Reversal of gene dysregulation in cultured cytotrophoblasts reveals possible causes of preeclampsia (original) (raw)
Ex vivo normalization of sPE CTB gene expression. We used global transcriptional profiling to explore mRNA changes that underlie CTB defects in various forms of sPE. Villous CTBs were isolated from sPE placentas and placentas of preterm labor patients with no signs of infection (nPTL), which served as gestation-matched controls. Our previous work showed that CTB invasion is essentially normal in the latter group (17), and we confirmed this finding for the samples in this study. However, we cannot eliminate the possibility of other placental pathologies relative to normal controls, which are not possible to collect at the relevant gestational ages. The newborns did not differ by birth weight or gestational age at delivery (Supplemental Table 1). The patients with sPE and nPTL had comparable BMIs and ages, but women with sPE had higher systolic/diastolic blood pressures and proteinuria. To better understand the CTB phenotype in the context of sPE variants, we included patients diagnosed with the most clinically significant forms of this condition that necessitated preterm delivery (18, 19): women with sPE with or without IUGR, superimposed hypertension, or HELLP syndrome with or without IUGR. We profiled the gene expression patterns of the case and control groups before plating (0 hour) and at 12, 24, and 48 hours after culture using the Affymetrix HG-U133Plus 2.0 GeneChip platform. With LIMMA, we identified numerous genes that were differentially expressed — a common CTB fingerprint — in nearly all the sPE samples at 1 or more time points (≥2 fold; P ≤ 0.05; Figure 1). Surprisingly, after 48 hours in culture most were expressed at control levels. Unsupervised hierarchical clustering showed that the gene expression patterns of sPE and nPTL samples, which segregated into their respective groups at 0 hours, merged at 48 hours (data not shown). The initially upregulated molecules included factors previously associated with PE (e.g., growth hormone 2, corticotropin-releasing hormone, inhibin A, KISS-1, ADAM-12) (20–23), a transcriptional regulator (HOPX), and an angiogenic factor (SEMA3B). In addition to growth hormone 2, other placenta-specific products, including PLAC1 and PLAC4, and 7 pregnancy-specific glycoprotein (PSG) family members, were also upregulated as was an enzyme involved in fat metabolism (oleoyl-ACP hydrolase). Many fewer genes were downregulated. A subset of the results was confirmed by qRT-PCR (Supplemental Figure 2). The fact that a common set of dysregulated genes was associated with a broad spectrum of the maternal signs suggested that a complex interplay between abnormal placentation and patient-specific factors ultimately determined the clinical features. The finding that most sPE-related aberrations in CTB gene expression normalized when the cells were cultured for 2 days supported the theory that an unfavorable in vivo environment contributed to placental defects in this syndrome. Next, we asked whether any of the dysregulated genes were autocrine regulators of the CTB phenotype that is the hallmark of PE.
sPE-associated aberrations in CTB gene expression returned to control values after 48 hours of culture. RNA was analyzed immediately after the cells were isolated (0 hour) and after 12, 24, and 48 hours in culture. The relative gene expression levels for CTBs isolated from placentas of patients who delivered due to nPTL (n = 5) or sPE (n = 5) are shown as a heat map, ranging from high (red) to low (blue). The sPE CTBs were from the following cases (tiled from left to right): (a) HELLP syndrome and IUGR; (b) sPE; (c) sPE and IUGR; (d) superimposed sPE; and (e) HELLP syndrome. One sample of nPTL CTBs collected at 48 hours was omitted for technical reasons. The fold changes for each time point (sPE vs. nPTL) are shown on the right. ns, no significant difference (LIMMA); t, no significant difference in expression (sPE vs. nPTL) by 48 hours (maSigPro).
Upregulated trophoblast expression of SEMA3B in sPE. In PE, trophoblast expression of angiogenic factors is dysregulated. This phenomenon plays a central role in restricting CTB invasion (6) and in the etiology of the maternal signs, including elevated blood pressure and proteinuria (14, 15). In this context, we addressed the functions of SEMA3B. SEMA3 family members play important roles in neuronal wiring (24), and SEMA3B is an angiogenesis inhibitor and tumor suppressor. As a first step, we profiled SEMA3B mRNA expression in a variety of human cells and organs. Placenta gave the strongest signal (Figure 2A). Northern blot analyses of mRNA from control (first, second, and third trimester) and experimental placentas from patients with sPE showed that the abundance of SEMA3B mRNA increased as a function of gestational age and was highest in sPE samples (Figure 2B). The 2 bands likely reflect alternative splicing. In situ hybridization of placental chorionic villi demonstrated that SEMA3B mRNA expression, which was limited to trophoblasts, was lower in normal second trimester and nPTL samples as compared with sPE chorionic villi (Figure 2C). Immunoblot analyses of CTB lysates showed that expression of SEMA3B was either undetectable or low during the second and third trimesters of normal pregnancy and in cells isolated from nPTL placentas (Figure 2D). In contrast, higher levels of SEMA3B were detected in sPE CTBs immediately after isolation. For these experiments, SEMA3A-Fc served as a negative control and recombinant SEMA3B protein served as a positive control for antibody specificity. Immunolocalization showed that trophoblasts of chorionic villi from control nPTL placentas had much lower anti-SEMA3B immunoreactivity as compared with samples of similar gestational ages from sPE placentas (Figure 2E). Within the basal plate, extravillous CTBs in the setting of sPE also exhibited stronger staining for SEMA3B as compared with the nPTL samples (Figure 2E).
SEMA3B expression was high in the placenta and upregulated in sPE. (A) Binding of a 32P-SEMA3B probe to a multiple tissue expression array revealed high placental expression (coordinate B8). (B) Northern hybridization of polyA+ RNA extracted from chorionic villi and pooled from 3 placentas showed that SEMA3B expression increased over gestation and was highest in sPE (n = 3 replicates). (C) In situ hybridization (3 placentas per group) confirmed enhanced SEMA3B mRNA expression in the STB layer of the chorionic villi in sPE (25 weeks) as compared with normal pregnancy (23 weeks) and nPTL (34 weeks). (D) Immunoblotting of CTB lysates (15 μg per lane) showed that SEMA3B protein expression was low to undetectable in control cells from normal placentas (15–39 weeks). In all cases, expression was higher in sPE (26–33 weeks) as compared with nPTL (30, 33 weeks). A protein of the expected _M_r was detected in COS-1 cells transfected with SEMA3B but not in those transfected with SEMA3A-Fc. Vertical lines denote noncontiguous lanes from the same gel. The relative intensity of the bands quantified by densitometry is also shown. The values for each sample type were averaged and expressed relative to the α-actin loading controls. The entire experiment was repeated twice. (E) Staining tissue sections with anti-SEMA3B showed a sPE-associated upregulation of immunoreactivity associated with the trophoblast components of chorionic villi and among extravillous CTBs within the basal plate (n = 5 per group). Trophoblasts were identified by staining adjacent tissue sections with anti–cytokeratin-8/18 (data not shown). Scale bars: 100 μm (C and E). NB, Northern blot; GA, gestational age; RP, recombinant protein.
Neuropilin expression and SEMA3B actions. Next, we assessed the expression of the SEMA3B receptors, neuropilin-1 (NRP-1) and NRP-2 (24), in tissue sections of the maternal-fetal interface. Costaining with anti–cytokeratin-8/18 identified trophoblasts (Figure 3, A, C, E, and G). Immunolocalization analyses of normal second trimester samples showed that NRP-1, which was expressed by villous trophoblasts, was upregulated as the CTBs invaded the uterine wall (Figure 3B). Strong staining was also detected in association with endovascular CTBs and the endothelial lining of uterine vessels (Figure 3D, arrow). NRP-2 immunoreactivity was associated with villous trophoblasts and invasive CTBs as well as the villous stroma (Figure 3F). In the uterine wall, NRP-2 expression was strongly upregulated on endovascular CTBs (Figure 3H) and a subset of endothelial cells (data not shown).
NRP-1 and NRP-2 (protein) expression at the maternal-fetal interface in normal pregnancy and in sPE. Tissue sections were double stained with anti–cytokeratin-8/18 (CK), which reacts with all trophoblast subpopulations, and anti–NRP-1 or NRP-2. (A and B) NRP-1 expression was detected in association with villous trophoblasts. Within the uterine wall, immunoreactivity associated with invasive CTBs was upregulated as the cells moved from the surface to the deeper regions. (C and D) Endovascular CTBs that lined a maternal blood vessel (BV) were also stained. (E–H) Anti–NRP-2 reacted with trophoblast and nontrophoblast cells in anchoring villi (AV) as well as interstitial and endovascular CTBs. Essentially the same staining patterns, but with weaker intensity, were observed in sPE (data not shown). CTBs were isolated from the placentas of control nPTL cases and from the placentas of women who experienced sPE. (I) Over 48 hours in culture, NRP-1 expression was upregulated in both instances but to a lesser degree in sPE. (J) Control nPTL CTBs also upregulated NRP-2. Expression of this receptor was reduced in sPE and the soluble form was more abundant. (A–J) The data shown are representative of the analysis of a minimum of 3 samples from different placentas. Scale bars: 100 μm. AV, anchoring villi.
Immunoblot analyses of CTB lysates from control nPTL placentas showed upregulation of NRP-1 expression over 48 hours of culture, which was blunted in sPE (Figure 3I). As for NRP-2, control nPTL CTBs also upregulated this receptor, and soluble forms were detected (Figure 3J). In sPE, NRP-2 was expressed at reduced levels, and the relative abundance of the major soluble form of this receptor increased as compared with the control nPTL CTBs. Together these data suggested that placenta-derived SEMA3B could have autocrine effects on CTBs and paracrine actions on uterine endothelial cells.
We tested this hypothesis in the context of VEGF actions using our in vitro model of CTB invasion. Previously, we showed that CTBs produce large amounts of VEGF and that its autocrine actions include promoting invasion and inhibiting apoptosis (6). Blocking VEGF signals (anti–VEGF-A) or the addition of recombinant SEMA3B protein reduced invasion by approximately 60% as compared with cells that were cultured with a control CD6-Fc protein (Figure 4A). In contrast, removal of both ligands (anti-VEGF plus NRP-1–Fc or NRP-2–Fc) restored invasion to control levels. The removal of VEGF-A or the addition of SEMA3B doubled the rate of apoptosis and the absence of both ligands returned levels to below control values (Figure 4B). Taken together, these results suggested that SEMA3B opposed the actions of VEGF to restrain CTB invasion by promoting apoptosis of these cells.
Exogenous SEMA3B mimicked the effects of sPE on CTBs and endothelial cells and inhibited angiogenesis. (A) The addition of anti-VEGF or SEMA3B protein significantly inhibited CTB invasion as compared with the addition of a control protein, CD6-Fc. The removal of both ligands (anti–VEGF/NRP1-Fc and anti–VEGF/NRP-2–Fc) restored invasion to control levels. (B) The variables tested in A had the opposite effects on CTB apoptosis, suggesting that increased programmed cell death contributed to decreased invasion. (C) Exogenous VEGF stimulated the migration of UtMVECs, which was inhibited by SEMA3B. (D) The results in C were quantified relative to the addition of CD6-Fc. (E) In UtMVECs, VEGF promoted survival and SEMA3B increased apoptosis relative to control levels. (F) In the chick chorioallantoic membrane angiogenesis assay, VEGF promoted angiogenesis by approximately 3 fold and SEMA3B inhibited this process approximately 5 fold relative to the effects of CD6-Fc. Arrows mark the edge of the filter paper used to apply the protein. The area of the CAM beneath the filter paper is shown in the bottom row. Scale bar: 200 μm (top row); 100 μm (bottom row). n = 6 replicates (A–D); n = 3 replicates (E and F). Mean ± SEM; 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.
As to vascular effects, a monolayer of uterine microvascular endothelial cells (UtMVECs) was disrupted with a scratch, and the effects of SEMA3B, in terms of migration, were tracked by video microscopy. The results of a typical experiment are shown in Figure 4C. As with many ECs, the addition of VEGF strongly promoted directed UtMVEC migration; exogenous SEMA3B decreased levels to approximately 50% of control values, with a loss of directionality. Figure 4D summarizes the results of 3 experiments. Under the same conditions, the opposite effects were observed with regard to apoptosis; VEGF was protective and SEMA3B was a strong inducer (Figure 4E). These findings suggested that SEMA3B is primarily antiangiogenic, as was previously proposed. To test this theory, we used the chick chorioallantoic membrane assay, in which filter paper discs delivered VEGF, SEMA3B, or CD6-Fc (Figure 4F, top row). Removal of the discs showed that SEMA3B markedly inhibited angiogenesis as compared with the positive control VEGF or CD6-Fc (Figure 4F, bottom row). Together, these results suggested that the autocrine effects of enhanced SEMA3B expression recapitulated aspects of the CTB phenotype in PE, with paracrine actions including impaired UtMVEC functions.
Exogenous SEMA3B alters CTB signaling, phenocopying sPE effects. Next, we investigated the CTB signaling pathways that were involved. First, we asked whether SEMA3B opposed VEGF signaling by inhibiting the activation of PI3K, as measured by the production of phosphatidylinositol 3,4,5-triphosphate (PIP3). The addition of SEMA3B to first or second trimester CTBs reduced PIP3 concentrations to levels that were comparable to those after addition of the PI3K inhibitor, wortmannin (Figure 5A). DMSO, the vehicle, had no effect. Addition of VEGF increased PIP3 production 2.5 fold over control levels. These results suggested SEMA3B as a negative regulator of PI3K (25).
SEMA3B inhibited PI3K/AKT and GSK3β signaling in CTBs and the same effects were observed in sPE. (A) SEMA3B and wortmannin (WM) inhibited PI3K activity, which was stimulated by VEGF. DMSO was used as a vehicle control. Mean ± SEM, 2-tailed Student’s t test. *P < 0.05, **P < 0.01. (B) The addition of SEMA3B to UtMVECs resulted in the dissociation of the p85 and the p110α subunits of PI3K, which was rescued by the addition of VEGF. (C) In COS-1 cells, SEMA3B inhibited AKT Ser473 phosphorylation (activation), which increased during CTB differentiation/invasion (0–12 hours). The addition of SEMA3B inhibited AKT phosphorylation, which was enhanced by exogenous VEGF. (D) In COS-1 cells, SEMA3B inhibited GSK3β Ser9 phosphorylation (inactivation), which increased during CTB differentiation/invasion (0–12 hours). Exogenous SEMA3B inhibited GSK3β phosphorylation, which was enhanced by VEGF. GSK3α Ser21 phosphorylation was variable. (E) In CTBs, sPE correlated with dissociation of the p85 and p110α (and γ) subunits of PI3K relative to control cells isolated from normal third trimester placentas. (F) In freshly isolated CTBs, sPE was associated with decreased phosphorylation of AKT Ser473 and GSK3β Ser9. α-Actin was used as a loading control. (G) In chorionic villi, sPE was associated with phosphorylation (inactivation) of β-catenin. (A–D) The same results were obtained in 3 separate experiments that used different preparations of cells. (F and G) The results shown are representative of analyses of a total of 6 CTB isolates from different placentas of women diagnosed with sPE.
Then we sought to explain the mechanisms involved. A previous study demonstrated that VEGF-mediated VEGFR-2 phosphorylation creates a docking site for the p85 subunit of PI3K (26). However, preliminary experiments showed that SEMA3B did not interfere with VEGFR-2 phosphorylation (data not shown). Thus, we studied the interactions between the regulatory subunits of PI3K. UtMVECs were cultured in medium containing SEMA3B and VEGF or in the absence of one or the other factor. Cell lysates were immunoprecipitated with an antibody that specifically recognized the p85 regulatory subunit of PI3K. The pull downs were immunoblotted with anti–VEGFR-2, anti–NRP-2, and anti-p110α PI3K (Figure 5B); NRP-1 was not expressed (data not shown). VEGFR-2 and NRP-2 levels remained constant under all the test conditions. The addition of SEMA3B resulted in the dissociation of p85 and p110α, which was rescued by the addition of VEGF. Taken together, these results suggested that SEMA3B inhibited PI3K activity (Figure 5A) by preventing the association of p85 and p110α, to our knowledge a novel mechanism.
Downstream of PI3K activation, AKT is phosphorylated at Thr308 and/or Ser473 (27, 28). Thus, we were interested in the effects of SEMA3B on this process (Figure 5C). In initial experiments, we failed to detect SEMA3B-associated changes in phosphorylation of Thr308. Thus, we focused on modification of Ser473. First, COS-1 cells were either transfected with an empty vector or SEMA3B. No Ser473 phosphorylation was observed in the latter case. Next, we evaluated AKT phosphorylation as a function of CTB differentiation in culture. Lysates of cells isolated from first and second trimester placentas were assayed immediately upon isolation (0 hour) and after 12 hours in culture, during which time a band with strong anti–p-Ser473 reactivity appeared. The time course was rapid. After initial CTB adhesion (1 hour), the addition of wortmannin for 30 minutes downregulated Ser473 phosphorylation as did addition of SEMA3B; addition of VEGF had the opposite effect. In each case, the results were compared with the total amount of AKT, which was determined by stripping the blots and reprobing with an antibody that recognized all forms of this molecule. Together, these data suggested that, in CTBs, SEMA3B strongly downregulated AKT signaling.
AKT inactivates GSK3α and GSK3β by phosphorylating Ser21 and Ser9 (29, 30), respectively. In COS-1 cells, expressing SEMA3B abolished phosphorylation of GSK3α and GSK3β (Figure 5D). In first and second trimester CTBs, GSK3β phosphorylation on Ser9 increased during 12 hours of culture, whereas GSK3α phosphorylation on Ser21 was variable (Figure 5D). LiCl, a GSK3 inhibitor, increased phosphorylation of Ser9 (data not shown), with wortmannin having the opposite effect (Figure 5D). Consistent with the AKT results, the addition of exogenous SEMA3B decreased phosphorylation of GSK3β, which was increased by the addition of VEGF. Immunoblot analysis with an antibody that recognized the GSK3 protein backbone showed that levels did not change under any of the experimental conditions. Since GSK3, often a negative regulator, intersects several critical signaling pathways (30, 31), it is likely that overexpression of SEMA3B has important consequences.
Based on our analysis of SEMA3B/VEGF effects on PI3K/AKT and GSK3 signaling, we predicted that this pathway would be dysregulated in sPE. CTBs from control placentas throughout gestation and from patients who were diagnosed with sPE were analyzed immediately after isolation. As with UtMVECs (Figure 5B), an IP/immunoblot strategy showed disassociation of the p85 and p110α subunits of PI3K in sPE (Figure 5E). In this case, the expression of P110γ was also detected and relative expression was reduced in sPE. The results of the AKT and GSK3 analyses were interpreted using the expression of α-actin as a control for protein loading (Figure 5F). The 2 bands observed in the 32-week sPE sample were attributed to proteolysis, which is sometimes observed in these samples. Two patterns were seen in sPE. In one (26 week), no differences were detected at the protein level, but phosphorylation was markedly decreased. The other was characterized by downregulation at both levels (25 and 32 week). In the case of AKT, p-Ser473 was either undetectable or nearly absent in the sPE samples (Figure 5F). Likewise, p-Ser9 of GSK3β was lower in abundance. Finally, we reasoned that an increase in GSK3 activity would have important effects on pathways that we know are critical to CTB invasion. In chorionic villi from sPE placentas, we observed a large increase in the phosphorylated form of β-catenin, which leads to ubiquitination and proteosomal degradation of this molecule (Figure 5G). Given that activation of β-catenin is associated with tumorigenesis (32), inhibiting this pathway could restrict CTB invasion perhaps by altering cell adhesion or Wnt signaling (33).
Based on these data, we propose a model that integrates SEMA3B and VEGF functions in normal pregnancy and in sPE (Figure 6). We found that SEMA3B competed with VEGF binding to neuropilins. High SEMA3B levels led to the dissociation of the p85 and p110α subunits of PI3K, a novel mechanism. The downstream consequences included inactivation of AKT and activation of GSK3, which led to apoptosis and degradation of β-catenin. Together, these data suggested an autocrine mechanism by which elevated SEMA3B levels contributed to the sPE-associated phenotype of invasive CTBs in terms of the signaling pathways that we analyzed.
Model of SEMA3B effects on CTBs in sPE and normal pregnancy.