ACVR2A Facilitates Trophoblast Cell Invasion through TCF7/c-JUN Pathway in Pre-eclampsia Progression

Reviewer #1 (Public review):

Summary:

This study has preliminarily revealed the role of ACVR2A in trophoblast cell function, including its effects on migration, invasion, proliferation, and clonal formation, as well as its downstream signaling pathways.

Strengths:

The use of multiple experimental techniques, such as CRISPR/Cas9-mediated gene knockout, RNA-seq, and functional assays (e.g., Transwell, colony formation, and scratch assays), is commendable and demonstrates the authors' effort to elucidate the molecular mechanisms underlying ACVR2A's regulation of trophoblast function. The RNA-seq analysis and subsequent GSEA findings offer valuable insights into the pathways affected by ACVR2A knockout, particularly the Wnt and TCF7/c-JUN signaling pathways.

Weaknesses:

The molecular mechanisms underlying this study require further exploration through additional experiments. While the current findings provide valuable insights into the role of ACVR2A in trophoblast cell function and its involvement in the regulation of migration, invasion, and proliferation, further validation in both in vitro and in vivo models is needed. Additionally, more experiments are required to establish the functional relevance of the TCF7/c-JUN pathway and its clinical significance, particularly in relation to pre-eclampsia. Additional techniques, such as animal models and more advanced clinical sample analyses, would help strengthen the conclusions and provide a more comprehensive understanding of the molecular pathways involved.

Reviewer #2 (Public review):

Summary:

ACVR2A is one of a handful of genes for which significant correlations between associated SNPs and the incidences of preeclampsia have been found in multiple populations. It is one of the TGFB family receptors, and multiple ligands of ACVR2A, as well as its coreceptors and related inhibitors, have been implicated in placental development, trophoblast invasion, and embryo implantation. This useful study builds on this knowledge by showing that ACVR2A knockout in trophoblast-related cell lines reduces trophoblast invasion, which could tie together many of these observations. Support for this finding is incomplete, as reduced proliferation may be influencing the invasion results. The implication of cross-talk between the WNT and ACRV2A/SMAD2 pathways is an important contribution to the understanding of the regulation of trophoblast function.

Strengths:

(1) ACVR2A is one of very few genes implicated in preeclampsia in multiple human populations, yet its role in pathogenesis is not very well studied and this study begins to address that hole in our knowledge.

(2) ACVR2A is also indirectly implicated in trophoblast invasion and trophoblast development via its connections to many ligands, inhibitors, and coreceptors, suggesting its potential importance.

(3) The authors have used multiple cell lines to verify their most important observations.

Weaknesses:

(1) There are a number of claims made in the introduction without attribution. For example, there are no citations for the claims that family history is a significant risk factor for PE, that inadequate trophoblast invasion of spiral arteries is a key factor, and that immune responses, and renin-angiotensin activity are involved.

(2) The introduction states "As a receptor for activin A, ACVR2A..." It's important to acknowledge that ACVR2A is also the receptor for other TGFB family members, with varying affinities and coreceptors. Several TGFB family members are known to regulate trophoblast differentiation and invasion. For example, BMP2 likely stimulates trophoblast invasion at least in part via ACVR2A (PMID 29846546).

(3) An alternative hypothesis for the potential role of ACVR2A in preeclampsia is its functions in the endometrium. In the mouse ACVR2A knockout in the uterus (and other progesterone receptor-expressing cells) leads to embryo implantation failure.

(4) In the description of the patient population for placental sample collections, preeclampsia is defined only by hypertension, and this is described as being in accordance with ACOG guidelines. ACOG requires a finding of hypertension in combination with either proteinuria or one of the following: thrombocytopenia, elevated creatinine, elevated liver enzymes, pulmonary, edema, and new onset unresponsive headache.

(5) I believe that Figures 1a and 1b are data from a previously published RNAseq dataset, though it is not entirely clear in the text. The methods section does not include a description of the analysis of these data undertaken here. It would be helpful to include at least a brief description of the study these data are taken from - how many samples, how were the PE/control groups defined, gestational age range, where is it from, etc. For the heatmap presented in B, what is the significance of the other genes/ why are they being shown? If the purpose of these two panels is to show differential expression specifically of ACVR2A in this dataset, that could be shown more directly.

(6) More information is needed in the methods section to understand how the immunohistochemistry was quantified. "Quantitation was performed" is all that is provided. Was staining quantified across the whole image or only in anchoring villous areas? How were HRP & hematoxylin signals distinguished in ImageJ? How was the overall level of HRP/DAB development kept constant between the NC and PE groups?

(7) In Figure 1E it is not immediately obvious to many readers where the EVT are. It is probably worth circling or putting an arrow to the little region of ACVR2A+ EVT that is shown in the higher magnification image in Figure 1E. These are actually easier to see in the pictures provided in the supplement Figure 1. Of note, the STB is also staining positive. This is worth pointing out in the results text.

(8) It is not possible to judge whether the IF images in 1F actually depict anchoring villi. The DAPI is really faint, and it's high magnification, so there isn't a lot of context. Would it be possible to include a lower magnification image that shows where these cells are located within a placental section? It is also somewhat surprising that this receptor is expressed in the cytoplasm rather than at the cell surface. How do the authors explain this?

(9) The results text makes it sound like the data in Figure 2A are from NCBI & Protein atlas, but the legend says it is qPCR from this lab. The methods do not detail how these various cell lines were grown; only HTR-SVNeo cell culture is described. Similarly, JAR cells are used for several experiments and their culture is not described.

(10) Under RT-qPCR methods, the phrase "cDNA reverse transcription cell RNA was isolated..." does not make any sense.

(11) The paragraph beginning "Consequently, a potential association..." is quite confusing. It mentions analyzing ACVR2A expression in placentas, but then doesn't point to any results of this kind and repeats describing the results in Figure 2a, from various cell lines.

(12) The authors should acknowledge that the effect of the ACVR2A knockout on proliferation makes it difficult to draw any conclusions from the trophoblast invasion assays. That is, there might be fewer migrating or invading cells in the knockout lines because there are fewer cells, not because the cells that are there are less invasive. Since this is a central conclusion of the study, it is a major drawback.

(13) The legend and the methods section do not agree on how many fields were selected for counting in the transwell invasion assays in Figure 3C. The methods section and the graph do not match the number of replicate experiments in Figure 3D (the number of replicate experiments isn't described for 3C).

(14) Discussion says "Transcriptome sequencing analysis revealed low ACVR2A expression in placental samples from PE patients, consistent with GWAS results across diverse populations." The authors should explain this briefly. Why would SNPs in ACVR2A necessarily affect levels of the transcript?

(15) "The expression levels of ACVR2A mRNA were comparable to those of tumor cells such as A549. This discovery suggested a potential pivotal role of ACVR2A in the biological functions of trophoblast cells, especially in the nurturing layer." Alternatively, ACVR2A expression resembles that of tumors because the cell lines used here are tumor cells (JAR) or immortalized cells (HTR8). These lines are widely used to study trophoblast properties, but the discussion should at least acknowledge the possibility that the behavior of these cells does not always resemble normal trophoblasts.

(16) The authors should discuss some of what is known about the relationship between the TCF7/c-JUN pathway and the major signaling pathway activated by ACVR2A, Smad 2/3/4. The Wnt and TGFB family cross-talk is quite complex and it has been studied in other systems.