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HYPERTENSION/2005/057463R2
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Generation of reactive oxygen species by neutrophils and endothelial cell injury in
normal and preeclamptic pregnancies
Kiyomi Tsukimori1, Kotaro Fukushima2, Akitoshi Tsushima1, and Hitoo Nakano1
1
Department of Obstetrics and Gynecology, Graduate School of Medical Sciences,
Kyushu University
2
Maternity and Perinatal Care Unit, Kyushu University Hospital, Fukuoka, Japan
Address for correspondence and reprint requests:
Kiyomi Tsukimori, M.D., Ph.D.
Department of Obstetrics and Gynecology, Graduate School of Medical Sciences,
Kyushu University
Maidashi 3-1-1, Higashi-ku, Fukuoka 812, Japan
Phone: +81-92-642-5395
Fax:
+81-92-642-5414
E-mail:
[email protected]
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HYPERTENSION/2005/057463R2
Expanded Materials and Methods
Chemicals
The chemicals required for the study were collagenase, penicillin, streptomycin,
N-formyl-methionyl-leucyl-phenylalanine (FMLP), ferricytochrome c, superoxide
dismutase (SOD), catalase, and Triton X-100 (all from Sigma Chemical Co., St. Louis,
MO, USA), RPMI 1640, fetal calf serum, and L-glutamine (GIBCO, Grand Island, NY,
USA), sulfanilamide and naphthyl ethylenediamine (Wako Chemical Co., Osaka, Japan),
and NG-nitro-L-arginine methyl ester (L-NAME) (Funakoshi Lab. Co., Tokyo, Japan).
Isolation of human umbilical vein endothelial cells (HUVEC)
The isolation and culture of human umbilical endothelial cells (HUVEC) was performed
according to a modified method of Jaffe et al.1 Human umbilical cords were treated with
collagenase. Harvested cells were suspended in RPMI 1640 medium supplemented with
20% fetal calf serum, penicillin, 200 U/mL, streptomycin, 200 ng/mL, and L-glutamine
and cultured in a 5% CO2 incubator at 37oC. The cultured endothelial cells were
prepared for transmission electron microscopy and immunofluorescence staining for
Factor VIII. The experiments were performed when the cells had reached confluence
after a second pass of the culture medium.
Preparation of neutrophils
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We collected blood early in the morning after at least 12 hours of fasting in order to
avoid the influence of dietary nitrate. A 25–30 mL heparinized blood sample was
obtained once from each subject, and neutrophils were isolated according to the
following procedure: dextran sedimentation, hypotonic lysis of erythrocytes, and the
Conray-Ficoll method.2 The cell population used for subsequent analysis had more than
98% neutrophils and less than 2% collectively of lymphocytes, monocytes, and
eosinophils.
Variable measurements
1. Superoxide measurement
Neutrophils (1×106 cells/mL) were suspended in a HEPES buffer solution containing
131 mmol/L NaCl, 4.7 mmol/L KCl, 1 mmol/L CaCl2, 5 mmol/L glucose, and 20
mmol/L HEPES (pH 7.4) for 5 min at 37 oC prior to the addition of FMLP (10-7 mol/L).
Release of O2- was measured by determining the rate of SOD-inhibitable reduction of
ferricytochrome c at 550–540 nm by means of a dual-wavelength spectrophotometer
(model Hitachi 557, Hitachi Co., Ibaragi, Japan).2 The mean O2- value for each subject
obtained from five independent measurements was used.
2. Nitrite measurement
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HYPERTENSION/2005/057463R2
Nitrite release by neutrophils was determined by accumulation of a stable nitrite end
product in cell-free culture supernatants using a modified Griess reaction method.3
Neutrophils were seeded at a density of 2×107 cells/mL in 24-well culture plates (Sigma
Chemical Co., St. Louis, MO, USA) in RPMI 1640 medium containing 1 mmol/L
L-arginine and supplemented with or without 10 mmol/L of the NO synthase inhibitor
L-NAME. Briefly, after a 12-hour incubation with 5% CO2 and 95% air at 37oC,
samples were reacted with 1% sulfanilamide and 0.1% naphthyl ethylenediamine at
room temperature for 10 min, after which the nitrite concentration was measured by
absorbance at 540 nm (model 3550 EIA reader, Bio-Rad Lab., Hercules, Calif., USA.),
referring to the standard curve of sodium nitrite (dissolved in culture medium solution)
within a concentration range of 1 μmol/L to 10 μmol/L. Nitrite release was confirmed to
depend on the number of neutrophils within the range of 2×106 to 2×107cells/mL. The
mean nitrite value for each subject obtained from three independent measurements was
used.
3. Measurement of the correlation between superoxide and nitrite production
The reaction between O2- and NO to form peroxynitrite has a very high rate constant. In
a system in which NO and O2- are produced at a constant rate, the amount of O2- or NO
detected can be increased if the other free radical is scavenged. Of the women included
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in Table 1, we selected 8 subjects with preeclampsia, 9 with a normal pregnancy, and 6
non-pregnant women. To evaluate production of reactive oxygen species (ROS) by
neutrophils, we measured O2- release by neutrophils after pretreatment with L-NAME in
RPMI 1640 medium for each group of women. After incubation with 5% CO2 and 95%
air at 37oC for 12 hours, neutrophils were washed three times with HEPES buffer
solution and subjected to O2- production measurement in the manner described above.
To evaluate nitrite production by neutrophils, nitrite release by neutrophils was
also measured in 10 subjects with preeclampsia and 12 subjects with normal pregnancy
(Table 1) in the manner described above after supplementation with O2- scavengers,
SOD, and catalase.
4. Endothelial cell injury assay
Neutrophil-mediated endothelial cell injury was measured according to Hardy et al.4
Briefly, HUVEC were seeded onto a 96-well flat bottom microtiter plate (Falcon,
Lincoln Park, NJ, USA) at a density of 2×104 cells/well. After overnight incubation, the
cells were labeled for 6 hr with 10 μCi/mL [51Cr]Na2CrO4 (Japan Radioisotope
Association), and then washed with culture medium. Suspensions of neutrophils were
added at a concentration of 4×105 cells/well (ratio of 20 neutrophils to 1 endothelial cell).
After 6 hrs, 51Cr release in the supernatant was determined by counting the radioactivity
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of a 0.1 mL aliquot from each well using a gamma counter (Aloka LSC 5100, Aloka,
Tokyo, Japan). Results were expressed as the percentage of
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Cr release calculated by
the following formula: %Cr release = (cpm test - cpm control) / (cpm maximum - cpm
control) × 100 (%). To determine maximum and control release, cells were incubated
with 1% Triton X-100 and control medium alone, respectively. All tests were performed
in triplicate. The intra- and inter-assay coefficients of variation were 6.2% (n=10) and
8.1% (n=10), respectively.
5. Effect of oxygen radical scavengers on endothelial cell injury
Of the women included in Table 1, we selected 6 subjects with preeclampsia, 8 with
normal pregnancy, and 6 non-pregnant women. Under the conditions stated previously
(ratio of 20 neutrophils to 1 endothelial cell), SOD, catalase, or L-NAME was added to
the HUVEC and neutrophil co-culture well before incubation. The amount of
51
Cr
release was measured after 6 hr of incubation.
In this study, more than 95% of neutrophils were proven to be viable when
assessed using the trypan blue dye exclusion method.
Statistical analysis
Results are expressed as mean ± standard deviation (SD). Differences in nitrite
concentration, O2- production, and %Cr release were first statistically evaluated using
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the Kruskal-Wallis H test and then analyzed by means of the Mann-Whitney U test to
compare subgroups.5 Differences in %Cr release when cells were pretreated with or
without free radical scavengers were analyzed by a paired, one-tailed t -test. A p value
less than 0.05 was considered statistically significant.
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References
1. Tsukimori K, Maeda H, Shingu M, Koyanagi T, Nobunaga M, Nakano H. The
possible role of endothelial cells in hypertensive disorders during pregnancy. Obstet
Gynecol. 1992;80:229-233.
2. Tsukimori K, Maeda H, Ishida K, Nagata H, Koyanagi T, Nakano H. The
superoxide generation of neutrophils in normal and preeclamptic pregnancies.
Obstet Gynecol. 1993;81:536–540.
3. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR.
Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Analytical
Biochem. 1982;126:131–138.
4. Hardy MM, Flickinger AG, Riley DP, Weiss RH, Ryan US. Superoxide dismutase
mimetics inhibit neutrophil-mediated human aortic endothelial cell injury in vitro. J
Biol Chem. 1994;269:18535-18540.
5. Glantz SA. Primer of Biostatistics. 5th ed. New York: McGraw-Hill; 2002.
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