Vascular endothelial growth factor (VEGF) is a potent regulator of placental vascular function. Endothelial dysfunction is a key factor associated with preeclampsia. In this study, we examined expression of VEGF, endocrine gland-derived VEGF (EG-VEGF), VEGF receptors 1 and 2 (VEGFR-1 and VEGFR-2), and neuropilin-1 and -2 (NP-1 and NP-2) in human placentas from women with normal and preeclamptic (PE) pregnancies using quantitative or semiquantitative PCR. We found that total VEGF mRNA expression was increased 2.8-fold (P < 0.05), along with increases in mRNA expression of VEGF121, 165, and 189 (P < 0.05; 1.7-, 1.9-, and 1.8-fold, respectively) in PE vs. normal placentas. Expression of VEGFR-1 mRNA, but not EG-VEGF and the other three VEGF receptors studied, was elevated (P < 0.05) 2.7-fold in PE vs. normal placentas. Protein expression of VEGF and its four receptors was determined using Western blot analysis. For VEGF, two major isoforms (VEGF165 and 189) were detected. For VEGFR-1, VEGFR-2, NP-1, and NP-2, one major band was observed at 180, 235, 130, and 130 kDa, respectively. All of these bands were corresponding to their positive controls. Of these five proteins studied, only VEGFR-1 levels were increased (P < 0.05; 1.7-fold) in PE placentas. The expression of VEGF and the four VEGF receptors was confirmed using immunohistochemistry. They were primarily present in syncytiotrophoblasts and endothelial cells of villous capillaries and large vessels. Thus, together with previous reports that VEGFR-1 mediates trophoblast function and inhibits VEGF-induced angiogenesis and endothelium-dependent vasodilation, these data suggest that the increased VEGFR-1 expression may alter VEGF- mediated function on trophoblast and endothelial cells in PE placentas.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, mediates many biological processes, including fetal development. In this study, we examined AhR protein expression in human placentas from normal (N) and severe preeclamptic (sPE) pregnancies, as well as human fetal tissues from the second trimester of pregnancy, using immunohistochemistry and/or Western blot analysis. In the placentas, the AhR immuno-reactivity was present primarily in syncytiotrophoblasts. The AhR staining was also seen in endothelium of large blood vessels in villi and endothelium of umbilical cord arteries and veins. No difference in AhR protein levels was found between N and sPE placentas. In fetal tissues, the AhR immunoreactivity was localized in lung, kidney, esophagus, pancreas, liver, testicle, thymus gland, retina, and choroid, mainly in epithelial cells, whereas it was absent in heart, brain, sclera, and thoracic aorta. These findings suggest that the AhR plays a critical role in syncytiotrophoblasts of human placentas and epithelium of many fetal organs. These data also imply that human placentas and those fetal organs with high AhR expression (e.g., lung, kidney, liver, pancreas, and thymus gland) during fetal development are highly susceptible to environmental toxicants such as dioxin.

Background: The placenta is an important organ for fetal and maternal health during pregnancy and impacts offspring health late in life. Defects in placental vasculature and trophoblast have been identified in several pregnancy complications. Thus, the detailed molecular profile and heterogeneity of endothelial cells and trophoblasts in placentas will aid us in better understanding placental behaviors and improving pregnancy outcomes. Methods: Single-cell RNA sequencing (scRNA-seq) was performed to profile the transcriptomics of human placental villous tissues from eleven patients with normal pregnancies in the first and second trimesters (6–16 weeks of gestation). Results: The transcriptomic landscape of 52,179 single cells was obtained, and the cells were classified as trophoblasts, fibroblasts, endothelial cells, erythroid cells, Hofbauer cells, and macrophages. Our analysis further revealed the three subtypes of placental endothelial cells, with distinct metabolic signatures and transcription factor regulatory networks. We also determined the transcriptomic features of the trophoblast subpopulations and characterized two distinct populations of progenitor cells in cytotrophoblasts, which were capable of differentiating to extravillous trophoblasts and syncytiotrophoblasts, respectively. Conclusions: Our study provided a high-resolution molecular profile of the human placenta between 6 and 16 weeks of gestation. Our data revealed the placental cell complexity and demonstrated the transcriptional networks and signaling involved in placental endothelial and trophoblast differentiation during early pregnancy, which will be a resource for future studies of the human placental development.

Context Preeclampsia is a leading cause of fetal and maternal morbidity and mortality during pregnancy. Although the etiology of preeclampsia is unknown, preeclampsia offspring have increased risks of developing cardiovascular disorders in adulthood, implicating that preeclampsia programs fetal vasculature in utero. Objective We hypothesize that preeclampsia alters expression profiles of endothelial microRNAs (miRNAs) in fetal endothelial cells and disturbs the vascular endothelial growth factor A (VEGFA)- and fibroblast growth factor 2 (FGF2)-induced endothelial function. Design and Setting Unpassaged (P0) human umbilical vein endothelial cells (HUVECs) were isolated immediately after cesarean-section delivery from normotensive (NT) and preeclamptic (PE) pregnancies. Differentially expressed miRNAs between P0-HUVECs from NT and PE pregnancies were identified using a miRNA polymerase chain reaction (PCR) array and confirmed using reverse transcription quantitative PCR. To determine the function of these differentially expressed miRNAs, miRNAs of interest were knocked down in NT-HUVECs following by cell functional assays. Results Sixteen miRNAs, including miR-29a/c-3p, were downregulated in P0-HUVECs from the PE group compared with the NT group. Bioinformatics analysis predicted the PI3K–v-akt murine thymoma viral oncogene homolog 1 (AKT) signaling pathway was dysregulated in P0-HUVECs from the PE group, which was associated with the miR-29a/c-3p downregulation. We further demonstrated that miR-29a/c-3p knockdown inhibited the VEGFA- and FGF2-induced endothelial migration as well as FGF2-induced AKT1 phosphorylation in HUVECs. However, miR-29a/c-3p knockdown did not alter the extracellular signal-regulated kinase 1/2 phosphorylation, cell proliferation, and endothelial monolayer integrity in response to VEGFA and FGF2 in HUVECs. Conclusions Preeclampsia-downregulated miR-29a/c-3p may impair fetal endothelial function by disturbing the FGF2-activated PI3K-AKT signaling pathway, hence inhibiting endothelial cell migration.