Decreased RSPO3 and β-Catenin in Preeclampsia: Correlation with Blood Pressure and Pregnancy Outcomes

Introduction

Preeclampsia (PE) is defined as the sudden onset of high blood pressure after 20 weeks of gestation, along with one other associated symptom of proteinuria, dizziness, or edema. Preeclampisa is regarded as one of the most serious complications of pregnancy, with a prevalence of 3% to 5% [1,2]. The cause of PE is still not fully understood, and there are several proposed hypotheses, including (1) placental or trophoblast ischemia-hypoxia, (2) oxidative stress, (3) immune imbalance, and (4) vascular endothelial injury [3,4]. The commonly accepted pathological changes are underdevelopment of the placenta due to insufficient blood flow and oxygen caused by inadequate remodeling of the arteries in the uterus, as well as various systemic inflammation, damage to the blood vessel lining, and microangiopathy in the placenta. These changes lead to placental insufficiency [5].

The Wnt/β-catenin pathway is crucial in embryonic and fetal development, maintaining tissue homeostasis and regulating various physiological processes, such as immunity, stress response, cellular carcinogenesis, apoptosis, differentiation, and maintenance of cellular morphology and function [6–8]. The Wnt pathways consist of 4 main pathways: the canonical Wnt/β-catenin pathway, non-canonical Wnt/Ca2+ pathway, Wnt/planar cell polarity pathway, and Wnt/protein kinase A pathway [9]. The components of the canonical Wnt/β-catenin pathway include ligands, transmembrane receptors and LDL receptor-associated proteins 5/6, cytoplasmic regulator proteins, adenomatous polyposis coli, axin, glycogen synthase kinase-3β, β-catenin, and nuclear transcription factors family [10]. The canonical pathway has been extensively researched in various diseases, including cervical cancer and liver cancer [11–13]. Furthermore, studies have indicated that the Wnt/β-catenin pathway is linked to miscarriage, as it inhibits the invasion and proliferation of trophoblast cells [14,15]. However, there is limited research on the role of the Wnt/β-catenin pathway in PE.

β-catenin is a multifunctional protein that mediates the Wnt/β-catenin pathway and holds a regulatory role in physiological processes, like embryonic development and implantation, placenta formation, and thrombosis [16–18]. The study by Ortega et al shows that an increased expression of the Wnt/β-catenin pathway in the placenta of patients with chronic venous disease leads to abnormal inflammation and a stressful environment [19]. Chen et al found that autophagy defects in the uterine vascular micro-environment cause hyperpermeability through dysregulation of b-catenin [20].

RSPO3 is an activator of the Wnt/β-catenin pathway. It enhances the pathway by working together with Wnt ligands, and it is one of the 4 secreted proteins in the RSPO protein family [21]. RSPO1 has been observed to be increased in ovarian cancer [22]. Over-expression of RSPO2 in the liver, along with deletion of transformation-associated protein 53 (Trp 53), leads to reduced expression of the Wnt/β-catenin pathway and promotes liver tumor development [23]. RSPO3 has been linked to endothelial vascular remodeling, and experiments with mice have shown that inducing endothelial RSPO3 deficiency (RSPO3-iecko) results in vascular defects [24]. In human endothelial cells, RSPO3 can directly regulate endothelial cells and promote new angiogenesis [25]. The cause of PE is still unknown, and some studies have shown that certain factors, like soluble fibrillar tyrosine kinase 1 and placental growth factor, can predict pre-eclampsia [26,27]. However, they have low sensitivity and positive predictive value. Further research is needed to identify more accurate predictive factors for PE.

The role and expression of RSPO3 and β-catenin in PE have not been extensively studied. In this research, our goal was to examine the RSPO3 and β-catenin levels in patients with PE and assess their influence on maternal and fetal outcomes.

Material and Methods

ETHICS APPROVAL:

The study was approved by the Medical Research Ethics Committee of Yongchuan Hospital, Chongqing Medical University (approval number: 2021 Research Ethics Review No. 192), and complied with the ethical standards for medical research involving human participants, such as the Ethical Review Measures for Biomedical Research Involving Human Beings, Helsinki Declaration, and International Ethical Guidelines for Research Related to Human Health. All patients provided written informed consent.

STUDY DESIGN AND STUDY PARTICIPANTS:

From November 2022 to April 2024, sixty patients with PE were diagnosed and treated at Chongqing Yongchuan Hospital. The inclusion criteria were: (1) meeting the diagnostic criteria for PE, (2) singleton pregnancy, and (3) no history of primary hypertension. The exclusion criteria were the following: (1) other pregnancies with combined medical and surgical diseases, such as cardiovascular and cerebrovascular diseases, (2) combined infectious diseases, such as HIV and syphilis, (3) combined diabetic pregnancies, and (4) incomplete maternity information during pregnancy.

Meanwhile, 60 pregnant women without PE were randomly selected as the control group. Exclusion criteria for all participants were as follows: (1) multiple pregnancies, (2) primary hypertension, kidney disease, and diabetes mellitus, (3) chronic infectious diseases and autoimmune diseases, and (4) therapeutic history of assisted reproductive technology. The diagnostic criteria of PE were by the guidelines [28].

EXPERIMENTAL REAGENTS:

The reagents used in the study were as follows: RevertAid first-strand cDNA synthesis kit (Sangon B300538-0100, Shanghai); 2xSG Fast qPCR premix (Sangon B639271-0005, Shanghai); RSPO3 primer, GAPDH primer, β-catenin primer (Sangon, Shanghai); phosphate-buffered saline (PBS; Beyotime C0221A Shanghai); bicinchoninic acid (BCA) protein quantification detection kit (Beyotime P0010 Shanghai); diethyl pyrocarbonate water (Beyotime R0022, Shanghai); DAB color development kit (ZSBG-BIO ZLI9810, China); immunohistochemistry rabbit antibody kit (ZSBG-BIO PV-9001, China); ethylenediaminetetraacetic acid (EDTA) antigen repair solution (Solarbio C1034, China); tris-buffered saline (Solarbio T1083, China); Tween-20 (Solarbio T8220, China); 5% goat blood (Solarbio SL038, China); hematoxylin staining fluid (Solarbio BP-DL007, China); Trizol (Thermofisher 15596026, USA); rabbit polyclonal antibody RSPO3 (Proteintech 17193-1-AP, USA); rabbit polyclonal antibody β-catenin (Proteintech 51067-2-AP, USA); horseradish peroxidase-labeled goat anti-rabbit IgG (Earthox E030120, USA); β-actin antibody (AF7018 Affinity, China); RAPI buffer (Topscience C0045, China); chemiluminuteescence kit (4A Biotech 4AW011, China); human β-catenin enzyme-linked immunosorbent assay (ELISA) kit (JingMei Biotechnology JM-5216H1-96, China); and human RSPO3 ELISA kit (JingMei Biotechnology JM-5520H1-96, China).

PATIENT TISSUE SAMPLES:

Basic information about the patients was collected upon admission, including age, height, weight, and pre-pregnancy body mass index (BMI), derived from data recorded in the maternity book. At the same time, the patients’ peripheral blood was collected, and a routine ultrasound was performed. Routine blood tests, liver function, renal function, and coagulation function tests were performed. Blood pressure was measured after 30 min of quiet rest. Umbilical cord blood was collected within 2 min after delivery of the fetus, and fetal information, such as birth weight, birth length, fetal outcome, and gestational age at delivery, were collected. All blood specimens were left for 30 min and centrifuged at 4000 rpm for 10 min, and plasma was collected and stored at −80 °C. Placental tissue was collected within 30 min after fetal delivery and then washed with PBS; one part was fixed with 4% formaldehyde for immunohistochemical staining, and the other part was stored at −80°C for quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis.

ENZYME-LINKED IMMUNOSORBENT ASSAY:

RSPO3 and β-catenin in peripheral blood and umbilical cord blood were measured by commercial kits according to the manufacturer’s instructions. First, 50 uL of standard and 40 uL of blood sample were added to the standard and sample wells, respectively. Then, 10 uL of sample diluent buffer was added to the sample well. Next, 100 uL of horseradish peroxidase-labeled detection antibody was added to each well. The plate was incubated at 37°C for 1 h away from light. The plates were washed 1 min with a washing buffer for 5 times. Then a stop solution was added to all the wells. Within 15 min after the color changed, the optical density values were measured with a microplate reader at a wavelength of 450 nm.

IMMUNOHISTOCHEMISTRY STAINING:

RSPO3 and β-catenin in the placenta were evaluated by immunohistochemistry staining. The placenta was fixed with 4% paraformaldehyde and was then paraffin-embedded and cut into 5-μm-thick sections. Sections were deparaffinized and hydrated in graded ethanol, followed by EDTA antigen repair solution. Endogenous peroxidase activity was reduced with an endogenous peroxidase blocker for 10 min. Sections were then incubated with 5% goat blood for 1 h at room temperature, followed by dropwise addition of primary antibodies (RSPO3 17193-1-AP 1: 400; β-catenin 51067-2-AP 1: 1000) at 4°C overnight. The next day, secondary antibody was added at room temperature for 30 min. Then, the sections were stained with DAB and counterstained with hematoxylin. Sections were dehydrated and dried, fixed with neutral gel, and examined by microscope.

WESTERN BLOT ANALYSIS:

Placental tissues were ground on ice, and total proteins were isolated from placental tissues using RAPI buffer. Protein concentration was determined using an enhanced BCA protein assay kit. The protein was heated at 100°C for 10 min to denature the protein for preparing the sampling protein. Proteins were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis and then transferred to the polyvinylidene fluoride membrane. The membrane was blocked with 5% milk for 1 h at room temperature, and then incubated with primary antibodies against the target proteins (RSPO3 17193-1-AP 1: 1000; β-catenin 51067-2-AP 1: 20 000) overnight at 4°C. The next day, secondary antibody (Earthox E030120 1: 20 000) was incubated for 1 h at room temperature. Membranes were visualized using an enhanced chemiluminescence kit and detected with a gel imaging system. Image J software was used to analyze the images and calculate the relative expression of target proteins. Statistical analysis was done after fold-change of the gray values, using GAPDH as an internal reference.

QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION:

The placenta was dissected and then quickly frozen at −80°C. Total RNA was extracted from placenta by using a Trizol reagent. To separate the chloroform phase, ice-cold isopropanol was used to precipitate the RNA, and then the RNA precipitates were washed with 75% ethanol. Centrifugation was at 12 000 rpm for 15 min at 4°C, and RNA was resuspended in 20 uL diethyl pyrocarbonate-treated water. In quantifying RNA concentration with Nanodrop, RNA was reverse-transcribed to cDNA by using a RevertAid first-strand cDNA synthesis kit. PCR reaction (final volume of 20 μL) was performed at 42°C for 60 min and 70°C for 5 min and then kept at 4°C. cDNA were used for qRT-PCR by using 2xSG Fast qPCR premix (final volume of 20 μL), which was repeated twice for each sample. BioRad CFX96 real-time PCR system was used for amplification. GAPDH was used for normalization, and its relative expression was analyzed using the 2−ΔΔCT method, then the data was displayed in fold-change. The primers used are shown in Table 1.

STATISTICAL ANALYSIS:

The statistical analysis for this study was conducted by using SPSS version 27.0. To compare groups, we used a 2-sample independent t test. The data were processed using GraphPad Prism 9.0, and we verified normal distribution using the Shapiro-Wilk test. For normally distributed measurements, expressed as mean±standard deviation, independent samples t test were used for pairwise comparisons. For non-normally distributed measurement data, we used quartiles and the Kruskal-Wallis test for pairwise comparison. Count data are presented as the number of cases. Correlations were conducted using Spearman analysis. The risk factors for PE were analyzed using univariate and multivariate logistic regression analyses. Statistically significant differences were considered when P<0.05.

Results

CLINICAL BASELINE CHARACTERISTICS:

The clinical and demographic characteristics of the study group are summarized in Table 2. There were no significant differences in age, BMI at delivery, weight gain, hemoglobin level, platelet count, non-bound bile acids, and aspartate aminotransferase level between the PE and control groups (all P>0.05). Maximum systolic blood pressure (SBP), maximum diastolic blood pressure (DBP), gestational age at delivery, pre-pregnancy BMI, uric acid level, creatinine level, total bile acid level, albumin level, alanine transaminase level, fetal umbilical artery blood flow parameters (S/D), and blood flow resistance index in the fetal umbilical artery (RI) were significantly different (all P<0.05).

PATIENTS WITH PE HAD MORE ADVERSE PREGNANCY OUTCOMES:

We compared adverse pregnancy outcomes between the control and PE groups. We discovered that patients with PE had a significantly higher likelihood of experiencing adverse pregnancy outcomes, such as cesarean delivery, Neonatal Intensive Care Unit (NICU) admission, low birth weight, stillbirth, low birth weight, low birth length, low 1-min Apgar score, and low 5-min Apgar score (all P<0.05, Table 3). There was no significant difference in the 10-min Apgar score (P=0.096, Table 3).

RSPO3 AND β-CATENIN IN MATERNAL AND CORD BLOOD WERE LOWER IN PE GROUP:

We measured RSPO3 and β-catenin levels in maternal blood and cord blood samples of PE and normal pregnancy by ELISA. The experimental results indicated that RSPO3 and β-catenin in maternal blood were significantly lower in the PE group than in the control group (Figure 1A, 1B, *** P<0.001, ** P<0.01). RSPO3 and β-catenin levels in the cord blood were significantly lower in the PE group than in the control group (Figure 1C, 1D, *** P<0.001, * P<0.05).

EXPRESSION OF RSPO3 AND β-CATENIN IN PLACENTA WAS LOWER IN PE GROUP:

The immunohistochemistry staining results indicated that RSPO3 and β-catenin were expressed in the trophoblast cells (Figure 2A–2D). Western blot analysis revealed that RSPO3 and β-catenin were lower in the PE group than in the control group (Figure 3A–3D, * P<0.05, *** P<0.001). Furthermore, qRT-PCR results demonstrated a significantly lower level of RSPO3 and β-catenin mRNA in the placenta of the PE group than in the control group (Figure 4A, 4B, * P<0.05, * P<0.05).

RSPO3 LEVEL IN CORD BLOOD WAS NEGATIVELY CORRELATED WITH SBP AND DBP:

We then conducted Spearman correlation analysis to investigate the relationship between RSPO3 levels in the cord blood and blood pressure of all participants. The results showed a negative correlation between RSPO3 levels and SBP and DBP, with values of SBP (r=−0.3706, P<0.001) and DBP (r=−0.3579, P<0.001; Figure 5A, 5B). Then we further analyzed the correlation of RSPO3 level in the PE group and found that RSPO3 level was also negatively correlated with SBP and DBP, with the values of SBP (r=−0.4654, P<0.001) and DBP (r=−0.4617, P<0.001; Figure 5C, 5D).

RSPO3 LEVEL IN MATERNAL BLOOD WAS NEGATIVELY CORRELATED WITH SBP AND DBP:

We analyzed the correlation of RSPO3 in the maternal blood of all participants, and the results demonstrated a negative correlation between RSPO3 levels and SBP and DBP, with values of SBP (r=−0.3513, P<0.05) and DBP (r=−0.3618, P<0.05; Figure 6A, 6B). We further analyzed the correlation of RSPO3 levels in the PE group and found that RSPO3 level was negatively correlated with SBP and DBP, with values of SBP (r=−0.5373, P<0.05) and DBP (r=−0.4898, P<0.05; Figure 6C, 6D).

FACTORS LEADING TO THE DEVELOPMENT OF PREECLAMPSIA:

The logistic regression analyses revealed that RSPO3 levels in maternal blood and uric acid were associated with OR of 0.983 (0.968–0.999), P=0.034, and 1.020 (1.003–1.038), P=0.023, respectively (Table 4). Furthermore, RSPO3 level in cord blood, uric acid, creatinine, and albumin were associated with OR of 0.985 (0.976–0.994), P<0.001; 1.019 (1.009–1.029), P<0.001; 1.139 (1.052–1.233), P=0.001; and 0.696 (0.569–0.850), P<0.001, respectively (Table 5).

Discussion

Previous studies have established that PE can lead to adverse outcomes [29], for example, stillbirth, low birthweight, and preterm birth. In our study, we observed that PE leads to adverse pregnancy outcomes, such as a higher chance of cesarean delivery, NICU admissions, low birth weight babies, and stillbirth. Furthermore, we noted that fetal length, 1-min Apgar scores, and 5-min Apgar scores were lower in the PE group than in the control group. Those results align with previous findings [30,31]. We observed higher levels of uric acid, creatinine, total bile acids, and alanine transaminase levels in the PE group than in the control group. We also observed elevated S/D and RI values in patients with PE, which we attribute to the decreased invasive capacity of trophoblast cells, resulting in insufficient remodeling of the spiral arteries, narrowing of the lumen, and increased pressure [32].

In PE, impaired remodeling of the spiral arteries in the uterus is a crucial factor in the development of PE. Huang et al suggested that the Wnt/β-catenin pathway plays a vital role in angiogenesis [33]. Therefore, placental angiogenesis is crucial for embryonic development. RSPO3 promotes the growth and development of human endothelial cells and may promote placental vascular remodeling through activation of the Wnt pathway [34]. Based on these results, we further explored the RSPO3 and β-catenin expression in PE. Our research indicated that the RSPO3 and β-catenin levels were lower in the maternal blood and cord blood in PE. Ueland et al found elevated levels of RSPO3, which could have been caused by the dysregulation of the Wnt pathway, resulting from the activation and interaction of Wnt signaling factors. This can inhibit the canonical Wnt pathway and subsequently activate noncanonical Wnt signaling to enhance Wnt signaling. As a result, a compensatory elevation in RSPO3 improves impaired vascular remodeling and helps improve placental development [35]. In the present study, most of the patients had developed the disease, and the vascular remodeling disorder had already occurred and was in a state of decompensation. Furthermore, the level of RSPO3 can fluctuate with gestational age. In the study by Tayyar et al, RSPO3 expression was increased in early-onset PE but was decreased in late-onset PE [36]. Most of the included patients were in the third trimester, leading to a decrease in RSPO3 level. However, further study is needed to investigate the changes in the expression level of RSPO3. Our results indicated β-catenin expression was lower in PE, which aligns with the results of previous studies [37,38].

The maternal-fetal interface refers to the direct contact between the mother and the fetus. The placenta holds a vital role in facilitating this interface. Remodeling of the uterine spiral artery is closely associated with trophoblast invasion and is a vital factor in creating the maternal-fetal interface. The placenta has the ability to exchange gases, nutrients, and metabolites, and it also produces various hormones and growth factors that are essential for supporting normal fetal development and maintaining a healthy pregnancy [39]. Based on our experimental results, we further explored the effects of RSPO3 and β-catenin at the maternal-fetal interface. We looked into the localization and differences in the levels of RSPO3 and β-catenin in the placentas of patients with PE. The results revealed that both RSPO3 and β-catenin were present in trophoblasts, and their expression was reduced. Our findings indicated that RSPO3 and β-catenin decreases from maternal to fetal circulation. The Wnt pathway is abnormally activated in PE, and its expression can impact placental trophoblast invasion [40]. In the mouse experiment by Aoki et al, RSPO3 affected the development of the mouse placenta by affecting the formation of blood vessels. This can result in the embryonic blood vessels failing to penetrate the chorion, ultimately leading to embryonic death [41]. Zhang et al demonstrated that β-catenin and Dickkopf-1 in the Wnt pathway were decreased in severe PE, leading to abnormal activation of the Wnt pathway and contributing to the development of PE [42]. Consequently, we hypothesized that reduced RSPO3 expression can hinder the proper remodeling of uterine spiral arteries. Simultaneously, this could lead to abnormal expression of the Wnt pathway, affecting the invasive functions of trophoblast cells. Consequently, this can result in placental dysfunction, ultimately leading to PE.

Based on the results presented above, we examined the relationship between clinical characteristics and RSPO3 and β-catenin levels. Our findings indicated that the expression of RSPO3 was negatively associated with SBP and DBP. Opichka et al and Staff et al investigated the development of PE in terms of vascular dysfunction and trophoblasts [43,44]. From this, we believe that insufficient expression of angiogenic factors results in impaired remodeling of the placental blood vessels. This leads to inadequate invasion of trophoblasts, which in turn causes placental dysfunction and triggers the release of inflammatory factors from the placenta. Ultimately, this cascade of events contributes to the development of clinical symptoms, such as elevated blood pressure. While no correlation was found with β-catenin, we suspect that RSPO3 may play more of a role in promoting the development of PE. To explore whether RSPO3 could contribute to PE, we performed logistic regression analysis, which showed different results due to the varying sample sizes of umbilical cord blood and maternal blood. Furthermore, we discovered that uric acid level was statistically significant in the regression analyses. In 1988, Voto et al found that uric acid levels were a good indicator of the severity of preeclampsia and assessment of perinatal prognosis [45]. Other studies have also revealed that uric acid can serve as a predictor for the development and severity of PE [46,47]. Our findings are consistent with previous studies indicating that uric acid levels are elevated in PE. This elevation is statistically significant, suggesting an important role for uric acid in PE. Additionally, Perni et al found that combining angiogenic factors with uric acid improved the accuracy of predicting preeclampsia [48]. We were not able to create a combined predictive model of RSPO3 and uric acid, due to sample size limitations. However, this will be the focus of our future research. Our results revealed that RSPO3 could be a risk factor for developing PE. Therefore, we propose that RSPO3 can serve as a potential predictor for PE in the future. Additionally, RSPO3 and β-catenin are part of the Wnt/β-catenin pathway, and reduced expression of RSPO3 might lead to decreased activity of the pathway, which could affect trophoblast invasion and the remodeling of uterine spiral arteries, resulting in PE. Hence, we propose that RSPO3 could be a promising target for regulating the Wnt/β-catenin pathway to modulate the development of PE.

This study had limitations. First, the case-control design did not account for certain predictive variables of preeclampsia, such as pre-pregnancy BMI, nulliparity, aspirin use, and calcium supplements. Second, the limited sample size of this single-center study prevented the adjustment for other clinical variables predictive of preeclampsia in the model. Third, our findings have not been utilized in clinical settings to validate their predictive effects. Therefore, further studies are necessary to confirm these effects. Lastly, for establishing prediction, this type of cross-sectional design is not ideal, as it does not evaluate causality.

Conclusions

In this study, we investigated the expression of RSPO3 and β-catenin in patients with normal pregnancy and in patients with PE. The results showed that RSPO3 levels were negatively correlated with SBP and DBP. Logistic regression indicated that RSPO3 level could be an independent factor to develop PE, suggesting its potential as a clinical indicator for PE. However, due to the limited sample size, short data collection period, and single-center nature of the study, our research is affected by selection bias, information bias, and confounding bias. Thus, it necessitates the implementation of prospective multi-center studies. We measured only RSPO3 and β-catenin levels, indicating that our results do not offer direct or fundamental insights into basic pathophysiology. Therefore, it is necessary to conduct additional molecular biology studies, including the exploration of related ligands of the Wnt canonical pathway. This will enhance the credibility of the results and help assess their diagnostic and prognostic value in early-onset PE.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author (XL) upon reasonable request.