Mast cells and histamine: do they influence placental vascular network and development in preeclampsia?
ABSTRACT The physiological course of pregnancy is closely related to adequate development of the placenta. Shallow invasion of trophoblast as well as decreased development of the placental vascular network are both common features of preeclampsia. To better understand the proangiogenic features of mast cells, in this study we aim to identify the potential relationship between the distribution of mast cells within the placenta and vascular network development.
Placentas from preeclampsia-complicated pregnancies (n = 11) and from physiological pregnancies (n = 11) were acquired after cesarean section. The concentration of histamine was measured, and immunohistochemical staining for mast cell tryptase was performed. Morphometric analysis was then performed.
We noticed significant differences between the examined groups. Notably, in the preeclampsia group compared to the control group, we observed a higher mean histamine concentration, higher mast cell density (MCD), lower mean mast cell (MMCA) and lower vascular/extravascular (V/EVT) index. In physiological pregnancies, a positive correlation was observed between the histamine concentration and V/VEVT index as well as MCD and the V/VEVT index. In contrast, a negative correlation was observed between MMCA and the V/EVT index in physiological pregnancies.
Based on the data from our study, we suggest that a differential distribution of mast cells and corresponding changes in the concentration of histamine are involved in the defective placental vascularization seen in preeclamptic placentas.
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ABSTRACT: Co-ordinated development of the fetal villous tree of the placenta is necessary for continued fetal growth and well-being. Before fetal viability, blood vessel development within the developing immature intermediate villi (IIV) is characterized by branching angiogenesis, such that the placenta expands to produce 10-16 generations of stem villi. Once fetal viability is attained, a developmental switch occurs to form large numbers of gas-exchanging terminal villi (TV) by non-branching angiogenesis in mature intermediate villi (MIV). Several growth factors, including vascular endothelial growth factor (VEGF), placenta growth factor (PlGF), angiopoietins, and angiostatins are produced within the villi and act locally, via their receptors, to control angiogenesis. Their relative contributions to placental vascular development are not fully understood at the present time. Severe early-onset intrauterine growth restriction (IUGR) is characterized by absent/reversed end-diastolic flow velocity (ARED) in the umbilical arteries, leading to fetal hypoxia, acidosis and a substantial rise in perinatal mortality and morbidity. The placentas from such cases show a deficit in peripheral villous development, which may be perpetuated by the effects of oxygen (delivered by maternal blood into the intervillous space) upon VEGF-directed angiogenesis, the so-called 'placental hyperoxia' theory of villous maldevelopment. Trophoblast apoptosis is a significant feature of early-onset IUGR and may explain poor flow-independent transfer of nutrients to the fetus. Finally, since transgenic mouse studies highlight the importance of trophoblast-derived transcription factors for placental villous (labyrinth) development, it is possible that the villous trophoblast controls the orderly development of the underlying mesoderm and blood vessels into the fetal villi.European Journal of Obstetrics & Gynecology and Reproductive Biology 10/2000; 92(1):35-43. · 1.84 Impact Factor
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ABSTRACT: In human pregnancy, placental cytotrophoblasts that invade the uterus downregulate the expression of adhesion receptors that are characteristic of their epithelial origin, and upregulate the expression of adhesion receptors that are expressed by vascular cells. We suggest that this transformation could be critical to endovascular invasion, the process whereby cytotrophoblasts invade the uterine spiral arterioles and line their walls (Zhou et al. J. Clin. Invest. 1997. 99: 2139-2151.). To better understand the in vivo significance of these findings, we tested the hypothesis that in preeclampsia, an important disease of pregnancy in which endovascular invasion is abrogated, cytotrophoblasts fail to adopt a vascular adhesion phenotype. In experiments described here we stained placental bed biopsy specimens from age-matched control pregnancies and from those complicated by preeclampsia with antibodies that recognize adhesion molecules that are normally modulated during this transformation. In preeclampsia, differentiating/invading cytotrophoblasts fail to express properly many of these molecules, including integrin, cadherin, and Ig superfamily members. These results suggest that preeclampsia is associated with failure of cytotrophoblasts to mimic a vascular adhesion phenotype. The functional consequences of this abnormality are unknown, but are likely to affect negatively cytotrophoblast endovascular invasion and uterine arteriole remodeling, thereby compromising blood flow to the maternal-fetal interface.Journal of Clinical Investigation 06/1997; 99(9):2152-64. · 12.81 Impact Factor
- Inflammation Research 04/1997; 46 Suppl 1:S7-8. · 1.96 Impact Factor
Hindawi Publishing Corporation
Mediators of Inflammation
Volume 2012, Article ID 307189, 5 pages
Mast CellsandHistamine: Do TheyInfluencePlacental Vascular
Grzegorz Szewczyk,1,2MichałPyzlak,1Jakub Klimkiewicz,3Wacław´Smiertka,2
Magdalena Miedzi´ nska-Maciejewska,2and DariuszSzukiewicz1
1Chair and Department of General and Experimental Pathology, Medical University of Warsaw, Ul. Krakowskie Przedmiescie 26/28,
00-927 Warsaw, Poland
2Department of Gynecological Oncology, National Institute of Oncology, Ul. Wawelska 15, 02-000 Warsaw, Poland
3Department of Anesthesiology and Intensive Care, Military Institute of Health, Ul. Szaser´ ow 128, 04-141 Warsaw, Poland
Correspondence should be addressed to Grzegorz Szewczyk, firstname.lastname@example.org
Received 16 February 2012; Revised 27 April 2012; Accepted 27 April 2012
Academic Editor: Felipe Vadillo-Ortega
Copyright © 2012 Grzegorz Szewczyk et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
well as decreased development of the placental vascular network are both common features of preeclampsia. To better understand
the proangiogenic features of mast cells, in this study we aim to identify the potential relationship between the distribution of mast
cells within the placenta and vascular network development. Material and Methods. Placentas from preeclampsia-complicated
pregnancies (n = 11) and from physiological pregnancies (n = 11) were acquired after cesarean section. The concentration of
histamine was measured, and immunohistochemical staining for mast cell tryptase was performed. Morphometric analysis was
then performed. Results. We noticed significant differences between the examined groups. Notably, in the preeclampsia group
compared to the control group, we observed a higher mean histamine concentration, higher mast cell density (MCD), lower
mean mast cell (MMCA) and lower vascular/extravascular (V/EVT) index. In physiological pregnancies, a positive correlation was
observed between the histamine concentration and V/VEVT index as well as MCD and the V/VEVT index. In contrast, a negative
correlation was observed between MMCA and the V/EVT index in physiological pregnancies. Conclusions. Based on the data from
our study, we suggest that a differential distribution of mast cells and corresponding changes in the concentration of histamine are
involved in the defective placental vascularization seen in preeclamptic placentas.
Angiogenesis is a crucial process for the growth and devel-
opment of new tissues. We can observe angiogenesis in neo-
plasms, during tissue repair after injury and in the placenta.
Proper placental angiogenesis is necessary for the normal
course of pregnancy and labor . The pathogenesis of pree-
clampsia is still unclear, but it is known that shallow spiral
artery invasion may contribute to preeclampsia develop-
ment. Shallow spiral artery invasion results in poor placental
perfusion and may lead to hypoxic stress in the fetus. Imma-
vascular network is defectively developed as well. In some
preeclampsia-complicated pregnancies, the placenta and
associated placental vascular network are diminished. Mast
cells are found in the placenta in every stage of placenta
development. Their potential role, apart from immuno-
logical properties, can be associated with proangiogenic
activity. Mast-cell-derived mediators of known angiogenetic
potential include vascularendothelial growth factor(VEGF),
transforming growth factor beta (TGF-β), histamine, tumor
necrosis factor alpha (TNF-α), interleukin-8, and basic
fibroblast growth factor . The activation and degranula-
tion of mast cells in the place of angiogenesis stimulate vessel
sprouting and sustain mast cell attraction and activation .
Data from the literature and our own experience suggest
that mast cells may be involved in the pathogenesis of
2Mediators of Inflammation
Table 1: The characteristics of patients included into the study.
PE group n = 11Control group n = 11
Weeks of gestation
Birth weight (g)
1st minute Apgar’s score
preeclampsia-complicated pregnancies [5, 6]. In this study,
we examined the relationship between mast cells (number
and morphological features), histamine concentration, and
microvascular density in placentas obtained after delivery
from normal and preeclampsia-complicated pregnancies.
The characteristics of the patients are detailed in Table 1.
Placental samples were obtained in a standardized manner
after the dissection of fetal membranes. Three samples were
excised from the maternal side of the placenta and two were
excised from the fetal side. Macroscopically changed areas,
large vessels, and fibrous tissues were avoided. Samples were
taken immediately after cesarean sections in each group:
preeclamptic women (PE, n = 11) and healthy women
(control group, n = 11). In the PE group, cesarean sections
were performed due to severe preeclampsia. In the control
group, cesareans were performed due to severe myopia
and breech presentation of the fetus. None of the patients
included in the study had contractile activity . The study
was reviewed and accepted by the local ethic committee.
The tissue fragments were fixed in formaldehyde solution,
dehydrogenized with 96% alcohol, acetone, and xylene and
then paraffinized. Next, they were cut in microscopic slides
and deparaffinized, and the intrinsic peroxidase activity was
blocked with hydrogenium superoxide. The samples were
then washed with PBS and incubated with normal human
serum for 20 minutes. Excess antibody was removed, and
the slides were incubated with mouse anti-tryptase antibody
(Novocastra, 1:3000), followed by secondary anti-mouse
biotinylated antibody and Novostain Super ABC Reagent
(Novocastra). Both incubations lasted for 30 minutes.
The slides were washed with PBS and exposed to 3,3?-
diaminobenzidine (Immunotech) for 3 minutes as an elec-
with Mayer’s hematoxylin (Sigma) for 1 minute. Finally,
the slides were mounted with DPX (Sigma). As a negative
control, the slides were incubated with PBS instead of the
A fluorimetric method was applied as previously described
. The determination of histamine was based on a
precolumn derivatization with o-phthaldialdehyde using
reversed-phase high-performance liquid chromatography in
perchloric acid extracts. A fluorescence detection system was
used, with the excitation set at 360nm and the emission read
at 455nm. The intra- and inter-assay coefficients of variation
were 8.5% and 10.0%.
Morphometric analysis was carried out with the computer
image analysis system Leica Quantimet 500C+ (Leica Cam-
Pentium computer operating at 120MHz equipped with an
ARK Logic 2000MT graphic card and graphic processor.
The computer was connected to a CCD video camera JVC
TK-1280E and Leica DMLB light microscope. Sections of
placentas were imaged using a 20:1 objective and a 10:1,20
ocular. The optical image was focused by a video camera,
and an analogue video signal was generated. An analogue
to digital converter (ADC) produced a digitized video with
distinct color level values in HSI system. The images were
processed, and mast cells and placental vessels were clearly
identified  (Figure 1).
Two independent researchers were responsible for image
acquisition and analysis. All measurements were recorded
in a blinded fashion. Neither researcher had previous
knowledge of the clinical data. For each case, 50 random
visual fields were analyzed. After system calibration, the
area of a single analyzed image (visual field) was defined
as approximately 0,14mm2. The following parameters were
analyzed: mast cell density (MCD), defined as number of
mast cells per mm2of placental tissue; mean mast cell area
(MMCA), the mean area of mast cells cross-sections; shape
of mast cells, defined as the ratio of long to short axis of a
cell (with perfectly round cells defined as having 1.00 index);
vascular/extravascular tissue index (V/EVT index), the ratio
of vessel cross-section area to remaining placental tissue.
Technical error caused by uniaxial sections of vessels was
eliminated by accepting the lowest value of Ferret’s diameter
as the diameter for a single lumen. Vessels between 10 and
70μm in diameter were included for analysis.
Statistical analysis was performed with Statistica 8.0 (Stat-
Soft, Poland). Groups were compared with Student’s t-
test. In each group analysis, correlation was measured
between the histamine concentration, V/EVT index, and
Mediators of Inflammation3
Figure 1: Process of mast cells identification with morphometric software. Stage 1: initial picture obtained from microscope and saved in
HSI colour system. Stage 2: binary image after detection of mast cells in distinct hue values. Stage 3: final reduction of noises and smoothing
of detected fields.
morphometric parameters of the mast cells. Differences were
deemed statistically significant if P < 0,05.
Specific differences were observed in several examined
parameters between the PE and control groups. The mean
histamine concentration (ng of histamine per 1g of tissue)
was significantly higher in the PE group compared to the
control group (245,6 ± SD 19,8 versus 175,1 ± SD 15,1; P =
0,002). MCD (in cells/mm2) was also significantly higher in
the PE group compared to the control group (7,67 ± SD 3,56
versus 2,89 ± SD 1,34; P = 0,004). In contrast, the MMCA
was significantly lower in the PE group in comparison to the
control group (62,25μm2±SD 18,91 versus 101,98μm2±
SD 57,91; P = 0,0428). We also observed some differences in
cell shape. Mast cells in the control group were longer than
mast cells in the PE group (shape index 1,88 ± SD 0,8 versus
1,52 ± SD 0,39; P = 0,051; refer to Table 2).
Morphometric assessment of placental circulature was
performed and revealed a decrease in the V/EVT index in the
PE group compared to the control group (0,15 ± SD 0,04
versus 0,23 ± SD 0,074; P = 0,005; refer to Table 2).
The analysis revealed a positive correlation between the
histamine concentration and the V/VEVT index as well as
existed between the MMCA and V/EVT index in the control
group, while the PE group showed no significant correlation
between these parameters. Specific values of correlation for
these parameters are provided in Table 3.
Angiogenesis is the process of vessel growth from preexisting
vessels, a process that requires stimulation by proangio-
genic factors. Important stimulants of placental angiogenesis
include VEGFs and placental growth factor, which act
through the VEGF receptor family. VEGF production is
stimulated by histamine acting through the H2 receptor
. Mast cells are pointed to as a potential source of
potent proangiogenic factors during angiogenesis, including
histamine, VEGF, bFGF, TGF-beta, TNF-alpha, and IL-8.
4 Mediators of Inflammation
Table 2: Morphometric parameters analyzed in the study compared with Student’s t-test. V/EVT index: vascular/extravascular tissue index,
MCD: mast cell density, and MMCA: mean mast cell area. The statistically significant results are in bold.
PE group Control group
Table 3: The indexes of correlation between histamine concentration and morphometric parameters of mast cells and V/EVT index in each
group. V/EVT index: vascular/extravascular tissue index, MCD: mast cell density, MMCA: mean mast cell area. The statistically significant
results are in bold.
PE group n = 11
Control group n = 11
Histamine concentration (ng/1g of tissue)
Additionally, mast cells are a source of extracellular matrix-
degrading proteinases .
In vitro models of angiogenesis observed in hypoxic
conditions provide us with information on increased
angiogenesis, which occurs mainly through increases in
VEGF synthesis . Histamine proangiogenetic action is
provided through H1- and H2-receptor-mediated VEGF
synthesis. Mast cell degranulation leads to a local increase
in histamine concentration and therefore an increase in
VEGF synthesis. Mast cells, however, synthesize and secrete
VEGF apart from histamine. The final effect is vigorous
formation of new vessels in place of mast cell degranulation
Decreases in mast cell density in connection with
decreased histamine concentration correlated with lower
V/EVT index values; nevertheless, this correlation was
observed only in the control group. Decreased mast cell area
may indicate changes in mast cell activation, perhaps as an
effect of degranulation. Hypoxia, which is dominant during
placenta formation, is a potent stimulator for mast cell
activation and new vessel formation. The most important
pathway through which hypoxia stimulates angiogenesis
is the activation of hypoxia inducible factor-1α (HIF-1α)
transcription and further synthesis of VEGF. It is also
observed that the synthesis of histamine within mast cells
and their degranulation is increased after stimulation with
HIF-1α that is achieved through histidine decarboxylase
(HDC, EC:22.214.171.124) .
Preeclampsia is a specific state of pregnancy associated
with hypertension and proteinuria. Shallow trophoblast
invasion of maternal spiral arteries results in an increase
in systemic blood pressure. The leading hypothesis for
preeclampsia pathogenesis suggests it may arise in order
to maintain placental perfusion pressure at a satisfactory
level . The vascular bed of the placenta is diminished
as a whole, with reduced branching and malformations
observed; blood vessels are characterized by decreased num-
ber, lumen diameter, and total lumen area . Data from
our study support this previous finding, as the V/EVT index
was decreased in the PE group compared to the control
group. The reduced proportion of vascular area may reflect
diminished placental angiogenesis in the first trimester of
pregnancy. The decreased vascular network development is
a result of a multifactorial pathogenetic course as well as
The differences in mast cell organization observed
between the PE and control groups suggest that mast cells
take part in the process of vessel development. Because
mast cells are observed to gather close to blood vessels just
before the process of angiogenesis begins (this is particularly
characteristic for neoplasm growth ), we expect an
expanded vascular network in preeclamptic placentas. In our
study, we observed an increase in mast cell density and an
increase in histamine concentration but a low V/EVT ratio.
We conclude that in PE, susceptibility to histamine and/or
other mast cell proangiogenic compounds may be decreased.
In PE placentas, the mast cells had a different shape and
smaller area in comparison to the control group. The
data suggest that we observed mast cells after an intensive
degranulation, as we also found an increased concentration
of histamine . Increased mast cell density and histamine
cular network development. On the other hand, we cannot
exclude impairments in histamine receptor configuration.
Functional predominance of intracellular histamine receptor
(HIC) over H1and H2receptors may be a causative factor in
the observed decreased angiogenesis .
The reason for the decreased V/EVT index in preeclamp-
tic placentas may be associated not only with decreased
angiogenesis but also with fibroblast proliferation and fibro-
sis in the extravascular area. In the examined material, the
V/EVT index was assessed in placentas obtained during the
Mediators of Inflammation5
third trimester. A remodeling of extravascular tissue during
the pregnancy should also be taken into consideration. Mast
cells are sources of matrix-degrading enzymes including
collagenases and gelatinases . Prolonged stimulation of
mast cells with hypoxia leads to an increase in collagenolytic
activity and an accumulation of low molecular collagen frag-
ments, thus providing a stimulatory factor to fibroblasts and
smooth muscle cell proliferation . A dominance of acti-
vated fibroblasts may lead to a decrease in the V/EVT index.
We conclude that mast cells are strongly involved in the
pathogenesis of preeclampsia, as their concentration and
tic placentas despite higher histamine concentration and
accumulation of mast cells suggests that mast cells fail
in their proangiogenic potential, concurrently increasing
 J. Kingdom, B. Huppertz, G. Seaward, and P. Kaufmann,
“Development of the placental villous tree and its conse-
quences for fetal growth,” European Journal of Obstetrics
Gynecology and Reproductive Biology, vol. 92, no. 1, pp. 35–43,
 Y. Zhou, C. H. Damsky, and S. J. Fisher, “Preeclampsia
is associated with failure of human cytotrophoblasts to
mimic a vascular adhesion phenotype: one cause of defective
endovascular invasion in this syndrome?” Journal of Clinical
Investigation, vol. 99, no. 9, pp. 2152–2164, 1997.
 K. Norrby, “Mast cells and de novo angiogenesis: angiogenic
capability of individual mast-cell mediators such as histamine,
TNF, IL-8 and bFGF,” Inflammation Research, vol. 46, supple-
ment 1, pp. S7–S8, 1997.
 K. Norrby, “Mast cells and angiogenesis: review article,”
APMIS, vol. 110, no. 5, pp. 355–371, 2002.
 W. M. Purcell and T. H. P. Hanahoe, “A novel source of mast
cells: the human placenta,” Agents and Actions, vol. 33, no. 1-2,
pp. 8–12, 1991.
 D. Szukiewicz, A. Szukiewicz, D. Maslinska, and M. W.
Markowski, “Placental mast cells (MC) and histamine (HA)
in pregnancy complicated by diabetes class C—relation to the
development of villous microvessels,” Trophoblast Research,
vol. 13, pp. 503–510, 1999.
 D. Szukiewicz, D. Maslinska, J. Stelmachow, and E. Wojtecka-
Lukasik, “Biogenetic amines in placental tissue: relation to
the contractile activity of the human uterus—preliminary
communication,” Clinical and Experimental Obstetrics and
Gynecology, vol. 22, no. 1, pp. 66–70, 1995.
 A. H. Anton and D. F. Sayre, “A modified fluorometric
procedure for tissue histamine and its distribution in various
animals,” Journal of Pharmacology and Experimental Thera-
peutics, vol. 166, no. 2, pp. 285–290, 1969.
 A. E. Jakobsson, K. Norrby, and L. E. Ericson, “A morpho-
metric method to evaluate angiogenesis kinetics in the rat
mesentery,” International Journal of Experimental Pathology,
vol. 75, no. 3, pp. 219–224, 1994.
 A. K. Ghosh, N. Hirasawa, and K. Ohuchi, “Enhancement by
histamine of vascular endothelial growth factor production
in granulation tissue via H2 receptors,” British Journal of
Pharmacology, vol. 134, no. 7, pp. 1419–1428, 2001.
 V. H. Shore, T. H. Wang, C. L. Wang, R. J. Torry, M. R.
Caudle, and D. S. Torry, “Vascular endothelial growth factor,
placenta growth factor and their receptors in isolated human
trophoblast,” Placenta, vol. 18, no. 8, pp. 657–665, 1997.
 J. Sorbo, A. Jakobsson, and K. Norrby, “Mast-cell histamine is
angiogenic through receptors for histamine1 and histamine2,”
International Journal of Experimental Pathology, vol. 75, no. 1,
pp. 43–50, 1994.
 V. Rizzo and D. O. DeFouw, “Mast cell activation accelerates
the normal rate of angiogenesis in the chick chorioallantoic
membrane,” Microvascular Research, vol. 52, no. 3, pp. 245–
 H. J. Jeong, P. D. Moon, S. J. Kim et al., “Activation of hypoxia-
inducible factor-1 regulates human histidine decarboxylase
expression,” Cellular and Molecular Life Sciences, vol. 66, no.
7, pp. 1309–1319, 2009.
 T. Naicher, S. M. Khedun, J. Moodley, and R. Pijnenborg,
“Quantitative analysis of trophoblast invasion in preeclamp-
sia,” Acta Obstetricia et Gynecologica Scandinavica, vol. 82, no.
8, pp. 722–729, 2003.
 K. Benirschke and P. Kaufmann, Pathology of the Human
Placenta, Springer, New York, NY, USA, 2000.
 M. K. Dabbous, R. Walker, L. Haney et al., “Mast cells
and matrix degradation at sites of tumour invasion in rat
no. 3, pp. 459–465, 1986.
 F. Levi-Schaffer, D. Slovik, L. Armetti, D. Pickholtz, and E.
Touitou, “Activation and inhibition of mast cells degranula-
tion affect their morphometric parameters,” Life Sciences, vol.
66, no. 21, pp. PL283–PL290, 2000.
 K. Norrby, “Evidence of a dual role of endogenous histamine
in angiogenesis,” International Journal of Experimental Pathol-
ogy, vol. 76, no. 2, pp. 87–92, 1995.
 H. Maxova, J. Herget, and M. Vizek, “Lung mast cells and
hypoxic pulmonary hypertension,” Physiological Research, vol.
61, pp. 1–11, 2012.