Intersection of regulatory pathways controlling hemostasis and hemochorial placentation

Hemochorial placentation is characterized by the development of trophoblast cells specialized to interact with the uterine vascular bed. We utilized trophoblast stem (TS) cell and mutant rat models to investigate regulatory mechanisms controlling trophoblast cell development. TS cell differentiation was characterized by acquisition of transcript signatures indicative of an endothelial cell-like phenotype, which was highlighted by the expression of anticoagulation factors including tissue factor pathway inhibitor (TFPI). TFPI localized to invasive endovascular trophoblast cells of the rat placentation site. Disruption of TFPI in rat TS cells interfered with development of the endothelial cell-like endovascular trophoblast cell phenotype. Similarly, TFPI was expressed in human invasive/extravillous trophoblast (EVT) cells situated within first-trimester human placental tissues and following differentiation of human TS cells. TFPI was required for human TS cell differentiation to EVT cells. We next investigated the physiological relevance of TFPI at the placentation site. Genome-edited global TFPI loss-of-function rat models revealed critical roles for TFPI in embryonic development, resulting in homogeneous midgestation lethality prohibiting analysis of the role of TFPI as a regulator of the late-gestation wave of intrauterine trophoblast cell invasion. In vivo trophoblast-specific TFPI knockdown was compatible with pregnancy but had profound effects at the uterine–placental interface, including restriction of the depth of intrauterine trophoblast cell invasion while leading to the accumulation of natural killer cells and increased fibrin deposition. Collectively, the experimentation implicates TFPI as a conserved regulator of invasive/EVT cell development, uterine spiral artery remodeling, and hemostasis at the maternal–fetal interface.

Impaired Sphingosine-1-Phosphate Synthesis Induces Preeclampsia by Deactivating Trophoblastic YAP (Yes-Associated Protein) Through S1PR2 (Sphingosine-1-Phosphate Receptor-2)-Induced Actin Polymerizations

Incomplete spiral artery remodeling, caused by impaired extravillous trophoblast invasion, is a fundamental pathogenic process associated with malplacentation and the development of preeclampsia. Nevertheless, the mechanisms controlling this regulation of trophoblast invasion are largely unknown. We report that sphingosine-1-phosphate synthesis and expression is abundant in healthy trophoblast, whereas in pregnancies complicated by preeclampsia the placentae are associated with reduced sphingosine-1-phosphate and lower SPHK1 (sphingosine kinase 1) expression and activity. In vivo inhibition of sphingosine kinase 1 activity during placentation in pregnant mice led to decreased placental sphingosine-1-phosphate production and defective placentation, resulting in a preeclampsia phenotype. Moreover, sphingosine-1-phosphate increased HTR8/SVneo (immortalized trophoblast cells) cell invasion in a Hippo-signaling–dependent transcriptional coactivator YAP (Yes-associated protein) dependent manner, which is activated by S1PR2 (sphingosine-1-phosphate receptor-2) and downstream RhoA/ROCK induced actin polymerization. Mutation-based YAP-5SA demonstrated that sphingosine-1-phosphate activation of YAP could be either dependent or independent of Hippo signaling. Together, these findings suggest a novel pathogenic pathway of preeclampsia via disrupted sphingosine-1-phosphate metabolism and signaling-induced, interrupted actin dynamics and YAP deactivation; this may lead to potential novel intervention targets for the prevention and management of preeclampsia.

Hyperosmolarity Impairs Human Extravillous Trophoblast Differentiation by Caveolae Internalization

We recently reported that an intact caveolar structure is necessary for adequate cell migration and tubulogenesis of the human extravillous trophoblast (EVT) cells. Emerging evidence supports that hyperosmolarity induces the internalization of caveolae into the cytoplasm and accelerates their turnover. Furthermore, signaling pathways associated with the regulation of trophoblast differentiation are localized in caveolae. We hypothesized that hyperosmolarity impairs EVT differentiation and caveolae/caveolin−1 (Cav-1) participates in this process. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 and exposed to hyperosmolar condition (generated by the addition of 100 mM sucrose). Hyperosmolarity altered the EVT cell migration and the formation of tube-like structures. In addition, cell invasion was decreased along with a reduction in the latent and active forms of matrix metalloproteinase-2 (MMP−2) secreted by these cells. With respect to Cav-1 protein abundance, we found that hyperosmolarity enhanced its degradation by the lysosomal pathway. Accordingly, in the hyperosmolar condition, we also observed a significant increase in the number of vacuoles and the internalization of the caveolae into the cytoplasm. Taken together, our findings suggest that hyperosmolarity may induce caveolae internalization and increase their turnover, compromising the normal differentiation of EVT cells.

Decorin: a multifunctional proteoglycan involved in oocyte maturation and trophoblast migration

Decorin (DCN) is a proteoglycan belonging to the small leucine-rich proteoglycan family. It is composed of a protein core containing leucine repeats with a glycosaminoglycan chain consisting of either chondroitin sulfate or dermatan sulfate. DCN is a structural component of connective tissues that can bind to type I collagen. It plays a role in the assembly of the extracellular matrix (ECM), and it is related to fibrillogenesis. It can interact with fibronectin, thrombospondin, complement component C1, transforming growth factor (TGF), and epidermal growth factor receptor. Normal DCN expression regulates a wide range of cellular processes, including proliferation, migration, apoptosis, and autophagy, through interactions with various molecules. However, its aberrant expression is associated with oocyte maturation, oocyte quality, and poor extravillous trophoblast invasion of the uterus, which underlies the occurrence of preeclampsia and intrauterine growth restriction. Spatiotemporal hormonal control of successful pregnancy should regulate the concentration and activity of specific proteins such as proteoglycan participating in the ECM remodeling of trophoblastic and uterine cells in fetal membranes and uterus. At the human feto-maternal interface, TGF-β and DCN play crucial roles in the regulation of trophoblast invasion of the uterus. This review summarizes the role of the proteoglycan DCN as an important and multifunctional molecule in the physiological regulation of oocyte maturation and trophoblast migration. This review also shows that recombinant DCN proteins might be useful for substantiating diverse functions in both animal and in vitro models of oogenesis and implantation.

Upregulation of circRNA hsa_circ_0008726 in Pre-eclampsia Inhibits Trophoblast Migration, Invasion, and EMT by Regulating miR-345-3p/RYBP Axis

Accumulating evidence shows that impaired spiral artery remodeling, placental dysfunction, and insufficient trophoblast infiltration contribute to the etiology and pathogenesis of pre-eclampsia (PE). circRNAs are a class of endogenous non-coding RNAs implicated in the pathogenesis of many diseases, including PE. This study aims to investigate the role of circRNA hsa_circ_0008726 in regulating the migration and invasion of extravillous trophoblast cells. RNase R assay was performed to confirm that circ_0008726 was a circular transcript. The expression of circ_0008726, RYBP, and miR-345-3p was examined by qRT-PCR. The functional interaction between miR-345-3p and circ_0008726 or RYBP was confirmed using dual-luciferase reporter assay and RNA immunoprecipitation (RIP). Cell migration and invasion ability was analyzed by Transwell assays. Western blot was used for the quantification of RYBP protein level. Circ_0008726 expression was significantly increased in PE placenta tissues as compared with normal placenta tissues. Circ_0008726 was resistant to RNase R digestion and was predominately located in the cytoplasm of HTR-8/SVneo cells. Silencing circ_0008726 promoted cell migration and EMT (epithelial-mesenchymal transition), while circ_0008726 overexpression suppressed these processes. Mechanistically, circ_0008726 sponged miR-345-3p to negatively regulate its expression, and miR-345-3p negatively modulated the expression of RYBP. In PE samples, the expression level of circ_0008726 was negatively correlated with miR-345-3p level, but was positively correlated with RYBP expression. Transfection of miR-345-3p mimic or RYBP knockdown counteracted the effects of circ_0008726 overexpression on cell migration and EMT. Our data demonstrate the upregulation of circ_0008726 in PE placenta, which inhibits the migration, invasion, and EMT of HTR-8/SVneo cells by targeting miR-345-3p/RYBP axis. These data suggest that circ_0008726 could be a potential biomarker and therapeutic target for PE.

Matrix Metalloproteinases in Human Decidualized Endometrial Stromal Cells

Cyclic changes, such as growth, decidualization, shedding, and regeneration, in the human endometrium are regulated by the reciprocal action of female hormones, such as estradiol (E2), and progesterone (P4). Matrix metalloproteases (MMPs) and tissue inhibitors of MMPs (TIMPs) control the invasion of extravillous trophoblast cells after implantation. Several MMPs and TIMPs function in the decidua and endometrial stromal cells (ESCs). Here, we aimed to systematically investigate the changes in MMPs and TIMPs associated with ESC decidualization. We evaluated the expression of 23 MMPs, four TIMPs, and four anti-sense non-coding RNAs from MMP loci. Primary ESC cultures treated with E2 + medroxyprogesterone acetate (MPA), a potent P4 receptor agonist, showed significant down-regulation of MMP3, MMP10, MMP11, MMP12, MMP20, and MMP27 in decidualized ESCs, as assessed by quantitative reverse transcription PCR. Further, MMP15 and MMP19 were significantly upregulated in decidualized ESCs. siRNA-mediated silencing of Heart and Neural Crest Derivatives Expressed 2 (HAND2), a master transcriptional regulator in ESC decidualization, significantly increased MMP15 expression in untreated human ESCs. These results collectively indicate the importance of MMP15 and MMP19 in ESC decidualization and highlight the role of HAND2 in repressing MMP15 transcription, thereby regulating decidualization.

Limited replication of human cytomegalovirus in a trophoblast cell line

Several viruses, including human cytomegalovirus (HCMV), are thought to replicate in the placenta. However, there is little understanding of the molecular mechanisms involved in HCMV replication in this tissue. We investigated replication of HCMV in the extravillous trophoblast cell line SGHPL-4, a commonly used model of HCMV replication in the placenta. We found limited HCMV protein expression and virus replication in SGHPL-4 cells. This was associated with a lack of trophoblast progenitor cell protein markers in SGHPL-4 cells, suggesting a relationship between trophoblast differentiation and limited HCMV replication. We proposed that limited HCMV replication in trophoblast cells is advantageous to vertical transmission of HCMV, as there is a greater opportunity for vertical transmission when the placenta is intact and functional. Furthermore, when we investigated the replication of other vertically transmitted viruses in SGHPL-4 cells we found some limitation to replication of Zika virus, but not herpes simplex virus. Thus, limited replication of some, but not all, vertically transmitted viruses may be a feature of trophoblast cells.

Regulation of Epigenetic Modifications in the Placenta During Preeclampsia: PPARγ Influences H3K4me3 and H3K9ac in Extravillous Trophoblast Cells

The aim of this study was to analyze the expression of peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RxRα), a binding heterodimer playing a pivotal role in the successful trophoblast invasion, in the placental tissue of preeclamptic patients. Furthermore, we aimed to characterize a possible interaction between PPARγ and H3K4me3 (trimethylated lysine 4 of the histone H3), respectively H3K9ac (acetylated lysine 9 of the histone H3), to illuminate the role of histone modifications in a defective trophoblast invasion in preeclampsia (PE). Therefore, the expression of PPARγ and RxRα was analyzed in 26 PE and 25 control placentas by immunohistochemical peroxidase staining, as well as the co-expression with H3K4me3 and H3K9ac by double immunofluorescence staining. Further, the effect of a specific PPARγ-agonist (Ciglitazone) and PPARγ-antagonist (T0070907) on the histone modifications H3K9ac and H3K4me3 was analyzed in vitro. In PE placentas, we found a reduced expression of PPARγ and RxRα and a reduced co-expression with H3K4me3 and H3K9ac in the extravillous trophoblast (EVT). Furthermore, with the PPARγ-antagonist treated human villous trophoblast (HVT) cells and primary isolated EVT cells showed higher levels of the histone modification proteins whereas treatment with the PPARγ-agonist reduced respective histone modifications. Our results show that the stimulation of PPARγ-activity leads to a reduction of H3K4me3 and H3K9ac in trophoblast cells, but paradoxically decreases the nuclear PPARγ expression. As the importance of PPARγ, being involved in a successful trophoblast invasion has already been investigated, our results reveal a pathophysiologic connection between PPARγ and the epigenetic modulation via H3K4me3 and H3K9ac in PE.

Prediction of Non-canonical Routes for SARS-CoV-2 Infection in Human Placenta Cells

The SARS-CoV-2 is the causative agent of the COVID-19 pandemic. The data available about COVID-19 during pregnancy have demonstrated placental infection; however, the mechanisms associated with intrauterine transmission of SARS-CoV-2 is still debated. Intriguingly, while canonical SARS-CoV-2 cell entry mediators are expressed at low levels in placental cells, the receptors for viruses that cause congenital infections such as the cytomegalovirus and Zika virus are highly expressed in these cells. Here we analyzed the transcriptional profile (microarray and single-cell RNA-Seq) of proteins potentially interacting with coronaviruses to identify non- canonical mediators of SARS-CoV-2 infection and replication in the placenta. Despite low levels of the canonical cell entry mediators ACE2 and TMPRSS2, we show that cells of the syncytiotrophoblast, villous cytotrophoblast, and extravillous trophoblast co-express high levels of the potential non-canonical cell-entry mediators DPP4 and CTSL. We also found changes in the expression of DAAM1 and PAICS genes during pregnancy, which are translated into proteins also predicted to interact with coronaviruses proteins. These results provide new insight into the interaction between SARS-CoV-2 and host proteins that may act as non-canonical routes for SARS-CoV-2 infection and replication in the placenta cells.