Journal of Clinical Laboratory Analysis 30: 7583 (2016)

HIF-1 Alpha and Placental Growth Factor in Pregnancies

Complicated With Preeclampsia: A Qualitative and Quantitative
Analysis
Gayatri Rath ,1 Ruby Aggarwal ,1 Poonam Jawanjal,1 Richa Tripathi,1 and Aruna Batra2
1
Department of Anatomy, Vardhman Mahavir Medical College & Safdarjung Hospital, New Delhi, India
2
Department of Obstetrics and Gynecology, Vardhman Mahavir Medical College & Safdarjung Hospital,
New Delhi, India

Background: The pathophysiology of mic expression of HIF-1 was noticed in

preeclampsia is not clearly understood syncytiotrophoblast (P = 0.0001) but in con-
worldwide. Hypoxia inducible factor 1 trol placenta, it was localized to cytoplasm
(HIF-1) is thought to be the preliminary (P = 0.0001). The intensity of PIGF expres-
factor for the hypoxic conditions prevailing sion was lower in syncytiotrophoblast cyto-
in preeclampsia, which causes imbalance plasm (P = 0.0001) in preeclamptic cases
in the expression of angiogenic proteins. as compared with control. Also, the signif-
A proangiogenic protein, placental growth icant upregulated concentration of HIF-1
factor (PIGF), is reported to be dysregu- and downregulated PIGF was observed
lated in preeclampsia. Therefore, this study in serum samples of preeclamptic woman
focuses on the investigation of HIF-1 and (P = 0.0001). Thus, there was a signif-
PIGF in preeclamptic conditions and a pos- icant direct negative correlation between
sible molecular association between them. HIF-1 and PIGF both at tissue and serum
Methods: Placental tissue (n = 45 + 45) and level (P < 0.01). Conclusion: The direct
serum samples (n = 80 + 80) of preeclamp- inverse association between HIF-1 and
tic patients and healthy control were col- PIGF in serum and placental tissues may
lected and processed for the analysis of be responsible for the low oxidative stress
HIF-1 and PIGF by immunohistochem- and endothelial dysfunction, leading to the
istry and enzyme-linked immunosorbent pathogenesis of preeclampsia. J. Clin.
assay (ELISA). Results: In preeclamptic Lab. Anal. 30:7583, 2016. 
C 2014 Wiley

group, the significant nuclear and cytoplas- Periodicals, Inc.

Key words: preeclampsia; hypoxia; HIF-1; PIGF

INTRODUCTION nal circulation, which causes endothelial dysfunction pre-

vailing in preeclampsia. The key mediator of the hypoxic
Preeclampsia is a heterogeneous pregnancy disorder
condition is the hypoxia inducible factor 1 (HIF-1). HIF-1
that is characterized by hypertension and proteinuria
is involved in transcription of many oxygen-dependent
mostly developing in late pregnancy. It affects 28%
genes that encode for proteins associated with
of pregnancies worldwide (1, 2). The incident rate of
preeclampsia is 2.8% higher in developing countries like
India as compared to that of developed nations (3). In Theseauthors contributed equally to this work.
spite of extensive research in the pathophysiology of this Grant sponsor: Indian Council of Medical Research (ICMR).
disease, the etiology is still poorly understood. It may be Correspondence to: Dr. Gayatri Rath, Director Professor Department
due to insufficient adaptation of decidual and intramy- of Anatomy, Vardhman Mahavir Medical College & Safdarjung Hos-
ometrial portion of spiral arterioles or due to shallow tro- pital, New Delhi 110029, India. E-mail: [email protected];
phoblastic invasion, resulting in reduced uteroplacental [email protected]
blood flow leading to placental hypoxia (4). Placental hy- Received 23 October 2013; Accepted 21 October 2014
poxia results in the release of several mediators into mater- DOI 10.1002/jcla.21819
Published online in Wiley Online Library (wileyonlinelibrary.com).


C 2014 Wiley Periodicals, Inc.
76 Rath et al.

angiogenesis and cell metabolism. HIF-1 consists of two from all the patients prior to sample collection. The pla-
dimeric subunits and . HIF-1 is hypoxia inducible, cental samples were rinsed with normal saline to remove
while HIF-1 is constitutively expressed (5). During low excess blood followed by perfusion with buffered 10% for-
oxygen conditions, HIF-1 is highly expressed and helps malin (pH 7.0) and then processed for paraffin embedding
in development of placenta in early gestation. Overex- sectioning for Immunohistochemistry (IHC). The serum
pression of HIF-1 has been observed in many inflam- was separated from blood samples by centrifugation at
matory disorders, including cancer and preeclampsia (6). 12,000 rpm for 15 min for enzyme-linked immunosorbent
Hypoxia induces angiogenesis by regulating angiogenic assay (ELISA) analysis and stored at 20C. The clini-
proteins such as vascular endothelial growth factor copathological parameters of patients are summarized in
(VEGF), placental growth factor (PIGF) and FLT-1. The Table 1. All the placental samples (n = 45 + 45) and serum
change in concentration of these proteins causes angio- samples (n = 80 + 80) were processed for IHC and ELISA,
genic imbalance, leading to endothelial damage and the respectively.
onset of preeclampsia. PIGF is a proangiogenic protein
and member of the vascular endothelial growth factor Immunohistochemistry
(VEGF) family. It is one of the key molecules in angio-
genesis and vasculogenesis especially during embryogen- Formalin-fixed and paraffin-embedded tissue sections
esis and placental trophoblast is the main source of PIGF of 5 thickness were collected and processed for con-
throughout the gestational period of pregnancy (7). It ventional histological assessment by Haematoxylin and
shares structural as well as amino acid sequence similarity Eosin (H & E) staining. The immunoexpression of HIF-1
with VEGF, but PIGF has binding affinity only for VEGF and PIGF was analyzed by immunohistochemistry. In
receptor 1 (VEGFR-1; (8)). The inter- and intramolecular
cross-talk between the VEGFR-1 and VEGFR-2 is regu- TABLE 1. Clinicopathological Characteristics of the Study
lated by PIGF. It binds to VEGFR-1 and displaces VEGF Groups
from this receptor, which results in activation and in-
Clinicopathological Control Preeclampsia
termolecular transphosphorylation of VEGFR-2 thereby parameters (n = 45) (n = 45) P-value
amplify the VEGF-induced angiogenesis (9). But soluble
form of VEGFR-1 (known as sFLT-1) inhibit the inter- Maternal age (years; 21.53 0.410 22.29 0.446 0.216
mean SE)
action of PIGF and its receptor that causes endothelial
Gestational age (weeks; 37.62 0.485 38.58 0.366 0.120
dysfunction, a manifestation of preeclampsia (10). Al- mean SE)
though extensive studies have been conducted to investi- Gravidity (no. /%)
gate the expression of these two proteins in preeclampsia, 1 22/48.9 23/51.1 0.891
but hardly any report is available regarding the serum 2 18/40.0 16/35.6
3 5/11.1 6/13.3
analysis of HIF-1 in preeclamptic patients as well as the
Parity (no. /%)
association of HIF-1 with the angiogenic protein PIGF 0 22/48.9 21/46.7 0.945
at tissue and serum level. Thus, this study focused on the 1 18/40.0 18/40.0
quantification of serum levels of HIF-1 and finding the 2 5/11.1 6/13.3
possible molecular link between the expression of HIF-1 Systolic blood pressure 117.73 0.368 145.07 1.730 0.0001*
and PIGF in preeclamptic Indian women compared with (mmHg; mean SE)
Diastolic blood pressure 77.40 0.520 100.13 1.247 0.0001*
normotensive pregnancy. (mmHg; mean SE)
Urine albumin (no. /%)
0 45/100 0/0.0 0.0001*
MATERIAL AND METHODOLOGY 1 0/0.0 15/33.3
2 0/0.0 19/42.2
Sample 3 0/0.0 11/24.4
Edema (no. /%)
Placental tissue as well as serum samples of preeclamp- 0 45/100 31/68.9 0.0001*
tic patients and age-matched controls were retrieved from 1 0/0.0 14/31.1
the department of Obstetrics and Gynaecology, Safdar- Baby weight (kg; 3.133 0.0360 2.016 0.0360 0.0001*
jung Hospital, New Delhi, India. All the preeclamptic pa- mean SE)
Mode of delivery (no. VD = 42/93.3 VD = 39/86.7 0.292
tients included in our study had hypertension (systolic BP
/%) CS = 3/6.7 CS = 6/13.3
> 140 mmHg and diastolic BP > 90 mmHg) and protein-
uria exceeding 0.3 g/day after 20th week of gestation. Eth- Data are represented as mean standard error or number/percentage as
ical approval was obtained from the institutional ethics per requirement. MannWhitney U-test and Wilcoxon W test (Asymp.
committee of VMMC and Safdarjung Hospital, New sig. (two-tailed]).
Delhi, India and written informed consent was obtained *P < 0.05 is considered significant.

J. Clin. Lab. Anal.

 HIF-1 and PIGF in Preeclampsia 77

brief, the sections were deparaffinized in xylene, dehy- Statistical Analysis

drated by graded alcohols followed by blocking of en-
Statistical analysis was performed using SPSS 18.0
dogenous peroxidises activity by 0.03% H2 O2 in methanol
statistics software (SPSS, Inc., Chicago, IL). These data
for 15 min. The slides were then retrieved for antigen
are represented as mean standard error. Chi-square
in 10 mM citrate buffer (pH 6.0) by heating the sec-
test was carried out to determine the significance of
tions at 900 W for 15 min and at 360 W for 10 min
protein expression among control and preeclamptic pla-
in microwave oven. Further, the sections were incubated
centa. The significance of clinicopathological parameters
overnight with primary antibodies for HIF-1 (concen-
of preeclamptic patients and control was determined with
tration 1:70 [mouse anti-human monoclonal HIF-1 anti-
Wilcoxons W-test and MannWhitneys U-test (Asymp.
body ab1250; Abcam, Inc., Cambridge, UK]) and PIGF
sig. [two-tailed]). The associations between proteins HIF-
(concentration 1:40 [rabbit anti-human polyclonal PIGF
1 and PIGF was explored using Pearson correlation test
antibody ab97618; Abcam, Inc.) in humid chamber, at
(two-tailed). The P values less than 0.05 were regarded as
4C. Next day, after brief washing with TBS (Tris buffer
significant for MannWhitneys U-test (Asymp. sig. [two-
saline, pH 7.4) , the serial sections were incubated with
tailed]) and less than 0.01 was considered significant for
EnvisionTM (dextran conjugated with peroxidase and in-
Pearson correlation test [two-tailed].
corporated with molecules of secondary antibody against
The receiver operating characteristic (ROC) analysis
both immunoglobulins of mouse and rabbit, Dako Cy-
was also carried out to find the significance of immunohis-
tomation, Glostrup, Denmark) for 1 hr at room tempera-
tochemistry result. For ELISA results, Wilcoxons W-test
ture. The 3,3-diaminobenzidine hydrochloride (DAB) was
and MannWhitneys U-test (Asymp. sig. [two-tailed]) as
used for the chromogenic visualization reaction, counter-
well as box plot were performed. Results were considered
stained with Mayers hematoxylin, and mounted. The se-
significant, when P was less than 0.05.
rial sections were then examined under light microscope
(Olympus BX-51, Japan). In the negative control, isotype-
specific immunoglobulin G was used in place of primary RESULTS
antibody.
The clinicopathological characteristics, such as baby
weight, urine albumin, edema, systolic and diastolic blood
ELISA pressure of preeclamptic mother, were found to be statis-
The serum samples of control (n = 80) and preeclamp- tically significant (P = 0.000), while there was no signif-
sia (n = 80) were quantitatively analyzed for both HIF-1 icant difference between control and preeclamptic group
and PIGF by sandwich ELISA. For HIF-1, Cusabio in terms of maternal age, gestational age, gravidity, and
ELISA kit was used and manufacturers protocol was fol- parity (P = 0.216, P = 0.120, P = 0.0.891, P = 0.945,
lowed. The minimum detectable dose of human HIF-1 respectively; Table 1).
was typically less than 15.6 pg/ml. For PIGF, the ELISA
kit of R&D System (Minneapolis, Minnesota) was used
Immunohistochemistry
and manufacturers protocol was followed. The minimum
detectable dose of human PIGF was typically less than 7 The IHC analysis revealed that HIF-1 was localized
pg/ml. both in nucleus and cytoplasm of syncytiotrophoblast in
preeclamptic placental tissue. The nuclear expression was
intense in 64.4% (29/45), moderate in 22.2% (10/45), and
Scoring of Immunohistochemical Staining
mild in 13.3% (6/45) cases. However, the intensity of HIF-
Evaluation of immunohistochemistry for all proteins 1 in cytoplasm was moderate in 8.9% (4/45) and mild in
was performed on two arbitrarily chosen slide of each 91.1% (41/45) of preeclamptic group. While in the con-
case (both preeclamptic and control). Result of immuno- trol group, cytoplasmic expression was noticed. The ex-
histochemistry was evaluated by two observers and fol- pression was intense in 42.2% (19/45), moderate in 40.0%
lowed the scoring criterion given by Tripathi et al. (11). (18/45), and mild in 17.8% (8/45). And only 6.6% (3/45)
Protein expression was semiquantified with regard to the cases showed the nuclear expression of HIF-1 in con-
intensity of cell staining graded as 03(0 for negative trol. Both nuclear and cytoplasmic expression was found
staining if there is total absence, 1 for mild staining, to be significant in preeclampsia as well as in control ones
2 for moderate, 3 for intense staining). H-score was (P = 0.0001; Fig. 1A to C; Table 2).
calculated for both preeclampsia and control using the The immunoreactivity of PIGF revealed a significant
formula, H-score = P(S + 1), where P represent the ag- downregulation in the cytoplasm of syncytiotrophoblast
gregate percentage of stained cells and S represents the of preeclamptic placenta. The intensity was mild in 95.6%
intensity of the cell. (43/45) and moderate in 4.4% (2/45) of the study group.

J. Clin. Lab. Anal.

78 Rath et al.

TABLE 2. Assessment of IHC for HIF-1 and PIGF in Control

and Preeclamptic Placenta

Preeclampsia Control
(N = 45) (N = 45)
Protein Expression n (%) n (%) P values

HIF-1 nuclear No staining 0 (0.0) 42(93.3) 0.0001*

Mild 06 (13.3) 3 (6.6) 0.0001*
Moderate 10 (22.2) 0 (0.0) 0.0001*
Intense 29 (64.4) 0 (0.0) 0.0001*
HIF-1 No staining 4(8.9) 0(0.0) 0.0001*
cytoplasmic Mild 41 (91.1) 8 (17.8) 0.0001*
Moderate 0 (0.0) 18 (40.0) 0.0001*
Intense 0 (0.0) 19(42.2) 0.0001*
PIGF Mild 43(95.6) 0 (0.0) 0.0001*
cytoplasmic Moderate 2 (4.4) 20(44.4) 0.0001*
Intense 0 (0.0) 25 (55.6) 0.0001*

Pearson chi-square test (Asymp. sig. [two-tailed]).

* P < 0.05 is considered significant.
N, no. of cases.

TABLE 3. H-Score Assessment of HIF-1 and PIGF in Control

and Preeclamptic Placenta

HIF-1 PIGF

Nucleus Cytoplasm Cytoplasm

Study group (mean SD) (mean SD) (mean SD)

Control 102.13 4.875 275.69 11.872 314.20 7.305

(N = 45)
Preeclampsia 312.22 11.132 167.62 4.353 177.51 3.275
(N = 45)
Pvalue 0.0001 0.0001 0.0001

Fig. 1. (A) Control placenta (40 weeks) showing moderate cytoplasmic MannWhitney U-test and Wilcoxon W test (Asymp. sig. [two-tailed]).
expression of HIF-1 in syncytiotrophoblast; (B and C) preeclamptic P < 0.05 is considered significant.
placenta (38 and 36 weeks) showing nuclear accumulation of HIF-1 in N, no. of cases.
syncytiotrophoblast; (D) control placenta (32 weeks) showing moderate
cytoplasmic expression of PIGF in syncytiotrophoblast; (E) preeclamp-
tic placenta (32 weeks) showing mild cytoplasmic expression of PIGF in
was more for cytoplasmic expression (275.69 11.872;
syncytiotrophoblast; (F) negative control incubated with IgG showing
placental villi. Arrow shows the expression of protein. Magnification: Table 3, Fig. 2A). In case of PIGF, the preeclamptic cy-
400. toplasmic expression was lower (177.51 3.275) than the
control group (314.20 7.305; Table 3, Fig. 2B).
The ROC curve analysis revealed the significant nuclear
However, the cytoplasmic expression was moderate in expression of HIF-1 in preeclamptic group as compared
44.4% (20/45) and intense in 55.6% (25/45) of the con- with controls (P = 0.0001, Fig. 3A) with area under curve
trol placenta. In both control and preeclamptic cases, of 0.989, specificity 88.9%, and sensitivity 88.9%. How-
PIGF expression was found to be significant (P = 0.0001; ever, PIGF was significantly downregulated in preeclamp-
Fig. 1D and E; Table 2). Although cytotrophoblast tic group as compared with controls (P = 0.0001, Fig. 3B)
was present in some of the studied preeclamptic cases, with area under curve of 0.962, specificity 82.2%, and
the immunoexpression of HIF-1 and PIGF was not sensitivity 82.2%.
significant.
The H-score analysis was carried out for control as
well as experimental group to assess the intensity of
ELISA
HIF-1 and PIGF at nuclear and cytoplasmic level. The
mean and standard error value of HIF-1 was higher The MannWhitney test showed that the serum concen-
(312.22 11.132) for nuclear expression in preeclamp- tration of HIF-1 was higher (mean = 6.581 pg/ml) in
tic group, but in control the mean and standard error preeclamptic cases than that of control group (mean

J. Clin. Lab. Anal.

 HIF-1 and PIGF in Preeclampsia 79

Fig. 2. Comparison of immunohistochemical H-score analysis. (A) HIF-1 expression in cytoplasm and nucleus of syncytiotrophoblast cells of
control and preeclamptic patients. (B) PIGF expression in cytoplasm of syncytiotrophoblast cells of control and preeclamptic group.

= 4.947 pg/ml). For PIGF, the serum concentration Correlation Between HIF-1 and PIGF
of control was increased (mean = 6.333 pg/ml), while
The significant negative association was noted between
that of preeclampsia was lower (mean = 3.939 pg/ml).
HIF-1 nuclear and PIGF cytoplasmic expression in pa-
The concentration of HIF-1 and PIGF in serum of
tients suffering from preeclampsia (r = 0.196, P < 0.05;
control and preeclamptic patients was represented as
Table 5). There was also a significant inverse correla-
mean standard error in Table 4.
tion between the serum levels of HIF-1 and PIGF in
According to Box plot analysis, the serum levels of
preeclamptic group (r = 0.220, P < 0.05; Table 6).
HIF-1 were higher (median 6.640 pg/ml) in preeclamptic
cases as compared with the control ones (median = 4.945
pg/ml; Fig. 4A); however, for PIGF, the serum levels were
DISCUSSION
found to be downregulated significantly (median = 3.434
pg/ml) in preeclamptic group than that of control (me- Hypoxic environment is essential for the invasion and
dian = 7.462 pg/ml; Fig. 4B). infiltration of cytotrophoblast into the maternal decidua

Fig. 3. ROCs curve showing the expression of HIF-1 and PIGF to differentiate preeclamptic group from control. (A) HIF-1 (AUC = 0.989,
sensitivity = 88.9%, specificity = 88.9%). (B) PIGF (AUC = 0.962, Sensitivity = 82.2%, specificity = 82.2%).

J. Clin. Lab. Anal.

80 Rath et al.

Fig. 4. Box plot showing the serum concentration (pg/ml) of (A) HIF-1 and (B) PIGF in control and preeclamptic patients. The solid bar
indicates median, upper, and lower limits of box, 75th and 25th percentiles; upper and lower bars, maximum and minimum values (P < 0.05).

TABLE 4. HIF-1 and PIGF Concentration (pg/ml) in Serum TABLE 6. Correlation of HIF-1 and PIGF in Serum Samples
of Control and Preeclamptic Cases of Preeclamptic Group

Study group HIF-1 (mean SE) PIGF (mean SE) Protein No. of cases HIF-1 PIGF

Control (N = 80) 4.947 0.045 6.335 0.093 HIF-1 80 1 r = 0.220*

Preeclampsia (N = 80) 6.581 0.030 3.939 0.179 P = 0.05
P-value 0.0001* 0.0001* PIGF 80 r = 0.220* 1
P = 0.05
MannWhitney U-test and Wilcoxon W test (Asymp. sig. [two-tailed]).
*Significant.
Pearson correlation test (two-tailed); r, correlation coefficient.
P < 0.05 is considered significant.
*Correlation is highly significant at 0.01 level.
N, no. of cases.

TABLE 5. Correlation of HIF-1 and PIGF in Placental Tissue gene that counteract angiogenesis (14). It also has an im-
of Preeclamptic Patients portant role in vascularization and survival of embryos,
Protein No. of cases HIF-1 nuclear PIGF cytoplasm pulmonary vascular remodeling, and vascularization of
tumors (15). Caniggia and Winter noticed the increased
HIF-1 nuclear 45 1 r = 0.752* expression of HIF-1 mRNA and protein in placental
P = 0.0001
tissues of preeclamptic patients (16). Several researchers
PIGF cytoplasm 45 r = 0.752* 1 also showed the overexpression of HIF-1 in preeclamp-
P = 0.0001
tic human placenta that modulates the pathogenesis of
Pearson correlation test (two-tailed); r, correlation coefficient. preeclampsia (1719). It has been observed that during
*Correlation is highly significant at 0.01 level. hypoxic conditions, HIF-1 gets stabilized and translo-
cates to nucleus from cytoplasm (20, 21). In the present
study, the immunoreactivity of HIF-1 in preeclamp-
for maintenance of materno-fetal circulation at early pe- tic placental tissue showed significant nuclear expression
riods of pregnancy. But its prevalence in later stages of (P = 0.0001) in syncytiotrophoblast. However, cytoplas-
pregnancy causes several complications that may lead mic expression of HIF-1 with low intensity was also ob-
to maternal and fetal morbidity and mortality (12, 13). served in preeclamptic tissues (P = 0.0001). While in the
Under hypoxic conditions, HIF-1 plays a vital role in control group, significant cytoplasmic immunoexpression
multiple physiological responses, such as erythropoiesis, was noticed in syncytiotrophoblast (P = 0.0001). Thus,
glycolysis, and also affects the transcription of VEGF there is possibility of significant translocation of HIF-1

J. Clin. Lab. Anal.

 HIF-1 and PIGF in Preeclampsia 81

from cytoplasm to nucleus, leading to low oxidative stress woman (P = 0.0001), which suggests that the decrease in
prevailing in preeclampsia. the serum concentration of PIGF may be responsible for
Many researchers have worked on serum concentration the improper trophoblast invasion, leading to endothelial
of HIF-1 in different pathological conditions, such as dysfunction in preeclampsia.
cancer. Zhang et al. observed the overexpression of HIF- Several researchers have proved that the overexpression
1 in serum of primary hepatocellular carcinoma (PHC) of HIF-1 is associated with the increased maternal serum
(22). To the best of our knowledge, no investigator has concentration of soluble Fms-like tyrosine kinase 1 (sFlt1)
evaluated the expression of HIF-1 in serum samples of during hypoxic conditions (33). High circulating levels of
preeclamptic patients. Therefore, in this study quantifica- sFlt1 exerts an antiangiogenic state that is associated with
tion of the serum concentration of HIF-1 in maternal low levels of proangiogenic factors, such as PIGF, and
circulation of both control and preeclamptic patients was inhibition of PIGF with its receptor VEGFR-1, caus-
carried out. The ELISA analysis showed that the mean ing endothelial dysfunction in preeclampsia (34). Other
serum concentration of HIF-1 was higher (mean = 6.581 researchers proved that VEGF and PIGF are dysregu-
pg/ml) in preeclamptic cases than that of control group lated in preeclampsia due to high levels of sVEGFR-1,
(mean = 4.947 pg/ml), which infer that the maternal which leads to impaired placental angiogenesis (35). The
serum levels of HIF-1 also get significantly elevated dur- downregulation of trophoblast PIGF gene expression was
ing pathogenesis of preeclampsia (P < 0.05). mediated by hypoxia, which is differentially regulated by
PIGF, an angiogenic protein, plays an important role HIF-1, and had been observed in previous studies (36,37).
in placental development and is expressed in villous cy- HIF-1 affects the expression of PIGF gene that is de-
totrophoblast and syncytiotrophoblasts in placenta (23). pendent on the type of cell and the conditions prevail-
Various researchers had noticed the increased expres- ing in a cell (38). Gobble et al. reported that hypoxia
sion of PIGF gene in normal trophoblast, while the ex- decreases PIGF gene transcription that results in the de-
pression was reduced in preeclampsia (24). Torry et al. creased value of PIGF via mechanisms independent of
found in normal pregnancy PIGF is highly expressed HIF-1 (24). In this study, the significant negative associa-
in trophoblasts and its expression is significantly down- tion was noted between HIF-1 nuclear and PIGF cyto-
regulated in preeclampsia by low oxygen tension (25). plasmic expression in patients suffering from preeclamp-
The same group in their another study observed the sia (r = 0.752, P = 0.0001). Also, the statistical anal-
decreased PIGF mRNA expression in preeclamptic tro- ysis of the serum levels of HIF-1 and PIGF showed a
phoblast (26). In the present study, the immunoreactivity significant negative correlation between the proteins in
of PIGF revealed a significant downregulation of PIGF preeclampsia (r = 0.220, P < 0.05). The expression of
(P = 0.0001) in the cytoplasm of syncytiotrophoblast of VEGFR-1 (sFLT-1) was also studied in another study and
preeclamptic placenta. However, the intensity of cytoplas- it was upregulated both at tissue and serum level. There
mic expression in the control placenta was lower in com- was no statistical association found between VEGFR-1
parison to preeclamptic cases (P = 0.0001). Therefore, and PIGF (data not shown in this report). Therefore, we
this study coincides with the reports provided by previ- hypothesized that the upregulation of HIF-1 and down-
ous authors (2426) regarding the decreased expression of regulation of PIGF in serum and placental tissues may be
PIGF in the preeclamptic conditions. Furthermore, ma- directly associated with the pathogenesis of preeclampsia.
ternal serum levels of PIGF were quantified to assess the Under pathological conditions, due to failure of replace-
quantity of PIGF in the preeclamptic conditions. Many ment of vascular smooth muscle and endothelial cells of
researchers had suggested that the abnormal serum levels spiral artery by cytotrophoblast, the spiral artery remod-
of PIGF in preeclampsia result in improper trophoblast eling do not occur, leading to the prevalence of hypoxic
invasion and the generalized maternal endothelial dys- conditions in late gestation of pregnancy. During this pe-
function, which leads to preeclampsia (27, 28). Some au- riod, HIF-1 may be responsible for the molecular modi-
thors also noticed the increased serum concentration of fication of several mediators involved in pathophysiology
PIGF in normal pregnancy and decreased concentration of pregnancy disorders. In addition, the abnormal expres-
in preeclampsia (29, 30). Livingston et al. also observed sion of angiogenic factor, such as PIGF, may be associated
that the median concentrations of PIGF were significantly with the hypoxic conditions prevailing in preeclampsia via
lower in pregnancies complicated by severe preeclampsia activation of HIF signaling pathway.
than in control (31). Torry et al. also reported the signifi- In light of the present study on preeclampsia, the
cantly reduced concentration of maternal serum placental inverse correlation of PIGF and HIF-1 may offer
growth factor (P < 0.0001) in women with preeclampsia tremendous promise in screening of preeclamptic pa-
than normotensive controls (32). In this study, the sig- tients. Several treatment strategies may be aimed at
nificant low concentration of serum PIGF was noticed preventing endothelial dysfunction by using PIGF.
in preeclamptic group as compared to normal pregnant Further studies on different angiogenic factors and

J. Clin. Lab. Anal.

82 Rath et al.

hypoxic proteins have also been designed on larger 16. Caniggia I, Winter JL. Adriana and luisa castellucci Award 2001.
cohorts to understand the exact molecular mechanisms Hypoxia-inducible factor 1: Oxygen regulation of trophoblast dif-
ferentiation in normal and preeclamptic pregnancies-a review. Pla-
regulating the pathogenesis of preeclampsia. This may
centa 2002;23:S47S57.
allow the development of new preventive strategies for 17. Rajakumar A, Brandon HM, Daftary A, Ness R, Conrad KP.
pregnancy complications, such as preeclampsia. Evidence for the functional activity of hypoxia-inducible tran-
scription factors overexpressed in preeclamptic placentae. Placenta
2004;25:763769.
18. Iwagaki S, Yokoyama Y, Tang L, Takahashi Y, Nakagawa Y,
ACKNOWLEDGEMENT Tamaya T. Augmentation of leptin and hypoxia-inducible fac-
The authors are grateful to Indian Council of Medical tor 1 mRNAs in the preeclamptic placenta. Gynecol Endocrinol
2004;18:263268.
Research (Ref. No. 5/7/587/11-RHN) New Delhi, India 19. Tal R, Shaish A, Barshack I, Charcon SP, et al. Effects of hypoxia-
for providing the grants without which the work was not inducible factor-1-overexpression in pregnant mice. Am J Pathol
possible. They thank their technician Rajeshwar Singh for 2010; 177:29502962
his support during the study. 20. Huang LE, Arany Z, Livingston DM, Bunn HF. Activation of hy-
poxia inducible transcription factor depends primarily upon redox-
sensitive stabilization of its alpha subunit. J Biol Chem 1996;271:53
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