Angiotensin Receptor Agonistic Autoantibody Is Highly

Prevalent in Preeclampsia
Correlation With Disease Severity
Athar H. Siddiqui, Roxanna A. Irani, Sean C. Blackwell, Susan M. Ramin,
Rodney E. Kellems, Yang Xia

Abstract—Preeclampsia (PE), a syndrome affecting 5% of pregnancies, characterized by hypertension and proteinuria, is

a leading cause of maternal and fetal morbidity and mortality. The condition is often accompanied by the presence of
a circulating maternal autoantibody, the angiotensin II type I receptor agonistic autoantibody (AT1-AA). However, the
prevalence of AT1-AA in PE remains unknown, and the correlation of AT1-AA titers with the severity of the disease
remains undetermined. We used a sensitive and high-throughput luciferase bioassay to detect AT1-AA levels in the
serum of 30 normal, 37 preeclamptic (10 mild and 27 severe), and 23 gestational hypertensive individuals. Here we
report that AT1-AA is highly prevalent in PE (⬇95%). Next, by comparing the levels of AT1-AA among women with
mild and severe PE, we found that the titer of AT1-AA is proportional to the severity of the disease. Intriguingly, among
severe preeclamptic patients, we discovered that the titer of AT1-AA is significantly correlated with the clinical features
of PE: systolic blood pressure (r⫽0.56), proteinuria (r⫽0.70), and soluble fms-like tyrosine kinase-1 level (r⫽0.71),
respectively. Notably, only AT1-AA, and not soluble fms-like tyrosine kinase-1, levels are elevated in gestational
hypertensive patients. These data serve as compelling clinical evidence that AT1-AA is highly prevalent in PE, and its
titer is strongly correlated to the severity of the disease. (Hypertension. 2010;55:386-393.)
Key Words: preeclampsia 䡲 gestational hypertension 䡲 angiotensin receptor autoantibodies 䡲 sFlt-1 䡲 proteinuria

P reeclampsia (PE) is a serious hypertensive disorder of

pregnancy that affects ⬇5% of pregnancies and remains
a leading cause of maternal and neonatal mortality and
soluble fms-like tyrosine kinase 1 (sFlt-1) leading to de-
creased angiogenesis in endothelial cells, increase plasmino-
gen activator inhibitor 1 production resulting in decreased
morbidity in the United States and the world.1–3 The disease trophoblast invasion, and increase NADPH oxidase produc-
is multifactorial and includes such clinical features as high tion in trophoblast cells resulting in oxidative stress.14 –17
blood pressure, proteinuria, inflammation, endothelial dys- However, these studies were restricted to the use of in vitro
function, vasoconstriction, and placental abnormalities.4 –7 cultured cell systems and, therefore, did not directly address
The clinical symptoms in the advanced stages of preeclamp- the relevance of AT1-AAs to hypertension and proteinuria,
sia include cerebral hemorrhage, renal failure, hemolysis, the defining features of PE. However, recent experiments
elevated liver enzymes, and low platelets syndrome. In have demonstrated that the injection of pregnant mice with
serious cases, termination of pregnancy is the only available AT1-AAs recapitulates the key features of PE, including
option to prevent further deterioration of the fetus and hypertension, proteinuria, renal and placental morphological
mother. Despite being a leading cause of maternal death and changes, and an increase in the concentration of antiangio-
a major contributor to maternal and perinatal morbidity, the genic factor sFlt-1.18 Thus, these in vivo studies provide the
triggering factors and underlying mechanisms responsible for first direct evidence for a pathophysiological role of AT1-AA
the pathogenesis of PE remain elusive. in PE and suggest that these autoantibodies contribute to the
Numerous studies have shown that women with PE possess pathogenesis of PE. However, the prevalence of AT1-AA in
angiotensin (Ang) II type 1 (AT1) receptor agonistic autoan- PE remains unknown, and the correlation of AT1-AA to the
tibodies (AAs) that bind to and activate the AT1 Ang receptor severity of the disease remains undetermined because of the
in multiple cellular systems and provoke biological responses lack of a sensitive and convenient assay to accurately mea-
that are relevant to the pathophysiology of PE.8 –13 For sure AT1-AA in human sera.
example, AT1-AAs increase the contraction rate of rat car- In this study, because of our newly developed sensitive and
diomyocytes, elevate levels of the antiangiogenic factor high-throughput luciferase bioassay, we were able to address

Received July 29, 2009; first decision August 16, 2009; revision accepted November 9, 2009.
From the Departments of Biochemistry and Molecular Biology (A.H.S., R.A.I., R.E.K., Y.X.) and Obstetrics, Gynecology, and Reproductive Sciences
(S.C.B., S.M.R.), University of Texas Medical School at Houston, Houston, Tex.
Correspondence to Yang Xia, MSB 6.200, 6431 Fannin St, Houston, TX 77030. E-mail Yang.Xia@uth.tmc.edu
© 2010 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.109.140061

386
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 Siddiqui et al AT1-AA and Disease Severity 387

2 important clinical questions regarding what percentage of Table. Clinical Features of Patients From Various Groups in
women with PE contain AT1-AA and whether the titer of the Present Study
AT1-AA correlates with the severity of disease. Using this Gestational
bioassay, we have provided the first compelling patient Patient Group Normotensive Hypertension Mild PE Severe PE
evidence that AT1-AA is highly prevalent in PE, and its titer Age, y 28⫾2 28⫾2 25⫾2 28⫾2
strongly correlates with the severity of the disease. These
Systolic blood 120⫾2 153⫾4 145⫾2 168⫾3
findings add support to the novel concept that PE is an pressure, mm Hg
autoimmune disease associated with AT1-AA.13 We believe Diastolic blood 73⫾2 88⫾3 91⫾4 98⫾2
that these initial clinical studies, coupled with our bioassay, pressure, mm Hg
have provided a strong foundation for us to perform a Urinary protein, 25⫾12 71⫾20 363⫾37 1201⫾250
large-scale clinical studies in the future. mg/24 h
Serum creatinine, 0.66⫾0.05 0.63⫾0.02 0.70⫾0.02 0.69⫾0.02
Methods mg/dL
Materials sFlt-1, ng/mL 5⫾1 7⫾1 11⫾2 20⫾2
Tissue culture medium (RPMI 1640), FBS, and antibiotics, such as Weeks’ 38.0⫾0.5 36.0⫾1.0 35.0⫾1.0 32.0⫾1.0*
penicillin-streptomycin (⫻100), and geneticin (G418, 50 mg/mL) gestational age
were purchased from Invitrogen Life Technologies. Human Ang II was
*For early onset preeclampsia (delivery ⬍32 weeks), weeks’ gestational
obtained from Sigma. Losartan (COZAAR) was a gift from Merck
age⫽26⫾2 (n⫽10). For late preeclampsia (delivery at ⬎32 weeks), weeks’
Research Laboratory. The 7 amino acid (7aa) peptide (7aaAFHYESQ)
is an epitope sequence present on the second extracellular loop of the gestational age⫽36.0⫾0.5 (n⫽17).
AT1 receptor that is recognized by AT1-AA. These peptides were
synthesized by the Baylor College of Medicine Protein Chemistry Core Preparation of the IgG Fraction
Laboratory. Protein G Sepharose 4 Fast Flow, used for IgG isolation, The IgG fraction was isolated by the batch purification method using
was purchased from Amersham Pharmacia Biotech. PathDetect Protein Sepharose G 4 Fast Flow, as described previously.17 The
nuclear factor of activated T cell (NFAT) cis-reporting system and purity of the isolated IgGs was ascertained using gel electrophoresis.
synthetic Renilla luciferase reporter vector were purchased from The presence of 2 bands at ⬇50 kDa and ⬇25 kDa indicated the
Stratagene and Promega Corp, respectively. presence of the heavy and light chains of the IgG.

Patients Transient Transfection Assay

Patients who were admitted to Memorial Hermann Hospital were CHO.AT1A cells were plated at a density of 1⫻105 cells in 24-well
identified by the obstetrics faculty of the University of Texas plates for 2 hours. Cells were transfected using 500 ng of the
Medical School at Houston. Twenty-seven patients were diagnosed NFAT-luciferase reporter construct containing 4 copies of the NFAT
with severe PE on the basis of the definition set by the National High binding element (PathDetect NFAT cis-reporting system), 20 ng of
Blood Pressure Education Program Working Group report.19 The phRTK, a synthetic Renilla luciferase reporter construct (for internal
criteria include the presence of high blood pressure of ⱖ160/ control), and 5 ␮L of Lipofectamine reagent (Invitrogen Life
110 mm Hg and urinary protein of 300 mg in a 24-hour period or a Technologies) for 5 hours. The cells were serum starved for 24 hours
dipstick value of ⱖ1⫹. These women had no previous history of and treated with Ang II overnight, where indicated. Similar experi-
hypertension. Other criteria included the presence of persistent ments were carried out using the 2⫻-Egf-luciferase reporter con-
headache, visual disturbances, epigastric pain, or the hemolysis, struct. The treated cells were lysed in 100 ␮L of passive lysis buffer
elevated liver enzymes, and low platelets syndrome in women with (Promega Inc) at room temperature for 45 minutes. Luciferase
blood pressure of ⱖ140/90 mm Hg. For patients with mild PE, the activity (measured in relative light units [RLUs]) was measured
blood pressure criteria were ⱖ140/90 mm Hg and urinary protein using 10 ␮L of lysate with a Dual Luciferase system (Promega Inc).
level of 300 mg per 24 hours or a dipstick value of ⱖ1⫹. Patients
with a blood pressure of ⱖ140/90 mm Hg appearing after 20 weeks’ Luciferase Activity
gestation and having ⬍300 mg of urinary protein per 24-hour period CHO.AT1A (1⫻105 cells) containing stably integrated copies of a
were classified as having gestational hypertension. Blood samples minigene encoding the rat AT1 receptor and a 4⫻NFAT-driven
collected from the patients were allowed to clot and were then luciferase construct were plated on 24-well plates overnight. The
centrifuged at 20 000g for 20 minutes, and the serum samples were next day, cells were changed to serum-free medium and treated with
stored at ⫺80°C. Patients were generally approached for the study IgG (1:10 dilution) for 24 hours. Luciferase activity in cell lysates
during the prepartum or early intrapartum period. Patient enrollment was measured using a luciferase assay kit (Promega). To test the
occurred from May 2007 to April 2009. The research protocol, reproducibility of our bioassay, we carried out the assay multiple
including the consent form, was approved by the Institutional times with different IgG isolations obtained from the same patient
Committee for the Protection of Human Subjects. The general and also carried out the assay with the same IgG sample multiple
clinical features of the patients involved in the study are shown in the times. We obtained very reproducible activation levels with the IgGs
Table. obtained from normotensive pregnant women and women with
severe PE. In general we observed no more than a ⫾10% variation
Cell Culture when assaying multiple IgG samples from the same patient.
Chinese hamster ovary cells stably transfected with rat Ang II
receptor type 1A (CHO.AT1A) were kindly provided by Dr Terry S. sFlt-1 Determination
Elton (Ohio State University, Columbus, OH). Cells were main- Commercially available ELISA kits (R&D Systems) were used
tained at 37°C and 5% CO2 and cultured in RPMI 1640 containing according to the manufacturer’s recommendations to determine the
5% FBS, 1% antibiotics, 8.75 g of L-proline, and 100 ␮g/mL of maternal serum sFlt-1 concentrations.
gentamicin. The CHO.AT1.luc cells were isolated by introducing the
4⫻NFAT luciferase construct bearing a hygromycin phosphotrans- Data Calculation
ferase gene. Stable transformants were isolated in the cell culture All of the data were calculated as a percentage of change (increase/
medium described above including hygromycin (100 ␮g/mL). decrease) of luciferase activity measured in terms of RLUs, as

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388 Hypertension February 2010

determined by monolight luminometer (Pharmingen) of (over) basal.

The average luciferase activity (RLUs) obtained for basal was
A Angiotensin II
AT1 receptor
250⫾50.

Statistical Analysis
Results are expressed as mean⫾SEM. All of the data were subjected Gq
to statistical analyses using GraphPad Prism 5. One-way ANOVA
and unpaired t tests were performed to determine the significance of
differences between groups. Data were also subjected to correlation
analysis using the same software to determine Spearman r values. PLC MAPKK
Statistical significance was set at P⬍0.05.
IP3 DAG
Results
Construction of a Cell Line That Reports the ERK, JNK, STAT
Activation of AT1 Receptors as Increased Intracellular PKC
Luciferase Activity Ca 2+
In view of known signaling events downstream of AT1
receptor activation (Figure 1A), we chose 2 luciferase re-
Downstream Factors NFAT EgR
porter constructs for potential use in monitoring AT1 receptor
activation. One reporter construct, termed “2⫻-Egr-luciferase,” Downstream Genes sFlt-1 Cyclin D
contains 2 copies of a consensus early growth response factor
response element followed by a cytomegalovirus promoter– 1.
driven firefly luciferase reporter gene. The other construct, 2x-EgR elements Fire-fly Luciferase
termed “4⫻-NFAT-luciferase,” is a cytomegalovirus promoter–
driven luciferase reporter plasmid under the control of 4 2.
NFAT cis-regulatory elements. These DNA constructs were 4x-NFAT elements Fire-fly Luciferase
transiently transfected into CHO.AT1 cells that were incu-
bated with a range of Ang II concentrations (10 to 1000 nM). Promoter constructs for Bioassay Strategies
After 24 hours, the cells were lysed and luciferase activity
determined in cell extracts. The results (Figure 1B) show a B 20 p2xcEgr-luc
dose-dependent increase in luciferase activity with both
LUCIFERASE ACTIVITY

p4xNFAT-luc
luciferase reporter genes after treatment with Ang II. How-
15
(RLU 1x10 4)

ever, the NFAT-luciferase construct was maximally activated

over a broader range of Ang II concentrations, and for this
10
reason it was chosen for use in subsequent experiments.
To convert the CHO.AT1 cell line to one that easily
reports the activation of AT1 receptors, we stably intro- 5
duced 4⫻-NFAT-luciferase expression plasmids using co-
transfection with a selectable marker. A schematic illustration 0
0.00 0.01 0.10 1.00
of the use of the genetically engineered cells to detect AT1
Ang II (µmoles)
receptor activation by measuring luciferase activity is shown
in Figure 2A. Stable transformants (termed “CHO.AT1.luc”) Figure 1. Signaling pathways downstream of AT1 receptor acti-
vation. A, Various downstream molecules involved in the AT1
were isolated, expanded, and tested for the ability to synthe- receptor signaling. PLC indicates phospholipase C; MAPKK,
size increased amounts of luciferase in response to increasing mitogen-activated protein kinase kinase; IP3, inositol (1, 4, 5)
concentrations of Ang II. The results (Figure 2B) show that P3; DAG, diacyl glycerol; EgR, immediate early growth response
luciferase activity increased over a concentration range of 0.1 factor. Reporter constructs used to detect AT1 receptor activa-
tion. B, Construction of a cell line that reports the activation of
nM to 10.0 ␮mol/L, reaching a maximum of ⬇5-fold over the AT1 receptors with increased luciferase activity Ang II regulation
basal (nontreated cells) at 100 nM. The increased luciferase of 2⫻cEgR and 4⫻NFAT luciferase reporter constructs in AT1
synthesis was completely blocked by the presence of receptor– expressing Chinese hamster ovary cells. CHO.AT1A
1 ␮mol/L of losartan, an AT1 receptor specific antagonist, cells were plated in either 12-well or 24-well plates and tran-
siently transfected with 2⫻cEgR and 4⫻NFAT reporter con-
and by FK506, an inhibitor of Ca2⫹/calmodulin-dependent structs along with synthetic Renilla luciferase reporter (internal
phosphatase 2C (calcineurin; Figure 2C). These results show control) using Lipofectamine (Invitrogen Life Technologies) for 6
that the Ang II–induced stimulation of luciferase activity in hours. Cells were serum starved for 24 hours, treated with dif-
ferent concentrations of Ang II over night, harvested, and mea-
CHO.AT1.luc cells was mediated through AT1 receptor acti- sured for relative luciferase activity using a luminometer. Graph
vation and downstream signaling through the calcineurin/ points denote the RLUs.
NFAT pathway.
stimulate luciferase activity, we treated these cells with a
Use of CHO.AT1.luc Cells to Measure 10-fold concentration range (1:50 to 1:5) of IgG from women
AT1-AA Activity with severe PE and from normotensive pregnant women.
To determine whether autoantibodies from women with PE After 24 hours, cells were lysed and extracts assayed for
are able to activate AT1 receptors on CHO.AT1.luc cells and luciferase activity. The results (Figure 3A) show a
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 Siddiqui et al AT1-AA and Disease Severity 389

A B
Ang II

(RLU-fold induction over basal)

AT1 AA IgG

LUCIFERASE ACTIVITY
6 Ang II + Losartan

5
AT1 receptor
4
Figure 2. Construction of a cell line that
3
* * reports the activation of AT1 receptors
2 * * * with increased luciferase activity. A, Sche-
1 matic illustration of genetically engineered
Ca2+ cell line, CHO.AT1.luc (4⫻NFAT-luciferase)
0
used in our bioassay to detect AT1 recep-

5
1

4
6
0

-0

-0
-1

-0

-0
-0
-1
tor activation by measurement of lucif-

-0
Cam activation

10

10
10

10

10
10

10
10
erase activity. B, Concentration-response

0x

0x
0x

0x

0x
0x

0x
0x
P

1.

1.
1.

1.

1.
1.

1.
1.
curve of Ang II with the CHO.AT1A cells.
Ang II showed a concentration-dependent
FK506 Calcineurin C luciferase activation, which was blocked

(RLU-fold induction over basal)

6
LUCIFERASE ACTIVITY significantly by losartan (1 ␮mol/L), an AT1
receptor antagonist. C, Ang II–induced
P luciferase activation is mediated via AT1
4
NFAT receptor activation and calcineurin/NFAT
signaling, as evidenced by the attenuation
2
of the Ang II–mediated luciferase activa-
tion in the presence of the AT1 receptor
antagonist, losartan, and Ca2⫹/calmodulin-
NFAT Nucleus 0
dependent phosphatase 2B (calcineurin)
inhibitor FK506.

6
n
l

II
tro

50
rta
g
on

An

FK
sa
NFAT
C

Lo
nM

+
II
+
0

g
II
10

An
g
An

nM

4xNFAT elements Fire-fly Luciferase

nM

0
10
0
10

concentration-dependent increase in luciferase activity when luciferase activity observed in IgG-treated CHO.AT1.luc cells
using IgG from women with PE that was much greater that is a measure of AT1-AA–mediated AT1 receptor activation.
that observed with IgG from normotensive pregnant women.
Maximal stimulation was achieved at a 1:10 antibody dilu- Prevalence and Abundance of AT1-AA in Women
tion, where the luciferase activity expressed as a percentage With Hypertensive Disorders of Pregnancy
increase over basal activity was found to be 9⫾3 for The CHO.AT1.luc cells were used to determine the preva-
normotensive versus 64⫾13 for the severe PE samples. The lence and abundance of AT1-AAs in normotensive pregnant
antibody-mediated stimulation of luciferase activity was women, women with gestational hypertension, and women
blocked by the presence of losartan. These results indicate with PE (mild and severe). Luciferase activity (in RLUs) was
that increased luciferase activity resulted from antibody- expressed as a percentage increase over basal activity. The
mediated AT1 receptor activation. Overall, the results indicate results (Figure 4A) show that the highest stimulation and the
that the synthesis of luciferase by CHO.AT1.luc cells served broadest range of activities was achieved with IgG isolated
as a bioassay to detect AT1-AAs present in the IgG of women from women with severe PE. The broad range of NFAT-
with severe PE. luciferase activation observed for this group was also asso-
A characteristic and defining feature of AT1-AAs is the ciated with a broad spectrum of clinical features of PE. It is
interaction with a 7aa peptide epitope present on the second noteworthy that 10 patients in this category fell into the
extracellular loop of the AT1 receptor. The presence of the 7aa severe early onset category of PE with delivery before 32
epitope peptide in the culture medium prevents the binding of weeks. The level of AT1-AA was not different between the
AT1-AAs to AT1 receptors. As a test for the specificity of the early and late-onset cases of severe PE. The stimulation of
NFAT-luciferase bioassay, we added the 7aa epitope peptide luciferase activity by IgG from the severe PE group was
(0.25 ␮g/mL) to CHO.AT1.luc cells before the addition of IgG inhibited by the 7aa epitope peptide, indicating that it resulted
from women with severe PE or normotensive pregnant from AT1-AA–mediated AT1 receptor activation. In this
women. The presence of the 7aa epitope peptide completely group, 26 of the 27 samples tested showed significant
blocked the antibody-mediated induction of luciferase activ- stimulation of luciferase activity, with an average stimulation
ity, including the relatively small increase in luciferase of 66⫾9% that was ⬇5-fold greater than that observed with
activity observed for cells treated with IgG from normoten- IgG from normotensive pregnant women.
sive pregnant women (Figure 3B). These results suggest that A significant increase in luciferase activity was also
the antibody-mediated induction of luciferase activity is observed with IgG from women with mild PE, although it
mediated through interaction with the common peptide was not as high as in the severe PE group. The range of
epitope associated with the second extracellular loop of the activities was more narrow for the mild PE group than for that
AT1 receptor. Overall, these results indicate that increased observed for the severe PE group. All 10 of the mild PE
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390 Hypertension February 2010

A A 250
80 PE 200
LUCIFERASE ACTIVITY
(RLU-percent over basal)
NT

LUCIFERASE ACTIVITY
150
*

(RLU-percent over basal)

60 * 100

80
40
*
60
20 *
40

0 20
1:50 1:25 1:10 1:5
Dilution of IgGs 0

a
a

E
H

PE
* p<0.05 compared to NT

PE
T

7a

7a
7a

7E
G
N

re
+

ild

+
+
B

+
H

ve

PE
T

PE
N

Se
80

re
ild
NT
LUCIFERASE ACTIVITY

ve
M
(RLU-percent over basal)

Se
PE
60 n=27
B 80 @$
40
*

LUCIFERASE ACTIVITY
(RLU-percent over basal)
60
20 *
n=23 n=10
0 40 *
*
n

n
a

a
G

G
rta

rta
7a

7a
Ig

Ig
sa

sa

n=30
+

+
Lo

Lo
G

20 #
Ig

Ig
+

# #
G

G
Ig

Ig

#
* p<0.05 compared to IgG alone
0
Figure 3. Measurement of AT1-AA activity by a luciferase assay

a
a

E
H

PE
PE
T

7a

7a
7a

7E
G
N

in CHO.AT1.Luc cells. A, Dose-dependent response profile of

re
+

ild

+
+

+
H

ve

PE
T

M
IgGs (AT1-AAs) isolated from the sera of women with PE and

PE
N

Se

re
women with normotensive (NT) pregnancies. n⫽6 to 10 for each

ild
*p<0.05 compred to NT

ve
M

Se
group. B, IgG (AT1-AA)-induced increase in luciferase activation @p<0.05 compared to GH
is significantly blocked by AT1 receptor antagonist losartan and $p<0.05 compared to Mild PE
7aa peptide corresponding with this common epitope, which #p<0.05 compared without 7aa
blocks the binding of AT1-AA to the AT1 receptor. n⫽8 for NT
and PE. Figure 4. Prevalence and abundance of AT1-AAs in women with
hypertensive disorders of pregnancy. A, Activation levels of IgGs
(AT1-AAs), expressed as luciferase activity, obtained from indi-
samples showed significant stimulation, with an average vidual serum samples from various groups of patients. The AT1-
value of 35⫾4% (Figure 4B). The stimulation of luciferase AA–induced luciferase activation is significantly blocked in the
activity was blocked by the 7aa epitope peptide, indicating presence of 7aa that blocks the binding of AT1-AAs to the AT1
receptor. Data are calculated according to the percentage
that this was because of AT1-AA–mediated AT1 receptor change (increase) in luciferase synthesis, determined as RLUs
activation. compared with the basal (no treatment). n⫽30 for normotensive
IgG isolated from the normotensive pregnant women (NT) pregnancies, 23 for gestational hypertension (GH), 10 for
showed the lowest range of activity analyzed, with an average mild PE, and 27 for severe PE. B, Average (mean⫾SEM) activa-
tion of luciferase activity induced by the IgGs (AT1-AAs), as
stimulation of only 14⫾3% over basal, a value that was determined by the luciferase activity, from various groups of
significantly less than that of all of the other groups. It is patients. The luciferase activation is significantly blocked by the
noteworthy that approximately half of the normotensive 7aa that blocks the binding of AT1-AAs to the AT1 receptor,
samples had no detectable activity in this assay. The low level thereby also establishing that the increase in luciferase synthe-
sis is indeed caused by the AT1-AAs. *P⬍0.05, significantly dif-
of activity displayed by most of the normotensive pregnancy ferent compared with NT, @P⬍0.05 significantly different com-
samples was inhibited by the presence of the 7aa epitope pared with GH, $P⬍0.05 significantly different compared with
peptide, indicating that the low level of activity observed in mild PE, #P⬍0.05 significantly different compared with the
these samples was likely the result of low titers of AT1-AA. absence of 7aa. Data were analyzed by unpaired t test.
We draw several conclusions from the data presented in
Figure 4. First, ⬎95% of women with PE (10 of 10 of those AT1-AA Activity Significantly Correlates With
with mild PE and 26 of 27 with severe PE) harbor AT1-AAs. Blood Pressure, Proteinuria, and sFlt-1 in
Second, normotensive pregnant women harbor low or unde- Severe PE
tectable levels of AT1-AAs, and the overall average antibody The wide distribution of AT1-AA levels among the women
levels in these women are ⬇5-fold less than those in women with severe PE (Figure 4) was associated with a wide range
with severe PE. Third, the average level of AT1-AA activity in the severity of clinical features of the disease. For this
for each of the 3 groups of women examined shows a reason, this group of patients provided a favorable opportu-
relationship to the clinical severity of the disease. nity to examine the relationships among the AT1-AA activity,
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 Siddiqui et al AT1-AA and Disease Severity 391

A B C
40
Systolic Blood Pressure

220 3000

sFlt-1 (ng/ml)
200 30

(mg/24 hrs)
2000

Protein
(mmHg)

180 20
1000
160 10

140 0 0
0 50 100 150 0 100 200 300 0 50 100 150
LUCIFERASE ACTIVITY LUCIFERASE ACTIVITY LUCIFERASE ACTIVITY
(RLU-percent over basal) (RLU-percent over basal) (RLU-percent over basal)
Figure 5. Positive correlation among AT1-AA, blood pressure, urinary protein, and sFlt-1 in severe PE. A, Correlation analysis between
AT1-AA activity and systolic blood pressure (r⫽0.51; P⬍0.05). B, The positive correlation between AT1-AA activity and urinary protein
(r⫽⫺0.70; P⬍0.05). C, The relation between AT1-AA and serum sFlt-1 (r⫽0.71; P⬍0.05).

blood pressure, urinary protein, and sFlt-1 levels. This was significantly elevated in the blood circulation of patients with
accomplished by plotting AT1-AA against blood pressure, gestational hypertension compared with those of normoten-
proteinuria, and sFlt-1 for individual patients in the severe PE sive controls. However, sFlt-1 levels were significantly in-
group. The results (Figure 5A) show that the concentration of creased in both mild and severe preeclamptic patients, as
AT1-AAs in the serum of these women shows a strong summarized in Table. Thus, patients with gestational hyper-
positive correlation with systolic blood pressure (r⫽0.56; tension show a discordance between AT1-AA and sFlt-1
n⫽21; P⬍0.05). The correlation analysis between the levels.
AT1-AA concentration and urinary protein is illustrated in
Figure 5B (r⫽0.70; n⫽15; P⬍0.05), and in Figure 5C we Discussion
show the positive correlation between the plasma sFlt-1 In this study, our newly developed sensitive and high-
levels with the concentration of AT1-AAs in women with throughput luciferase bioassay allowed us to address 2
severe PE (r⫽0.71; n⫽16; P⬍0.05). Thus, among women important questions. First, what percentage of women with
with severe PE, there is a strong positive correlation between PE have AT1-AA? Second, does the titer of AT1-AA correlate
the abundance of AT1-AAs and blood pressure, urinary with the severity of the disease? Our results show the
protein, and sFlt-1 levels. following: (1) ⬎95% of women with PE harbor significantly
elevated levels of AT1-AAs; (2) the level of AT1-AA activity
AT1-AA Is Significantly Increased in Women With increases with the severity of the disease; (3) there is a strong
Gestational Hypertension correlation of AT1-AA activity to hypertension, proteinuria,
We also examined IgG from women with gestational hyper- and sFlt-1 in severe PE; (4) elevated levels of AT1-AA are
tension for the presence of AT1-AAs. These women are present in women with gestational hypertension, lacking
characterized by hypertension appearing after 20 weeks’ proteinuria; and (5) normotensive pregnant women harbor
gestation and the absence of proteinuria. The results (Figure low or undetectable levels of AT1-AAs, and the average
4A) show that IgG obtained from women with gestational antibody level in these women is ⬇5-fold less than that in
hypertension showed an average stimulation of 33⫾4% women with severe PE. In summary, our findings show that
(Figure 4B) in the NFAT-luciferase bioassay. The activation AT1-AA is highly prevalent in PE and that its titer strongly
of luciferase was inhibited by the presence of the 7aa epitope correlates with the severity of the disease.
peptide, indicating that the luciferase activation resulted from We have recently extended multiple in vitro findings to in
AT1-AA–mediated AT1 receptor activation. The AT1-AA vivo studies showing that the introduction of AT1-AA from
activity levels obtained with the IgG from the gestational preeclamptic patients into pregnant mice results in key
hypertension and mild PE groups were quite similar (Figure features of PE. These findings provide support for the
4), and the degree of blockage obtained with the 7aa epitope hypothesis that AT1-AAs contribute to pathophysiology in
peptide was also similar (10⫾2 versus 10⫾3; P⬍0.05 com- PE.18 However, the prevalence of AT1-AAs in PE is largely
pared with respective activation without 7aa). These findings undetermined because of the lack of a sensitive bioassay to
indicate that the abundance of AT1-AAs is similar in the 2 accurately measure autoantibody activity. In this study, be-
hypertensive groups (gestational hypertension and mild PE) cause of successful establishment of a sensitive and conve-
and likely contributes to hypertension by mimicking the nient bioassay to quantify AT1-AA activity in patients, we are
vasoconstrictive actions of Ang II. able to provide the first compelling evidence that AT1-AA is
present in nearly all women diagnosed with PE (both mild
sFlt-1 Levels Are Not Significantly Elevated in and severe). These studies complement our recent animal
Gestational Hypertension studies showing that AT1-AAs cause features of PE when
We also measured sFlt-1 concentrations in patients with mild injected into pregnant mice. More importantly, we also
PE, gestational hypertension, and in normotensive pregnant discovered that AT1-AA activity is significantly higher in
women. To our surprise, we found that sFlt-1 levels were not patients with severe PE compared with those with mild PE.
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392 Hypertension February 2010

Notably, we found that there is a significant correlation of the for the autoantibody to induce sFlt-1 levels. This possibility
titer of AT1-AAs to hypertension, proteinuria, and sFlt-1 will be addressed in future experiments.
levels in patients with severe PE. The significant correlation In summary, we have provided initial patient studies
of AT1-AA activity with severity of the disease in hu- showing that AT1-AA is highly prevalent in PE, and its titer
mans5,20,21 is in good agreement with our mouse studies increases with disease severity. This study adds additional
showing that AT1-AA induces preeclamptic-like features in a support to the novel hypothesis that PE is an autoimmune
dose-dependent way in pregnant mice.18 In addition, the disease in which AT1-AAs contribute to the pathophysiology
correlation of AT1-AA with sFlt-1 levels seen in severe PE is of the disease.13
also consistent with earlier reports that link sFlt-1 production
with AT1 receptor activation.14,22 Thus, the results of both Clinical Perspectives
human and animal studies show that the levels of AT1-AA Considerable evidence indicates that a circulating maternal
increase with the severity of the disease. autoantibody, AT1-AA, is associated with PE and contributes
In contrast to high prevalence of AT1-AAs in PE, we found to the pathogenesis of the disease. Here we report the use of
that normotensive patients were characterized by low to a convenient and sensitive bioassay to show that these
nondetectable levels of AT1-AAs. The average AT1-AA autoantibodies are present in nearly all women diagnosed
activity in normotensive pregnant women was much lower with PE and that the titer of the autoantibodies increases with
than that of women with mild PE and severe PE. However, the severity of the disease. Overall, our experimental evi-
the low levels of AT1-AA activity in these samples presum- dence supports the novel concept that PE is an autoimmune
ably represent a low titer of AT1-AAs, because the activity disease in which disease symptoms result from autoantibody-
was blocked by either losartan or the 7aa epitope peptide. induced AT1 receptor activation. Our findings have signifi-
These findings imply that, among normotensive pregnant cant prognostic, diagnostic, and therapeutic implications with
individuals, the titer of AT1-AAs is not high enough or has regard to the medical management of this devastating disease
not been present for sufficient duration to cause the clinical for both mom and fetus.
features seen in PE. Notably, 2 normotensive individuals
contain a relatively high AT1-AA activity, similar to the Sources of Funding
average observed in patients with PE. One possible explana- The present work was supported by National Institutes of Health
tion is that AT1-AAs may not have been present long enough grants (HL076558 to Y.X. and HD34130 to R.E.K.), March of
Dimes grant 6-FY06-323, and the Texas Higher Education Coordi-
to cause symptoms. Thus, it will be critical to perform a nating Board (grant ARP011618-0012-2006).
prospective clinical study to determine when AT1-AA occurs
in both normal and preeclamptic patients. Disclosures
Among 23 patients with gestational hypertension, we None.
found that the average concentration of AT1-AAs was signif-
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 Angiotensin Receptor Agonistic Autoantibody Is Highly Prevalent in Preeclampsia:
Correlation With Disease Severity
Athar H. Siddiqui, Roxanna A. Irani, Sean C. Blackwell, Susan M. Ramin, Rodney E. Kellems
and Yang Xia

Hypertension. 2010;55:386-393; originally published online December 7, 2009;

doi: 10.1161/HYPERTENSIONAHA.109.140061
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2009 American Heart Association, Inc. All rights reserved.
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