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Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014

399

Methods to Observe and Weaken the Influence of

Pregnancy-Associated Plasma Protein-A on Endothelial Cell
Function
Li-ping Peng1, Kan Yang1, Wei-hong Jiang1, and Bin Yi2
1Department of Cardiology and 2Department of Nephrology, Third Xiangya Hospital of Central South University,
Changsha, China

Abstract. Objective: To investigate the effect of pregnancy-associated plasma protein-A (PAPP-A) on hu-
man umbilical vein endothelial cell (HUVEC) contraction and relaxation function. We also investigated
some effects of pregnancy serum, which was voluntarily provided by women who were 9 months pregnant,
on PAPP-A function and endothelial cell function. Methods: HUVECs were cultured in vitro with PAPP-
A at 0, 50, 100, and 200 ng/mL for 0, 12, 24, 48 hours. HUVECs were also pretreated for 12 hours with
pregnancy serum before in vitro culture in the presence or absence of PAPP-A (50 ng/mL). Nitric oxide
(NO) levels in cell supernatants were assessed using spectrophotometry. Endothelin-1 (ET-1) levels were
determined using immunohistochemistry assays and quantified by mean optical density (MOD). Results:
The NO levels of HUVECs in the PAPP-A groups were significantly lower compared to the control group
(P<0.05), whereas ET-1 levels in PAPP-A HUVECs were significantly higher compared to the control
group (P<0.05.) Additionally, these effects were dose-dependent. The NO levels of HUVECs in the PAPP-
A groups with pregnancy serum were higher compared to the control group (P<0.05). ET-1 levels in PAPP-
A HUVECs with pregnancy serum were lower than in the control group (P<0.05). Conclusions: PAPP-A
may affect HUVEC contraction and relaxation by reducing NO secretion and increasing ET-1 levels. In
addition, pregnancy serum may affect PAPP-A function on vascular endothelial cells.

Key words: pregnancy-associated plasma protein-A (PAPP-A), nitric oxide, vascular endothelial cell, endo-
thelin-1, pregnancy serum

Introduction regulated by C Reactive Protein and TNF-α

Pregnancy-associated plasma protein-A (PAPP-A)
is a type of matrix metalloproteinase. Some studies
[1-3] have found that PAPP-A is highly expressed
in atherosclerosis lesions, inducing lesion deteriora-
tion and rupture. In the early phase of acute coro-
nary syndrome (ACS) attack, elevated PAPP-A is
an independent risk factor for the occurrence of
adverse cardiovascular events. The function of
PAPP-A in atherosclerosis has two parts. First,
PAPP-A concentration may be a marker of ACS [4]
[5] because it has been reported that human pe-
ripheral blood mononuclear cells (PBMCs) express
PAPP-A in vitro. PAPP-A expression may be
Address correspondence to Kan Yang, M.D., Ph.D., Department
of Cardiology, Third Xiangya Hospital, Central South University,
138 Tong Zi Po RoadChangsha, 410013, Hunan Province,
China; phone:+86 731 8861 8213; fax: : +86 731 8861 8072; e
mail:yangkanxy3@gmail.com
through the NF-κB pathway. This mechanism may
play a significant role in the observed increase in
serum PAPP-A levels in ACS. Second, PAPP-A
could contribute to the development of atheroscle-
rosis indirectly via insulin-like growth factor (IGF)
[6]. PAPP-A has been reported to regulate the ac-
tivity of IGF signaling through proteolytic degrada-
tion of IGF-binding proteins (IGFBPs), thereby
increasing the local concentration of free IGFs
available to receptors [7,8]. However, whether
PAPP-A affects the function of vascular endothelial
cells and thus negatively influences atherosclerosis
at the time of its onset is not yet known. The aim of
the present study was to determine whether PAPP-A
affects the systolic and diastolic function of vascular
endothelial cells. We then evaluated whether the
antigen-antibody immune response could affect
PAPP-A function, since PAPP-A is a proteinase
that increases during pregnancy.

0091-7370/14/0400-399. © 2014 by the Association of Clinical Scientists, Inc.

400 Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014

Table 1. The effect of PAPP-A on NO concentrations in HUVECs ( χ± SD,n =5).

Group 0h 12 h 24 h 48 h
Control(µmol/L) 61.31±2.78 60.55±1.78 61.56±1.45 58.20±3.03
50 ng/mL PAPP-A(µmol/L) 62.50±1.73 43.31±2.54*∆ 53.84±4.44*∆ 49.28±2.58*∆
100 ng/mL PAPP-A(µmol/L) 61.79±3.65 37.09±2.27*∆! 42.11±3.08*∆! 40.11±1.96*∆!
200 ng/mL PAPP-A(µmol/L) 61.05±4.83 30.69±1.35* # 34.12±4.23*∆#
∆ 33.50±5.99*∆#

In contrast to the same time of the control group, *P<0.05; In contrast to the same group at 0 h, ∆P<0.05; In contrast
to the 50 ng/mL PAPP-A group, !P<0.05; In contrast to the 100 ng/mL PAPP-A group, #P<0.05.

Table 2. The effect of PAPP-A on ET-1 in vascular endothelial cells (MOD) ( χ± SD, n=5).

Group 0h 12 h 24 h 48 h
Control 0.094±0.005 0.092±0.004 0.090±0.002 0.087±0.004
50 ng/mL PAPP-A 0.095±0.007 0.284±0.020*∆ 0.211±0.013*∆ 0.207±0.012*∆
100 ng/mL PAPP-A 0.091±0.005 0.399±0.009*∆! 0.482±0.010*∆! 0.414±0.025*∆!
200 ng/mL PAPP-A 0.093±0.003 0.658±0.020*∆# 0.622±0.018*∆# 0.644±0.032*∆#

In contrast to the same time of the control group, *P<0.05; In contrast to the same group of the 0 h, ∆P<0.05; In
contrast to the 50 ng/mL PAPP-A group, !P<0.05; In contrast to the same time of the 100 ng/mL PAPP-A group,
#P<0.05.

Table 3. The effect of PAPP-A on NO and ET-1 levels in vascular endothelial cells ( χ± SD, n =5).

Group NO concentration (µmol/L) ET-1 MOD

Control 65.08±3.21 0.019±0.021
PAPP-A 41.2±1.06* 0.648±0.022*
pregnancy serum with PAPP-A 49.21±4.31*∆ 0.473±0.031*∆

In contrast to the same time of the control group, *P<0.05; In contrast to the same group of the PAPP-A only,
∆P<0.05.

Materials and Methods
Experimental cells. Experimental human umbilical
vein endothelial cells were provided by the Cell Center
at the Xiangya Medical College of Central South
University.
Reagents and apparatuses. Pregnancy-related serum
protein-A was purchased from R&D Systems (molecu-
lar mass, 120 ~ 160 kD). The DAB chromogenic re-
agent kit was provided by Sangon Biotech Co., Ltd
(Shanghai, China).
The ET-1 immunohistochemical kit was purchased
from The United States R&D Biotechnology Company,
and the immunohistochemical double reagent box was
provided by the Beijing Fir Jinqiao Biotechnology
Company. The raw materials of fluvastatin were pro-
vided by Novartis. Nitric oxide kits were purchased
from Nanjing Building Research Institute of Biological
Engineering. The HF151UV CO2 incubator was pur-
chased from Shanghai’s Scientific Instrument Limited
Company. The YJ-875 medical purification table was
from Jiangyin health beauty. The ELX80ONB Kejunior
enzyme mark instrument is from Bio-TEK Instrument
Company. The XDS-1 B inverted microscope was pur-
chased from Chongqing Photoelectric Science
Instrument Limited Company; The 722 grating spec-
trophotometer was from the Shanghai Precision
Scientific Instruments Limited Company.
Culturing Endothelial Cells. Using human umbilical
vein endothelial cells (HUVECs) and microscopy, we
observed that the cells were arranged tightly like cobble-
stones, without overlap. Cells were grown in DMEM
(high glucose) containing fetal calf serum (10%) and
streptomycin and cultured at 37°C with 5% CO2 and
saturated humidity. After 2-3 days, when the cells con-
fluent the bottom of the bottle, cells were passaged us-
ing 0.25% trypsin digestion.
Observation of Cell morphology. We used an inverted
microscope to observe the growth and proliferation of
cells directly.
 Influence of pregnancy-associated plasma protein-A on endothelial cells 401

Optimum concentration and

time point determination for
PAPP-A intervention. At dif-
ferent concentrations of
PAPP-A and after different
treatment times, we evaluated
NO concentration. In order to
make the intervention time the
horizontal axis and the concen-
tration of NO longitudinal,
the NO concentration levels
were generated at different
concentrations and times after
the intervention of PAPP-A. At
the same time, we acquired the
endothelial cell binding/ET-1
results.

Determination of NO concen-
tration in the supernatant.
We determined NO levels us-
Figure 1. The effect of different PAPP-A concentrations on ET-1 levels in vascular ing nitrate reductase assays.
endothelial cells after 12 h (×200) A: The control group; B: 50 ng/mL PAPP-A; C: NO concentrations in islets
100 ng/mL PAPP-A; D: 200 ng/mL PAPP-A.
were estimated using the Griess
reagent (0.1% naphthyl ethyl-
enediamine dihydrochoride
1% sulfanilamide).
Conditioned media (100 mL)
from treated and untreated is-
let groups was incubated with
an equal volume of freshly pre-
pared Griess reagent in the
dark for 30 min. Optical den-
sity was measured at 550 nm
using a spectrophotometer.
Values are expressed as micro-
moles per 150 islets. NaNO2
was used as the standard.

DAB staining to endothelial

cell ET-1. A clean coverslip was
placed into high-pressure
steam for sterilization, washed
three times with PBS, and
washed in cell culture liquid
Figure 2. The effect of different PAPP-A concentrations on ET-1 levels in vascular once. Coverslips were placed in
endothelial cells after 24 h (×200) A: The control group; B: 50 ng/mL PAPP-A; C: a 6 well plate and inoculated
100 ng/mL PAPP-A; D: 200 ng/mL PAPP-A.
with the HUVEC suspension.
APP-A experimental groups. HUVEC cell suspensions After 2 days, serum-free media was added, and we con-
were inoculated into a 6 well culture plate. After 2 days tinued intervention experiments. The culture media was
in serum-free media, cells were stimulated with 0, 50, then removed, and cells were washed in PBS 3 times, for
100, or 200 ng/mL PAPP-A for 0, 12, 24, or 48 hours. 2 min each. Cells were fixed in 4% paraformaldehyde
Supernatants and cell lysates were examined. Each group for 20 min. After removing the fixing solution, cells
and each concentration had 5 wells. were again washed three times in PBS. The absorption
402 Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014
Statistical analyses.
SPSS13.0 statistical software
was used for all statistical
analyses. Data are represented
by the mean ± standard devia-
tion (χ± SD). The difference
between groups was evaluated
using one-way analysis of vari-
ance (ANOVA), and groups
were compared using Q tests.
P < 0.05 indicates statistical
significance.

Results

The effect of PAPP-A on

NO levels of HUVECs.
HUVECs were cultured in
vitro with different concen-
trations of PAPP-A for var-
Figure 3. The effect of different PAPP-A concentrations on ET-1 levels in vascular ious time points. HUVEC
endothelial cells after 48 h (×200) A: The control group; B: 50 ng/mL PAPP-A; C: 100 NO levels did not change
ng/mL PAPP-A; D: 200 ng/mL PAPP-A. at any point in time
(P>0.05, Table 1).
liquid adhered to the endothelial cells, which were then However, the NO levels of HUVECs with PAPP-A
incubated together with immunohistochemistry primary at different concentrations for 12, 24, and 48 h
antibody overnight at 4°C. Cells were washed three times were lower than the same concentration group at 0
in PBS. Immunohistochemistry secondary antibody was h (p<0.05). The lowest NO levels were observed at
added to each slide and incubated for 60 min at 37°C.
12 h.
After being washed in PBS, slides were stained with DAB
for 10 min and flushed with water. After the second dye-
ing and dehydration step, we sliced gum with neutral The concentration of NO was lower at higher con-
sheet. We used a microscope equipped with a camera to centrations of PAPP-A after 12, 24, and 48 h
observe the endothelial cells ET-1 and obtain photos. (p<0.05).

Measurement of ET-1 expression in endothelial cells
(gray value). We used an image software analysis system
to detect the expression of ET-1 in endothelial cells’ gray
value with the formula: average optical density (MOD) =
LG (positive products / background gray value).
Increasing MOD values indicated increased ET-1 expres-
sion in endothelial cells.
Obtaining pregnancy serum. This trial was reviewed
and approved by the Ethics Committee of the Third
Xiangya Hospital of Central South University on May
17, 2012. We obtained permission from six pregnant
women to take blood samples. Peripheral venous blood
(4 mL) was collected into EDTA-containing tubes and
processed within 2 h after venipuncture. To ensure cell-
free plasma collection, all EDTA-blood samples were
centrifuged in two steps (3,000 rpm for 10 min, followed
by 12,000 rpm for 10 min). Cell-free plasma was stored
at -20°C until extraction. The concentration of pregnan-
cy serum was 1% during culture with HUVECs.
The effect of PAPP-A on HUVEC ET-1 levels. The
effect of different concentrations of PAPP-A on
ET-1 levels in HUVECs after 12, 24, and 48 h was
clear. We observed yellow pellets in the cell with
higher concentrations of PAPP-A. However, in the
control group, we did not observe any yellow pellets
in the cells (Figures 1, 2, and 3).
The ET-1 MOD [9] from each group is shown in
Table 2. In the control group, there were no obvi-
ous changes in ET-1 at any time point (P>0.05).
When HUVECs were cultured with PAPP-A at dif-
ferent concentrations, we observed increased MOD
after 12, 24, and 48 h (P<0.05). In the 50 ng/ml
PAPP-A group, the highest MOD was at 12 h. In
the 100 ng/ml group, the highest MOD was at 24
h. In the 200 ng/ml PAPP-A group, the highest
MOD was at 12 h.
 Influence of pregnancy-associated plasma protein-A on endothelial cells
Figure 4. The effect of PAPP-A and pregnancy serum plus PAPP-A on ET-1 levels in vascular endothelial cells after 12 h
(×200) A: Control group; B: PAPP-A group C: Pregnancy serum + PAPP-A group.
The effect of PAPP-A on NO levels of HUVECs
that were pre-cultured with pregnancy serum. As
shown in Table 3, when HUVECs were cultured
with pregnancy serum, followed by culture with
PAPP-A for 12 h, NO levels were lower than they
were in the control group (P<0.05). However, NO
levels of this group were higher than in the group
cultured with PAPP-A but without pregnancy se-
rum (P>0.05).
The effect of PAPP-A on ET-1 levels in HUVECs
that were pre-cultured with pregnancy serum. As
shown in Figure 4, there were fewer yellow pellets
in the group that was cultured with pregnancy se-
rum first, compared to cells cultured with PAPP-A
but without pregnancy serum.
Discussion
Endothelial cell dysfunction is an important patho-
physiological change in the early arterial atheroscle-
rotic process [10,11]. There are many ways to avoid
atherosclerosis at physiological NO concentrations,
including dilation of blood vessels, inhibition of
vascular smooth muscle cell (VSMC) hyperplasia,
and actions affecting platelet adhesion [12].
Decreased levels of NO secretion suggests damage
to endothelial cell (EC)-dependent vasodila-
tion[13,14]. Because ET-1 is the strongest active
vasoconstrictor constituent, increased ET-1 levels
were due to vascular endothelial injury or stimula-
tion [15-17]. ET-1 secretion in vascular endothelial
cells that were damaged was significantly higher
compared to normal control subjects [18,19].
The results of this study show that after 12, 24, and
48 h, the supernatant NO concentration decreased
gradually with increasing concentrations of
PAPP-A. The MOD value, expressed by the ET-1
levels of endothelial cells, increased gradually with
403
increasing concentrations of PAPP-A after 12, 24,
and 48 h. These data suggest that decreased NO
secretion levels in endothelial cells exist concomi-
tantly with increased ET-1 levels and are dependent
on PAPP-A concentration. Because the decomposi-
tion activity of MMP-9 could increase as the con-
centration increases [20,21] and because PAPP-A
could specifically cleave IGFBP-4 [22-24], PAPP-A
may degrade NO synthase as a protease [25,26]. In
our trial, we performed two experimental data sets
to investigate both the effect of pregnancy serum
alone on the HUVEC cells and the effect of non-
pregnant women serum on the HUVEC cells. We
found that the results were the same as the controls.
PAPP-A is an important regulatory component of
the IGF system. Through proteolysis of inhibitory
IGF binding proteins (IGFBPs), PAPP-A acts as a
positive modulator of local IGF signaling in a vari-
ety of biological systems. Thus, eliminating its ac-
tivity may lead to decreased NO secretion, followed
by weak inhibition of ET-1 and a corresponding
increase of ET-1. When the concentration of
PAPP-A increases, more NO synthases are degrad-
ed, and changes in NO concentration are more
pronounced.
Initially, Bonno et al. found that PAPP2A is synthe-
sized by placental X cells and syncytiotrophoblasts.
Plasma levels increased with gestational age, reach-
ing a peak of 324±277 ng/mL at full-term preg-
nancy and subsequently declining postnatally
[27,28]. Because PAPP-A is a metalloproteinase
and an antigen, we questioned whether it produced
antibodies in the body. First, we incubated endo-
thelial cells and full-term maternal serum (from
pregnant women with no complications) together
and then added PAPP-A. The results show that NO
secretion and endothelin both decreased, compared
with the control group. These results suggest that
maternal serum contains an antibody that can
404 Annals of Clinical & Laboratory Science, vol. 44, no. 4, 2014
protect endothelial cells from PAPP-A damage.
This prompted us to question whether we can pro-
tect humans from the damaging effects of PAPP-A
(or even matrix metalloproteinases), using induc-
tion or immunity.
Because this study used in vitro experiments with
an enzyme, PAPP- A activity may have been affect-
ed over time, due to degradation. Therefore, we did
not observe clear time dependence. Further studies
in animals or humans are required to confirm
whether the results of this study can be applied in
the clinic in the future.
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