Recombinant Vascular Endothelial Growth Factor 121 Attenuates Autoantibody-Induced Features of Pre-eclampsia in Pregnant Mice
AMERICAN JOURNAL OF HYPERTENSION |
Recombinant Vascular Endothelial Growth Factor 121 Attenuates Autoantibody-Induced Features of Pre-eclampsia in Pregnant Mice
Athar H. Siddiqui 1
Roxanna A. Irani 1
Yujin Zhang 1
Yingbo Dai 1
Sean C. Blackwell 0
Susan M. Ramin 0
Rodney E. Kellems 1
Yang Xia 1
0 Department of Obstetrics, Gynecology and Reproductive Sciences, The University of Texas medical School at , USA
1 Department of Biochemistry and molecular Biology, The University of Texas medical School at Houston , Houston, Texas, USa , USA
Bacgkround Pre-eclampsia (PE) is a serious hypertensive disorder of pregnancy characterized by excessive production of a soluble form of the vascular endothelial growth factor (VEGF) receptor-1, termed soluble fms-like tyrosine kinase-1 (sFlt-1). This placental-derived factor is believed to be a key contributor to the clinical features of PE. Women with PE are also characterized by the presence of autoantibodies, termed angiotensin type 1 receptor activating autoantibody (aT1-aa ), that activate the major angiotensin receptor, aT1. These autoantibodies cause clinical features of PE and elevated sFlt-1 when injected into pregnant mice. The research reported here used this autoantibody-injection model of PE to assess the therapeutic potential of recombinant VEGF121, a relatively stable form of the natural ligand.
angiotensin receptor agonistic autoantibody; blood pressure; hypertension; pre-eclampsia; recombinant VEGF121; sFlt-1
Immunoglobulin G (IgG) from women with PE was injected into
pregnant mice with or without continuous infusion of recombinant
VEGF121. Injected mice were monitored for symptoms of PE.
Pre-eclampsia (PE) is a serious complication of pregnancy
and a leading cause of maternal and neonatal morbidity and
mortality.1–3 It is a multisystem disorder generally appearing
after the 20th week of gestation and characterized by
hypertension, proteinuria, inflammation, and endothelial
dysfunction.4–6 Despite intensive research efforts and several large
clinical trials, the underlying cause of PE remains a mystery
and treatment options continue to be unsatisfactory. It is a
commonly held belief that “toxic factors” secreted by the
placenta into the maternal circulation are responsible for
systemic endothelial dysfunction, hypertension and multi-organ
damage.7,8 Prominent among such factors is soluble
fmslike tyrosine kinase-1 (sFlt-1), a soluble form of the vascular
endothelial growth factor (VEGF) receptor-1. While the data
linking enhanced sFlt-1 production in the pathogenesis of PE
are compelling, the mechanisms accounting for enhanced
production and release of sFlt-1 from placentas of women with PE
are not well understood.
We initially showed that angiotensin II stimulates sFlt-1
synthesis and secretion by the placenta during pregnancy.9
Realizing that angiotensin II levels are not increased in women
with PE over that occurring during normotensive (NT)
pregnancies, we tested the possibility that angiotensin type 1
receptor agonistic autoantibodies (AT1-AAs) harbored by women
with PE contribute to increased production of sFlt-1. We found
that immunoglobulin G (IgG) from these women, in contrast
to IgG from NT pregnant women, stimulates the synthesis
and secretion of sFlt-1 via AT1 receptor (AT1R) activation in
pregnant mice, human placental explants, and in human
trophoblast cells.10 Similar findings have been recently reported
by Parrish et al.11 In addition, in women with PE, AT1-AA
titers are proportional to serum levels of sFlt-1.12 Overall our
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published studies suggest that angiotensin II is a significant
physiological regulator of sFlt-1 synthesis and secretion
during normal pregnancy and that the excessive accumulation
of sFlt-1 observed in women with PE is due to the additional
activation of AT1Rs mediated by AT1-AAs. Excessive
production of sFlt-1 is considered harmful because, as a soluble form
of the VEGF receptor-1, it binds to free VEGF and thereby
disrupts normal pro-angiogenic signaling. It is this antiangiogenic
feature of sFlt-1 that is believed to contribute to the clinical
symptoms of PE.13,14 Although PE is a human condition that
has not been observed to occur naturally in animals, a number
of experimentally-induced animal models, characterized with
excessive production of sFlt-1, have been described.15–18 The
research reported here used an autoantibody-injection model
of PE in pregnant mice to assess the therapeutic potential of
VEGF121 infusion to prevent autoantibody-induced clinical
features of PE.
Chemicals. Recombinant mouse VEGF121 (cat. no. CYT-574)
was purchased from ProSpec-Tany TechnoGene Ltd. (Rehovot,
Israel). Losartan was a generous gift from Merck Research
Laboratory (Rahway, NJ). The 7-amino acid (7-aa) peptide
(AFHYESQ), an epitope sequence present on the second
extracellular loop of the AT1R that is recognized by AT1-AA,
was synthesized at the Baylor College of Medicine, Protein
Chemistry Core Laboratory (Houston, TX). The specificity of
this 7-aa peptide in neutralizing the AT1-AA mediated actions
has been verified previously.16,19 Protein G Sepharose 4 Fast
Flow, used for IgG isolation, was purchased from Amersham
Pharmacia Biotech (Uppsala, Sweden). All other chemicals
used in the present study were of high quality grade and were
obtained from Sigma Aldrich (St. Louis, MO).
Animals. Eight-week-old timed pregnant C57BL/6 mice (mated
with syngeneic males) were obtained from Harlan Laboratories
(Indianapolis, IN). The day the copulation plug was detected is
designated E0. The mice were housed in the animal care
facility of the University of Texas, Houston and had access to food
and water ad libitum. Blood was collected from the heart at
the time of killing on day E18. All the protocols involving
animal studies were reviewed and approved by the Institutional
Animal Welfare Committee of the University of Texas Houston
Health Science Center.
IgG injection, VEGF121 infusion, losartan and 7-aa peptide
administration. We have used our established adoptive transfer
animal model of PE as previously described.16 For IgG
injections, mice were anesthetized with isofluorane and concentrated
IgG (800 µg) purified from 200 µl of patient’s serum (NT or
preeclamptic) was introduced into embryonic day 13 and 14
pregnant mice by retro orbital injection. Some of the mice (n = 5)
injected with the IgG from pre-eclamptic patients were infused
with recombinant mouse VEGF121 (180 µg/kg body weight/day
on E13) for 5 days using Alzet osmotic minipumps implanted
subcutaneously (model 1007D, Alzet, Cupertino, CA). The dose
was chosen based on the report by Gilbert et al.20 showing that
this dose had pronounced therapeutic benefit in a rat model of
PE. Losartan (0.24 mg), an AT1R antagonist, or a 7-aa epitope
peptide (1.5 mg) that prevents autoantibody-induced AT1R
activation were co-administered with the IgGs. Beginning with
the first IgG injection blood pressure was measured daily by the
tail cuff method as described below.
Blood pressure measurement. Blood pressure was monitored
at the same time daily using a carotid catheter-calibrated eight
chamber tail-cuff system (CODA, Kent Scientific, Torrington,
CT). Mice were kept warm using a warming pad. After an
initial acclimatization of the mice for five cycles, blood pressure
was monitored over a period of 20 cycles and averaged for the
final blood pressure measurement.
Renal function studies. Urine for creatinine, protein and sodium
analysis was collected by placing mice in metabolic cages for
24 h. Total protein in the urine was determined after suitable
dilution by the BCA method using a kit (Pierce, Rockford, IL).
Creatinine, sodium (urinary and blood) and blood urea nitrogen
(BUN) levels were determined at the University of Texas, MD
Anderson Cancer Center, Laboratory of Veterinary Medicine.
Hematoxylin and eosin (H&E) staining and quantification in
kidneys and placenta. Kidneys and placentas were harvested
from the mice on E18, fixed in 4% formaldehyde, dehydrated,
and embedded in paraffin. Sections of 4µm were cut, stained
with H&E using standard techniques and analyzed by light
microscopy. The extent of renal damage was assessed by
quantifying the glomeruli that showed characteristic features of
damage in PE: decreased Bowman’s space and occlusion of
capillary loop spaces. To examine those features, the glomeruli
were counted in 6–9 fields of randomized and blinded slides
(×10 magnification), with each field having at least 16–22
glomeruli. The glomeruli in each field were given a score based
on the amount of capillary space evident within the Bowman’s
capsule. A highest score of 5 was accorded to glomeruli with
a normal amount of capillary space within Bowman’s
capsule. A score of 1 was assigned to the glomeruli that showed
complete loss of capillary space and an intermediate score of
3 was assigned to the glomeruli that displayed reduced, but
not completely obliterated, capillary space. The scores for each
field were divided by the number of glomeruli to get an average
score per glomerulus for each field.
Similarly placental histological quantification was carried by
quantifying the number of calcifications/field under ×10
magnification. Placental sections were examined under the
microscope and the number of calcifications was counted in each field
and then plotted as number of calcifications recorded per field.
CD34 immunostaining in placenta and quantification.
Placentas were harvested from the mice on E18, fixed in 4%
formaldehyde, dehydrated, and embedded in paraffin. Sections
of 4 µm were cut and stained with anti mouse CD34 (cat. no.
553731, BD Pharmingen, San Diego, CA) at a dilution of 1:100
in a humidified chamber at 37 °C for 2 h. Following the primary
antibody incubation, anti rat IgG HRP detection kit (cat. no.
551013, BD Pharmingen) was used was used to detect the CD34
staining. The immunohistochemical staining for CD34 positive
stains (brown stains) in the labyrinth zone of the placenta was
quantified by Image-Pro Plus software (Media Cybernetics,
Bethesda, MD). The density of the brown staining (positive for
CD34) was measured. The average densities of 6–10 areas per
placenta was determined and averaged to get a mean value.
Statistical analysis. Results are expressed as mean ± s.e.m. All
the data were subjected to statistical analysis using one-way
analysis of variance followed by the Newman Keuls post hoc
test or student’s t-test to determine the significance between
groups. Statistical programs were run by GraphPad Prism 5,
statistical software (GraphPad, San Diego, CA). Statistical
significance was set at P < 0.05.
Veg F121 infusion attenuates at 1-aa induced
hypertension in pregnant mice
To assess the potential role of VEGF therapy to blunt
autoantibody-induced features of PE, we treated autoantibody-injected
pregnant mice with recombinant murine VEGF121
administered by an osmotic minipump implanted subcutaneously. We
found that blood pressure increased significantly in the mice
injected with IgG isolated from women with PE (PE-IgG)
relative to that of mice injected with IgG from NT pregnant women
(NT-IgG) (Figure 1a). In contrast, the increased blood
pressure seen in PE-IgG-injected pregnant mice was completely
abolished by co-injection with losartan or a 7-aa epitope
peptide (Figure 1b). These findings demonstrate the autoantibody
in pre-eclamptic women is capable of increasing blood
pressure in pregnant mice via AT1R activation. Additional
findings show that the autoantibody-induced hypertension was
almost completely blocked by infusion of VEGF121 (159 ± 5 to
124 ± 5 mm Hg, P < 0.05) in pregnant mice (Figure 1a,b).
In vivo effect of Veg F121 on renal dysfunction seen
in autoantibody-injected pregnant mice
We assessed the effect of VEGF121 on proteinuria, another
key maternal feature of PE. The ratio of total urinary
protein:creatinine was significantly higher in pregnant mice
injected with IgG from women with PE (111 ± 16 mg/ml)
compared to mice injected with IgG from NT pregnant women
(22 ± 1 mg/ml, P < 0.05) (Figure 2a). Co-injection with losartan
or the 7-aa epitope peptide completely blocked
autoantibodyinduced proteinuria (Figure 2a), indicating the requirement
for AT1R activation. VEGF121 infusion significantly decreased
UN /gd 20
urinary protein excretion (40 ± 5 mg/ml, P < 0.05) in mice
injected with IgG from women with PE (Figure 2a).
An increase in BUN is a physiological response to decreased
blood flow in the kidneys and may indicate renal damage. BUN
levels were significantly increased in pregnant mice injected
with IgG from women with PE (31 ± 1 mg/dl) compared to
mice injected with IgG from NT pregnant women (24 ± 2 mg/
dl, P < 0.05, Figure 2b). The autoantibody-induced increase in
BUN was prevented by co-injection with losartan or the 7-aa
epitope peptide (18 ± 2 mg/dl and 20 ± 2 mg/dl respectively,
P < 0.05 compared to PE, Figure 2b). Continuous infusion
with VEGF121 prevented the increase in BUN that
accompanied injection of pregnant mice with IgG from women with PE
(18 ± 2 mg/dl, P < 0.05 compared to PE).
a utoantibody-induced changes in renal histology
are prevented by Veg F121 treatment
The impaired renal function associated with PE is also
accompanied with characteristic alterations in renal histology, especially
that of the glomeruli.5,15,21,22 These renal histological changes
are also seen in AT1-AA injected pregnant mice.16 To determine
whether VEGF treatment can prevent autoantibody-induced
renal histological changes, we injected pregnant mice on days 13
and 14 of gestation with IgG from women with PE in the
presence or absence of infused VEGF121. Kidneys were harvested on
embryonic day 18 for histological analysis. H&E staining revealed
extensive renal damage as evidenced by narrowing or loss of
Bowman’s capillary space in the majority of glomeruli (Figure 3a)
seen in PE-IgG-injected pregnant mice. The kidneys of pregnant
mice injected with IgG from NT pregnant women rarely showed
signs of glomerular damage. VEGF121 infusion prevented the
renal abnormalities in the mice that resulted from injection with
IgG from women with PE (Figure 3a). Co-injection of losartan
or the 7-aa epitope peptide also prevented the renal damage seen
in the kidneys of the autoantibody-injected mice. The
histomorphometric quantification analysis showed that the extent of
glomerular damage was significantly greater in PE-IgG-injected
pregnant mice compared to that in NT-IgG-injected pregnant
mice and that VEGF121 treatment attenuated the renal damage
observed in autoantibody-injected mice (Figure 3b).
In addition to the H&E staining, we also performed periodic
acid–Schiff staining of the kidney sections obtained from the
mice from all groups. As with H&E stained sections we noted
pathological alterations in glomerular structure, especially loss
of Bowman’s space that was prevented by VEGF121, losartan or
the 7-aa epitope peptide (Figure 3a, bottom panel). However,
no periodic acid–Schiff-positive materials were observed in
glomeruli in all of those groups. Overall, histological studies
demonstrate typical renal damage in PE-IgG-injected pregnant
mice as seen in humans and that these changes were prevented
by infusion of VEGF121.
Veg F121 treatment reduces autoantibody-induced placental
damage and improves placental endothelial cell content
in autoantibody-injected mice
Impaired placental development is often associated with PE
and has been observed in AT1-AA-injected pregnant mice.23
In agreement with previous reports23,24 histological analyses
of placentas of antibody-injected pregnant mice revealed
extensive cellular disorganization of the labyrinth zone and
increased intraparenchymal calcifications in pregnant mice
injected with IgG from women with PE (Figure 4). These
histological changes were not seen in pregnant mice injected with
IgG obtained from NT pregnant women. Continuous
infusion of VEGF121 prevented the placental pathological changes
induced by IgG from women with PE.
To determine the role of autoantibody-induced
placentaderived antiangiogenic factors in placenta impairment,
we analyzed the vasculature of isolated mouse placentas
by immunostaining using antibody recognizing CD34, an
endothelial cell-specific marker. The results show that CD34
staining was decreased in the labyrinth zone of the
placentas of mice injected with IgG from women with PE
compared to those injected with IgG from NT pregnant women
(Figure 4). Continuous infusion with VEGF121 attenuated
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on 10 June 2018
autoantibody-induced reduction in angiogenesis as
evidenced by improved CD34 staining. Similarly, co-injection
of antibody with losartan or the 7-aa epitope peptide also
showed increased CD34 staining (Figure 4). Taken together,
our findings demonstrate that VEGF121 treatment can
prevent AT1-AA-induced placental impairment and decreased
endothelial cell content.
We report here that continuous infusion of VEGF121 in
pregnant mice attenuated AT1-AA-induced hypertension and
proteinuria, the hallmark features of PE. In addition, we
demonstrate that infusion of VEGF121 blunted other features
induced by PE-IgG in the pregnant mice including kidney
damage, renal dysfunction featured with increased BUN and
placental impairment coupled with decreased endothelial cell
content. Overall, our findings reveal that VEGF121 infusion is
maY 2011 | VOLUmE 24 NUmBER 5 | AMERICAN JOURNAL OF HYPERTENSION
a promising therapeutic possibility to attenuate
autoantibodyinduced features of PE.
VEGF is important in renal function where it is highly
expressed by glomerular podocytes25 and targets VEGF
receptors present on glomerular endothelial cells.26 Initial evidence
for the potential importance of VEGF signaling in renal–
vascular function came indirectly from clinical trials of
antiVEGF therapy for the treatment of certain cancers, where
unexpected side effects included hypertension and
proteinuria.27 Maynard et al.5 realized that excessive sFlt-1, because of
its anti-VEGF properties, could account for the same features
in PE and tested this possibility by introducing recombinant
adenoviruses encoding sFlt-1 into rats. Their results showed
that sFlt-1 mediated a dose-dependent increase in
hypertension, proteinuria and characteristic renal findings termed
glomerular endotheliosis when overexpressed in pregnant or
nonpregnant rats. Clinical studies have shown that the increase
in sFlt-1 in pregnant women precedes the onset of symptoms
of PE by several weeks28 and that the levels of circulating sFlt-1
achieved are proportional to disease severity. Other studies
have shown that infusion of sFlt-1 via controlled release by
osmotic minipumps resulted in hypertension and proteinuria
in pregnant rats.29 In a rat model of PE based on
experimentally-induced placental ischemia, excessive sFlt-1 production
has also been implicated in hypertension and proteinuria.30
We have previously shown that increased sFlt-1 is also
associated with the autoantibody-injection model of PE in pregnant
mice. It is likely that the beneficial effects of VEGF in these
animal models of PE results from neutralizing the antiangiogenic
effects of excessive sFlt-1. Thus, the evidence implicating sFlt-1
in the pathogenesis of PE is considerable and compelling and
the use of VEGF therapy to neutralize the effects of excessive
sFlt-1 is especially attractive.
The placental ischemia model of PE in rats (reduced
uteroplacental perfusion pressure (RUPP)) and the
autoantibody-injection model in pregnant mice share a number of
important features including elevated sFlt-1, tumor necrosis
factor-α, interleukin-6, and AT1-AA.30–33 Additional
evidence shows that all of these molecules can induce
hypertension and proteinuria when introduced into pregnant
animals.5,11,16,31–35 It has also been shown that antagonism
of each of these molecules significantly reduces hypertension
and proteinuria, indicating that each contributes to disease
pathology. Recent evidence supports a role for endothelin-1
as a mediator of sFlt-1-induced hypertension.36 In the latter
studies infusion of sFlt-1 resulted in increased production of
preproendothelin-1 mRNA in the kidney cortex and
hypertension that was prevented by antagonism of the endothelin
A receptor, the target of endothelin-1. Thus the hypertensive
and proteinuric effects of sFlt-1 are observed in pregnant and
nonpregnant animals and are mediated by endothelin-1
signaling pathways. The antihypertensive effects of VEGF therapy
may be mediated by stimulating endothelial cells to produce
nitric oxide and vasodilatory prostacyclins, resulting in
vascular smooth muscle relaxation.37 Because VEGF stimulates
the production of vasodilatory substances such as nitric oxide
and prostacyclins it is also possible that VEGF121 therapy can
function as a general antihypertensive agent to counteract the
hypertensive effects of a number of molecules in addition to
The major focus of the research reported in this study
was to evaluate the therapeutic potential of VEGF121 in our
autoantibody-injection model of PE in mice. For this reason
we examined the potential therapeutic effect of VEGF121 with
regard to the defining clinical features of the disease including
hypertension, proteinuria, renal abnormalities, and
endothelial damage. We have shown here that VEGF121 infusion
prevents these autoantibody-induced features of PE. In view of
the data presented here showing that VEGF infusion reduced
autoantibody-induced placental damage and improves
placental endothelial cell content it will be of interest to examine
the effects of VEGF121 therapy on fetal and placental weights
and development. This issue has been addressed in two
experimental models of PE in rat, an adenovirus-induced sFlt-1 over
expression model and the RUPP model. No reduction in
placental and fetal size was reported for the sFlt-1 over expression
model and no effect of VEGF treatment was observed.17 The
RUPP model is characterized by reduced fetal weight and no
reduction in placental size. The therapeutic dose of VEGF121
did not result in a change in either of these parameters.20 Thus,
in the RUPP model of PE the reduction in fetal weight was not
corrected by the dose of VEGF121 therapy that was most
effective in preventing the maternal features of PE. We have recently
reported that the introduction of AT1-AA into pregnant mice
resulted in smaller placentas and fetuses.23 It will be
interesting to examine the effects of VEGF121 therapy on placental and
fetal weights and development in the autoantibody-injection
model of PE in pregnant mice.
In conclusion, we provide for the first time in vivo
evidence that infusion of VEGF121 significantly attenuates key
features of PE induced by AT1-AA in pregnant mice. In
addition to the antibody-injection model of PE presented here, the
therapeutic benefit of VEGF121 infusion has also been
demonstrated in two other animal models of PE. The first was a
rat model of PE generated by adenoviral over expression of
sFlt-1. In this model, VEGF121 therapy attenuates
hypertension and prevents kidney damage.17 More recently the
benefit of VEGF121 therapy was shown in an animal model of PE
based on experimentally-induced RUPP20 Both of these
models, as with the antibody-injection model used in the present
report, are characterized by excessive production of sFlt-1.
However, the RUPP model and the antibody-injection model
are also characterized by increased production of other
molecules that have been associated with PE, including tumor
necrosis factor-α, interleukin-6, and sEng. Our findings are in
agreement with those from the RUPP model and show that
VEGF121 prevents hypertension and renal dysfunction in the
presence of increased levels of multiple other factors
associated with PE. Thus, the potential benefit of VEGF therapy has
now been shown in three preclinical models of PE justifying
serious consideration of this as a therapeutic approach for use
in women with PE.
a cknowledgments: This work was supported by National Institute of
Health Grants HL076558, HD34130, a merican Heart a ssociation Grant
10GRNT3760081 and grants from the march of Dimes Foundation and the
Texas Higher Education Coordinating Board.
Disclosure: The authors declared no conflict of interest
1. Roberts JM , Pearson G , Cutler J , Lindheimer M ; NHLBI Working Group on Research on Hypertension During Pregnancy. Summary of the NHLBI Working Group on Research on Hypertension During Pregnancy. Hypertension 2003 ; 41 : 437 - 445 .
2. Sibai B , Dekker G , Kupferminc M . Pre-eclampsia . Lancet 2005 ; 365 : 785 - 799 .
3. Lindheimer MD , Umans JG . Explaining and predicting preeclampsia . N Engl J Med 2006 ; 355 : 1056 - 1058 .
4. Young BC , Levine RJ , Karumanchi SA . Pathogenesis of preeclampsia . Annu Rev Pathol 2010 ; 5 : 173 - 192 .
5. Maynard SE , Min JY , Merchan J , Lim KH , Li J , Mondal S , Libermann TA , Morgan JP , Sellke FW , Stillman IE , Epstein FH , Sukhatme VP , Karumanchi SA . Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia . J Clin Invest 2003 ; 111 : 649 - 658 .
6. Maynard SE , Venkatesha S , Thadhani R , Karumanchi SA . Soluble Fms-like tyrosine kinase 1 and endothelial dysfunction in the pathogenesis of preeclampsia . Pediatr Res 2005 ; 57 : 1R - 7R .
7. Barden A . Pre-eclampsia: contribution of maternal constitutional factors and the consequences for cardiovascular health . Clin Exp Pharmacol Physiol 2006 ; 33 : 826 - 830 .
8. de Groot CJ , Taylor RN . New insights into the etiology of pre-eclampsia . Ann Med 1993 ; 25 : 243 - 249 .
9. Zhou CC , Ahmad S , Mi T , Xia L , Abbasi S , Hewett PW , Sun C , Ahmed A , Kellems RE , Xia Y. Angiotensin II induces soluble fms-Like tyrosine kinase-1 release via calcineurin signaling pathway in pregnancy . Circ Res 2007 ; 100 : 88 - 95 .
10. Zhou CC , Ahmad S , Mi T , Abbasi S , Xia L , Day MC , Ramin SM , Ahmed A , Kellems RE , Xia Y. Autoantibody from women with preeclampsia induces soluble Fms-like tyrosine kinase-1 production via angiotensin type 1 receptor and calcineurin/ nuclear factor of activated T-cells signaling . Hypertension 2008 ; 51 : 1010 - 1019 .
11. Parrish MR , Murphy SR , Rutland S , Wallace K , Wenzel K , Wallukat G , Keiser S , Ray LF , Dechend R , Martin JN , Granger JP , LaMarca B . The effect of immune factors, tumor necrosis factor-alpha, and agonistic autoantibodies to the angiotensin II type I receptor on soluble fms-like tyrosine-1 and soluble endoglin production in response to hypertension during pregnancy . Am J Hypertens 2010 ; 23 : 911 - 916 .
12. Siddiqui AH , Irani RA , Blackwell SC , Ramin SM , Kellems RE , Xia Y. Angiotensin receptor agonistic autoantibody is highly prevalent in preeclampsia: correlation with disease severity . Hypertension 2010 ; 55 : 386 - 393 .
13. Xu B , Thornton C , Tooher J , Hennessy A . Exogenous soluble VEGF receptor-1 (sFlt1) regulates Th1/Th2 cytokine production from normal placental explants via intracellular calcium . Hypertens Pregnancy 2009 ; 28 : 448 - 456 .
14. Karumanchi SA , Epstein FH . Placental ischemia and soluble fms-like tyrosine kinase 1: cause or consequence of preeclampsia? Kidney Int 2007 ; 71 : 959 - 961 .
15. Mutter WP , Karumanchi SA . Molecular mechanisms of preeclampsia . Microvasc Res 2008 ; 75 : 1 - 8 .
16. Zhou CC , Zhang Y , Irani RA , Zhang H , Mi T , Popek EJ , Hicks MJ , Ramin SM , Kellems RE , Xia Y. Angiotensin receptor agonistic autoantibodies induce pre-eclampsia in pregnant mice . Nat Med 2008 ; 14 : 855 - 862 .
17. Li Z , Zhang Y , Ying Ma J , Kapoun AM , Shao Q , Kerr I , Lam A , O'Young G , Sannajust F , Stathis P , Schreiner G , Karumanchi SA , Protter AA , Pollitt NS . Recombinant vascular endothelial growth factor 121 attenuates hypertension and improves kidney damage in a rat model of preeclampsia . Hypertension 2007 ; 50 : 686 - 692 .
18. Isler CM , Bennett WA , Rinewalt AN , Cockrell KL , Martin JN Jr, Morrison JC , Granger JP . Evaluation of a rat model of preeclampsia for HELLP syndrome characteristics . J Soc Gynecol Investig 2003 ; 10 : 151 - 153 .
19. Wallukat G , Homuth V , Fischer T , Lindschau C , Horstkamp B , Jüpner A , Baur E , Nissen E , Vetter K , Neichel D , Dudenhausen JW , Haller H , Luft FC . Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor . J Clin Invest 1999 ; 103 : 945 - 952 .
20. Gilbert JS , Verzwyvelt J , Colson D , Arany M , Karumanchi SA , Granger JP . Recombinant vascular endothelial growth factor 121 infusion lowers blood pressure and improves renal function in rats with placentalischemia-induced hypertension . Hypertension 2010 ; 55 : 380 - 385 .
21. Henao DE , Saleem MA , Cadavid AP . Glomerular disturbances in preeclampsia: disruption between glomerular endothelium and podocyte symbiosis . Hypertens Pregnancy 2010 ; 29 : 10 - 20 .
22. Strevens H , Wide-Swensson D , Hansen A , Horn T , Ingemarsson I , Larsen S , Willner J , Olsen S. Glomerular endotheliosis in normal pregnancy and pre-eclampsia . BJOG 2003 ; 110 : 831 - 836 .
23. Irani RA , Zhang Y , Blackwell SC , Zhou CC , Ramin SM , Kellems RE , Xia Y. The detrimental role of angiotensin receptor agonistic autoantibodies in intrauterine growth restriction seen in preeclampsia . J Exp Med 2009 ; 206 : 2809 - 2822 .
24. Irani RA , Zhang Y , Zhou CC , Blackwell SC , Hicks MJ , Ramin SM , Kellems RE , Xia Y . Autoantibody-mediated angiotensin receptor activation contributes to preeclampsia through tumor necrosis factor-alpha signaling . Hypertension 2010 ; 55 : 1246 - 1253 .
25. Hakroush S , Moeller MJ , Theilig F , Kaissling B , Sijmonsma TP , Jugold M , Akeson AL , Traykova-Brauch M , Hosser H , Hähnel B , Gröne HJ , Koesters R , Kriz W. Effects of increased renal tubular vascular endothelial growth factor (VEGF) on fibrosis, cyst formation, and glomerular disease . Am J Pathol 2009 ; 175 : 1883 - 1895 .
26. Schrijvers BF , Flyvbjerg A , De Vriese AS . The role of vascular endothelial growth factor (VEGF) in renal pathophysiology . Kidney Int 2004 ; 65 : 2003 - 2017 .
27. Kuo CJ , Farnebo F , Yu EY , Christofferson R , Swearingen RA , Carter R , von Recum HA , Yuan J , Kamihara J , Flynn E , D'Amato R , Folkman J , Mulligan RC . Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer . Proc Natl Acad Sci USA 2001 ; 98 : 4605 - 4610 .
28. Levine RJ , Maynard SE , Qian C , Lim KH , England LJ , Yu KF , Schisterman EF , Thadhani R , Sachs BP , Epstein FH , Sibai BM , Sukhatme VP , Karumanchi SA . Circulating angiogenic factors and the risk of preeclampsia . N Engl J Med 2004 ; 350 : 672 - 683 .
29. Gilbert JS , Ryan MJ , LaMarca BB , Sedeek M , Murphy SR , Granger JP . Pathophysiology of hypertension during preeclampsia: linking placental ischemia with endothelial dysfunction . Am J Physiol Heart Circ Physiol 2008 ; 294 : H541 - H550 .
30. Gilbert JS , Babcock SA , Granger JP . Hypertension produced by reduced uterine perfusion in pregnant rats is associated with increased soluble fms-like tyrosine kinase-1 expression . Hypertension 2007 ; 50 : 1142 - 1147 .
31. Gilbert JS , Gilbert SA , Arany M , Granger JP . Hypertension produced by placental ischemia in pregnant rats is associated with increased soluble endoglin expression . Hypertension 2009 ; 53 : 399 - 403 .
32. Zhou CC , Irani RA , Zhang Y , Blackwell SC , Mi T , Wen J , Shelat H , Geng YJ , Ramin SM , Kellems RE , Xia Y. Angiotensin receptor agonistic autoantibody-mediated tumor necrosis factor-alpha induction contributes to increased soluble endoglin production in preeclampsia . Circulation 2010 ; 121 : 436 - 444 .
33. LaMarca B , Wallukat G , Llinas M , Herse F , Dechend R , Granger JP . Autoantibodies to the angiotensin type I receptor in response to placental ischemia and tumor necrosis factor alpha in pregnant rats . Hypertension 2008 ; 52 : 1168 - 1172 .
34. LaMarca BB , Bennett WA , Alexander BT , Cockrell K , Granger JP . Hypertension produced by reductions in uterine perfusion in the pregnant rat: role of tumor necrosis factor-alpha . Hypertension 2005 ; 46 : 1022 - 1025 .
35. LaMarca B , Speed J , Fournier L , Babcock SA , Berry H , Cockrell K , Granger JP . Hypertension in response to chronic reductions in uterine perfusion in pregnant rats: effect of tumor necrosis factor-alpha blockade . Hypertension 2008 ; 52 : 1161 - 1167 .
36. Murphy SR , LaMarca BB , Cockrell K , Granger JP . Role of endothelin in mediating soluble fms-like tyrosine kinase 1-induced hypertension in pregnant rats . Hypertension 2010 ; 55 : 394 - 398 .
37. Shibuya M. Structure and function of VEGF/VEGF-receptor system involved in angiogenesis . Cell Struct Funct 2001 ; 26 : 25 - 35 .