Objective
The aim of this study was to investigate a possible association between hydrogen peroxide and human leukocyte antigen G (HLA-G) in preeclampsia.
Study Design
We explored the correlation between HLA-G and hydrogen peroxide in preeclamptic (n = 30) and normotensive (n = 30) placentas, which was confirmed by in vitro experiments. Furthermore, RNA interference was used to explore the influence of HLA-G on the proliferation, apoptosis and invasion of HTR-8/SVneo cells when exposed to hydrogen peroxide.
Results
We found an inverse correlation between hydrogen peroxide and HLA-G expression in preeclamptic placentas. High levels of hydrogen peroxide could down-regulate HLA-G expression in HTR-8/SVneo. Compared with HLA-G knockdown HTR-8/SVneo, increased proliferation inhibition, higher apoptosis, and decreased cell invasion were found in the cell expressing HLA-G when exposed to hydrogen peroxide.
Conclusion
Our findings highlight that high levels of hydrogen peroxide can down-regulate HLA-G expression in trophoblasts during preeclampsia and trophoblasts expressing HLA-G are vulnerable to oxidative stress.
Preeclampsia (PE), a major contributor to maternal and neonatal mortality and morbidity, is a pregnancy-specific syndrome characterized by new-onset hypertension (blood pressure ≥140/90 mm Hg in previously normotensive women after the 20th week of pregnancy) and proteinuria (300 mg/24 hours urine collection).
The precise cause of PE is still open to debate, but it is believed to be multifactorial, caused by a combination of immunologic, environmental and genetic factors. In particular, oxidative stress and immune maladaptation seem to attract people’s attention in the development of PE.
The cause of PE is unclear but oxidative stress seems to play an important role because it can cause widespread endothelial dysfunction and vasospasm. Hydrogen peroxide (H 2 O 2 ), a terminal metabolite of oxidative stress, is an oxidative stress marker and a member of reactive oxygen species (ROS) family. It has been reported that circulating levels of H 2 O 2 are increased in women with PE.
One prominent hypothesis considers that immune maladaptation is also involved in PE. Human leukocyte antigen G (HLA-G) is a major histocompatibility complex (MHC)-derived (nonclassical) class Ib molecule. HLA-G gene encodes 2 isoforms: membrane-bound HLA-G (HLA-G1, HLA-G2, HLA-G3, and HLA-G4) and soluble HLA-G (HLA-G5, HLA-G6, and HLA-G7). The 2 isoforms are both present at the placental interface by trophoblast cells except HLA-G7. Decreased HLA-G expression could induce immune destruction of extravillous trophoblast. The abnormal HLA-G expression may play a pathogenetic role in PE. It has been reported that an aberrant or reduced expression of HLA-G protein was found in preeclamptic compared with control placentas. Another study found that PE group had no difference in the blood serum levels of soluble HLA-G (sHLA-G) in the third trimester but lower sHLA-G levels in the second trimester. Jauniaux et al have suggested that extravillous trophoblast cells expressing HLA-G are more vulnerable to mutations . But this hypothesis has not been experimentally verified.
This study sought to answer the following questions: is there an interaction between H 2 O 2 and HLA-G in PE? Are trophoblast cells expressing HLA-G more susceptible to oxidative stress? This study was the first to simultaneously examine the correlation between H 2 O 2 and HLA-G in normotensive and preeclamptic placentas and to use in vitro experiments to confirm the relationship. To further investigate the effects of HLA-G on H 2 O 2 -induced injury , a knockdown of HLA-G was used, and the functional biology of the human trophoblast-derived cell line HTR-8/SVneo was determined when exposed to H 2 O 2 .
Materials and Methods
Collection of placental samples
The acquisition of placental tissue was approved by the Medical Ethics Review Committee of Nanjing Medical University. All the placenta samples were obtained from the Department of Obstetrics of the First Affiliated Hospital of Nanjing Medical University. Thirty placentas were obtained from nulliparous women with PE who delivered by cesarean section without labor, and 30 placentas were obtained from nulliparous women with normal pregnancies undergoing cesarean section at term without labor (control group) ( Table ).
| Variable | Group | P value a Control vs PE | |
|---|---|---|---|
| Control (n = 30) | PE (n = 30) | ||
| Maternal age, y | 27.5 ± 5.2 | 30.6 ± 7.2 | > .05 |
| Maternal weight, kg | 74.3 ± 4.0 | 79.7 ± 11.2 | > .05 |
| Gestational age, wk | 38.2 ± 2.7 | 35.4 ± 3.2 | > .05 |
| Birthweight, g | 3356 ± 521 | 3102 ± 326 | > .05 |
| Systolic blood pressure, mm Hg | 108 ± 12.3 | 159 ± 21.3 | < .01 |
| Diastolic blood pressure, mm Hg | 69 ± 8.2 | 104 ± 17.5 | < .01 |
a Obtained by 1-way analysis of variance using SPSS 13.0 software (SPSS Inc, Chicago, IL).
PE was diagnosed according to the following standard criteria: the systolic blood pressure was greater than 140 mm Hg and/or the diastolic blood pressure was greater than or equal to 90 mm Hg in 2 successive measurements 4 to 6 hours apart, and urinary protein was greater than 0.3 g/24 hours in a 24-hour urine collection.
Placental samples were collected immediately after extraction of the placenta from the uterus. Three biopsies were taken from the center of the placenta, avoiding areas of infarcts and thrombosis. One biopsy (4 cm 2 ) was immediately fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for immunohistochemistry, another biopsy (1 cm 2 ) was homogenized to collect the fresh tissue supernatant according to a previous protocol and assessed for spontaneous production of H 2 O 2 . The tissue protein supernatant produced from the other biopsy (1 cm 2 ) was stored at −80°C for use in western blot analysis.
H 2 O 2 determination
Placental levels of H 2 O 2 were measured based on the colorimetric H 2 O 2 assay (R&D Systems, Minneapolis, MN). This assay uses a color reagent, which contains xylenol orange dye in an acidic solution with sorbitol and ammonium iron sulfate that reacts with H 2 O 2 to produce a purple color in proportion to the concentration of H 2 O 2 in the sample. Briefly, the fresh placenta tissue supernatant was 64-fold diluted with sample diluents and then added hydrogen peroxide color reagent. After mixed for 10 seconds and incubated for 30 minutes at room temperature, the optical density (OD) of the mixture was measured at 550 nm and compared with a standard curve generated from dilutions of a reference solution of H 2 O 2.
Placental tissue immunohistochemistry
Placental samples were fixed in 4% paraformaldehyde in PBS for 24 hours at 4°C, then washed in PBS and embedded in paraffin at 56°C. Cross-sections (5 μm thick) were cut and prepared for immunohistochemical staining. Then the sections were mounted on glass slides and air-dried at room temperature for 16 hours, followed by 30 minutes at 60°C. After dewaxed in xylene for 30 minutes, the sections were washed with serial concentrations of ethanol (95, 80, 70, and 60%) and PBS. To inhibit endogenous peroxidase activity, the sections were incubated in a solution of 3% H 2 O 2 for 15 minutes, and then washed with PBS and incubated overnight at 4°C with mouse anti-HLA-G (used at a 1/50 dilution, 4H84, sc-21799). After 3 washes in PBS, the reactions were detected with Vectastain Elite ABC Kit (no. PK7200; Vector Laboratories, Burlingame, CA) and visualized by Vector Nova Red substrate kit (no. SK4800; Vector Laboratories), according to the manufacturers’ protocol. Negative control staining was performed without primary antibodies. The immunoreactivity was detected on a light microscope. The staining intensity was recorded as positively stained target cells in each of 4 intensity categories that were recorded as: – (no staining), 1+ (weak but detectable), 2+ (distinct), and 3+ (intense) for HLA-G immunoreactivity.
Cell culture and treatment
The human trophoblast-derived cell line HTR-8/SVneo, purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China), was cultured at 37°C in a 5% CO 2 incubator in 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). HTR-8/SVneo cells were treated with a range of H 2 O 2 (Sigma-Aldrich, St. Louis, MO) concentrations (from 25 to 250 μM) for 48 hours and the viability of cells was assessed by 3-(4, 5-Dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT). We found that H 2 O 2 decreases cell survival in a dose-dependent manner. Cell viability ranged from 82.4% to 25.6%, respectively. In the presence of 175 μM of H 2 O 2 , there were 54.8% + 2.1% (n = 3) viable cells compared with control cells. Therefore, treatment with 175 μM H 2 O 2 was performed in the subsequent experiments to provide a maximum dynamic range for quantifying oxidative stress damage. Then HTR-8/SVneo cells were exposed to 175 μM H 2 O 2 in complete medium to study the effects of oxidative stress on the expression of HLA-G protein and the influence of HLA-G on H 2 O 2 -induced injury to HTR-8/SVneo cells. The expression of HLA-G protein and the functional biology of HTR-8/SVneo cells were determined.
HLA-G siRNA
The siRNA template insert for HLA-G (5′-TCG AGA GAA GAG CTC AGA TTG AAA TTC AAG AGA TTT CAA TCT GAG CTC TTC TTT A-3′) and the control siRNA template insert (5′-TCG AGA TAG AAG AGA CGA GTT AAA TTC AAG AGA TTT AAC TCG TCT CTT CTA TTT A-3′) were chemically synthesized by GenePharma (Shanghai, China). HTR-8/SVneo cells at 70% confluence were transfected using Oligofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, except that the transfection was repeated after 24 hours.
Cell viability assay
The cytotoxic effects of H 2 O 2 were examined using the cell proliferation reagent MTT. The transfected HTR-8/SVneo cells were plated in 96-well plates at 10 4 cells per well with or without H 2 O 2 and each condition had 6 replicate wells. After exposure to 175 μM H 2 O 2 for 24 hours, 48 hours, and 72 hours, 100 μg MTT was added to each well and incubated for approximately 2 hours. The cell medium was removed and the cells were lysed in dimethylsulfoxide (DMSO) to solubilize the purple forman crystals produced by the reduction of MTT in active mitochondria. The absorbance at 492 nm was measured using an enzyme-linked immunosorbent assay plate reader. All data points represent the mean of a minimum of 6 wells. The viability of untreated cells was considered to be 100%. Experiments were performed in triplicate.
Early apoptosis detection assay
Cells were trypsinized, washed with PBS (4°C), and suspended in 500 μL binding buffer (4°C) containing 5 μL Annexin V-PE and 5 μL 7-AAD (eBioscience, San Diego, CA), then incubated for 15 minutes in the dark at room temperature according to the manufacturer’s instructions. The cells were then analyzed by FACScan (Becton Dickinson, Franklin Lakes, NJ). The data were analyzed using FCS Express V3 software (De Novo Software, Thornhill, Canada). Annexin V-PE − and 7-AAD − cells were used as controls. Annexin V-PE + and 7-AAD − cells were designated early apoptotic, and Annexin V-PE + and 7-AAD + cells were designated late apoptotic and necrotic. Experiments were performed in triplicate. In this research, we wanted to observe the effects of HLA-G on H 2 O 2 -induced early apoptosis of HTR-8/SVneo cells.
In vitro invasion assay
Invasion assays were performed using Transwell membranes coated with Matrigel (BD Biosciences, San Jose, CA). The transfected cells (5 × 10 4 ) were plated in the upper chamber and incubated at 37°C in 5% CO 2 humidified air. When attached overnight, the cells were exposed to 175 μM H 2 O 2 for 48 hours. The lower compartment was filled with 20% FBS as a chemoattractant. After a 24-hour incubation, cells remaining in the upper chamber were carefully removed with cotton swabs, and invaded cells were fixed with 3% paraformaldehyde, stained with crystal violet, counted, and photographed with a microscope in 5 independent 10× fields for each well, then the samples were evaluated for the percentages of invading cells out of the total number of viable cells (invasion index). Experiments were performed in triplicate.
Gelatin zymography
Gelatin zymography was performed with the Gelatin zymography kit (GENMED, Shanghai, China). Briefly, after exposure to 175 μM H 2 O 2 in serum-free Dulbecco’s Modified Eagle’s Medium for 48 hours, equal amounts of supernatant from transfected cells were electrophoresed on a 5-10% sodium dodecyl sulfate-polyacrylamide gel containing gelatin (1 mg/mL). The gel was incubated in washing buffer (50 mM Tris-HCl, pH 7.5; 2.5% Triton X-100) for 1 hour and incubated in reaction buffer (50 mM Tris-HCl, pH 7.5, containing 5 mM CaCl 2 , 150 mM NaCl, and 0.02% NaN 3 ) for 36 hours at 37°C, according to the manufacturer’s instructions. Enzyme activity was visualized by staining with 0.05% Coomassie Blue and analyzed using Quantity One software (Bio-Rad, Hercules, CA).
RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was extracted from the transfected HTR-8/SVneo cell line using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Two micrograms of total RNA was reverse-transcribed into cDNA in a total volume of 25 μL using the RT-PCR kit (TaKaRa Biotechnology, Dalian, China). HLA-G was specifically amplified with the following primer pair: 5′-CTG ACC CTG ACC GAG ACC TGG-3′ (forward) and 5′-GTC GCA GCC AAT CAT CCA CTG GAG-3′ (reverse). β-actin was amplified with the following primer pair: 5′-AAG ACC TGT ACG CCA ACA CAG T-3′ (forward) and 5′-AGA AGC ATT TGC GGT GGA CGA T-3′ (reverse), with amplification using the reverse primer serving as an internal control.
Western blot analysis
Proteins were extracted from placental tissues and from the HTR-8/SVneo cell line. To determine HLA-G protein expression levels, soluble proteins were isolated with lysis buffer (137 mM NaCl, 15 mM EGTA, 0.1 mM sodium orthovanadate, 15 mM MgCl 2 , 0.1% Triton X-100, 25 mM MOPS, 100 μM phenylmethylsulfonyl fluoride, and 20 μM leupeptin, adjusted to pH 7.2). A total protein/lane of 50 μg was separated by 12% sodium dodecyl sulfate-polyacrylamide and transferred to a polyvinylidene difluoride membrane, then subjected to immunoblot analysis with mouse anti-HLA-G (used at a 1/200 dilution, 4H84, sc-21799). Antigen-antibody reactions were detected and visualized with enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL). To check for equal loading of the gel, glyceraldehyde phosphate dehydrogenase (1/2000, sc-47724) was reprobed on the same membrane after washing, as an internal control. The intensity of HLA-G protein bands was quantified by Quantity One software and normalized with glyceraldehyde phosphate dehydrogenase protein.
Cell immunofluorescence
Cells were fixed in phosphate buffer containing 0.02% sodium azide, 2% glucose, and 2% paraformaldehyde; permeabilized in 100% methanol; and blocked in 10% normal goat serum. Cells were then incubated in mouse anti-HLA-G (used at a 1/50 dilution, 4H84, sc-21799), washed in PBS and incubated in goat-anti-mouse antibody (used at a 1/200 dilution, sc-3798). Images were obtained using a fluorescence microscope.
Statistical analysis
All statistical analysis was performed using SPSS Graduate Pack 13.0 statistical software (SPSS, Chicago, IL). Data are presented as mean ± standard deviation. P < .05 was considered statistically significant.
Results
The correlation between HLA-G and H 2 O 2 levels in preeclamptic placentas
There were no significant differences in maternal age, weight, infant’s gestational age, and birthweight between the 2 matched patient groups. The clinical characteristics of the study groups are shown in the Table .
After adjusted to total protein concentration, levels of H 2 O 2 were higher in preeclamptic placentas than in normotensive placentas (102.3 ± 16.4 vs 58.2 ± 16.6 nmol/mg· protein; P < .05) ( Figure 1 , B).
The placenta immunohistochemistry studies showed that HLA-G expression was restricted in trophoblast cells. In general, the immunohistochemical staining was weaker in the preeclampsia biopsies compared with normotensive placentas ( Figure 1 , A).
The relative expression of HLA-G protein in preeclamptic placentas was lower than in normotensive placentas (0.23 ± 0.07 vs 1.13 ± 0.58; P < .05) ( Figure 1 , C).
Finally, there was an inverse correlation between HLA-G and H 2 O 2 levels in preeclamptic placentas and the correlation coefficient was r1 = − 0.52, P < .05 ( Figure 1 , D-1). We also found an inverse correlation between HLA-G and H 2 O 2 levels in normotensive placentas and the correlation coefficient was r2 = −0.79, P < .05 ( Figure 1 , D-2).
Expression of HLA-G protein in HTR-8/SVneo cells exposed to H 2 O 2
To study the effects of H 2 O 2 on the in vitro expression of HLA-G in trophoblast cells, 5 experimental replicates were performed (n = 5). Compared with the relative expression of HLA-G protein in the control group (3.21 ± 0.33), the relative expression in HTR-8/SVneo cells after exposure to H 2 O 2 for 24 hours (1.94 ± 0.25) and 48 hours (0.64 ± 0.08) was reduced by 39% ( P < .05) and 80% ( P < .05), respectively. The relative expression of HLA-G protein in HTR-8/SVneo cells after exposure to H 2 O 2 for 48 hours was reduced by 67% ( P < .05) compared with the level after exposure for 24 hours. Cells exposed to H 2 O 2 for 48 hours displayed weaker staining under the fluorescence microscope than did the control group ( Figure 2 ) .
Expression of HLA-G is down-regulated by targeting siRNA in HTR-8/SVneo cells
The siRNA used in the current study, targeting exons 5 and 6, should interfere with the expression of mRNAs from HLA-G1 to HLA-G6, but not HLA-G7, and the primer set used in RT-PCR can recognize all siRNA-influenced isoforms. The anti-HLA-G antibody (4H84) used in this study, reactive with HLA-G heavy chain should recognize all isoforms of HLA-G.
After 48 hours of infection with HLA-G siRNA or control siRNA oligofectamine in HTR-8/SVneo cells, HLA-G mRNAs and proteins were extracted from control siRNA transfected cells, and HLA-G siRNA transfected cells, and then were analyzed by RT-PCR and Western blot. As shown in Figure 3 , HLA-G mRNA and protein in HLA-G siRNA group were nearly down-regulated by 90% compared with the cells infected with the control siRNA oligofectamine.
