Preeclampsia, an idiopathic obstetric disease, is a main cause of maternal and perinatal morbidity and mortality.1,2 It is characterized by the onset of hypertension and is accompanied by proteinuria after 20 weeks of gestation.2 Although the etiology and pathogenesis of preeclampsia remain largely unknown, it is widely accepted that the main mechanisms of preeclampsia, especially early-onset preeclampsia, actually include altered angiogenic balance, immunological intolerance, inflammation/oxidative stress, and placental ischemia/hypoxia as a consequence or cause of inadequate trophoblast invasion and incomplete uterine spiral artery remodeling.3,4
Chemokine, a superfamily of structurally related molecules that can be divided into four subfamilies: the C, CC, CXC, and CX3C subfamily, are recognized as mediators of cell recruitment, angiogenesis, immunity and stem cell trafficking.5,6 Recently, an increasing number of researchers have confirmed that CXC chemokines play a role in the genesis of preeclampsia. The CXC chemokine subfamily can be further divided into two different subgroups by the presence or absence of the amino acid sequence Glu-Leu-Arg (ELR motif).7,8 CXC chemokines that contain the ELR motif (ELR+) are potent inducers of angiogenesis, which achieve by bonding to the receptor CXCR2; in contrast, CXC chemokines that lack the ELR motif (ELR−) are potent antiangiogenic factors and can inhibit neovascularization mediated by ELR+ CXC chemokines.7,8 It has been reported that CXCL8 (ELR+) stimulates trophoblast cell migration and invasion by increasing the levels of matrix metalloproteinases (MMP) 2 and MMP-9.9 In addition, our previous study showed that the placental expression of CXCL3 (ELR+), which was identified in syncytiotrophoblasts and the vascular endothelium is decreased in severe preeclampsia.10 It has been further verified that exogenous CXCL3 is able to promote the proliferation and invasion profile of HTR-8/SVneo cells in vitro.10
However, the role of CXCR2, the common receptor of the chemokine ELR+ CXC, has not been fully researched in preeclampsia.10 Therefore, this study was aimed to determine changes in CXCR2 expression in the preeclampsia placenta and its correlation with clinical parameters.
Materials and methods
Sixty-four gravidas ranging in age from 25 to 42 years referred to the obstetrics unit of the West China Second University Hospital (Chongqing, China), from April 2012 to October 2012 were recruited in this case-control study; the group contained 22 patients with early-onset severe preeclampsia (<34 weeks, early-onset group), 22 patients with late-onset severe preeclampsia (≥34 weeks, late-onset group) and 20 normal term pregnant women (as the healthy group). In the early-onset group, there were 21 cases of preterm delivery and only one case of term delivery; in the late-onset group, there were 10 cases of preterm delivery and 12 cases of term delivery; and in the healthy group, all cases were term deliveries. Inclusion criteria: all the participants delivered by elective cesarean section, with the indications of previous cesarean section, breech presentation, and social indications. Exclusion criteria included vaginal delivery (avoiding potential influence on protein levels), multiple pregnancy, diabetes, heart diseases, chronic hypertension, fetal malformation, chronic nephritis and hemolysis, elevated liver enzymes and low platelet count (HELLP) syndrome. The study was approved by the ethics committee of the West China Second University Hospital, Sichuan University (decision number: 2019 No.010). Informed consent was obtained from all the patients in accordance with the Declaration of Helsinki.
Preeclampsia was defined as blood pressure that measured ≥140/90 mm Hg on two separate occasions 4 hours apart, and the association with proteinuria ≥1+ (via dipstick testing) or proteinuria ≥300 mg per 24 hour after 20 weeks of gestation. Patients with severe preeclampsia were further classified as either having early-onset (<34 weeks) or late-onset (≥34 weeks) disease according to the gestational age at which onset preeclampsia was diagnosed.11
Gestational age was based on the last menstrual period and/or was confirmed by ultrasound examination, which was conducted at an early stage of pregnancy. Blood pressure was measured on the right arm with a mercury sphygmomanometer; subjects were seated, and the measurement was collected after a 5-minutes rest. Three measurements were taken, and the values were averaged. Systolic blood pressure and diastolic blood pressure were recorded as phase I and V Korotkoff sounds.
Human placenta tissues were collected from participants in each group as described previously.12 Approximately 1.0 cm3 biopsy tissues in the center zone (avoiding the vessels and/or calcium deposits) were obtained from both the fetal and the maternal side after delivery as immediately as possible. The tissue specimens were washed for three times in normal saline, and prepared for the placental homogenization and immunohistochemistry (IHC) assays.
Preparation of placental tissues and homogenates
Tissues of human placenta were collected from participants in each group as described previously.12 Placental tissues in the placenta central zone (umbilical cord attached to the contralateral maternal side) of women undergoing elective cesarean section were collected immediately after the operation. The tissue specimens were washed for three times in normal saline, and then were stored at −80°C until used.
Five hundred micrograms of the tissue samples were transferred to a 5 mL polypropylene tube containing 500 μL ice-cold homogenizing buffer with protease inhibitor added (250 mmol/L sucrose, 0.7 μmol/L pepstatin A, 1.6 μmol/L antipain, 80 μmol/L aprotinin, 1 mmol/L ethylene diamine tetraacetic acid, 10 mmol/L Hydroxyethyl piperazine ethanesulfonic acid (HEPES)-Tris (pH = 7.4)). The placental samples were then homogenized with a Powergen homogenizer (Fisher Scientific, Pittsburgh, PA, USA) for 30 seconds on ice. Then the homogenates were centrifuged at 5 000 ×g for 20 minutes at 4°C, and the collected supernatants were stored at −80°C until enzyme-linked immunosorbent assay (ELISA).
IHC was conducted with a biotin-streptavidin-peroxidase kit (Gene Tech, Shanghai, China) according to the manufacturer's instructions. The sections of placental samples were deparaffinized completely, rehydrated in decreasing series of alcohol and subjected to antigen retrieval in citrate buffer (10 mmol/L citrate sodium, 10 mmol/L citric acid, pH = 6.0) in a microwave oven at 92–98°C for 15 minutes followed by washing with 1× phosphate-buffered saline three times at room temperature for 5 minutes each. Sections were then incubated with mouse anti-human CXCR2 monoclonal antibody (1:50, Abcam, Cambridge Science Park in Cambridge, UK), at 4°C overnight. The tissue sections were further incubated with biotinylated antibodies and horseradish peroxidase-labeled streptavidin. The substrate was stained with diaminobenzidine, and counterstained with hematoxylin.
The placental homogenates of early-onset, late-onset and control groups were analyzed by ELISA with commercially available ELISA kits (Uscn Life Science, Wuhan, China). The concentrations of CXCR2 were determined by comparison with the standard curve using a spectrophotometer, which was set at a wavelength of 450 nm (TECAN, Männedorf, Switzerland).
Real-time fluorescent quantitative polymerase chain reaction (PCR)
Total RNA was extracted using a TRNzol-A+ reagent (Tiangen Biotech Co., Beijing, China), and its integrity was verified via agarose gel electrophoresis. Reverse transcription reactions were performed using the ReverTra Ace MMLV reverse transcriptase RnaseH- (Toyobo, Japan). Real-time PCR analyses were performed using Mastercycler epgradient S (Eppendorf, Germany) to determine the number of complementary DNA (cDNA) molecules in the reverse-transcribed samples. PCR was performed using 8.7 μL of 2× Maxima SYBR Green qPCR Master Mix (Fermentas Inc., Canada), 0.15 μL of each 5′ and 3′ primer, 1 μL of the cDNA samples, and 10 μL of H2O to a final volume of 20 μL. After the samples were denatured at 95°C for 10 minutes, amplification and fluorescence determination were performed in three steps, that is, denaturation at 95°C for 15 seconds, annealing at 60°C for 1 minute, and extension at 72°C for 30 seconds. Moreover, SYBR Green fluorescence was detected at the end of extension, and this value reflects the amount of double-stranded DNA. The amplification cycle number was 40. A melting curve was obtained at the end of each run to discriminate specific from nonspecific cDNA products. The data were normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in the samples. The primer sequences used for real-time PCR were listed respectively as follows: forward: 5′-AGC AGG TCA CAG CTG CTC TT-3′, reverse: 5′-TCT TCA AAG CTG TCA CTC TCC A-3′ for the CXCR2; the primers for GAPDH (internal control) were: forward: 5′-GAA GGT GAA GGT CGG AGT C-3′, reverse: 5′-GAA GAT GGT GAT GGG ATT TC-3′ (synthesized by Beijing DNAchem Biotechnology Co., Ltd., China).
Data were processed by using the SPSS 16.0 software package (IBM, Armonk, NY, USA). Continuous variables were recorded as mean ± standard deviation. Clinical and demographic characteristics of women were compared by one-way analysis of variance or the Wilcoxon Test. The correlation analyses were evaluated by Pearson and Spearman tests of correlation with Bonferroni correction. P < 0.05 (bilateral) was regarded as statistically significant.
Demographic characteristics of the subjects
The demographic characteristics of the subjects are summarized in Table 1. No significant difference was noted in maternal age and body mass index at early pregnancy among the three groups (P > 0.05). Gestational age at delivery and birth weight in the early-onset group were significantly lower than those in the late-onset group and healthy group (P < 0.05), moreover, the difference in gestational age between the late-onset group and healthy group was also significant (P < 0.05).
Localization of CXCR2 in the placenta
The localization of CXCR2 in the placenta was detected by IHC. The immunoreactivity of CXCR2 was observed in the cytoplasm of placental syncytiotrophoblasts and vascular endothelial cells in all groups (Fig. 1).
Placental CXCR2 expression and CXCR2-messenger ribonucleic acid (mRNA) expression
The protein expression levels of CXCR2 in placental homogenates were examined by ELISA. As shown in Figure 2A, the placental CXCR2 protein levels were significantly lower in the early-onset group (38.17 ± 13.04) ng/mL than they were in the late-onset (62.76 ± 16.79) ng/mL and healthy groups (58.72 ± 9.06) ng/mL, P < 0.05. However, the late-onset group (62.76 ± 16.79) ng/mL did not differ from the healthy group (58.72 ± 9.06) ng/mL, P > 0.05. Real-time fluorescent quantitative PCR was performed on placental homogenate to detect the mRNA levels among the groups. mRNA levels were normalized to those of the house-keeping gene GAPDH by the 2-ΔΔCt method.13 By comparison, we concluded that the CXCR2-mRNA expression level in the early-onset group (0.14 ± 0.08) was significantly lower than that of the late-onset group (0.93 ± 1.00) and normal pregnant group (1.10 ± 0.82), P < 0.05. However, there was no significant difference in CXCR2-mRNA levels between the late-onset group and the normal group, P > 0.05 (Fig. 2B).
Correlation analysis between CXCR2 level and clinical parameters
In the early-onset group, the CXCR2 levels in placental homogenates were negatively correlated with systolic blood pressure (r = −0.51, P < 0.05) and the levels of lactate dehydrogenase (LDH) (r = −0.43, P < 0.05) (Figs. 3A, 3B).
It is widely accepted that the pathogenesis of preeclampsia is associated with inadequate trophoblast invasion and failed spiral artery remodeling, resulting in endothelial dysfunction, an exaggerated inflammatory response and angiogenic imbalance.4 Notably, an imbalance of angiogenic and anti-angiogenic factors is considered to be a key event in the genesis of preeclampsia, especially early-onset preeclampsia, which is typically associated with placental dysfunction.3,4,14 For instance, plasma levels of vascular endothelial growth factor (VEGF) and placental growth factor (PLGF), both of which are potent angiogenesis factors, are significantly reduced in women with preeclampsia.14 However, the expression levels of soluble fishlike tyrosine kinase-1, (sFlt-1) a circulating anti-angiogenic protein that acts as an antagonist by binding VEGF and PLGF, appear to increase in women with preeclampsia even before the onset of the disease.14,15 In addition, the ratio of sFlt-1 to PLGF is thought to be a clinical predictor of the early-onset preeclampsia .14,15
Interestingly, the subgroups of the CXC chemokine family behave in a disparate manner in the regulation of angiogenesis. Generally, CXC chemokines such as CXCL1, 2, 3, 5, 6, 7, and 8, which contain the ELR motif (ELR+), are potent inducers of angiogenesis.7,8,16 By contrast, CXC chemokines that lack the ELR motif (ELR−), such as CXCL4, 9, 10, 11, and 12, are antiangiogenic factors that may inhibit the neovascularization induced by the ELR+ CXC chemokines.7,8 Although, ELR+ CXC chemokines signal through CXCR1 and CXCR2 in humans, the latter one is regarded as the primary receptor of angiogenesis and can bind to all ELR+ CXC chemokines, whereas only CXCL8 and CXCL6 bind to CXCR1.16 CXCR2 has been demonstrated to be expressed by endothelial cells and is required for endothelial cell chemotaxis.16,17 The present study confirmed that CXCR2 was present in syncytiotrophoblasts and vascular endothelial cells, indicating that it may perform a role in placentation.
CXC chemokines and their receptors play important regulatory roles in the migration, invasion, and metastasis of tumor cells in a variety of cancers, such as melanoma, colon, breast and gastric cancer.18–20 In gastric cancer tissue, increased expression of CXCR2 is associated with tumor progression, and advanced stages.18 In addition, CXCR2 may contribute to the progression of gastric cancer via the Ras and STAT3 pathways.18 Trophoblasts are known as pseudotumor cells that share some features with tumor cells but are strictly temporally and spatially controlled.21 Therefore, the role of CXC chemokines in the process of trophoblast invasion is being increasingly investigated. CXCL10 (ELR−) is expressed in vascular endothelial and vascular smooth muscle cells in the placenta and extravillous trophoblast-induced CXCL10 expression contributes to the reshaping of the spiral arteries by altering the motility and differentiation status of vascular smooth muscle cells in blood vessels. Regarding ELR+ CXC chemokines, exogenous CXCL3 can promote the proliferation and invasion of HTR-8/Svneo cells in vitro, and CXCL8 can stimulate trophoblast cell migration and invasion by increasing the expression levels of MMP-2 and MMP-9. The present study confirmed that the expression of CXCR2 protein, as well as mRNA expression, was decreased in early-onset preeclampsia, indicating that the process of trophoblast invasion that is regulated by ELR+ CXC chemokines may be blocked by the downregulation of CXCR2, although the exact mechanism remains unclear.
Correlations between placental CXCR2 expression and clinical indicators were observed in early-onset preeclampsia, nonetheless, these correlations were not found in either the late-onset group or the healthy pregnancy group. This may be because that the early-onset preeclampsia is a placental disease, whereas the late-onset form is considered a maternal disorder that is the result of an underlying maternal constitutional disorder.3
In short, significant abnormalities in placental CXCR2 expression in early-onset preeclampsia, and correlations between placental CXCR2 protein expression in early-onset preeclampsia and some clinical parameters of early-onset severe preeclampsia were discovered in our study, suggesting that CXCR2 may play a role in the pathogenesis of early-onset preeclampsia.
This work was supported by the National Natural Science Foundation of China (No. 81571465, No. 81871175).
Conceived and designed the experiments: Rong Zhou and Li Li. Performed the experiments: Xijing Liu and Yanping Zhang. Analyzed the data: Xijing Liu, Yanping Zhang and Jin Jia. Contributed reagents/materials/analysis tools: Tao Wang. Wrote the paper: Yanping Zhang and Xijing Liu.
Conflicts of Interest
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