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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

through the NF-κB pathway. This mechanism may
Pregnancy-associated plasma protein-A (PAPP-A) play a significant role in the observed increase in
is a type of matrix metalloproteinase. Some studies serum PAPP-A levels in ACS. Second, PAPP-A
[1-3] have found that PAPP-A is highly expressed could contribute to the development of atheroscle-
in atherosclerosis lesions, inducing lesion deteriora- rosis indirectly via insulin-like growth factor (IGF)
tion and rupture. In the early phase of acute coro- [6]. PAPP-A has been reported to regulate the ac-
nary syndrome (ACS) attack, elevated PAPP-A is tivity of IGF signaling through proteolytic degrada-
an independent risk factor for the occurrence of tion of IGF-binding proteins (IGFBPs), thereby
adverse cardiovascular events. The function of increasing the local concentration of free IGFs
PAPP-A in atherosclerosis has two parts. First, available to receptors [7,8]. However, whether
PAPP-A concentration may be a marker of ACS [4] PAPP-A affects the function of vascular endothelial
[5] because it has been reported that human pe- cells and thus negatively influences atherosclerosis
ripheral blood mononuclear cells (PBMCs) express at the time of its onset is not yet known. The aim of
PAPP-A in vitro. PAPP-A expression may be the present study was to determine whether PAPP-A
affects the systolic and diastolic function of vascular
Address correspondence to Kan Yang, M.D., Ph.D., Department endothelial cells. We then evaluated whether the
of Cardiology, Third Xiangya Hospital, Central South University,
138 Tong Zi Po RoadChangsha, 410013, Hunan Province, antigen-antibody immune response could affect
China; phone:+86 731 8861 8213; fax: : +86 731 8861 8072; e
mail:yangkanxy3@gmail.com
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 Company. The YJ-875 medical purification table was
from Jiangyin health beauty. The ELX80ONB Kejunior
Experimental cells. Experimental human umbilical enzyme mark instrument is from Bio-TEK Instrument
vein endothelial cells were provided by the Cell Center Company. The XDS-1 B inverted microscope was pur-
at the Xiangya Medical College of Central South chased from Chongqing Photoelectric Science
University. Instrument Limited Company; The 722 grating spec-
trophotometer was from the Shanghai Precision
Reagents and apparatuses. Pregnancy-related serum Scientific Instruments Limited Company.
protein-A was purchased from R&D Systems (molecu-
lar mass, 120 ~ 160 kD). The DAB chromogenic re- Culturing Endothelial Cells. Using human umbilical
agent kit was provided by Sangon Biotech Co., Ltd vein endothelial cells (HUVECs) and microscopy, we
(Shanghai, China). observed that the cells were arranged tightly like cobble-
stones, without overlap. Cells were grown in DMEM
The ET-1 immunohistochemical kit was purchased (high glucose) containing fetal calf serum (10%) and
from The United States R&D Biotechnology Company, streptomycin and cultured at 37°C with 5% CO2 and
and the immunohistochemical double reagent box was saturated humidity. After 2-3 days, when the cells con-
provided by the Beijing Fir Jinqiao Biotechnology fluent the bottom of the bottle, cells were passaged us-
Company. The raw materials of fluvastatin were pro- ing 0.25% trypsin digestion.
vided by Novartis. Nitric oxide kits were purchased
from Nanjing Building Research Institute of Biological Observation of Cell morphology. We used an inverted
Engineering. The HF151UV CO2 incubator was pur- microscope to observe the growth and proliferation of
chased from Shanghai’s Scientific Instrument Limited 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 The effect of PAPP-A on HUVEC ET-1 levels. The
(gray value). We used an image software analysis system effect of different concentrations of PAPP-A on
to detect the expression of ET-1 in endothelial cells’ gray ET-1 levels in HUVECs after 12, 24, and 48 h was
value with the formula: average optical density (MOD) = clear. We observed yellow pellets in the cell with
LG (positive products / background gray value).
higher concentrations of PAPP-A. However, in the
Increasing MOD values indicated increased ET-1 expres-
sion in endothelial cells. control group, we did not observe any yellow pellets
in the cells (Figures 1, 2, and 3).
Obtaining pregnancy serum. This trial was reviewed
and approved by the Ethics Committee of the Third The ET-1 MOD [9] from each group is shown in
Xiangya Hospital of Central South University on May Table 2. In the control group, there were no obvi-
17, 2012. We obtained permission from six pregnant ous changes in ET-1 at any time point (P>0.05).
women to take blood samples. Peripheral venous blood When HUVECs were cultured with PAPP-A at dif-
(4 mL) was collected into EDTA-containing tubes and ferent concentrations, we observed increased MOD
processed within 2 h after venipuncture. To ensure cell- after 12, 24, and 48 h (P<0.05). In the 50 ng/ml
free plasma collection, all EDTA-blood samples were
PAPP-A group, the highest MOD was at 12 h. In
centrifuged in two steps (3,000 rpm for 10 min, followed
by 12,000 rpm for 10 min). Cell-free plasma was stored the 100 ng/ml group, the highest MOD was at 24
at -20°C until extraction. The concentration of pregnan- h. In the 200 ng/ml PAPP-A group, the highest
cy serum was 1% during culture with HUVECs. MOD was at 12 h.
 Influence of pregnancy-associated plasma protein-A on endothelial cells 403

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 increasing concentrations of PAPP-A after 12, 24,
that were pre-cultured with pregnancy serum. As and 48 h. These data suggest that decreased NO
shown in Table 3, when HUVECs were cultured secretion levels in endothelial cells exist concomi-
with pregnancy serum, followed by culture with tantly with increased ET-1 levels and are dependent
PAPP-A for 12 h, NO levels were lower than they on PAPP-A concentration. Because the decomposi-
were in the control group (P<0.05). However, NO tion activity of MMP-9 could increase as the con-
levels of this group were higher than in the group centration increases [20,21] and because PAPP-A
cultured with PAPP-A but without pregnancy se- could specifically cleave IGFBP-4 [22-24], PAPP-A
rum (P>0.05). may degrade NO synthase as a protease [25,26]. In
our trial, we performed two experimental data sets
The effect of PAPP-A on ET-1 levels in HUVECs to investigate both the effect of pregnancy serum
that were pre-cultured with pregnancy serum. As alone on the HUVEC cells and the effect of non-
shown in Figure 4, there were fewer yellow pellets pregnant women serum on the HUVEC cells. We
in the group that was cultured with pregnancy se- found that the results were the same as the controls.
rum first, compared to cells cultured with PAPP-A PAPP-A is an important regulatory component of
but without pregnancy serum. the IGF system. Through proteolysis of inhibitory
IGF binding proteins (IGFBPs), PAPP-A acts as a
Discussion positive modulator of local IGF signaling in a vari-
ety of biological systems. Thus, eliminating its ac-
Endothelial cell dysfunction is an important patho- tivity may lead to decreased NO secretion, followed
physiological change in the early arterial atheroscle- by weak inhibition of ET-1 and a corresponding
rotic process [10,11]. There are many ways to avoid increase of ET-1. When the concentration of
atherosclerosis at physiological NO concentrations, PAPP-A increases, more NO synthases are degrad-
including dilation of blood vessels, inhibition of ed, and changes in NO concentration are more
vascular smooth muscle cell (VSMC) hyperplasia, pronounced.
and actions affecting platelet adhesion [12].
Decreased levels of NO secretion suggests damage Initially, Bonno et al. found that PAPP2A is synthe-
to endothelial cell (EC)-dependent vasodila- sized by placental X cells and syncytiotrophoblasts.
tion[13,14]. Because ET-1 is the strongest active Plasma levels increased with gestational age, reach-
vasoconstrictor constituent, increased ET-1 levels ing a peak of 324±277 ng/mL at full-term preg-
were due to vascular endothelial injury or stimula- nancy and subsequently declining postnatally
tion [15-17]. ET-1 secretion in vascular endothelial [27,28]. Because PAPP-A is a metalloproteinase
cells that were damaged was significantly higher and an antigen, we questioned whether it produced
compared to normal control subjects [18,19]. antibodies in the body. First, we incubated endo-
thelial cells and full-term maternal serum (from
The results of this study show that after 12, 24, and pregnant women with no complications) together
48 h, the supernatant NO concentration decreased and then added PAPP-A. The results show that NO
gradually with increasing concentrations of secretion and endothelin both decreased, compared
PAPP-A. The MOD value, expressed by the ET-1 with the control group. These results suggest that
levels of endothelial cells, increased gradually with 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. 13. Toda N, Tanabe S, Nakanishi S. Nitric oxide-mediated coro-
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