Abstract

Background

   Pre-eclampsia is a pregnancy-related disorder characterized by
   hypertension and proteinuria, severely affecting the health and quality
   of life of patients. However, the molecular mechanism of macrophages in
   pre-eclampsia is not well understood.

Methods

   In this study, the key biomarkers during the development of
   pre-eclampsia were identified using bioinformatics analysis. The
   [36]GSE75010 and [37]GSE74341 datasets from the GEO database were
   obtained and merged for differential analysis. A weighted gene
   co-expression network analysis (WGCNA) was constructed based on
   macrophage content, and machine learning methods were employed to
   identify key genes. Immunoinfiltration analysis completed by the
   CIBERSORT method, R package “ClusterProfiler” to explore functional
   enrichment of these intersection genes, and potential drug predictions
   were conducted using the CMap database. Lastly, independent analysis of
   protein levels, localization, and quantitative analysis was performed
   on placental tissues collected from both preeclampsia patients and
   healthy control groups.

Results

   We identified 70 differentially expressed NETs genes and found 367
   macrophage-related genes through WGCNA analysis. Machine learning
   identified three key genes: FNBP1L, NMUR1, and PP14571. These three key
   genes were significantly associated with immune cell content and
   enriched in multiple signaling pathways. Specifically, these genes were
   upregulated in PE patients. These findings establish the expression
   patterns of three key genes associated with M2 macrophage infiltration,
   providing potential targets for understanding the pathogenesis and
   treatment of PE. Additionally, CMap results suggested four potential
   drugs, including Ttnpb, Doxorubicin, Tyrphostin AG 825, and
   Tanespimycin, which may have the potential to reverse pre-eclampsia.

Conclusion

   Studying the expression levels of three key genes in pre-eclampsia
   provides valuable insights into the prevention and treatment of this
   condition. We propose that these genes play a crucial role in
   regulating the maternal-fetal immune microenvironment in PE patients,
   and the pathways associated with these genes offer potential avenues
   for exploring the molecular mechanisms underlying preeclampsia and
   identifying therapeutic targets. Additionally, by utilizing the
   Connectivity Map database, we identified drug targets like Ttnpb,
   Doxorubicin, Tyrphostin AG 825, and Tanespimycin as potential clinical
   treatments for preeclampsia.

   Keywords: pre-eclampsia, macrophages, immune infiltration, GSEA, CMAP

1 Background

   Pre-eclampsia (PE) is a common hypertensive disorder occurring
   typically after 20 weeks of pregnancy, characterized primarily by
   elevated blood pressure (>140/90 mmHg, 1 mmHg = 0.133 kPa) and
   proteinuria (>300 mg/L). It is a major cause of morbidity and mortality
   for both the perinatal period and pregnant women and newborns
   worldwide, affecting 2%–8% of pregnancies, particularly in low- and
   middle-income countries ([38]Yang et al., 2013; [39]González-Garrido et
   al., 2017). Epidemiological studies have shown that 5%–7% of pregnant
   women worldwide suffer from this disease, posing a significant threat
   to the life and health of pregnant women and newborns ([40]Kuc et al.,
   2011; [41]Firoz et al., 2022). Therefore, understanding the risk
   factors of PE is crucial for its prevention and treatment.

   As is well known, PE originates from placental dysfunction,
   characterized by fundamental pathophysiological changes such as
   endothelial injury, systemic arterial spasm, inadequate invasion of
   maternal spiral arteries by placental trophoblast cells, and
   alterations in placental development, perfusion, and nutrient transport
   ([42]Founds et al., 2018). However, the exact etiology and pathogenesis
   of PE remain incompletely understood, making it a critical research
   topic in obstetrics. Many studies have shown that the pathogenesis of
   PE may be related to the interaction of various genetic and
   environmental factors ([43]ACOG, 2019; [44]ACOG, 2020; [45]Cunningham,
   2021)For example, the placenta relies heavily on energy provided by
   mitochondria, and mitochondrial damage caused by circulating bioactive
   factors released from the placenta may lead to endothelial dysfunction
   and subsequent maternal hypertension ([46]Xu et al., 2019; [47]Hu and
   Zhang, 2022). Additionally, PE may occur in pregnant women with
   underlying conditions such as hypertension or other high-risk factors
   like multiple pregnancies and advanced maternal age ([48]Gao et al.,
   2020; [49]Saito et al., 2023).

   Systemic vascular dysfunction in the mother forms the basis of the
   clinical features of PE. Severe complications such as HELLP syndrome
   and placental abruption, if left untreated, can be life-threatening for
   PE patients ([50]Marshall et al., 2018). The only known treatment for
   PE is the delivery of the fetus and placenta ([51]Brown, 2020).
   Maternal-fetal immune tolerance is a complex phenomenon, with semi
   allogeneic fetuses exposed to maternal immune tolerance for up to
   10 months during pregnancy. This crucial immune phenomenon involves
   three maternal-fetal interfaces ([52]Bürk et al., 2001): firstly,
   maternal uterine decidual cells interact with fetal trophoblasts;
   secondly, the decidua is located in the maternal part of the placenta
   and is infiltrated by fetal extravillous trophoblast cells; thirdly,
   the villous chorion is the main component of the placental cells,
   formed by fetal-derived syncytiotrophoblast cells that directly
   interact with maternal blood circulation. Therefore, a healthy mother
   needs to develop immune tolerance to avoid immune attacks on fetal
   tissues while retaining the ability to defend against pathogens. At
   these three interfaces, several mechanisms of immune cell regulation
   have been elucidated ([53]Escribese et al., 2012), and it is known that
   decidual macrophages in the maternal decidua possess innate immune
   function ([54]Yang et al., 2017). Macrophages are well-known
   antigen-presenting cells that may play a role in immune regulation
   through phagocytosis and cytokine secretion. During pregnancy, decidual
   macrophages are associated with the secretion of immune-suppressive
   cytokines, maintaining immune tolerance ([55]Fujihara et al., 2003).
   Macrophages, such as Th1 and Th2 lymphocytes, can be classified into
   different types based on their distinct functions. M1 macrophages are
   involved in the classical functions of host defense against foreign
   pathogens, while M2 macrophages participate in immune regulation and
   tolerance ([56]Schonkeren et al., 2011). In the decidua, maternal
   macrophages expressing DC-SIGN exhibit a CD14+/CD163+ phenotype and are
   considered M2 macrophages ([57]Kim et al., 2007).

   Increasing evidence suggests that various genes and cellular pathways
   are involved in the occurrence and development of PE ([58]Chelbi and
   Vaiman, 2008). Therefore, high-throughput platforms such as microarrays
   are increasingly used for the analysis of miRNA and gene expression in
   PE ([59]Luo et al., 2017). Many recent studies have explored the
   pathogenesis of PE using various bioinformatics tools. The VEGF
   signaling pathway is believed to be most related to PE. The VEGF, FLT1
   and elements involved in arginine metabolism, including NO production,
   are considered the main mechanisms by which the placenta participates
   in PE ([60]Noris et al., 2005; [61]Cindrova-Davies et al., 2011;
   [62]Sundrani et al., 2013). Additionally, microRNAs are associated with
   PE by targeting key genes that regulate the differentiation of human
   trophoblast cells ([63]Zhu et al., 2015; [64]Zhang et al., 2016). Given
   the importance of immune cell infiltration in the pathogenesis of PE,
   analysis of immune cell infiltration can be used to select hub genes in
   PE as molecular markers for different subtypes of PE, providing new
   insights into the mechanism of PE and potential biomarkers for its
   diagnosis and treatment. In this study, we discussed the relationship
   between immune cell infiltration and PE based on WGCNA screening and
   GSEA analysis, as well as the differences in immune cell infiltration
   between PE and healthy pregnant women. We identified hub genes for PE
   molecular subtyping. The differences in hub genes between different PE
   subtypes suggest that these genes may serve as potential markers for PE
   subtyping, providing new insights into the pathogenesis of PE and
   potential biomarkers for its diagnosis and treatment.

   In this study, we discussed the relationship between immune cell
   infiltration and pre-eclampsia based on WGCNA screening and GSEA
   analysis. Through these methods, we were able to gain valuable insights
   into the distinct patterns of immune cell infiltration observed in
   women with pre-eclampsia compared to their healthy counterparts during
   pregnancy. We identified hub genes for PE molecular subtyping. These
   genes exhibit significant differences across various PE subtypes,
   indicating their potential as markers for PE subtyping. This novel
   finding not only sheds new light on the pathogenesis of PE but also
   opens up possibilities for the development of more precise diagnostic
   and prognostic biomarkers, paving the way for improved clinical
   management and treatment strategies for this complex disorder.

2 Materials

2.1 Clinical sample collection

   The samples used in this study were all obtained from the First
   Affiliated Hospital of Chongqing Medical University. Placental tissues
   from seven pregnant women diagnosed with PE and seven normal pregnant
   women were collected immediately after cesarean section. Individuals
   with diabetes, kidney disease, metabolic syndrome, infections,
   chromosomal abnormalities, or structural anomalies were excluded from
   the study. All patients underwent elective cesarean section, and they
   were informed and signed informed consent forms. Approximately 1 cm^2
   of placental tissue was collected immediately after delivery, washed,
   and divided. A portion was snap-frozen in liquid nitrogen, another
   portion was placed in RNA storage solution, and both were stored
   long-term at −80°C. The remaining portion was fixed in 4%
   paraformaldehyde and embedded in OCT. This study followed the
   principles outlined in the Helsinki Declaration and obtained approval
   from the Research Ethics Committee of the First Affiliated Hospital of
   Chongqing Medical University.

2.2 Data acquisition

   Details are provided in [65]Supplementary Table S1. The Series Matrix
   File data file of [66]GSE75010 were downloaded from the NCBI GEO public
   database. The annotation file is [67]GPL6244. A total of expression
   profile data of 157 patients were included, including 77 in the control
   group and 80 in the disease group. The Series Matrix File data file of
   [68]GSE74341 were downloaded from the NCBI GEO public database. The
   annotation file is [69]GPL16699. A total of expression profile data of
   25 patients were included, including 10 cases in the control group and
   15 cases in the disease group.

2.3 Immune cell infiltration analysis

   The CIBERSORT method is a widely used method to evaluate immune cell
   types in the microenvironment. This method is based on the principle of
   support vector regression and performs deconvolution analysis on the
   expression matrix of immune cell subtypes. It contains 547 biomarkers
   that distinguish 22 human immune cell phenotypes, including T cells, B
   cells, plasma cells, and myeloid cell subsets. This study used the
   CIBERSORT algorithm to analyze patient data to infer the relative
   proportions of 22 types of immune infiltrating cells. CIBERSORT and
   each algorithm/tool used in this study and references are included in