VE-cadherin enables trophoblast endovascular invasion and spiral artery remodeling during placental development

  1. Derek C Sung
  2. Xiaowen Chen
  3. Mei Chen
  4. Jisheng Yang
  5. Susan Schultz
  6. Apoorva Babu
  7. Yitian Xu
  8. Siqi Gao
  9. TC Stevenson Keller IV
  10. Patricia Mericko-Ishizuka
  11. Michelle Lee
  12. Ying Yang
  13. Joshua P Scallan
  14. Mark L Kahn  Is a corresponding author
  1. Cardiovascular Institute, Department of Medicine, University of Pennsylvania, United States
  2. Department of Radiology, Hospital of the University of Pennsylvania, United States
  3. University Laboratory Animal Resources, University of Pennsylvania, United States
  4. Department of Molecular Pharmacology and Physiology, University of South Florida, United States

Abstract

During formation of the mammalian placenta, trophoblasts invade the maternal decidua and remodel spiral arteries to bring maternal blood into the placenta. This process, known as endovascular invasion, is thought to involve the adoption of functional characteristics of vascular endothelial cells (ECs) by trophoblasts. The genetic and molecular basis of endovascular invasion remains poorly defined, however, and whether trophoblasts utilize specialized endothelial proteins in an analogous manner to create vascular channels remains untested. Vascular endothelial (VE-)cadherin is a homotypic adhesion protein that is expressed selectively by ECs in which it enables formation of tight vessels and regulation of EC junctions. VE-cadherin is also expressed in invasive trophoblasts and is a prime candidate for a molecular mechanism of endovascular invasion by those cells. Here, we show that VE-cadherin is required for trophoblast migration and endovascular invasion into the maternal decidua in the mouse. VE-cadherin deficiency results in loss of spiral artery remodeling that leads to decreased flow of maternal blood into the placenta, fetal growth restriction, and death. These studies identify a non-endothelial role for VE-cadherin in trophoblasts during placental development and suggest that endothelial proteins may play functionally unique roles in trophoblasts that do not simply mimic those in ECs.

Editor's evaluation

Understanding molecular and cellular pathways for endovascular invasion and pathogenesis of preeclampsia are important topics for current vascular biology and Ob/Gyn biology, making this study timely and important. It shows for the first time a causal link for the need of VE-cadherin on trophoblasts in vivo for the invasion of these cells into the decidua and for their role in vascular remodeling. The conclusions of this paper are well supported by data. It may provide a novel avenue for the prevention or treatment of preeclampsia.

https://doi.org/10.7554/eLife.77241.sa0

Introduction

During placental development in mice and humans, fetal trophoblasts invade the maternal decidua by a process known as endovascular invasion to remodel and connect to maternal spiral arteries (SAs) (Velicky et al., 2016; Soares et al., 2014; Hu and Cross, 2011). This connection allows the flow of maternal blood through trophoblast-lined sinuses characteristic of hemochorial placentation (Soares et al., 2018; Rai and Cross, 2014). Shallow trophoblast invasion, deficient SA remodeling, and poor remodeling of the maternal decidua are features of placental dysfunction such as preeclampsia, a hypertensive condition of pregnancy that can lead to maternal and fetal complications (Lyall et al., 2013; Roberts and Escudero, 2012). Despite the broad and clinically significant impacts of placental dysfunction, the mechanisms controlling trophoblast endovascular invasion and SA remodeling remain poorly defined in vivo.

Invasive trophoblasts are believed to adopt an endothelial-like state by expressing endothelial specific genes (Nelson et al., 2016; Zhou et al., 1997b; Govindasamy et al., 2021), a process that has also been termed ‘vascular mimicry’ (reviewed in Rai and Cross, 2014). Invasive trophoblasts in human and mouse placentas express vascular endothelial (VE)-cadherin (gene name Cdh5) during remodeling of SAs (Zhou et al., 1997b; Govindasamy et al., 2021; Zhou et al., 1997a). Invasive trophoblasts in preeclamptic placentas lack VE-cadherin (Zhou et al., 1997a), and loss of VE-cadherin reduces trophoblast invasion in vitro (Cheng et al., 2013). These studies suggest a functional role for VE-cadherin in endovascular invasion and vessel formation. VE-cadherin is a well-studied cell-cell adhesion protein in the vascular endothelium where it regulates vascular integrity and growth and endothelial barrier function (Corada et al., 1999; Crosby et al., 2005; Carmeliet et al., 1999). In vitro studies have suggested that VE-cadherin may regulate trophoblast-endothelial interactions (Bulla et al., 2005), but the requirement for VE-cadherin in trophoblasts during placental development in vivo remains unknown.

In the present study, we functionally tested the role of VE-cadherin in trophoblasts during placental development in mice. We find that conditional deletion of VE-cadherin from trophoblasts disrupts trophoblast invasion into the decidua and SA remodeling, resulting in placental insufficiency and fetal growth restriction. We show that VE-cadherin is important for trophoblasts to interact with and displace SA endothelium. Additionally, trophoblast invasion is important for triggering multiple changes in the decidual extracellular matrix (ECM) and immune cell microenvironment. These studies identify a molecular mechanism by which fetal trophoblasts use VE-cadherin to invade and remodel the maternal environment for successful pregnancy that is relevant to preeclampsia pathogenesis. They also provide a first functional test of the concept of trophoblasts utilizing endothelial programs during endovascular invasion in vivo, and suggest that canonical endothelial proteins may be used by vascular trophoblasts in the placenta in ways that are specific for their function and do not merely mimic endothelial cell (EC) use.

Results

Trophoblast-specific deletion of VE-cadherin restricts placental and fetal growth and causes embryonic lethality

To understand the role of VE-cadherin in trophoblasts, we generated CYP19A1(Tg)Cre; Cdh5fl/fl (‘Cdh5 knockout’) placentas and mice in which VE-cadherin (encoded by Cdh5) is deleted specifically in fetal trophoblasts. Immunostaining for VE-cadherin and the trophoblast marker Cytokeratin 8 (CK8) demonstrated efficient deletion of VE-cadherin in trophoblasts but not ECs in the Cdh5 knockout placenta (Figure 1—figure supplement 1A, B). Cdh5 knockout embryos were present at the expected Mendelian ratio at E10.5–12.5 (Table 1, p = 1.0000). However, there was an almost complete loss of trophoblast Cdh5 knockout embryos at E14.5–16.5 and postnatal day 21 (Table 1, p < 0.05 and p < 0.005, respectively). Examination of placentas at E12.5 revealed that trophoblast Cdh5 knockout placentas were smaller and paler than those of control littermates in the same uterus (Figure 1A–D). Cdh5 knockout embryos exhibited marked fetal growth restriction and variable degrees of hemorrhage at E12.5 (green arrowheads, Figure 1E–H). No differences in weights were observed between Cre-negative (Cdh5fl/+ or Cdh5fl/fl) and Cre-positive heterozygous (CYP19A1(Tg)Cre; Cdh5fl/+) placentas and embryos (Figure 1—figure supplement 1C, D). Histological and immunofluorescence analysis of E12.5 knockout embryos showed growth defects in numerous organs, including the heart, brain, and liver (Figure 1—figure supplement 2A-D). Immunofluorescence staining for VE-cadherin in CYP19A1(Tg)Cre; Cdh5fl/fl embryos show that VE-cadherin is retained in the embryonic vasculature of affected organs, including the brain, heart, liver, lungs, and thorax (Figure 1—figure supplement 3A-D). These data demonstrate that trophoblast-specific loss of VE-cadherin confers fetal growth restriction and lethality. Importantly, since CYP19A1(Tg)Cre activity is present only in placental trophoblasts (Wenzel and Leone, 2007; López-Tello et al., 2019), these embryonic defects are secondary to placental defects.

Table 1
Decreased survival of CYP19A1(Tg)Cre; Cdh5fl/fl mutants in late gestation.

Cdh5fl/fl male mice were crossed with CYP19A1(Tg)Cre; Cdh5fl/+ females to generate litters with mixed genotypes. The expected percentage is listed under the genotype label. The observed number of each genotype is shown with the corresponding percentage given in parentheses. P-values were calculated using Fisher’s exact test at stages E10.5-12.5 (pooled), E14.5-16.5 (pooled), and P21. E designates embryonic day and P designates postnatal day.

CYP19A1(Tg)Cre; Cdh5fl/fl (25%)CYP19A1(Tg)Cre; Cdh5fl/+ (25%)Cdh5fl/fl (25%)Cdh5fl/+ (25%)Total (100%)Fisher Exact Test
E10.5-E12.513* (26%)13 (26%)12 (24%)12 (24%)50 (100%)P=1.0000
E14.5-E16.51 (2.9%)13 (38.2%)12 (35.3%)8 (23.5%)34 (100%)P=0.0033
P211 (2.2%)11 (24.4%)15 (33.3%)18 (40.0%)45 (100%)P=0.0333
  1. *

    One embryo at E12.5 was dead.

Figure 1 with 3 supplements see all
Deletion of VE-cadherin in trophoblasts disrupts cell migration resulting in placental and fetal growth restriction.

(A–D) Gross examination and quantification of E12.5 Control and CYP19A1(Tg)Cre; Cdh5fl/fl placentas and weights. Control n = 11, CYP19A1(Tg)Cre; Cdh5fl/fl n = 5. (E–H) Gross examination and quantification of E12.5 Control and CYP19A1(Tg)Cre; Cdh5fl/fl embryos and weights. Control n = 11, CYP19A1(Tg)Cre; Cdh5fl/fl n = 5. Green arrowheads point to areas of hemorrhage. (I–L) Hematoxylin and eosin (H&E) staining and immunofluorescence staining for Endomucin (green), CK8 (red), and TER119 (gray) of E12.5 Control (I, J) and CYP19A1(Tg)Cre; Cdh5fl/fl (K, L) serial placenta sections. Dotted lines demarcate the different placental regions. Red arrowheads indicate trophoblasts. Green arrowheads indicate spiral arteries (SA). White arrowheads indicate maternal red blood cells (RBCs). Note fewer non-nucleated, maternal TER119+ cells in the labyrinth region of CYP19A1(Tg)Cre; Cdh5fl/fl placentas. Boxes on the left correlate with magnified images on the right, and boxes in H&E and immunofluorescence images are of the same region. Scale bars = 500 μm. Dec (decidua), JZ (junctional zone), Lab (labyrinth). (M–O) Quantification of number of trophoblasts in the decidua (M), trophoblast invasion depth (N), and percent labyrinth Endomucin+ area (O). Control n = 11, CYP19A1(Tg)Cre; Cdh5fl/fl n = 5. Statistical analysis was performed using two-tailed, unpaired Welch’s t-test. Data are shown as means ± SD.

Figure 1—source data 1

Excel file containing quantification for embryo weights, placenta weights, trophoblast density, trophoblast migration distance, and fetal labyrinth vasculature in Figure 1.

https://cdn.elifesciences.org/articles/77241/elife-77241-fig1-data1-v2.xlsx

Loss of VE-cadherin disrupts trophoblast endovascular invasion but not formation of the fetal vasculature

To characterize the placental defects conferred by trophoblast loss of VE-cadherin, we performed histological staining with hematoxylin and eosin (H&E) and immunofluorescence staining for CK8 (trophoblasts), Endomucin (endothelial cells), and TER119 (erythrocytes) on serial control and Cdh5 knockout placenta sections (Figure 1I–L). H&E and immunofluorescence staining both showed abundant trophoblasts, marked by CK8 positivity, surrounding Endomucin+ SAs within the decidua in control placentas (red arrowheads, Figure 1J and J’). Notably, trophoblasts surrounding maternal SAs were absent in knockout placentas (Figure 1L and L’), and quantification of number of trophoblasts and invasion depth into the decidua showed fewer and shallower invasion of trophoblasts overall (decidua, Figure 1M and N).

The findings described above suggested that Cdh5 knockout placentas were less able to carry maternal blood to nourish the growing embryo. Fetal and maternal red blood cells (RBCs) can be differentiated by the presence of nuclei in fetal RBCs. The labyrinth in knockout placentas had fewer enucleated TER119+ RBCs, indicating less maternal blood and consistent with paler placentas observed by gross examination (white arrowheads, Figure 2J” vs. L”). To determine whether loss of VE-cadherin affects the fetal placental vasculature, we quantified Endomucin+ vascular area in the labyrinth. We detected no differences in Endomucin+ staining, demonstrating that loss of VE-cadherin from trophoblasts does not disrupt formation of the fetal placental capillary plexus (Figure 1J, L and O). These findings suggest that VE-cadherin is important for trophoblast migration and for the association with SAs required to channel maternal blood into the placenta.

VE-cadherin is required in trophoblasts to remodel spiral arteries (SAs) and to displace SA endothelium.

(A) Immunofluorescence staining of E12.5 Control and CYP19A1(Tg)Cre; Cdh5fl/fl placentas for Endomucin (green) and alpha-smooth muscle actin (αSMA) (red). White arrowheads indicate αSMA+ cells. Scale bars = 25 μm. (B, C) Quantification of αSMA+ cells per SA and SA diameter. Control n = 11, CYP19A1(Tg)Cre; Cdh5fl/fl n = 5. (D) Immunofluorescence staining of E12.5 Control and CYP19A1(Tg)Cre; Cdh5fl/fl placentas for Endomucin (green) and CK8 (red). White arrowheads indicate Endomucin+ SA endothelial cells (ECs). Positive signal in small, rounded cells in the lumen is the result of erythrocyte autofluorescence. (E) Schematic demonstrating differences in trophoblast and SA EC contact with the vessel lumen. Scale bars = 25 μm. (F) Quantification of the percent trophoblast-lumen contact, which was calculated by measuring the circumference of the vessel lumen and then measuring the length of CK8+ trophoblasts in contact with the lumen. Each point represents the average of at least three SAs from an individual placenta. Control n = 10, CYP19A1(Tg)Cre; Cdh5fl/fl n = 5. (G, H) Maximum intensity projections of whole-mount immunofluorescence of the decidua (G) and labyrinth (H) from 200 μm thick placenta sections stained for Endomucin (green), TER119 (red), and CK8 (magenta). Double-headed arrows indicate differences in lumen size. White arrowheads indicate Endomucin+ SA ECs. Red arrowheads indicate maternal red blood cells within the trophoblast-lined vessels. Yellow arrowheads indicate fetal red blood cells within fetal capillaries. Dotted white line demarcates the decidua from the junctional zone. Scale bars = 50 μm. Statistical analysis was performed using two-tailed, unpaired Welch’s t-test. Data are shown as means ± SD.

Figure 2—source data 1

Excel file containing quantification for smooth muscle cells per spiral artery, spiral artery diameter, and trophoblast-endothelial cell displacement in Figure 2.

https://cdn.elifesciences.org/articles/77241/elife-77241-fig2-data1-v2.xlsx

Loss of trophoblast VE-cadherin blocks displacement of SA ECs and SA remodeling

A critical early step in establishing maternal circulation to the placenta is trophoblast invasion of the decidua and its SAs. The observation that there were fewer VE-cadherin-deficient trophoblasts adjacent to SAs and decreased maternal blood within the labyrinth suggested that trophoblast VE-cadherin may play a requisite role in SA remodeling. Since loss of vascular smooth muscle is a key step in SA remodeling, we stained control and knockout placentas for alpha-smooth muscle actin (αSMA). SAs in Cdh5 knockout placentas exhibited persistent vascular smooth muscle coverage and reduced SA diameter compared to control placentas (white arrowheads, Figure 2A–C). These studies reveal that loss of trophoblast VE-cadherin disrupts SA remodeling, which likely contributes to reduced maternal blood within the placenta.

During the process of SA remodeling, invasive trophoblasts displace the endothelial layer of SAs to direct maternal blood flow through trophoblast-lined sinuses into the labyrinth. In vitro studies have suggested that trophoblast expression of VE-cadherin may enable these cells to adhere to SA ECs (Bulla et al., 2005). The finding that trophoblast Cdh5 knockout placentas fail to remodel SAs suggested that there may be defects in trophoblast-SA interactions. To address the role of VE-cadherin at the site of trophoblast-SA connection, we first sought to image the site at which trophoblasts connect to SAs. Close inspection of this region in control placentas revealed a clear demarcation from luminal Endomucin+ SA endothelium to luminal CK8+ trophoblasts (white arrowheads, Figure 2D and E). In contrast, we found that SAs in VE-cadherin knockout placentas maintained a layer of intact ECs despite being surrounded by trophoblasts (white arrowheads, Figure 2D and E). Quantification of the percent of trophoblasts immediately in contact with the lumen demonstrated that knockout placentas had a lower percentage of trophoblasts and higher percentage of ECs covering the lumen compared to controls (Figure 2F). To better appreciate differences in placental architecture, we additionally performed whole-mount immunofluorescence of thick placental sections. Consistent with our data above, Cdh5 knockout placentas maintained a layer of ECs in SAs surrounded by trophoblasts and had smaller lumens (Figure 2G). Fewer maternal RBCs were seen in trophoblast-lined sinuses in the labyrinth of Cdh5 knockout placentas, but we found no differences in the fetal capillary plexus (Figure 2H). Together, these data suggest that VE-cadherin is required for trophoblast displacement of SA ECs during endovascular invasion for efficient maternal-fetal circulatory connection.

Defective trophoblast invasion and SA remodeling cause placental insufficiency and fetal distress

The finding that Cdh5 knockout placentas have less maternal blood and are associated with mid-gestation embryonic lethality suggested that failed SA remodeling restricts maternal blood flow into the placenta, thus causing fetal demise. Human placental insufficiency is typically assessed with ultrasound measurements of placental hemodynamics and fetal heart rate, a readout for overall fetal health. We therefore utilized Doppler ultrasound to measure peak systolic (PSV) and end diastolic velocities (EDV) in the umbilical arteries (Figure 3A) and calculated resistance and pulsatility indices (RI and PI) and fetal heart rates to assess placental vascular resistance and fetal wellbeing in individual concepti. Elevated RI and PI values are clinical indicators of placental insufficiency in humans and are associated with conditions such as preeclampsia and fetal growth restriction. Fetal heart rate is used as a clinical parameter for fetal wellbeing, with fetal bradycardia indicative of fetal distress. While control embryos had RIs, PIs, and fetal heart rates within range of previously published values (Galaz et al., 2020), we found that trophoblast Cdh5 knockout embryos exhibited significantly increased RIs and PIs (Figure 3B–D) and significantly reduced fetal heart rates (Figure 3B and E), consistent with placental insufficiency and fetal distress. Knockout embryos also exhibited reversal of end-diastolic flow as shown by the directional change of velocity from negative (peak systole) to positive (end of diastole) (Figure 3B), indicative of high vascular resistance. These hemodynamic data demonstrate placental insufficiency that contributes to fetal growth restriction and fetal demise following loss of trophoblast invasion and SA remodeling.

Spiral artery remodeling defects result in placental insufficiency and fetal distress.

(A) Schematic of workflow for Doppler ultrasound of pregnant dams. The embryo, UA/UV (yellow arrow), and placenta (dotted white outline) are labeled. (B) Representative umbilical artery Doppler waveforms from two Control and two CYP19A1(Tg)Cre; Cdh5fl/fl placentas. The placenta is outlined in a dotted white line. Reversal of end-diastolic flow is evident by the change of directional velocity at the end of diastole compared to peak systole (i.e., negative to positive velocity). (C–E) Quantification of resistance index (RI), pulsatility index (PI), and fetal heart rate. PSV (peak systolic velocity), EDV (end diastolic velocity), UA/UV (umbilical artery/umbilical vein). Note that red/blue colors in color Doppler images do not indicate UA/UV, which can only be differentiated based on the Doppler waveform. Control n = 10, CYP19A1(Tg)Cre; Cdh5fl/fl n = 4. Statistical analysis was performed using two-tailed, unpaired Welch’s t-test. Data are shown as means ± SD.

Figure 3—source data 1

Excel file containing quantification for ultrasound studies (resistance index, pulsatility index, fetal heart rate) in Figure 3.

https://cdn.elifesciences.org/articles/77241/elife-77241-fig3-data1-v2.xlsx

Loss of trophoblast VE-cadherin alters decidual ECM remodeling and uterine natural killer cell clearance

Since trophoblast invasion occurs in conjunction with decidual changes, we hypothesized that failed trophoblast migration might affect other placental processes involved in that process. To identify such effects, we performed bulk RNA-sequencing (RNA-seq) on deciduas of E12.5 knockout and control placentas (Figure 4A). Analysis of the top 100 differentially expressed genes showed that genes highly expressed in invasive trophoblasts (Prl4a1, Pla2g4f, Pla2g4d, Nos1, Ncam1, Aldh1a3, Ascl2, Car2, Tfap2c) (Nelson et al., 2016; Müller et al., 1999; Simmons et al., 2008; Marsh and Blelloch, 2020; Outhwaite et al., 2015; Varberg et al., 2021; Bogutz et al., 2018; Sharma et al., 2016) were downregulated in knockout placentas (Figure 4B), consistent with reduced trophoblasts present in the decidua. Genes previously associated with trophoblast invasion (Gabrp, Mmp15) (Lu et al., 2016; Majali-Martinez et al., 2020) and differentiation (Cdx2) (Ralston and Rossant, 2008) were also differentially expressed, as were multiple genes related to defective decidualization (Ccl28, Slc27a2, Klk1, Csf1, Tmem132e, Ermap, Pappa2, Tmc5) (Woods et al., 2017; Goolam et al., 2020; Sun et al., 2013; Figure 4B). Thus RNA-seq data are consistent with defects in trophoblast invasion and suggest the presence of non-cell autonomous effects in the decidua that may contribute to a preeclamptic-like phenotype.

Figure 4 with 2 supplements see all
RNA-sequencing reveals defects in the decidual extracellular matrix and immune microenvironment.

(A) Schematic of bulk RNA-sequencing (RNA-seq) workflow on deciduas from Control (WT) and CYP19A1(Tg)Cre; Cdh5fl/fl (KO) E12.5 placentas. (B) Heatmap of the top 100 differentially regulated genes shown by z-score (n = 3 biological replicates). (C) Gene ontology (GO) term analysis of top 10 upregulated cellular components in CYP19A1(Tg)Cre; Cdh5fl/fl placentas. Purple bars indicate GO terms related to the extracellular matrix. (D) Heatmap of significantly differentially expressed genes from GO terms related to the extracellular matrix. (E) GO term analysis of top 10 upregulate biological processes in CYP19A1(Tg)Cre; Cdh5fl/fl placentas. Red bars indicate GO terms related to immune processes. (F) Heatmap of significantly differentially expressed genes from GO terms related to immune processes.

We next analyzed gene ontology (GO) related to cellular components and biological processes. We found that four of the top ten upregulated cellular component GO terms in knockout placentas were related to the ECM (Figure 4C). Many of the significantly upregulated genes were secreted ECM proteins (Vwa2, Ntn1, Fbln1) (Figure 4D). Several extracellular proteases previously linked to abnormal human pregnancies were also differentially expressed. These included increased expression of Adamts9 (associated with preterm birth; Li et al., 2020), reduced Adamts13 (associated with preeclampsia; Aref and Goda, 2013; Stepanian et al., 2011), and reduced Mmp15 (associated with reduced trophoblast invasion; Majali-Martinez et al., 2020). GO analysis of biological processes revealed that seven of the top ten upregulated GO terms were related to the immune system and immune activation, specifically the innate immune response (Figure 4E). Many of the significantly upregulated genes were involved in the complement factor pathway (C3, Cfb, Cfh) (Figure 4F). Thus GO analysis suggests that defects in ECM remodeling and changes in immune cells may contribute to the placental defects associated with reduced trophoblast invasion of the decidua.

We next aimed to directly assess the impact of trophoblast-specific deletion of VE-cadherin on (1) ECM remodeling and (2) immune cells. Our RNA-seq data showed a >2.5-fold decrease in Mmp15 (Figure 4D and Figure 4—figure supplement 1A). MMP15 (also known as MT2-MMP) is a membrane metalloprotease that is expressed in invasive trophoblasts in first trimester human placentas that promotes trophoblast invasion and degrades laminin as gestation progresses (Majali-Martinez et al., 2020; Pollheimer et al., 2014; Majali-Martinez et al., 2016; Turpeenniemi-Hujanen et al., 1995). Examination of MMP15 expression and its target laminin using immunofluorescence revealed increased laminin in the decidual stroma of Cdh5 knockout placentas but no differences in MMP15 expression in trophoblasts (Figure 4—figure supplement 1B, C). Additionally, we evaluated vinculin, a focal adhesion protein that regulates cell-matrix adhesion and associates with VE-cadherin (Huveneers et al., 2012). Vinculin is required for cell polarization and invasion (Thievessen et al., 2015; Mierke et al., 2010), and vinculin levels were indeed decreased in invasive trophoblasts of Cdh5 knockout placentas (Figure 4—figure supplement 1D). Together, these results suggest that VE-cadherin cell autonomously controls focal adhesions but not MMP activity and that persistent laminin in the decidua of Cdh5 knockout placentas is likely a consequence of fewer MMP15-expressing trophoblasts.

Since uterine natural killer (uNK) cells are the most abundant innate immune cell in the decidua, we stained placenta sections with the uNK cell-specific marker DBA. We found markedly increased uNK cells in knockout placentas compared to controls (Figure 4—figure supplement 2A, B). In order to determine the cause of increased uNK cells, we stained for the apoptosis marker cleaved caspase-3 and found numerous apoptotic uNK cells in control placentas; however almost no uNK cells in knockout placentas were positive for cleaved caspase-3 (Figure 4—figure supplement 2A, C). Lastly, we also found multiple significantly upregulated NK cell-related GO terms reflecting increased NK cell activity (Figure 4—figure supplement 2D). Together, these results indicate that loss of trophoblast invasion has non-cell autonomous effects that impact that maternal microenvironment of the placenta.

Discussion

The invasion of the maternal decidua by trophoblasts and their fusion to maternal SAs is a critical step in establishing placental circulation. However, the mechanisms by which trophoblast migration and endovascular invasion are accomplished remain largely unknown. Trophoblasts express endothelial molecular and genetic programs during invasion of SAs (Soares et al., 2018). However, the function of specific endothelial genes in trophoblasts has not been functionally assessed in vivo, and this model remains untested. In blood and lymphatic ECs, VE-cadherin is used to maintain vascular integrity (Corada et al., 1999; Crosby et al., 2005; Carmeliet et al., 1999), restrict endothelial migration (Crosby et al., 2005; Hägerling et al., 2018), and regulate angiogenic growth (Gaengel et al., 2012). While it is an attractive concept that trophoblasts may form vascular sinuses using similar genetic programs, the findings that loss of VE-cadherin decreases trophoblast cell migration and prevents SA remodeling suggest that trophoblasts utilize VE-cadherin in a manner distinct from ECs. Our work characterizing mechanisms of endovascular invasion in the placenta suggests that the use of endothelial proteins by trophoblasts may be relatively specific to their role in the placenta and not a simple reflection of vascular EC function.

VE-cadherin is primarily an adhesive receptor that acts in a homotypic manner to establish strong EC-cell junctions. Evidently, VE-cadherin is required in trophoblasts to invade the maternal decidua and remodel the maternal microenvironment. Our findings that loss of VE-cadherin decreases vinculin but not MMP15 in trophoblasts suggest that VE-cadherin mainly regulates cell invasion and that ECM remodeling defects are likely secondary consequences of decreased trophoblast invasion. A second interesting aspect of Cdh5 knockout placentas is persistent innate immune cells within the decidua. uNK cells (called decidual NK cells or dNK cells in humans) are present in the mouse decidua at E6.5, prior to formation and invasion of trophoblasts, and decline in number beginning at E12.5 (Rajagopalan, 2014; Sojka et al., 2018). uNK cells also secrete factors such as VEGF-C that promote SA remodeling (Pawlak et al., 2019). Significantly, increased dNK cells is also characteristic of preeclamptic placentas (Zhang et al., 2019), similar to our mouse model. Our findings raise the possibility that trophoblast migration into the decidua may coordinate decidual matrix and immune changes that promote SA remodeling.

In humans, defective SA remodeling and shallow trophoblast invasion are hallmarks of preeclampsia. Preeclampsia is a complex and heterogeneous disease with maternal and fetal contributions to its pathogenesis, and many in vitro models fail to fully recapitulate many aspects of its pathophysiology. Most rodent models of preeclampsia utilize maternal genetic or pharmacological perturbations (Gatford et al., 2020; Marshall et al., 2018), and there have been few in vivo models in which preeclamptic features are recapitulated with fetal modulation of trophoblasts. Previous studies of human placentas showed that invasive trophoblasts in severely preeclamptic placentas exhibit reduced expression of VE-cadherin (Zhou et al., 1997a) however whether this is a cause or consequence of placental dysfunction has been unclear. Our mouse model utilizing trophoblast-specific knockout of VE-cadherin exhibits many histopathological and clinical features of preeclampsia and suggests that loss of VE-cadherin in trophoblasts may be a primary contributor to preeclampsia pathogenesis. Additionally, we observe secondary defects in organogenesis and vascular development in the embryo (Perez-Garcia et al., 2018), which has also been linked to placentation defects. Interestingly, VEGF-A is known to induce VE-cadherin expression in cultured trophoblasts (Chang et al., 2005) and may therefore be a useful strategy in treating preeclampsia. Trophoblast-specific loss of VE-cadherin may serve as a useful model for studying fetal contributions to preeclampsia.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculcus)CYP19A1(Tg)-CreWenzel and Leone, 2007
Genetic reagent (Mus musculcus)Cdh5 floxYang et al., 2019
AntibodyAnti-Endomucin (goat polyclonal)R&DAF4666IF(1:400)
AntibodyAnti-Endomucin (rat monoclonal)Abcamab106100IF(1:300)
AntibodyAnti-TER119 (rat monoclonal)Abcamab91113IF(1:300)
AntibodyAnti-VE-cadherin (goat polycloncal)R&DAF1002IF(1:200)
AntibodyAnti-CK8 (rabbit monoclonal)Abcamab53280IF(1:300)
AntibodyAnti-CK8 (rat monoclonal)DSHBTROMA-1IF(1:400)
AntibodyAnti-αSMA-Cy3 (mouse monoclonal)SigmaC6198IF(1:300)
AntibodyAnti-Cleaved Caspase-3 (rabbit polyclonal)Millipore SigmaAB3623IF(1:100)
AntibodyAnti-MMP15 (rabbit polyclonal)Thermo Fisher ScientificPA5-13184IF(1:200)
AntibodyAnti-Laminin (rabbit polyclonal)SigmaL9393IF(1:200)
AntibodyAnti-Vimentin (goat polyclonal)R&DAF2105IF(1:300)
AntibodyAnti-Vinculin (mouse monoclonal)SigmaV9131IF(1:200)
Commercial assay or kitDirect-zol RNA Miniprep KitsZymo ResearchR2053
Software, algorithmImageJNIH, Bethesda, MD, USARRID:SCR_003070
Software, algorithmGraphPad PrismGraphPadRRID:SCR_002798
Software, algorithmPicard v2.17.11PicardRRID:SCR_006525
OtherDBA-BiotinVector LabsB-1035(1:500)

Generation of mutant mice

Request a detailed protocol

CYP19A1(Tg)-Cre mice have been previously described (Wenzel and Leone, 2007) in which the transgene relies on Cre expression under a 501 bp region with the first exon of human CYP19A1 containing regulatory elements for trophoblast-specific expression, as CYP19A1 is not endogenously expressed in trophoblasts. VE-cadherin (Cdh5) floxed mice have been previously described (Yang et al., 2019) and were generated with LoxP sites flanking exons 3 and 4. Mice were bred according to standard protocols and maintained on a mixed background. Male Cdh5fl/fl mice were mated to female CYP19A1(Tg)Cre; Cdh5fl/+ mice due to the influence of parental inheritance on Cre expression, with maternal inheritance providing the most robust and consistent expression (Wenzel and Leone, 2007). Mating pairs were set up in the afternoon and vaginal plugs checked in the morning. Presence of a vaginal plug indicated embryonic day (E)0.5. Cre-negative (Cdh5fl/+ and Cdh5fl/fl) and Cre-positive heterozygous (CYP19A1(Tg)Cre; Cdh5fl/+) littermates were used as controls. All procedures were conducted using an approved animal protocol (806811) in accordance with the University of Pennsylvania Institutional Animal Care and Use Committee.

Intrauterine Doppler ultrasound

Request a detailed protocol

In utero Doppler ultrasound was performed by a trained technician using the VEVO2100 Ultrasound System equipped with the MS-400 transducer (30 MHz). E12.5 pregnant mice were lightly anesthetized using 2% isoflurane. Hair was removed from the abdomen using chemical hair remover (Nair), and the animals were placed on a warming pad. Maternal heart rate and temperature were continuously monitored and consistently within 400–500 bpm and 37°C. Ultrasound gel was applied to the abdomen and the transducer applied to visualize embryos and placentas using the maternal bladder as an anatomical landmark. Color Doppler was used to visualize the umbilical vessels, and pulse wave (angle of insonation <60°) measurements were made at the point where the umbilical artery inserts into the placenta. From the Doppler waveforms, PSV and EDV were measured and used to calculate resistance index [RI = 1 – PSV/EDV] and pulsatility index [PI = (PSV – EDV)/mean velocity, where mean velocity = (PSV +EDV)/2]. Heart rate was calculated as beats per minute by dividing 60 s by the systolic + diastolic time.

Histology and immunofluorescence staining and analysis

Request a detailed protocol

Whole mouse embryos or placentas were collected and fixed in 4% paraformaldehyde (PFA) overnight at 4°C prior to dehydration in alcohol and paraffin embedding. Tissue sections underwent to dewaxing and rehydration through xylene and ethanol treatment and were then subject to H&E staining or processed for immunofluorescence. For immunodetection, 10 mM citrate buffer (pH 6) was used for antigen retrieval, and sections were blocked with 10% donkey serum in 1% BSA prior to primary antibody treatment overnight at 4°C. A list of antibodies can be found below. Fluorescence-conjugated Alexa Fluor secondary antibodies were used (1:500, Invitrogen) according to the primary antibody species and counterstained with DAPI (1:1000). Sections or tissues were mounted on slides with ProLong Gold Antifade reagent. Signals were detected and images collected using a Zeiss LSM 880 confocal microscope and Zeiss Axio Observer 7 widefield microscope. Images were visualized using ImageJ/FIJI software (NIH).

Whole-mount immunofluorescence

Request a detailed protocol

Whole mouse placentas were collected and fixed in 4% PFA overnight at 4°C and then placed in 1× PBS. Placentas were embedded in 3% low-melt agarose and cut into 200 μm thick sections using a vibratome. Sections were permeabilized with 0.2% Triton X-100 and blocked with 10% donkey serum in 1% BSA prior to primary antibody treatment overnight at 4°C. Fluorescence-conjugated Alexa Fluor secondary antibodies were used (1:500, Invitrogen) according to the primary antibody species and counterstained with DAPI (1:1000). Sections were mounted on a glass slide in a silicone isolator and filled with ProLong Gold Antifade reagent. Signals were detected and images collected using a Zeiss Axio Observer 7 widefield microscope with the Apotome 3 attachment for optical sectioning. Raw images were deconvoluted using ZEN Blue. Maximum intensity projections were generated using ImageJ/FIJI software (NIH).

Quantification of immunofluorescence images

Request a detailed protocol

Number of CK8+ trophoblasts were manually counted and divided by total decidual area. Trophoblast invasion depth was measured as the distance the farthest CK8+ trophoblast was found in the decidua relative to the junctional zone. Percent labyrinth EC coverage was measured by quantifying Endomucin-positive area as a percentage of total labyrinth area. Number of αSMA+ smooth muscle cells were manually counted and divided by number of SAs. SA diameter was manually measured using only circular SAs, taking the average of the long- and short-axis diameters, and averaging at least five SAs per section per placenta. Trophoblast-endothelial displacement was quantified by measuring the circumference of the vessel lumen and then measuring the length of CK8+ trophoblasts in contact with the lumen. Percent displacement was calculated by dividing the trophoblast length by the lumen circumference. VE-cadherin deletion was quantified by measuring VE-cadherin+CK8+ area (determined by Colocalization Threshold plugin in FIJI) and calculating it as a percent of CK8+ area. The Colocalization Threshold plugin was also used to generate colocalization images in Figure 1—figure supplement 3C. Laminin and vinculin mean fluorescence intensity was calculated by dividing total fluorescence intensity by area. All images were analyzed using ImageJ/FIJI software.

Bulk RNA-seq

Request a detailed protocol

Total RNA was extracted from E12.5 deciduas (n = 3 per genotype) using Direct-zol RNA miniprep kit (Zymo Research). Quality assessment, cDNA library synthesis, and sequencing using Illumina HiSeq with a 2 × 150 configuration were conducted through GeneWiz. Fastq files were assessed for quality control using the FastQC program (v0.11.7). They were then aligned against the mouse reference genome (mm39) using the STAR aligner (v2.7.8a) (Dobin et al., 2013). Duplicate reads were flagged using the MarkDuplicates program from Picard tools (v2.17.11) (http://broadinstitute.github.io/picard/). Per gene read counts for Ensembl (GRCm39) gene annotations were computed using the R package Rsubread (Liao et al., 2019) and duplicate reads were removed. Gene counts were normalized as counts per million (CPM) using the R package edgeR (Robinson et al., 2010) and genes with CPM < 1 in 25% of samples were filtered out. The data was transformed using the VOOM function from the limma R package (Law et al., 2014). Differential gene expression was performed as a paired analysis using limma. p-Values were adjusted for multiple comparisons using Benjamini-Hochberg procedure. Genes with adjusted p-values less than 0.05 and an absolute log2-fold change >1 were considered significantly differentially expressed genes. The RNA-seq data set has been deposited in the NCBI GEO under accession ID number GSE189408.

Statistical analysis

Request a detailed protocol

All data are reported as means with n ≥ 3 independent experiments or mice, and error bars represent standard deviation. Each data point in the figures represents one individual placental or embryo. The explicit number of samples is indicated in the figure legends. No explicit power analyses were used to predetermine sample size, and no randomization was used. No samples were excluded for analysis. Statistical significance was determined using Welch’s t-test. Differences between means were considered significant at p < 0.05. Significant differences in expected genotypes was calculated using two-tailed Fisher’s exact test and considered statistically significant at p < 0.05.

Data availability

Source Data files have been included for Figure 1, Figure 1—figure supplement 1, Figure 1—figure supplement 2, Figure 1—figure supplement 3, Figure 2, Figure 3, Figure 4—figure supplement 1, and Figure 4—figure supplement 2. All reagents have been listed in the Methods section in this paper. The RNA-seq data set has been deposited in the NCBI GEO under accession ID number GSE189408. Investigators interested in the animals used in this study should contact Dr. Jeremy Veenstra-Vanderweele (Columbia University), Dr. Gustsavo Leone (Medical University of South Carolina), and Dr. Joshua Scallan (University of South Florida).

The following data sets were generated
    1. Derek C S
    2. Mark L K
    3. Apoorva B
    (2021) NCBI Gene Expression Omnibus
    ID GSE189408. VE-cadherin is essential for trophoblast migration and endovascular invasion.

References

Decision letter

  1. Gou Young Koh
    Reviewing Editor; Institute of Basic Science and Korea Advanced Institute of Science and Technology (KAIST), Republic of Korea
  2. Ricardo Azziz
    Senior Editor; University at Albany, SUNY, United States
  3. Lena Claesson-Welsh
    Reviewer; Uppsala University, Sweden
  4. Gou Young Koh
    Reviewer; Institute of Basic Science and Korea Advanced Institute of Science and Technology (KAIST), Korea (South), Republic of

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Vascular mimicry by VE-cadherin enables trophoblast endovascular invasion and spiral artery remodeling during placental development" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Gou Young Koh as Reviewing Editor and Reviewer #3, and the evaluation has been overseen by Ricardo Azziz as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Lena Claesson-Welsh (Reviewer #1);

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

The comments of all three reviewers are in good agreement. While the reviewers found this study is well-designed and well-presented, high quality, and to be of importance as a translational research, they raised concerns about the lack of mechanistic insights to support the authors' claims. The authors are required to carefully address the comments point-by-point in a data-driven manner or with further analyses and discussions. Specifically, the authors are encouraged to pay attention to the major comment 1 of reviewer 1, comments 1 and 3 of reviewer 2, and comment 3 of reviewer 3.

Reviewer #1 (Recommendations for the authors):

1. I would like to question the use of the term "Vascular mimicry" in the context of trophoblast invasion and spiral artery remodelling since it is so strongly associated with the formation of non-endothelial channels in cancer, independently of normal blood vessels or angiogenesis. VE-cadherin is clearly not uniquely expressed in blood vascular endothelial cells. Moreover, as stated by the authors, the role of VE-cadherin in trophoblasts appears to be quite different from that in blood vascular endothelial cells. Perhaps endothelial mimicry is a better term, alternatively, just use "endovascular invasion".

2. Deletion of Cdh5 from trophoblasts is shown in Figure supplement 1 but it would be important to also have the data quantified.

3. The authors state that Cre-negative (Cdh5fl/+ and Cdh5fl/fl) and Cre-positive heterozygous (Cyp19Cre; Cdh5fl/+) littermates were used as controls. Since Cre toxicity is a well-known problem, it should be shown which type of control mice were used in which experiments.

Reviewer #2 (Recommendations for the authors):

1) It should be demonstrated that Cyp19-Cre driven gene inactivation of VE-cadherin does not affect VE-cadherin expression in endothelial cells of the embryo in tissues where bleeding was observed.

2) The results shown in figures 2 and 3 should be confirmed by whole mount staining of either vibratome sections or cleared tissue.

3) It should be attempted to get some mechanistic insights by analyzing the effects of VE-cadherin silencing on the expression of genes that could be relevant for cell migration, collective migration or association of trophoblast cell clusters.

4) The discussion is rather short and does not attempt to debate mechanistic aspects of the findings.

Reviewer #3 (Recommendations for the authors):

The questions and suggestions below are therefore not intended to trigger significant additional experimental work for the present paper, but to perhaps increase clarity and make the study more accessible to readers.

1. Please explain why the Cdh5 knockout embryos exhibited marked hemorrhage at E12.5 (Figure 1E).

2. What about the diameter of the spiral artery in the Cdh5 knockout placentas versus WT placentas?

3. It would be nice if the authors performed scRNA-seq of the trophoblasts of WT and Cdh5 knockout placentas and compared them to provide molecular insight into how the lack of Cdh5 in the trophoblasts led to such abnormalities.

4. Please describe any plausible ways to normalize Cdh5 level in the trophoblasts for enhanced endovascular invasion, which can be an alternative way for prevention of preeclampsia.

https://doi.org/10.7554/eLife.77241.sa1

Author response

Reviewer #1 (Recommendations for the authors):

1. I would like to question the use of the term "Vascular mimicry" in the context of trophoblast invasion and spiral artery remodelling since it is so strongly associated with the formation of non-endothelial channels in cancer, independently of normal blood vessels or angiogenesis. VE-cadherin is clearly not uniquely expressed in blood vascular endothelial cells. Moreover, as stated by the authors, the role of VE-cadherin in trophoblasts appears to be quite different from that in blood vascular endothelial cells. Perhaps endothelial mimicry is a better term, alternatively, just use "endovascular invasion".

We agree that the term “vascular mimicry” is not appropriate given its use in cancer biology and non-endothelial functions of trophoblasts. To avoid confusion, we have removed it from the title of our manuscript (which now reads as “VE-cadherin enables trophoblast endovascular invasion and spiral artery remodeling during placental development”) and from our revised discussion.

2. Deletion of Cdh5 from trophoblasts is shown in Figure supplement 1 but it would be important to also have the data quantified.

In the revised manuscript we provide new data quantifying VE-cadherin deletion in trophoblasts. Briefly, we utilized the Colocalization Threshold plugin in FIJI to determine area of VE-cadherin+CK8+ positivity in the decidua. This area was divided by the total CK8+ area and taken as a percentage. We found an approximately 80% decrease in VE-cadherin expression in trophoblasts in knockout placentas vs. controls. These data are now included as Figure 1—figure supplement 1A, B and the detailed in the Methods section. As noted in our response to Reviewer 3’s comments, the alternative approach of measuring CDH5 using flow cytometry of trophoblasts is technically difficult due to their large cell size.

3. The authors state that Cre-negative (Cdh5fl/+ and Cdh5fl/fl) and Cre-positive heterozygous (Cyp19Cre; Cdh5fl/+) littermates were used as controls. Since Cre toxicity is a well-known problem, it should be shown which type of control mice were used in which experiments.

The controls in every experiment and analysis consists of embryos and placentas of both Cre-negative (Cdh5fl/+ and Cdh5fl/fl) and Cre-positive heterozygous (Cyp19Cre; Cdh5fl/+) genotypes. Cyp19Cre; Cdh5fl/+ mice are born at expected mendelian ratios, survive until adulthood, and do not exhibit any notable defects compared to Cdh5fl/+ and Cdh5fl/fl mice. To further assess an effect of Cre expression on placentation we have compared the weights of Cdh5fl/+ and Cdh5fl/fl vs. Cyp19Cre; Cdh5fl/+ E12.5 placentas and embryos. These studies revealed no differences (now shown as Figure 1—figure supplement 1C, D). Lastly, we have included a Source Data File containing exact quantification and statistical values for each graph in the figures along with specific genotypes, including a specific breakdown of Cre-negative and Cre-positive heterozygous controls.

Reviewer #2 (Recommendations for the authors):

1) It should be demonstrated that Cyp19-Cre driven gene inactivation of VE-cadherin does not affect VE-cadherin expression in endothelial cells of the embryo in tissues where bleeding was observed.

The Cyp19-Cre is a very interesting Cre transgene. It was shown in the original paper describing these mice that the 501bp region within the first untranslated exon contains regulatory sequences specific for trophoblasts, whereas exons 2-10 encode the actual cytochrome P450 protein coding sequences that are expressed in liver, brain, gonads, etc1. The authors in this original paper specifically chose a clone in which Cre activity was present in trophoblasts and not in the embryo by LacZ reporter and X-gal staining, and multiple studies since then using fluorescent reporters also show no Cre activity in the embryo2–4.

Reviewer 2’s concerns regarding fetal hemorrhage due to embryonic loss of VE-cadherin were shared by Reviewer #3. Several lines of evidence support the conclusion that embryonic hemorrhage is secondary to placental defects rather than loss of VE-cadherin in embryo endothelial cells. First, staining of E12.5 Cyp19Cre; Cdh5fl/fl E12.5 embryos for VE-cadherin shows that VE-cadherin levels are unaffected in endothelial cells within the embryo, including in the heart, liver, lungs, thorax, and at sites of hemorrhage in the brain. These new data are shown in Figure 1—figure supplement 3A-D. Second, we observed highly variable degrees of hemorrhaging in knockout embryos, consistent with a secondary event due to placental defects. To better demonstrate this, we have included additional images of an embryo and placenta in Figure 1. Finally, placental defects have been shown to confer secondary defects in vascular development5. We conclude that hemorrhage observed in knockout embryos likely results from global ischemia secondary to reduced maternal blood flow rather than loss of VE-cadherin within the embryonic vasculature. We have revised the results and discussion to better address the potential causes of embryonic hemorrhage.

2) The results shown in figures 2 and 3 should be confirmed by whole mount staining of either vibratome sections or cleared tissue.

This is an excellent suggestion by the reviewer. We performed whole-mount immunofluorescence of thick vibratome sections from control and Cdh5 knockout placentas. Optical sectioning and reconstruction with maximum intensity projections further support data from thin paraffin sections. In contrast to control placentas, Cdh5 knockout placentas maintain a layer of spiral artery endothelial cells despite being surrounded by trophoblasts and have decreased lumen size compared with control, trophoblast-lined spiral arteries. In the labyrinth, fewer maternal RBCs are observed in Cdh5 knockout placentas while the fetal vasculature is unaffected. These data are now included as Figure 2G, H. We thank the reviewer for these and other valuable suggestions.

3) It should be attempted to get some mechanistic insights by analyzing the effects of VE-cadherin silencing on the expression of genes that could be relevant for cell migration, collective migration or association of trophoblast cell clusters.

More detailed mechanistic insight obtained through analysis of gene expression was also requested by Reviewer #3, who suggested scRNA-seq to look at trophoblast gene expression. As discussed below, technical limitations precluded us from specifically isolating invasive trophoblasts for transcriptional analysis. We instead performed bulk RNA-seq on whole control and Cdh5 knockout deciduas. Analysis of the top 100 differentially regulated genes showed multiple genes related to decidual stromal dysfunction6. Additionally, GO analysis showed upregulation in pathways related to the ECM and immune cells in Cdh5 knockout placentas. These data are now included as Figure 4 in the manuscript.

Based on these RNA-seq data, we further investigated the membrane metalloprotease MMP15, which is associated with trophoblast invasion, and its ECM target laminin7–9. We also examined vinculin, a focal adhesion protein that promotes cell invasion. While we observed increased laminin in the knockout decidua, we found this was not due to a cell autonomous loss of MMP15. However, vinculin levels in trophoblasts were decreased which likely contributes to diminished trophoblast invasion. These data are now included as Figure 4—figure supplement 1. Based on GO analysis, we also evaluated uterine natural killer (uNK) cells in the decidua, which are the only innate immune cells found in the maternal decidual compartment. We found that Cdh5 knockout placentas had elevated uNK cells due to decreased apoptosis of those cells. As discussed in the revised manuscript, we believe that the uNK phenotype is secondary to failure of trophoblast migration into the decidua. These data are now included as Figure 4—figure supplement 2 in the manuscript. Together, these studies suggest that VE-cadherin contributes to focal adhesion formation in trophoblasts, enabling their migration and remodeling of the decidual ECM and immune cell microenvironment.

4) The discussion is rather short and does not attempt to debate mechanistic aspects of the findings.

In light of our new results, we have revised the discussion to incorporate more mechanistic aspects of our work, such as how VE-cadherin regulates vinculin expression in trophoblasts to promote invasion to trigger decidual ECM remodeling and immune changes that affects spiral artery remodeling. Additionally, we have incorporated discussion of potential strategies for promoting VE-cadherin expression in trophoblasts as suggested by Reviewer 3.

Reviewer #3 (Recommendations for the authors):

The questions and suggestions below are therefore not intended to trigger significant additional experimental work for the present paper, but to perhaps increase clarity and make the study more accessible to readers.

1. Please explain why the Cdh5 knockout embryos exhibited marked hemorrhage at E12.5 (Figure 1E).

As discussed above in response to Reviewer 2’s first comment, placental defects have been reported to cause secondary defects in vascular development5. In the revised manuscript we analyze VE-cadherin expression in control and knockout embryo vessels and demonstrate that hemorrhage and growth restriction are not due to loss of endothelial VE-cadherin within the embryo (Figure 1—figure supplement 3A-D). VE-cadherin is present in the vessels of all organs in control and knockout embryos, including the brain, heart, liver, lungs, and thorax. Thus, hemorrhage observed in knockout embryos likely results from tissue degradation associated with global ischemia secondary to reduced maternal blood flow rather than loss of VE-cadherin within the embryonic vasculature. Additionally, we wished to clarify that we observed highly variable degrees of hemorrhaging in knockout embryos. To better demonstrate this, we have included additional images of a knockout placenta and embryo in. We have revised the results and discussion to better explain potential causes of embryonic hemorrhage.

2. What about the diameter of the spiral artery in the Cdh5 knockout placentas versus WT placentas?

We thank Reviewer 3 for this excellent suggestion. Indeed, previous studies have shown that reduced remodeling of the spiral arteries and retention of vascular smooth muscle cells results decreased spiral artery lumen diameter10. Since the spiral arteries are morphologically tortuous, we measured diameters of spiral arteries only from those with circular cross sections (detailed in the Methods section). We found that Cdh5 knockout placentas had significantly smaller spiral artery diameters compared to controls, consistent with defects in spiral artery remodeling (revised Figure 2C).

3. It would be nice if the authors performed scRNA-seq of the trophoblasts of WT and Cdh5 knockout placentas and compared them to provide molecular insight into how the lack of Cdh5 in the trophoblasts led to such abnormalities.

We agree with the reviewer that single cell RNA-seq of placentas would be very insightful. Transcriptional analysis of trophoblasts at the single cell level in mice is technically challenging since trophoblast cells are extremely large and not amenable to single cell isolation. Some groups have overcome this with single nucleus RNA-seq; however this is also technically challenging since some trophoblasts are polyploid. We therefore performed bulk RNA-seq on whole deciduas from WT and Cdh5 knockout placentas. Bulk RNA-seq of whole deciduas from WT and Cdh5 knockout placentas revealed that several processes are disrupted in knockout placentas. Analysis of the top 100 differentially regulated genes showed multiple genes related to decidual stromal dysfunction6. Additionally, GO analysis showed upregulation in pathways related to the ECM and immune cells in Cdh5 knockout placentas. These data are included as a new Figure 4 in the manuscript.

Based on these RNA-seq data, we further investigated the membrane metalloprotease MMP15, which is associated with trophoblast invasion, and its ECM target laminin7–9. We also examined vinculin, a focal adhesion protein that promotes cell invasion. While we observed increased laminin in the decidua, we found this was not due to a cell autonomous loss of MMP15. However, vinculin levels in trophoblasts were decreased which likely contributes to diminished trophoblast invasion. These data are now included as Figure 4—figure supplement 1. Based on GO analysis, we also evaluated uterine natural killer (uNK) cells in the decidua, which are the only innate immune cells found in the maternal decidual compartment. We found that Cdh5 knockout placentas had elevated uNK cells due to decreased apoptosis. These data are now included as Figure 4—figure supplement 2 in the manuscript. Together, these studies suggest that loss VE-cadherin disrupts focal adhesions in trophoblasts, resulting in decreased migration and failure to remodel the decidual ECM and immune cell microenvironment.

4. Please describe any plausible ways to normalize Cdh5 level in the trophoblasts for enhanced endovascular invasion, which can be an alternative way for prevention of preeclampsia.

We agree that identification of strategies that promote Cdh5 expression in trophoblasts may be a valuable opportunity for treatment of preeclampsia. One study has shown that treatment of primary rat trophoblasts with VEGF-A induces VE-cadherin expression in vitro11. Thus, VEGF-A administration may be a useful therapy in promoting trophoblast invasion through upregulation of VE-cadherin. We have incorporated these potential strategies into our discussion. We thank Reviewer 3 for this and the other valuable suggestions.

References

1. Wenzel PL, Leone G. Expression of Cre recombinase in early diploid trophoblast cells of the mouse placenta. genesis. 2007;45(3):129-134. doi:10.1002/dvg.20276

2. López-Tello J, Pérez-García V, Khaira J, et al. Fetal and trophoblast PI3K p110α have distinct roles in regulating resource supply to the growing fetus in mice. eLife. 2019;8:1-25. doi:10.7554/eLife.45282.001

3. Sandovici I, Georgopoulou A, Pérez-García V, et al. The imprinted Igf2-Igf2r axis is critical for matching placental microvasculature expansion to fetal growth. Dev Cell. 2022;57(1):63-79.e8. doi:10.1016/j.devcel.2021.12.005

4. Vacher CM, Lacaille H, O’Reilly JJ, et al. Placental endocrine function shapes cerebellar development and social behavior. Nat Neurosci. 2021;24(10):1392-1401. doi:10.1038/s41593-021-00896-4

5. Perez-Garcia V, Fineberg E, Wilson R, et al. Placentation defects are highly prevalent in embryonic lethal mouse mutants. Nature. 2018;555(7697):463-468. doi:10.1038/nature26002

6. Woods L, Perez-Garcia V, Kieckbusch J, et al. Decidualisation and placentation defects are a major cause of age-related reproductive decline. Nat Commun. 2017;8(1):1-14. doi:10.1038/s41467-017-00308-x

7. Majali-Martinez A, Hoch D, Tam-Amersdorfer C, et al. Matrix metalloproteinase 15 plays a pivotal role in human first trimester cytotrophoblast invasion and is not altered by maternal obesity. FASEB J. 2020;34(8):10720-10730. doi:10.1096/fj.202000773R

8. Pollheimer J, Fock V, Knöfler M. Review: The ADAM metalloproteinases – Novel regulators of trophoblast invasion? Placenta. 2014;35(SUPPL):57-63. doi:10.1016/j.placenta.2013.10.012

9. Majali-Martinez A, Hiden U, Ghaffari-Tabrizi-Wizsy N, Lang U, Desoye G, Dieber-Rotheneder M. Placental membrane-type metalloproteinases (MT-MMPs): Key players in pregnancy. Cell Adhes Migr. 2016;10(1-2):136-146. doi:10.1080/19336918.2015.1110671

10. Burke SD, Barrette VF, Bianco J, et al. Spiral arterial remodeling is not essential for normal blood pressure regulation in pregnant mice. Hypertension. 2010;55(3):729-737. doi:10.1161/HYPERTENSIONAHA.109.144253

11. Chang CC, Chang TY, Yu CH, Tsai ML. Induction of VE-cadherin in rat placental trophoblasts by VEGF through a NO-dependent pathway. Placenta. 2005;26(2-3):234-241. doi:10.1016/j.placenta.2004.06.002

https://doi.org/10.7554/eLife.77241.sa2

Article and author information

Author details

  1. Derek C Sung

    Cardiovascular Institute, Department of Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Conceptualization, Investigation, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6966-2596
  2. Xiaowen Chen

    Cardiovascular Institute, Department of Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Investigation, Validation
    Competing interests
    No competing interests declared
  3. Mei Chen

    Cardiovascular Institute, Department of Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Jisheng Yang

    Cardiovascular Institute, Department of Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  5. Susan Schultz

    Department of Radiology, Hospital of the University of Pennsylvania, Philadelphia, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  6. Apoorva Babu

    Cardiovascular Institute, Department of Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Data curation, Software
    Competing interests
    No competing interests declared
  7. Yitian Xu

    Cardiovascular Institute, Department of Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  8. Siqi Gao

    Cardiovascular Institute, Department of Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Formal analysis, Investigation, Methodology
    Competing interests
    No competing interests declared
  9. TC Stevenson Keller IV

    Cardiovascular Institute, Department of Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  10. Patricia Mericko-Ishizuka

    Cardiovascular Institute, Department of Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Methodology, Project administration
    Competing interests
    No competing interests declared
  11. Michelle Lee

    University Laboratory Animal Resources, University of Pennsylvania, Philadelphia, United States
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  12. Ying Yang

    Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, United States
    Contribution
    Resources
    Competing interests
    No competing interests declared
  13. Joshua P Scallan

    Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, United States
    Contribution
    Resources
    Competing interests
    No competing interests declared
  14. Mark L Kahn

    Cardiovascular Institute, Department of Medicine, University of Pennsylvania, Philadelphia, United States
    Contribution
    Conceptualization, Investigation, Methodology, Supervision, Validation, Writing - original draft, Writing - review and editing
    For correspondence
    markkahn@pennmedicine.upenn.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6489-7086

Funding

National Institutes of Health (T32 HL007439)

  • Derek C Sung

National Institutes of Health (F30 HL158014)

  • Derek C Sung

American Heart Association (Postdoctoral Fellowship 35200213)

  • Xiaowen Chen

National Institutes of Health (T32 HL007971)

  • Thomas C Stevenson Keller

American Heart Association (Postdoctoral fellowship 836238)

  • Thomas C Stevenson Keller

National Institutes of Health (HL142905)

  • Joshua P Scallan

National Institutes of Health (HL145397)

  • Ying Yang

National Institutes of Health (HL142976)

  • Mark L Kahn

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank members of the Kahn laboratory and Mainigi laboratory for thoughtful discussions during the course of these studies, the CDB Microscopy Core for support with microscopy, and the Small Animal Imaging Facility Core for their support with ultrasound imaging. We thank Dr Jeremy Veenstra-Vanderweele (Columbia University) and Dr Gustsavo Leone (Medical University of South Carolina) for kindly providing the CYP19A1(Tg)Cre mice. This work was supported by NIH grants T32 HL007439 and F30 HL158014 (to DCS), American Heart Association Postdoctoral Fellowship No. 35200213 (to XC), NIH grant T32 HL007971 and American Heart Association Postdoctoral Fellowship No. 836,238 (to TCSK), NIH R01 grant HL142905 (to JPS), NIH R01 grant HL145397 (to YY), and NIH R01 grant HL142976 (to MLK).

Ethics

All procedures were conducted using an approved animal protocol (806811) in accordance with the University of Pennsylvania Institutional Animal Care and Use Committee.

Senior Editor

  1. Ricardo Azziz, University at Albany, SUNY, United States

Reviewing Editor

  1. Gou Young Koh, Institute of Basic Science and Korea Advanced Institute of Science and Technology (KAIST), Republic of Korea

Reviewers

  1. Lena Claesson-Welsh, Uppsala University, Sweden
  2. Gou Young Koh, Institute of Basic Science and Korea Advanced Institute of Science and Technology (KAIST), Korea (South), Republic of

Publication history

  1. Received: January 21, 2022
  2. Preprint posted: February 16, 2022 (view preprint)
  3. Accepted: April 28, 2022
  4. Accepted Manuscript published: April 29, 2022 (version 1)
  5. Version of Record published: May 13, 2022 (version 2)

Copyright

© 2022, Sung et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,380
    Page views
  • 316
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Derek C Sung
  2. Xiaowen Chen
  3. Mei Chen
  4. Jisheng Yang
  5. Susan Schultz
  6. Apoorva Babu
  7. Yitian Xu
  8. Siqi Gao
  9. TC Stevenson Keller IV
  10. Patricia Mericko-Ishizuka
  11. Michelle Lee
  12. Ying Yang
  13. Joshua P Scallan
  14. Mark L Kahn
(2022)
VE-cadherin enables trophoblast endovascular invasion and spiral artery remodeling during placental development
eLife 11:e77241.
https://doi.org/10.7554/eLife.77241

Further reading

    1. Developmental Biology
    2. Stem Cells and Regenerative Medicine
    Jakke Neiro, Divya Sridhar ... Aziz Aboobaker
    Research Article Updated

    Planarians have become an established model system to study regeneration and stem cells, but the regulatory elements in the genome remain almost entirely undescribed. Here, by integrating epigenetic and expression data we use multiple sources of evidence to predict enhancer elements active in the adult stem cell populations that drive regeneration. We have used ChIP-seq data to identify genomic regions with histone modifications consistent with enhancer activity, and ATAC-seq data to identify accessible chromatin. Overlapping these signals allowed for the identification of a set of high-confidence candidate enhancers predicted to be active in planarian adult stem cells. These enhancers are enriched for predicted transcription factor (TF) binding sites for TFs and TF families expressed in planarian adult stem cells. Footprinting analyses provided further evidence that these potential TF binding sites are likely to be occupied in adult stem cells. We integrated these analyses to build testable hypotheses for the regulatory function of TFs in stem cells, both with respect to how pluripotency might be regulated, and to how lineage differentiation programs are controlled. We found that our predicted GRNs were independently supported by existing TF RNAi/RNA-seq datasets, providing further evidence that our work predicts active enhancers that regulate adult stem cells and regenerative mechanisms.

    1. Developmental Biology
    Javier Solivan-Rivera, Zinger Yang Loureiro ... Silvia Corvera
    Research Article Updated

    Mechanisms that control ‘beige/brite’ thermogenic adipose tissue development may be harnessed to improve human metabolic health. To define these mechanisms, we developed a species-hybrid model in which human mesenchymal progenitor cells were used to develop white or thermogenic/beige adipose tissue in mice. The hybrid adipose tissue developed distinctive features of human adipose tissue, such as larger adipocyte size, despite its neurovascular architecture being entirely of murine origin. Thermogenic adipose tissue recruited a denser, qualitatively distinct vascular network, differing in genes mapping to circadian rhythm pathways, and denser sympathetic innervation. The enhanced thermogenic neurovascular network was associated with human adipocyte expression of THBS4, TNC, NTRK3, and SPARCL1, which enhance neurogenesis, and decreased expression of MAOA and ACHE, which control neurotransmitter tone. Systemic inhibition of MAOA, which is present in human but absent in mouse adipocytes, induced browning of human but not mouse adipose tissue, revealing the physiological relevance of this pathway. Our results reveal species-specific cell type dependencies controlling the development of thermogenic adipose tissue and point to human adipocyte MAOA as a potential target for metabolic disease therapy.