Vascular endothelial growth factor ligands and receptors that regulate human cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome

Abstract

Human placental development combines elements of tumorigenesis and vasculogenesis. The organ's specialized epithelial cells, termed cytotrophoblasts, invade the uterus where they reside in the interstitial compartment. They also line uterine arteries and veins. During invasion, ectodermally derived cytotrophoblasts undergo pseudovasculogenesis, switching their adhesion molecule repertoire to mimic that of vascular cells. Failures in this transformation accompany the pregnancy complication preeclampsia. Here, we used a combination of in situ and in vitro analyses to characterize the cell's expression of vascular endothelial growth factor (VEGF) family ligands and receptors, key regulators of conventional vasculogenesis and angiogenesis. Cytotrophoblast differentiation and invasion during the first and second trimesters of pregnancy were associated with down-regulation of VEGF receptor (VEGFR)-2. Invasive cytotrophoblasts in early gestation expressed VEGF-A, VEGF-C, placental growth factor (PlGF), VEGFR-1, and VEGFR-3 and, at term, VEGF-A, PlGF, and VEGFR-1. In vitro the cells incorporated VEGF-A into the surrounding extracellular matrix; PlGF was secreted. We also found that cytotrophoblasts responded to the VEGF ligands they produced. Blocking ligand binding significantly decreased their expression of integrin alpha1, an adhesion molecule highly expressed by endovascular cytotrophoblasts, and increased apoptosis. In severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome, immunolocalization on tissue sections showed that cytotrophoblast VEGF-A and VEGFR-1 staining decreased; staining for PlGF was unaffected. Cytotrophoblast secretion of the soluble form of VEGFR-1 in vitro also increased. Together, the results of this study showed that VEGF family members regulate cytotrophoblast survival and that expression of a subset of family members is dysregulated in severe forms of preeclampsia.

Figure 1.

Diagram of the histological organization of the human maternal-fetal interface at mid-gestation. In this location cytotrophoblasts, specialized (fetal) epithelial cells of the placenta, differentiate and invade the uterine wall, where they also breach maternal blood vessels. The basic structural unit of the placenta is the chorionic villus, composed of a stromal core with blood vessels, surrounded by a basement membrane, and overlain by cytotrophoblast stem cells. As part of their differentiation program, these stem cells detach from the basement membrane and adopt one of two lineage fates. They either fuse to form the syncytiotrophoblasts that cover floating villi (FV), or join a column of extravillous cytotrophoblasts at the tips of anchoring villi (AV). The syncytial covering of floating villi mediates the nutrient, gas, and waste exchange between fetal and maternal blood. The anchoring villi, through the attachment of cytotrophoblast columns, establish physical connections between the fetus and the mother. Invasive cytotrophoblasts penetrate the uterine wall up to the first third of the myometrium. A portion of the extravillus cytotrophoblasts home to uterine spiral arterioles and remodel these vessels by destroying the muscular wall and replacing the endothelial lining. To a lesser extent they also remodel uterine veins. At term, few cytotrophoblast stem cells remain, the syncytiotrophoblast layer thins and the stromal cores expand. (Diagram modified from Hoang and colleagues.61 )

Figure 2.

Cytotrophoblast staining for VEGF-A was up-regulated as the cells differentiated and invaded the uterus in situ. Serial paraffin sections of the maternal-fetal interface were stained with anti-cytokeratin (CK) to identify all of the trophoblast populations (A, C, and E), and with anti-VEGF-A (B, D, and F). Essentially the same staining pattern was observed during the first and second trimesters (ie, 6 and 16 weeks of gestation). A few cytotrophoblast (CTB) stem cells and cells in the proximal column (PCOL) region stained with anti-VEGF-A (B), but much more intense staining was observed in association with a majority of cytotrophoblasts in the distal regions of columns (DCOL) and with those that invaded the uterine wall (D). Cytotrophoblasts within the lumina of uterine blood vessels (BV) also exhibited intense staining (arrowheads), as did the maternal endothelial cells (EC; F). In contrast, some cytotrophoblasts in the vessel wall failed to react with anti-VEGF-A (arrows). The cells continued to stain for VEGF-A at term (see Figure 12B▶ ). AV, anchoring villus; STB, syncytiotrophoblast; VS, villous stroma; COL, cytotrophoblast column.

Figure 3.

By the second trimester, cytotrophoblasts in all stages of differentiation stained with anti-VEGF-C in situ. Serial paraffin sections of the maternal-fetal interface were stained with anti-cytokeratin (CK) to identify the various trophoblast populations (A, C, and E), and with anti-VEGF-C (B, D, and F). In the first trimester (6 weeks), most of the cytotrophoblast populations stained with anti-VEGF-C, although cells in the distal portions of columns (COL) sometimes exhibited stronger antibody reactivity (B). In the second trimester (16 weeks), staining tended to be stronger than in the first trimester, with cytotrophoblast stem cells, cells in columns, and cytotrophoblasts within the uterine wall exhibiting similar levels of antibody reactivity (D). By term (38 weeks), little or no staining for VEGF-C was observed (F). AV, anchoring villus; VS, villus stroma; PCOL, proximal column.

Figure 4.

Cytotrophoblast staining for PlGF was up-regulated as the cells differentiated and invaded the uterus in situ. Frozen sections of the maternal-fetal interface in the first trimester (10 weeks) and the second trimester (25 weeks) were double-stained with anti-cytokeratin (CK; A, C, and E) and with anti-PlGF (B, D, and F). Essentially the same staining pattern was observed in the first and second trimesters. Staining for PlGF was first detected on cytotrophoblasts (CTBs) in the distal column (DCOL; B). Anti-PlGF reactivity was also observed in association with cytotrophoblasts that performed both interstitial (D) and endovascular invasion (F). At term cytotrophoblasts within the uterine wall either failed to stain or stained weakly with anti-PlGF (data not shown). AV, anchoring villus; PCOL, proximal column (arrowhead); BV, blood vessel.

Figure 5.

Cytotrophoblast subsets at the maternal-fetal interface differentially stain with antibodies that recognize individual VEGF receptors. Frozen sections of the first trimester maternal-fetal interface were double-stained with anti-cytokeratin (CK; A, C, and E) and antibodies that specifically reacted with one of three VEGF receptors (B, D, and F). Cytotrophoblast (CTB) stem cells and those in the proximal regions of cytotrophoblast columns (PCOL) stained with anti-VEGFR-2; little or no staining was detected in the distal column (DCOL) region and in association with cytotrophoblasts that invaded the uterine wall (B). Staining with anti-VEGFR-1 (D) and anti-VEGFR-3 (F) revealed a different pattern. Cytotrophoblast stem cells and those in the portion of the column immediately adjacent to the anchoring villus (AV) failed to react with either antibody, whereas cells in the rest of the column and within the uterine wall stained brightly with both. Syncytiotrophoblasts (STB) also stained for VEGFR-1 and VEGFR-3 (D and F). Essentially the same pattern was observed in the second trimester (data not shown). At term, only VEGF-R1 staining was detected in association with cytotrophoblasts (see Figure 13B▶ ).

Figure 6.

Endovascular cytotrophoblasts differentially stain for individual VEGF receptors. Frozen sections of the uterine wall from samples collected in the second trimester of pregnancy (19 to 20 weeks of gestation) were double-stained with anti-cytokeratin (CK; A, C, and E) and antibodies that specifically reacted with one of three VEGF receptors (B, D, and F). Cytotrophoblasts (CTBs) within the lumina of blood vessels (BV) stained for VEGFR-2 (B; arrowhead) and VEGFR-3 (F; arrowhead), whereas cytotrophoblasts that were embedded in the vessel wall usually stained much less intensely (B and D; arrows). In contrast, endovascular cytotrophoblasts showed little or no staining for VEGFR-1 (D; arrowheads). When staining was detected, reactivity was usually associated with cells embedded in the vessel wall (arrow). Essentially the same pattern was observed in blood vessels that were modified during the first trimester.

Figure 7.

Cytotrophoblasts produce the same repertoire of VEGF family members in vivo and in vitro. Cytotrophoblasts (CTBs) isolated from human placentas were examined for their production of VEGF mRNA (A) and protein (B and C). mRNA, purified from cells before they were plated (0 hours) and after 12 hours of culture on a Matrigel substrate, was analyzed by Northern blot hybridization. First (I) and second (II) trimester samples expressed approximately equal amounts of RNAs encoding VEGF-A and VEGF-C; VEGF-B mRNA levels were much lower and VEGF-D was not detected (A, left). Isolated placental fibroblasts also produced VEGF-A and VEGF-C mRNAs and low levels of VEGF-B mRNA. In contrast, cytotrophoblasts, not fibroblasts, produced PlGF mRNA (A, right). Cytotrophoblast production of VEGF-A and PlGF was assessed by ELISA (B). No VEGF-A was detected in conditioned medium from either first, second, or third (III) trimester cytotrophoblasts that were plated on Matrigel substrates, whereas all of the choriocarcinoma cell lines produced amounts of this growth factor that were easily detected by ELISA (B, left). In contrast, PlGF was secreted by both cytotrophoblasts and choriocarcinoma cells (B, right). The ELISA data in B are the means and standard deviations of three experiments. If the cells were plated on a laminin substrate instead of Matrigel, approximately equal amounts of VEGF-A partitioned in the cell and conditioned medium (CM) fractions as determined by immunoblotting (C). The isoforms produced by cytotrophoblasts migrated more slowly than recombinant VEGF₁₆₅.

Figure 8.

Cytotrophoblasts modulate VEGF receptor expression at the protein level as they differentiate in vitro. Cell lysates were prepared from first and second trimester cytotrophoblasts, either immediately after isolation (0 hours) or after they had been plated for 20 hours on Matrigel. Receptor expression was analyzed by immunoblotting. First and second trimester cells down-regulated expression of VEGFR-2 and up-regulated the expression of VEGFR-1. VEGFR-3 expression was higher in first trimester than in second. As a control, uterine vein endothelial cell (UtVEC) expression of the receptors was analyzed in parallel. Multiple bands may be due to receptor phosphorylation in some of the cytotrophoblast samples. Different glycoforms could also be present.

Figure 9.

VEGFs regulate cytotrophoblast morphology and some aspects of adhesion molecule expression. Isolated cytotrophoblasts were plated on Matrigel-coated substrates and cultured for 12 hours in the presence of VEGFR-1(1-3)-Fc (15 μg/ml) or VEGFR-3(1-3)-Fc (16 μg/ml) or, as a control, purified preimmune IgG. VEGFR-1(1-3)-Fc blocks VEGF-A, PlGF, and VEGF-PlGF heterodimer binding to endogenous VEGFR-1, and VEGFR-3(1-3)-Fc blocks VEGF-C binding to endogenous VEGFR-3. After 12 hours in culture, control cells that were originally plated as a monolayer formed discrete aggregates that were connected by cellular processes (A). Aggregate formation was impaired by the addition of VEGFR-1(1-3)-Fc (D) and primarily absent in the presence of VEGFR-3(1-3)-Fc (G). The morphological changes suggested that the cell’s adhesion molecule expression might also be affected. Thus, we double-stained cells plated under the same control and experimental conditions with antibodies that recognized the cytotrophoblast marker cytokeratin (CK; B, E, and H) and integrin α1, an adhesion molecule that is crucial for cytotrophoblast adhesion and invasion (C, F, and I). Staining for integrin α1 was reduced in the experimental as compared to the control conditions.

Figure 10.

Immunoprecipitation confirmed that blocking ligand binding to VEGFR-3 or VEGFR-1 down-regulated cytotrophoblast integrin α1 expression. Cells cultured under the control and experimental conditions for 12 hours were labeled with biotin. Cell lysates were prepared and subjected to immunoprecipitation with anti-integrin α1 as described in Materials and Methods. Control cells expressed higher levels of integrin α1β1 than did cytotrophoblasts cultured in the presence of either fusion protein (A). In contrast, immunoblotting showed that inhibiting ligand binding to VEGFR-1 or VEGFR-3 did not change the staining pattern for VE-cadherin (B).

Figure 11.

Blocking ligand binding to VEGFR-1 or VEGFR-3 decreased cytotrophoblast invasion and increased cytotrophoblast apoptosis. Cytotrophoblasts from first and second trimester chorionic villi were plated on Matrigel-coated filters in Transwell inserts and cultured for 48 hours in the presence of VEGFR-1(1-3)-Fc, VEGFR-3(1-3)-Fc, or control IgG. Invasion was quantified by counting the number of cell processes and cells that reached the undersides of the filter. Addition of the fusion proteins, but not the control IgG, significantly inhibited invasion (A). Data are means and standard deviations of seven experiments. The mechanism includes a significant increase in apoptosis as detected by the TUNEL method (B). Data are means and standard deviations of six experiments.

Figure 12.

In preeclampsia, staining of invasive cytotrophoblasts with anti-VEGF-A decreased. In the third trimester of normal pregnancy (38 weeks), cytotrophoblasts (CTBs) in the interstitium (arrow) and in the walls of blood vessels (BVs) (arrowheads) stained for VEGF-A (B). In preeclampsia (PE), staining of invasive cytotrophoblasts for VEGF-A was strikingly down-regulated (D and F). CK, cytokeratin; VS, villus stroma.

Figure 13.

In preeclampsia, staining of invasive cytotrophoblasts with anti-VEGFR-1 decreased. In the third trimester of normal pregnancy (36 weeks), cytotrophoblasts (CTBs) in the interstitium (arrows) exhibited strong staining with anti-VEGFR-1 (B). In preeclampsia (PE), staining of invasive cytotrophoblasts for VEGFR-1 was either absent or weak. In some areas of biopsies, no staining was detected (−; D). In other areas of the same biopsy, weak antibody reactivity was evident (+/−; F).

Figure 14.

In preeclampsia, cytotrophoblasts produce higher levels of soluble VEGFR-1 (sVEGFR-1) in vitro as compared to control cells. Cytotrophoblasts were isolated from control first, second, or third trimester (TM) placentas or from the placentas of women whose pregnancies were complicated by preeclampsia (PE). Then the cells were cultured on Matrigel substrates for 48 hours. Quantification of sVEGFR-1 in the conditioned medium by ELISA showed that preeclampsia is associated with a significant elevation of cytotrophoblast sVEGFR-1 secretion as compared to control cells. Data are means and standard deviations of five experiments.