr) were generously provided by Dr Christopher
Kontos (Duke University, USA) and AdCMV (empty vector used for
control infections) adenoviruses were amplified in HEK-293A cells
and purified using the BD Adeno-XTMpurification kit (BD Bio-
sciences). Viral titres were estimated by using the BD Adeno-XTM
rapid titer kit. Human umbilical vein endothelial cells were infected
by incubation with adenovirus in M199 containing 5% fetal calf
serum (FCS) overnight at 378C prior to addition of stimulants or
vehicle control for up to 24 h. Optimal multiplicity of infection for
the adenoviruses was determined by western blotting. The recombi-
nant, replication-deficient adenovirus encoding rat HO-1 (AdHO-1)
was used as described previously.27
Human umbilical vein endothelial cells were trypsinized and ?1 × 106
cells electroporated with ?3 mg of HO-1,28Akt-1, or control siRNA
using the HUVEC kit II and Nucleofector (Amaxa GmbH, Cologne,
Germany) as described previously.29
Quantitative real-time PCR
Sample preparation and real-time PCR was performed as described
previously.29Briefly, mRNA was prepared using TRIzol and DNase-1
digestion/purification on RNAeasy columns (Qiagen), and reverse
transcribed with the cDNA Synthesis Kit (Promega). Triplicate
cDNA samples and standards were amplified in SensiMix containing
SYBR green (Quantace) with primers specific for endoglin (Forward:
TCC-ATG-TTG-AGG-CAG-T) or b-actin. The mean threshold cycle
(CT) for HO-1 was normalized to b-actin and expressed relative to
Proteins were extracted from HUVEC with RIPA buffer and subjected
to SDS–PAGE on 10% gels, transferred to nitrocellulose membranes
(Amersham-Pharmacia,UK). Membranes wereincubated with
Akt and HO-1 suppress sEng release
appropriate antibodies at 48C overnight. Antibody reactions were
detected using the ECL detection kit (Amersham-Pharmacia, UK).
Ratios of protein expression to loading control were determined by
densitometry using ImageJ software.
Placental tissue and serum collection and
Institutional Ethics Committee approved the tissue and serum collec-
tion and written informed consent was obtained. Eligible cases were
singleton pregnancies with a diagnosis of preeclampsia. Preeclampsia
as diagnosed if a previously normotensive woman had two repeat
(4 h apart) diastolic blood pressure measurements of ≥90 mmHg
after week 20 of gestation, together with proteinuria of .300 mg in
a 24-h urine specimen or 2+ protein dipsticks in two repeat measure-
ments (4 h apart). Human placental tissue and serum were obtained
from normal pregnancies and gestationally matched pregnancies com-
plicated by preeclampsia.
All procedures and animal care were approved by Institutional Ethics
Committees and were in accordance with UK Home Office licensing
regulations. C57/Bl6J animals with targeted deletion of the HO-1
gene by neomycin resistance gene insertion30,31were supplied by
Prof Anupam Agarwal (University of Alabama, Birmingham, USA)
and rederived in accordance with local regulations. Mice were injected
in the tail vein with AdAktmyr(5 × 109pfu), AdAktdn or control
Adbgal. Five days post-injection blood was harvested by cardiac punc-
ture and organs collected for histology, western blotting, and liver
Ex vivo liver explant culture
Mice were sacrificed and their livers excised and cut into 1 mm2
pieces. Six to ten pieces of liver were equilibrated for 4 h in phenol
red-free DMEM containing 5% FCS in 24-well plate. Medium was
changed to fresh phenol red-free DMEM containing 5% FCS and
after 24 h conditioned medium was collected and stored at and liver
explants were collected and stored at –808C prior to assay for sEng
by ELISA. The explant protein was also harvested and protein
All data are expressed as the mean (+SEM). Statistical comparisons
were performed using one-way ANOVA followed by the Student–
Figure 1 Survival factors and Akt activation repress soluble endoglin release from endothelial cells. All experiments were conducted using
confluent human umbilical vein endothelial cells. Cells were incubated with (A) VEGF-A (20 ng/mL), (B) FGF-2 (20 ng/mL); angiopoieitn-1
(Ang-1;400 ng/mL), or insulin (200 nmol/L). (C) Human umbilical vein endothelial cells infected with adenoviruses encoding PTEN
(AdPTEN), a dominant-negative PTEN mutant (AdPTENdn) or empty vector (AdCMV). All experiments were conducted in M199/5%FCS
for 24 h and cell supernatants collected for soluble endoglin (sEng) quantification by ELISA. All results are the mean (+SEM) of three exper-
iments performed in triplicate (n ¼ 9). ***P , 0.001, *P , 0.05 vs. vehicle. **P , 0.01 and ***P , 0.001 vs. AdCMV.
M.J. Cudmore et al.
Newman–Keuls test as appropriate. Statistical significance was set at a
value of P , 0.05.
Soluble endoglin release is suppressed by
survival factors via activation of the
phosphoinositide 3-kinase/Akt pathway in
To assess the impact of pro-survival factors on sEng release in a
model system, isolated endothelial cells were incubated with
VEGF-A, FGF-2, Ang-1, and insulin, which all activate the PI3K/
Akt signalling.22–25These factors reduced the release of sEng
from endothelial cells (Figure 1A and B) suggesting that vascular
protection reduces shedding of endothelial membrane-bound
endoglin. To examine whether the PI3K/Akt pathway regulates
endoglin shedding, HUVEC were infected with adenoviruses
encoding phosphatase and tensin homolog (PTEN), the phospha-
tase that inhibits PI3K signalling (AdPTEN) or inactive PTEN
(AdPTENdn)32(see Figures 3C and 5A for overexpression).
AdPTENdn, which potentiates the PI3K pathway, thus activating
Akt, significantly decreased the release of sEng (Figure 1C),
whereas overexpression of PTEN, which depletes the cell of phos-
phatidylinositol 3,4,5-trisphosphate, the substrate required for Akt
activation, induced a two-fold increase in sEng release (Figure 1C).
Inhibition of Akt activity using an adenovirus encoding a
dominant-negative Akt construct, (Ad-Aktdn)33increased endoglin
mRNA expression in HUVEC (Figure 2A) and prevented
However, when AdAktdn was co-infected with AdPTENdn, the
ability of AdPTENdnto inhibit sEng release was lost (Figure 2C)
suggesting that PI3K is acting via Akt to modulate sEng release.
Tail vein injection of AdAktdninto mice resulted in increased Akt
expression in liver tissue after six days (Figure 2D inset). Liver
explants established from AdAktdninfected mice showed increased
secretion of sEng (Figure 2D). These data demonstrate that inhi-
bition of the survival protein, Akt, in vitro and in vivo, augments
the levels of cleaved endoglin released from cells.
To further investigate this phenomenon, we went on to examine
whether positive modulation of Akt, could suppress sEng release.
Interestingly, overexpression of constitutively active myristilated
Akt (Aktmyr) did not inhibit endoglin mRNA expression, nor did
it inhibit basal sEng release from HUVEC (Figure 3A and B).
However, when co-expressed with PTEN, Aktmyrcompletely
abrogated PTEN-mediated upregulation of endoglin mRNA
Figure 2 Akt inhibition induces the release of soluble endoglin. (A) Relative endoglin mRNA levels in human umbilical vein endothelial cells
after infection with an adenovirus encoding dominant-negative Akt (AdAktdn) or b-galactoside control adenovirus (Adbgal). **P , 0.01 vs.
Adbgal. Soluble endoglin (sEng) level from Aktdnoverexpressing human umbilical vein endothelial cells (B) stimulated with VEGF-A (20 ng/
mL) or (C) co-infected with AdPTENdn.(D) C57/B6J mice were injected i.v. with AdAktdnor Adbgal and 5 days later blood and organs analysed.
Soluble endoglin levels in conditioned medium of liver explants from mice at 24 h. Soluble endoglin release was normalized to total protein
content of the explants and soluble endoglin quantified by ELISA. All results are the mean (+SEM) of three experiments performed in triplicate
(n ¼ 9). **P , 0.01 vs. Adbgal. Inset—immunoblot of mouse liver lysates for Akt.
Akt and HO-1 suppress sEng release
Figure 4 Phosphorylation of Akt is decreased in preeclamptic placenta and correlates inversely with soluble endoglin. (A) Lysates of placenta
from normal or preeclamptic pregnancies were immunoblotted with antibodies against phosphorylated Akt (pAkt-ser 473) and b-actin. (B)
Densitometric analysis showing ration of pAkt:b-actin in A. (C) Correlation between plasma soluble endoglin and pAkt:b-actin ratio of placenta.
*P , 0.05.
Figure 3 Akt activation inhibits the release of soluble endoglin. (A) Relative endoglin mRNA and (B) soluble endoglin protein levels after
human umbilical vein endothelial cells were infected with AdCMV, AdPTEN, and/or myristylated Akt (AdAktmyr). (C) Immunoblot with anti-
bodies against; endoglin (N-terminus), endoglin (C-terminus), phosphorylated Akt [pAkt (ser 473)], Akt, PTEN, and b-actin. (D) Aktmyrover-
expressing cells incubated with interferon-g (IFN-g;10 ng/mL) or tumour necrosis factor-a (TNF-a; 50 ng/mL). All experiments (unless stated
otherwise) were conducted in M199/5%FCS for 24 h and cell supernatants collected for soluble endoglin quantification by ELISA. All results are
the mean (+SEM) of three experiments performed in triplicate (n ¼ 9). *P , 0.05, **P , 0.01. Mice were injected i.v. with AdAktmyror Adbgal
and 5 days later blood and organs analysed. (E) Endoglin levels in liver from mice at 24 h. Endoglin was normalized to total protein and quanti-
fied by ELISA. All results are the mean (+SEM) of three experiments performed in triplicate (n ¼ 9). ***P , 0.001 vs. Adbgal.
M.J. Cudmore et al.
(Figure 3A) and release of the soluble protein (Figure 3B). Human
umbilical vein endothelial cell lysates immunoblotted for the N
and C termini of endoglin confirmed the upregulation of endoglin
following PTEN overexpression and Aktmyr, which increased the
level ofphosphorylated Akt,
(Figure 3C). Consistent with these findings, the pro-inflammatory
cytokine-mediated release of sEng was inhibited by Aktmyr
(Figure 3D) and systemic administration of AdAktmyrto mice
resulted in decreased endoglin protein in liver tissues compared
with control animals (Figure 3E).
Akt is reduced in the preeclamptic
placenta and inversely correlates with
maternal soluble endoglin
Preeclampsia is characterized by widespread endothelial cell dys-
function and the progressive elevation of circulating sEng. Western
of Akt was significantly reduced in preeclamptic placenta compared
with placenta from normal pregnancies (Figure 4A and B). Further-
with maternal plasma sEng levels (Figure 4C). Thus, the rise in circu-
lating sEng paralleled the fall in Akt activity.
Haeme oxygenase-1 suppresses soluble
endoglin release via Akt
We previously demonstrated that HO reduces the release of sEng
under basal and cytokine-stimulated conditions.19This has been
recently confirmed in a study showing that TNF-a-induced sEng
release from endothelial cells and placental explants could be abro-
gated by upregulation of HO activity by haemin.20Interestingly, acti-
vation of Akt by overexpression of AdPTENdn, which induced Akt
phosphorylation, also upregulated HO-1 protein in endothelial
cells (Figure 5A). Knockdown of Akt1, using siRNA, significantly
induced sEng release (Figure 5B) and more importantly, prevented
the inhibition of sEng caused by overexpression of HO-1
(Figure 5B). In addition, HO-1 expression in HUVEC was also pre-
viously demonstrated that knockdown of HO-1, using siRNA,
induced sEng release,19here we show that knockdown of HO-1
(Figure 5D). In addition, overexpression of Aktmyrcould not
prevent the upregulation of sEng after loss of HO-1 (Figure 6A),
suggesting thatHO-1 and
Akt regulate sEngrelease
Figure 5 Haeme oxygenase-1 requires Akt to inhibit soluble endoglin release. (A) Lysates from human umbilical vein endothelial cells infected
with AdCMV, AdPTENdn, and/AdAktdnwere immunoblotted with antibodies against phosphorylated Akt [pAkt (ser 473)], Akt, PTEN, HO-1,
and b-actin. (B) Knockdown of Akt1 (siAkt) in human umbilical vein endothelial cells overexpressing HO-1 or bgal. (C) Immunoblot of Akt
siRNA treated human umbilical vein endothelial cells for HO-1 and b-actin. (D) Knockdown of HO-1 (siHO-1) in human umbilical vein endo-
thelial cells and stimulation with interferon-g (10 ng/mL) or tumour necrosis factor-a (TNF-a; 50 ng/mL). All experiments were conducted in
M199/5%FCS for 24 h and cell supernatants collected for soluble endoglin (sEng) quantification by ELISA. All results are the mean (+SEM) of
three experiments performed in triplicate (n ¼ 9). *P , 0.05, **P , 0.01.
Akt and HO-1 suppress sEng release
HO-1-null mice exhibit elevated circulating sEng19and western
blotting of organs from HO-1 null mice showed a decrease in
phosphorylation of Akt (Figure 6B). Furthermore, Aktmyroverex-
pression in WT animals resulted in reduced circulating sEng
(Figure 6C). Moreover, Aktmyroverexpression failed to suppress
the circulating levels of sEng in HO-1 null mice (Figure 6C). To
investigate this further, liver explants were established from
adenoviral-infected mice, cultured for 24 h and supernatants
assayed for sEng. Explants from AdAktmyrinfected WT and
HO-1 heterozygous mice, produced significantly less sEng com-
pared with Adbgal-infected controls (Figure 6D). Liver explants
from HO-1 null mice released significantly more sEng than WT
and heterozygous mice and consistent with our in vitro studies,
overexpression of AdAktmyrhad no effect on sEng release from
liver explants in HO-1 null mice (Figure 6D) demonstrating that
HO-1 and Akt play pivotal, interdependent roles in suppressing
the release of sEng in vivo.
Serum sFlt-1 and sEng are increased in pregnant women prior to
the clinical symptoms of preeclampsia.10Inhibition of VEGF or
TGF-b signalling by high circulating sEng activates the endothelium
to promote vascular dysfunction.1,6The salient finding highlighted
by this study is the identification of PI3K/Akt signalling, in concert
with HO-1, as a central negative regulator of endoglin shedding in
vivo. The significance of this finding is reinforced by the observation
that Akt phophorylation is decreased in the preeclamptic placenta
and inversely correlates with the maternal circulating levels of sEng.
In addition, survival factors that exert their protective effects via
Akt, such as VEGF-A, FGF-2, Ang-1, and insulin, all negatively regu-
late sEng release from endothelial cells. Our finding that VEGF sup-
presses sEng release from endothelial cells suggests that in
preeclampsia, the increase in placental sEng6,19is likely to be
further compounded by the loss of VEGF-A activity due to the
concomitant rise in its antagonist, sFlt-1, in the maternal
Knockdown of HO-2 reduces Akt phosphorylation in vivo34and
carbon monoxide, the gaseous product of HO, stimulates Akt
phosphorylation in hepatocytes35and endothelial cells36support-
ing a positive feedback loop between the HO and PI3K/Akt path-
ways. In this regard, it is important to remember that the loss of
HO activity may be a causative factor in preeclampsia, as HO-1
protects againstTNF-a-induced placentaldamage37
Figure 6 Akt requires haeme oxygenase-1 to inhibit soluble endoglin release. (A) Soluble endoglin (sEng) release from human umbilical vein
endothelial cells after knockdown of haeme oxygenase-1 (siHO-1), and control (siCtrl) in human umbilical vein endothelial cells overexpressing
Aktmyror bgal. (B) pAkt levels in organs of haeme oxygenase-1 wild-type and haeme oxygenase-1-deficient mice. Mice were injected i.v. with
AdAktmyror Adbgal and 5 days later blood and organs analysed. (B) Plasma soluble endoglin levels in wild-type and haeme oxygenase-1-
deficient mice. (C) Soluble endoglin levels in conditioned medium of liver explants from haeme oxygenase-1 wild-type, heterozygous, and
haeme oxygenase-1-deficient mice at 24 h. In explant studies, soluble endoglin release was normalized to total protein content of the explants
and soluble endoglin quantified by ELISA. All results are the mean (+SEM) of three experiments performed in triplicate (n ¼ 9). *P , 0.05,
**P , 0.01, ***P , 0.001.
M.J. Cudmore et al.
suppresses cytokine-mediated sEng and sFlt-1 release.19The most
compelling evidence for this comes from a recent study using faetal
placental cells from women at 11 weeks gestation. Farina et al.38
showed that the expression of HO-1 mRNA decreased in chorio-
nic villous samples (faetal cells) from women who went on to
develop preeclampsia. This very early decrease in HO-1 could
explain, at least in part, the elevated levels of anti-angiogenic
factors seen later in pregnancy in preeclamptic women. Transform-
ing growth factor-b1 stimulates HO-1 expression via the PI3K/Akt
pathway in human lung epithelial cells.39Thus, loss of TGF-b1 sig-
nalling, due to the rise in sEng in preeclampsia, may further com-
promise maternal endothelial HO activity. Our data show that
PI3K/Akt activation is decreased in the organs of HO-1 null mice
and that increased PI3K/Akt activation induces HO-1 expression
in endothelial cells and loss of such a positive feedback system
may lead to greater loss of endothelial integrity under conditions
of high circulating sEng, observed in a number of vascular dis-
orders. The predominant upstream regulator of HO-1 expression
is Nuclear factor-like 2 (Nrf-2). Nuclear accumulation of Nrf-2 and
HO-1 expression was shown to be PI3K-dependent and
MEK-MAPK independent in the endothelium.40The interdepen-
dency between PI3K/Akt and HO-1 identified in this study needs
further investigation to determine whether they are regulated at
the level of Nrf-2.
It has been shown that an increase in circulating sEng has direct,
significant, negative effects on endothelial health in vivo.1It abro-
gates TGF-b-mediated signalling, enhances lung and liver microvas-
cular permeability, causes focal endotheliosis in kidney glomeruli
and blocks TGF-b1-induced vasodilation.6The involvement of
sEng in a number of wide-ranging pathologies demonstrates that
sEng is not only marker of endothelial integrity but also a
contributing factor of endothelial dysfunction. Our discovery of a
co-dependency between HO-1 and Akt in relation to sEng
release implies that dysfunction of only one of these factors in
the endothelium may explain the resultant increase in sEng in
these disorders. Interestingly, both TNFa and sEng are elevated
in malaria41and the level correlates with disease severity,9
whereas HO-1 and CO protect against malaria progression.42
In conclusion, the discovery that PI3K/Akt and HO-1 provide
key co-dependent and inhibitory signals required to suppress
sEng release strongly suggests that the positive manipulation of
PI3K/Akt and/or HO pathways would provide potential thera-
peutic targets in preventing excessive sEng release in vascular dis-
orders including preeclampsia.
This work was supported by grants from the Medical Research Council
(G0601295 and G0700288) and British Heart Foundation (RG/09/001/
25940). Funding to pay the Open Access publication charges for this
article was provided by Medical Research Council.
Conflict of interest: none declared.
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