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Reproductive Endocrinology |
Magee-Womens Research Institute and the Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
Address all correspondence and requests for reprints to: Kirk P. Conrad, M.D., Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, Pennsylvania 15213.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF) and interleukin-1
(IL-1) are inflammatory cytokines capable of eliciting endothelial cell
dysfunction, we investigated whether the production of these
inflammatory cytokines by cultured villous explants from the human
placenta was affected by incubation in reduced oxygen (2%
O2). The term placenta produced TNF, IL-6, and low
levels of IL-1 and IL-1ß under standard tissue culture conditions.
Hypoxia significantly increased TNF, IL-1, and IL-1ß production
by 2-, 6-, and 23-fold, respectively, but did not affect IL-6
production. Further, cytokines were immunolocalized to the
syncytiotrophoblast layer as well as to some villous core cells.
Hypoxic regulation of placental TNF and IL-1ß production also
appeared to differ based on gestational age. Finally, treatment with
either cobalt chloride or an iron chelator mimicked the hypoxic
response, suggesting that stimulation of placental cytokine production
may involve a heme-containing, O2-sensing protein. These
results suggest that placental hypoxia can lead to the elaboration of
inflammatory cytokines, which may contribute to the pathophysiology of
preeclampsia. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The identity of deleterious factors elaborated by the placenta that
presumably compromise endothelial function during preeclampsia is
unknown. The inflammatory cytokines, tumor necrosis factor- (TNF)
and interleukin-1ß (IL-1ß), are notorious for producing endothelial
dysfunction (5), and interestingly, synthesis of these cytokines, as
well as IL-6, by trophoblast and other cells of the normal human
placenta has been documented (6, 7, 8). The regulation of placental
cytokine expression and, in particular, the potential impact of hypoxia
are unknown. As placentas from women with preeclampsia are likely to
contain ischemic foci, and the placenta has the capacity to produce
inflammatory cytokines, the current investigation was designed to
determine whether hypoxia further stimulates the production of
inflammatory cytokines by the placenta. To this end, placental tissues
were placed as explants into culture, and the elaboration of cytokines
was evaluated in response to reduced O2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Placentas were obtained from women undergoing elective cesarian
section with normal pregnancies at term or from women undergoing
elective pregnancy termination at 8–11 gestational weeks under
approval of the institutional internal review board of Magee-Women’s
Hospital. For term placenta, 3–5 cotelydons were extracted at random
and rinsed extensively with sterile saline. Decidual tissue and large
vessels were removed from villous placenta by blunt dissection. The
villous tissue was then finely dissected into 5- to 10-mg pieces while
in a bath of Hanks’s Balanced Salt Solution (Mediatech, Herndorn, VA)
containing 25 mmol/L HEPES (Sigma Chemical Co., St. Louis, MO), 10%
FBS (Summit Technology, Ft. Collins, CO), and antibiotics (penicillin,
streptomycin, and gentamicin, Mediatech). The pieces of tissues were
then washed twice in the above solution, and 5 pieces (30–50 mg
tissue) were placed into 24-well plates (Becton Dickinson, Franklin
Lakes, NJ) containing 1 mL phenol-free medium 199 (Life Technologies,
Gaithersburg, MD) supplemented with 2% Nutridoma HS (Boehringer
Mannheim, Indianapolis, IN) and antibiotics. First trimester villous
tissue was prepared in a comparable fashion. For the culture of decidua
basalis from term placenta, the basal plate was carefully trimmed from
the underlying villous tissue and placed into culture as a sheet of
tissue 
40–70 mg/well. Explants were incubated at 37 C for a 24-h
preincubation period on an orbital shaker (60 rpm) under standard
tissue culture conditions of 5% CO2-95% room air
(normoxia; 21% O2). Cultures were gently shaken because it
has been reported in stationary cultures of hepatoma cells maintained
in 21% O2 that the formation of an unstirred layer leads
to reduced pericellular pO2 and, consequently, elevated
erythropoietin secretion (9, 10) (our unpublished results). After a
medium change and the addition of any treatment, the plates were placed
on an orbital shaker at 37 C for various times in either normoxia or
reduced O2 (hypoxia; 2.1% O2-5%
CO2-92.8% N2). The treatments of cobalt
chloride (CoCl2; 100 µmol/L; Sigma) and the iron chelator
Tiron (30 mmol/L; Acros, Pittsburgh, PA) were performed for a 24-h
incubation period under normoxic conditions. Using a Tucker chamber and
a radiometer O2 electrode (11), the pO2 of the
hypoxic incubator was routinely determined to be 15–20 torr. At the
end of the experiment, tissue weight was recorded so that cytokine
values could be corrected per g wet weight, and the conditioned medium
was stored at -80 C.
Jar choriocarcinoma cell culture
The trophoblast-derived human choriocarcinoma Jar cell line
(American Type Culture Collection, Rockville, MD) was maintained in
RPMI 1640 (Mediatech) with 10% FBS and antibiotics. Approximately 5000
cells/cm2 were seeded in 6-well plates. After incubation
for 48 h under standard culture conditions, cells were further
incubated in either normoxia or hypoxia for 72 h. Conditioned
medium was collected and stored at -80 C for the measurement of
TNF, and the cell monolayer was digested in 1 N NaOH for
protein determination by the Lowry method (12).
Cytokine enzyme-linked immunosorbant assays (ELISAs)
All ELISAs were performed using kits obtained from R&D Systems
(Minneapolis, MN). For TNF and IL-ß, high sensitivity kits were
used, with sensitivities of 0.5 and 0.125 pg/mL, respectively, whereas
the sensitivities for the IL-1 and IL-6 assays were 3.9 and 3.13
pg/mL, respectively. When sample dilution was required, either the
manufacturer’s serum or culture diluent, or villous explant medium was
used.
L929 cytotoxicity bioassay
This bioassay is based on the cytotoxic response of actinomycin
D-treated L929 fibrosarcoma cells (American Type Culture Collection) to
TNF (13). L929 cells were seeded on 96-well plates at a density of
2.5 x 104 cells and allowed to attach overnight.
Cells were then treated for 30–60 min with actinomycin D (10 µg/mL;
Sigma). Subsequently, doses of recombinant human TNF (R&D) ranging
from 1–500 pg/mL or 100 µL conditioned medium from villous explants
cultured in either normoxia or hypoxia were added to the cells. After a
24-h incubation at 37 C, a 10% Alamar blue solution (Alamar
Biosciences, Sacramento, CA) was added, and fluorescence was measured
at 2, 4, and 6 h (530 nm; 590 nm emission). The percent
cytotoxicity was calculated as [control (untreated cells) -
conditioned medium or TNF standard]/control (untreated cells) x
100 (%) and was plotted against the log TNF concentration of the
standards to interpolate TNF levels of unknown samples.
Immunoabsorption of TNF activity was performed by coincubation of
medium samples with either goat antihuman TNF neutralizing antibody
(0.5 µg/mL; R&D) or mouse antihuman TNFß monoclonal antibody (5
µg/mL; PharMingen, San Diego, CA) for 1 h at 37 C before
addition to the L929 bioassay. At the concentrations used, these
antibodies were capable of completely neutralizing up to 200 pg/mL of
the respective cytokine.
Lactate dehydrogenase (LDH) cytotoxicity assay
Assessment of explant viability was routinely monitored by measuring the release of LDH into medium relative to a 1% Triton X-100 (Sigma)-lysed positive control. The assay employed a modification of the TOX-7 viability kit supplied by Sigma and entailed adding a 20-fold volume excess of assay mixture (160 µL) to the medium sample tested (8 µL). The assay mixture consisted of equal volumes of substrate (lactate), enzyme (diaphorase), and dye (nitro blue tetrazolium) solutions, and the reaction was allowed to proceed for 20 min in a microtiter plate before the addition of 0.1 vol 1 N HCl. Color change was quantified by reading the absorbance at 490 nm, and the percent cell death calculated as [OD of sample - OD of medium (background)]/(OD of Triton X-100-lysed positive control - OD of background) x 100 (%), where OD is the optical density.
Immunohistochemistry
After culture, villous explant tissues were embedded in OCT
compound (Baxter Scientific, McGraw Park, IL) and flash-frozen in
liquid nitrogen. Twelve-micron sections were cut on a cryostat and
mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA).
Slides were then rinsed in Dulbecco’s phosphate-buffered saline (DPBS)
and fixed in 4% paraformaldehyde for 20 min at room temperature. After
another DPBS wash, endogenous peroxidase activity was quenched by a
0.6% hydrogen peroxide treatment for 15 min, and then tissues were
permeablized with 0.3% Triton X-100 for 10 min, followed by a DPBS
wash. Immunodetection was performed using the Vectastain ABC Elite kit
protocol (Vector Laboratories, Burlingame, CA). The primary antibodies
were incubated with the tissue sections for 2 h at room
temperature and were all monoclonal antibodies generated against the
following human proteins: TNF (10–20 µg/mL; clone 2C8, Biodesign,
Kennebunk, ME), IL-1ß (10 µg/mL; Genzyme, Boston, MA), IL-1
(10–20 µg/mL; clone C12, Antigenix America, Franklin Square, NY),
IL-6 (20 µg/mL; Genzyme), CD68 (3.8 µg/mL; Dako, Carpenteria, CA),
and cytokeratin (identifies cells of epithelial origin; 3.5 µg/mL;
Sigma). For the negative control, the mouse IgG1 isotype (Sigma) was
substituted for the primary antibody at matched concentrations. After
incubation with biotinylated antimouse secondary antibody and the
avidin-biotin-peroxidase complex (Vector), hydrogen peroxide and
3,3'-diaminobenzidine substrates (Sigma) were used to detect the
peroxidase conjugate, which yielded a brown reaction product.
Data analysis
The time course of cytokine production and the response to hypoxia were analyzed by split plot design ANOVA. If significant main effects were observed, then differences between group means were assessed by orthogonal contrasts. P < 0.05 was considered to be significant. The effects of CoCl2 and Tiron treatments were assessed by randomized block design ANOVA using Fischer’s least significant difference post-hoc test. Wilcoxon’s signed rank test was used to analyze fold change in hypoxic stimulation of cytokine production.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Under standard culture conditions, villous placental explants
elaborated the inflammatory cytokines TNF, IL-1ß, and IL-1 as
well as IL-6 throughout a 24-h incubation period (Fig. 1
, A–D). Both TNF and IL-6 levels increased over
time in culture (P < 0.05), whereas IL-1 and
IL-1ß production remained at low levels (<200 pg/g wet wt). Hypoxia
stimulated TNF production by 4 h of incubation, and TNF
concentrations remained elevated through the 24-h incubation period
(P < 0.05; Fig. 1A). At 10 h of incubation in the
reduced O2 environment, there was a marked stimulation of
IL-1ß production (P < 0.001; Fig. 1B), and IL-1
levels were elevated by 24 h in hypoxia (P <
0.001; Fig. 1C). In contrast, hypoxia did not affect IL-6 synthesis at
any of the time points studied (Fig. 1D). Finally, the TNF produced
by villous explants was bioactive, as assessed by the L929 cytotoxicity
assay, and bioactivity approximated immunoreactive levels (Table 1). The TNF bioactivity was completely neutralized by
immunoabsorption with a polyclonal antibody against human TNF, but
was not affected by coincubation with the TNFß antibody (n = 3;
data not shown).
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Tissue localization
Localization of TNF, IL-1, IL-1ß, and IL-6 to specific
cell types comprising the explanted villous tissues was documented by
immunohistochemistry. For IL-1ß, slight immunoreactivity was found in
the syncytiotrophoblast layer of villous explants incubated in normoxia
(Fig. 2A), which was much more intense in tissues
incubated for 24 h in hypoxia (Fig. 2B). The syncytiotrophoblast
layer was identified as cytokeratin-positive cells (Fig. 2C). Prominent
TNF, IL-1, and IL-6 immunoreactivity was also detected in the
syncytiotrophoblast cell layer (data not shown); however, no obvious
differences in immunoreactivity for these cytokines between tissues
incubated in normoxia or hypoxia could be detected by
immunohistochemistry. In addition, immunoreactivity for all cytokines
was observed in some cells within the villous core in areas where
CD-68+ cells resided (fetoplacental macrophages or Hofbauer
cells; data not shown).
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production by cells of
trophoblast origin, the trophoblast-derived Jar choriocarcinoma cell
line was incubated under reduced O2 conditions. After
72 h of incubation, there was a 4-fold increase in TNF levels
in conditioned medium from Jar cells cultured in hypoxia compared to
those in medium from cells cultured in normoxia (145.2 ± 34.6
vs. 37.4 ± 8.5 pg/mg protein, respectively;
P < 0.05; n = 4 experiments).
Figure 3 depicts TNF (Fig. 3A) and IL-1ß (Fig. 3B)
production by explants of villous placenta compared to that produced by
decidual tissues. Although TNF production after 24 h in
normoxia was similar between villous and decidual explants, the
responsiveness of the villous tissues to hypoxia was 2.8-fold greater
than that of the decidua (P < 0.05). For IL-1ß
production, the villous placenta elaborated 7-fold more IL-1ß into
the culture medium over a 24-h incubation in normoxia compared to
decidual tissues (P < 0.05); both tissues, however,
responded to hypoxia with a 10-fold increase in IL-1ß levels.
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Cytokine production over a 24-h culture period in normoxia as well
as responsiveness to hypoxia varied according to gestational age. There
was no hypoxic stimulation of either TNF or IL-1ß production by
villous explants until after the ninth week of gestation, when the
production of both cytokines was increased by hypoxia
(P < 0.05; Fig. 4). In fact, TNF
elaboration by villous tissues obtained during weeks 8–9 of gestation
was actually reduced by 50% in response to hypoxia (P
< 0.02). Of note, however, is that production of TNF over 24 h
in normoxia was greater for placental villi of 8–9 gestational weeks
(1776.0 ± 265.3 pg/g wet wt) compared to 11 weeks (384.5 ±
146.3; P < 0.02) and that TNF levels produced by
the 11-week gestation villi were similar to those of the term villous
placental explants (refer to Fig. 1A
). IL-1ß production after 24
h in normoxia by tissues collected at either time in the first
trimester (8 weeks, 784.7 ± 196.7 pg/g wet wt; 11 weeks,
752.5 ± 452.3) was greater than that elaborated by the term
placenta (129.2 ± 24.8).
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A final objective of this study was to assess whether hypoxic
regulation of inflammatory cytokine production by placental tissues
involved an O2-sensing, heme-containing protein as
described in other hypoxia-sensitive systems (14). For this study,
tissues were incubated for 24 h in normoxia with and without
CoCl2 or the iron chelator, Tiron. These agents are
believed to displace iron in the heme protein and thus mimic a state of
reduced tissue O2 (14, 15). Indeed, treatment with either
CoCl2 or Tiron significantly increased TNF, IL-1ß, and
IL-1 production (Fig. 5). However, CoCl2
increased TNF production above normoxic values (22-fold) to a
greater extent than either hypoxia or Tiron treatment (each 3-fold;
P < 0.03; Fig. 5A). In contrast, the stimulation of
IL-1ß synthesis by either CoCl2 or Tiron was
significantly greater than that by hypoxia (P < 0.05;
Fig. 5B), whereas the degree of stimulation of IL-1 synthesis was
similar between CoCl2 treatment and hypoxia (Fig. 5C).
Finally, CoCl2 treatment resulted in a 5.5-fold stimulation
of IL-6 production by villous explants (P < 0.02; data
not shown), although hypoxia was without effect.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, IL-1ß, and IL-6.
Further, the production of TNF, IL-1, and IL-1ß, but not IL-6,
was markedly stimulated by reduced O2 tension. The
expression of these inflammatory cytokines by the trophoblast and other
cells in the normal placenta has been previously reported (6, 7, 8, 16, 17), and immunoreactivity was observed in the syncytiotrophoblast layer
of cultured villous explants in the present study. Heightened
production of TNF by the trophoblast-derived Jar choriocarcinoma
cells further suggests that the trophoblast cell is capable of
responding to reduced O2 with stimulated cytokine
production. In addition, TNF from the conditioned medium of cultured
villous explants was cytotoxic in the L929 bioassay. Only weak IL-1ß immunoreactivity in the syncytiotrophoblast layer of term placenta has been previously reported by others (7, 16), and no IL-1ß messenger ribonucleic acid (mRNA) was detected in freshly isolated tissues by Northern analysis (18, 19). However, IL-1ß mRNA and protein could be induced in trophoblast cells using cell isolation procedures (19). In the present study, very low levels of IL-1ß were detected by immunohistochemistry and high sensitivity ELISA in the conditioned medium of tissues incubated in normoxia. However, IL-1ß production was greatly stimulated by exposure to hypoxia. Therefore, it may be that at term the trophoblast produces little IL-1ß in situ, but focal areas of placental hypoxia could serve as a potent stimulus for trophoblast IL-1ß production.
As it is known that the maternal decidua has a relatively large
population of immune cells (20) and that the decidua expresses IL-1ß
mRNA (18) and produces TNF (21, 22), it was of interest to compare
the relative contributions of the maternal decidual and villous
placental compartments with respect to the production of these
inflammatory cytokines. When corrected for tissue weight, comparable
levels of TNF were produced by placental and decidual tissues over a
24-h culture period in standard O2 conditions, whereas
IL-1ß levels were greater in the villous placenta. A previous study
suggested that decidual production of TNF was slightly greater than
that by term chorionic villi (22); however, the levels reported were
highly variable and were analyzed using assay procedures 500-fold less
sensitive than those used in the present study. It is clear from the
present results that the capacity to respond to hypoxia with elevated
TNF and IL-1ß production resides primarily in the villous
placental compartment. Although decidual explants produced IL-1ß in
response to hypoxia, the absolute level of production was low and may
represent contamination by anchoring villi and extravillous trophoblast
cells, as it is difficult to prepare term decidua completely devoid of
these placental cells.
There was an interesting dichotomy in placental cytokine production
dependent on gestational age, such that villous tissues collected at
8–9 weeks gestation elaborated more TNF and IL-1ß in a 24-h
culture period than the term placenta. Others have shown that TNF
bioactivity (22) and IL-1ß immunoreactivity (7, 18) were greater in
first trimester than term placenta. Further, IL-1, IL-1ß, and IL-6
mRNA levels were high in cultured cytotrophoblast cells and declined as
the cells differentiated in vitro into syncytiotrophoblasts
(23). Thus, the greater number of villous cytotrophoblast cells in
first trimester placenta could contribute to the higher cytokine
levels. The ability of the villous placenta to respond to hypoxia with
heightened cytokine production also appeared to be dependent on
gestational age. Specifically, only placenta obtained after 11 weeks
gestation responded to hypoxia with elevated TNF and IL-1ß
production; TNF levels were actually decreased after a 24-h hypoxic
incubation of villous tissues obtained at 8 weeks gestation. A recent
study on the effects of hypoxia on early gestation human trophoblast
differentiation demonstrates that in placenta from 10–12 weeks
gestation, hypoxic incubation inhibits cell adhesion molecule
expression and cytotrophoblast invasion, but that before 7 weeks
gestation, the cytotrophoblast is insensitive to hypoxic incubation
in vitro (24). Notably, before 10–12 weeks gestation, the
placental environment is relatively hypoxic in situ (18
torr) (25), as the intervillous space is largely devoid of blood flow
(26), and after 10–12 weeks gestation, intervillous placental blood
flow begins, and pO2 rises (25, 26). Perhaps because the
placenta from 8–9 weeks gestation developed in a low O2
environment in situ, hypoxic incubation ex vivo
does not lead to an elevation in cytokine synthesis as it does in
tissues collected after 10 weeks gestation.
The inflammatory cytokines TNF and IL-1 can be included in a growing
number of genes regulated by low O2 tension (reviewed in
Ref. 27). Incubation of human peripheral blood mononuclear cells (28)
and monocytic cell lines (29) under hypoxic conditions have increased
TNF and IL-1ß synthesis, and hypoxic endothelial cells in culture
produce IL-1 (30) and IL-6 (31). Further, cerebral ischemia produced
by carotid constrictures in rats leads to enhanced TNF and IL-1
gene expression (32), and placement of humans in hypobaric hypoxia
leads to increased monocyte number and cytokine release (33). In
addition to enhanced secretion of inflammatory cytokines, low
O2 tension also increases receptors for TNF and IL-1 on
leukocytes (34). In the present study, the production of TNF and
IL-1 by villous explants was likewise increased by hypoxia, whereas
reduced O2 failed to increase IL-6 production. However, as
IL-6 levels were 3 orders of magnitude greater than those for the other
inflammatory cytokines under normoxic conditions, it may be that
trophoblast IL-6 production is already maximally activated.
Although several genes have been identified to be regulated by
O2 tension, the mechanisms by which O2 levels
are sensed by mammalian cells and the subsequent signaling pathways
have not been completely elucidated. The classical gene to be induced
by hypoxia is erythropoietin (EPO), and this hormone, which was
recently found to be expressed by the human placenta (35), has served
as a model for the study of O2-sensing pathways (14;
reviewed in Ref. 27). From those studies, it has been hypothesized that
the O2 sensor is a heme-containing protein, because the
induction of conformational changes in the heme protein to mimic a
deoxygenated state, such as the irreversible displacement of the iron
center by cobalt chloride or the removal of the iron center by
chelation can elicit EPO production (14). In the present study,
treatment of villous explants with either CoCl2 or the iron
chelator Tiron markedly enhanced the production of inflammatory
cytokines and thus mimicked the hypoxic response. However, the
magnitude of this stimulatory effect on TNF and IL-1ß, but not
IL-1, expression was far greater than stimulation by hypoxia,
suggesting that additional mechanisms might be activated by
CoCl2 or Tiron treatment. Further evidence in support of
this contention is that incubation with zinc (ZnCl2; 100
µmol/L) increased villous TNF production to the same extent as
CoCl2 treatment (17-fold; n = 3), yet
ZnCl2 did not interact with the O2 sensor to
stimulate EPO synthesis in cultured hepatoma cells (14) (our
unpublished data). As both zinc and cobalt ions are known to regulate
the metallothionein gene via specific metal response elements (MRE)
(36), we searched the TNF, IL-1, IL-1ß, and IL-6 gene sequences
listed in GenBank for possible MRE sequences. This computer-aided
analysis identified a single MRE consensus sequence (CT.CGCCC) (37) in
the human TNF gene 15 bp upstream from the start site, but not in
the IL-1, IL-1ß, or IL-6 sequences screened. Thus, other
mechanisms may be involved in the stimulation of cytokine production by
metals.
In conclusion, the results of this study demonstrate that reduced
O2 stimulates placental production of the inflammatory
cytokines TNF, IL-1, and IL-1ß. Besides its effects on cytokine
synthesis, hypoxic incubation of placental tissues has also been shown
to promote hyperplasia of the villous cytotrophoblast stem cell layer
(24, 38, 39) and to attenuate the programmed expression of the cell
adhesion markers required for appropriate trophoblast invasion (24).
These effects of hypoxia in vitro mimic some of the
pathologies documented in placenta from preeclamptic women (40, 41). If
another consequence of uteroplacental ischemia in vivo is
elevated placental cytokine production, as suggested by this study,
then the chronic overproduction of inflammatory cytokines may be
capable of eliciting detrimental effects on the maternal systemic
endothelium that most likely mediate the disease manifestations of
preeclampsia.
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Acknowledgments |
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Footnotes |
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Received November 11, 1996.
Revised January 3, 1997.
Accepted January 30, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and interleukin-1ß proteins in human
placentas. J Reprod Immunol. 22:257–268.[CrossRef][Medline]
and
IL-1ß in normal human placenta. Lymphokine Cytokine Res. 10:443–448.[Medline]
, and transforming
growth factor-ß expression in endometrium, placenta, and placental
membranes. Am J Obstet Gynecol. 163:1430–1437.[Medline]
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 X. Zhu, L. M. A. Heunks, E. M. M. Versteeg, H. F. M. van der Heijden, L. Ennen, T. H. van Kuppevelt, J. Vina, and P. N. R. Dekhuijzen Hypoxia-induced dysfunction of rat diaphragm: role of peroxynitrite Am J Physiol Lung Cell Mol Physiol, January 1, 2005; 288(1): L16 - L26. [Abstract] [Full Text] [PDF]
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 S. Ahmad and A. Ahmed Elevated Placental Soluble Vascular Endothelial Growth Factor Receptor-1 Inhibits Angiogenesis in Preeclampsia Circ. Res., October 29, 2004; 95(9): 884 - 891. [Abstract] [Full Text] [PDF]
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G. J. Burton and E. Jauniaux Placental Oxidative Stress: From Miscarriage to Preeclampsia Reproductive Sciences, September 1, 2004; 11(6): 342 - 352. [Abstract] [PDF]
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M. A. Turner, S. Vause, and S. L. Greenwood The Regulation of Interleukin-6 Secretion by Prostanoids and Members of the Tumor Necrosis Factor Superfamily in Fresh Villous Fragments of Term Human Placenta Reproductive Sciences, April 1, 2004; 11(3): 141 - 148. [Abstract] [PDF]
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 T.-H. Hung, D. S. Charnock-Jones, J. N. Skepper, and G. J. Burton Secretion of Tumor Necrosis Factor-{alpha} from Human Placental Tissues Induced by Hypoxia-Reoxygenation Causes Endothelial Cell Activation in Vitro: A Potential Mediator of the Inflammatory Response in Preeclampsia Am. J. Pathol., March 1, 2004; 164(3): 1049 - 1061. [Abstract] [Full Text] [PDF]
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S. Bauer, J. Pollheimer, J. Hartmann, P. Husslein, J. D. Aplin, and M. Knofler Tumor Necrosis Factor-{alpha} Inhibits Trophoblast Migration through Elevation of Plasminogen Activator Inhibitor-1 in First-Trimester Villous Explant Cultures J. Clin. Endocrinol. Metab., February 1, 2004; 89(2): 812 - 822. [Abstract] [Full Text] [PDF]
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 M. von Wolff, S. Stieger, K. Lumpp, J. Bucking, T. Strowitzki, and C.J. Thaler Endometrial interleukin-6 in vitro is not regulated directly by female steroid hormones, but by pro-inflammatory cytokines and hypoxia Mol. Hum. Reprod., December 1, 2002; 8(12): 1096 - 1102. [Abstract] [Full Text] [PDF]
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 A. Grosfeld, J. Andre, S. Hauguel-de Mouzon, E. Berra, J. Pouyssegur, and M. Guerre-Millo Hypoxia-inducible Factor 1 Transactivates the Human Leptin Gene Promoter J. Biol. Chem., November 1, 2002; 277(45): 42953 - 42957. [Abstract] [Full Text] [PDF]
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 J. J. Zulueta, R. Sawhney, U. Kayyali, M. Fogel, C. Donaldson, H. Huang, J. J. Lanzillo, and P. M. Hassoun Modulation of Inducible Nitric Oxide Synthase by Hypoxia in Pulmonary Artery Endothelial Cells Am. J. Respir. Cell Mol. Biol., January 1, 2002; 26(1): 22 - 30. [Abstract] [Full Text] [PDF]
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J. R. Mellembakken, P. Aukrust, M. K. Olafsen, T. Ueland, K. Hestdal, and V. Videm Activation of Leukocytes During the Uteroplacental Passage in Preeclampsia Hypertension, January 1, 2002; 39(1): 155 - 160. [Abstract] [Full Text] [PDF]
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D. F. Benyo, A. Smarason, C. W. G. Redman, C. Sims, and K. P. Conrad Expression of Inflammatory Cytokines in Placentas from Women with Preeclampsia J. Clin. Endocrinol. Metab., June 1, 2001; 86(6): 2505 - 2512. [Abstract] [Full Text] [PDF]
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B. T. Alexander, A. N. Rinewalt, K. L. Cockrell, M. B. Massey, W. A. Bennett, and J. P. Granger Endothelin Type A Receptor Blockade Attenuates the Hypertension in Response to Chronic Reductions in Uterine Perfusion Pressure Hypertension, February 1, 2001; 37(2): 485 - 489. [Abstract] [Full Text] [PDF]
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 A. Rajakumar and K. P. Conrad Expression, Ontogeny, and Regulation of Hypoxia-Inducible Transcription Factors in the Human Placenta Biol Reprod, August 1, 2000; 63(2): 559 - 569. [Abstract] [Full Text]
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 N. TAKABATAKE, H. NAKAMURA, S. ABE, S. INOUE, T. HINO, H. SAITO, H. YUKI, S. KATO, and H. TOMOIKE The Relationship between Chronic Hypoxemia and Activation of the Tumor Necrosis Factor-alpha System in Patients with Chronic Obstructive Pulmonary Disease Am. J. Respir. Crit. Care Med., April 1, 2000; 161(4): 1179 - 1184. [Abstract] [Full Text]
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A. Many, C. A. Hubel, S. J. Fisher, J. M. Roberts, and Y. Zhou Invasive Cytotrophoblasts Manifest Evidence of Oxidative Stress in Preeclampsia Am. J. Pathol., January 1, 2000; 156(1): 321 - 331. [Abstract] [Full Text] [PDF]
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 C. A. Hubel Oxidative Stress in the Pathogenesis of Preeclampsia Experimental Biology and Medicine, December 1, 1999; 222(3): 222 - 235. [Abstract] [Full Text]
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S. W. Kauma, V. Bae-Jump, and S. W. Walsh Hepatocyte Growth Factor Stimulates Trophoblast Invasion: A Potential Mechanism for Abnormal Placentation in Preeclampsia J. Clin. Endocrinol. Metab., November 1, 1999; 84(11): 4092 - 4096. [Abstract] [Full Text]
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H. Mise, N. Sagawa, T. Matsumoto, S. Yura, H. Nanno, H. Itoh, T. Mori, H. Masuzaki, K. Hosoda, Y. Ogawa, et al. Augmented Placental Production of Leptin in Preeclampsia: Possible Involvement of Placental Hypoxia J. Clin. Endocrinol. Metab., September 1, 1998; 83(9): 3225 - 3229. [Abstract] [Full Text]
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 C. H. Graham, T. E. Fitzpatrick, and K. R. McCrae Hypoxia Stimulates Urokinase Receptor Expression Through a Heme Protein-Dependent Pathway Blood, May 1, 1998; 91(9): 3300 - 3307. [Abstract] [Full Text] [PDF]
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