O3310: Impaired Endothelial Angiogenesis via Extracellular Vesicles Originating from Human Trophoblast Stem Cells in a Nutrient Restriction Model of Fetal Growth

Background: Fetal growth restriction (FGR) accounts for 50% of stillbirths and is defined by a fetal weight below the 10th percentile. Successful placentation is necessary to develop a maternal-fetal interface that supplies adequate nutrients and oxygen to a developing fetus. Critical to this process is placental endothelial angiogenesis, and placental pathology of pregnancies complicated by FGR demonstrate reduced angiogenesis with impaired villous vascular branching. The molecular pathways underpinning the FGR phenotype are poorly understood. Using nutrient deprivation to model FGR in vivo, we have previously shown that angiogenic pathways are dysregulated within the mouse placenta. To determine cell specific signaling mechanisms that may impair angiogenesis in FGR, we developed an in vitro model exposing human trophoblast stem cells (hTSCs) to nutrient-restricted media. As extracellular vesicles (EVs) contain microRNAs, which have been suggested to drive the FGR phenotype, and serve as a mediator for cell-cell communication, we designed a set of experiments to determine their role in impaired angiogenesis. We hypothesize that nutrient restriction in hTSCs simulate the release of EVs that communicate with placental endothelial cells to suppress placental angiogenesis.

Methods: hTSCs were plated in either nutrient complete or a 50% nutrient-restricted media and EVs released from hTSCs were isolated after 48 hours. Human umbilical vein endothelial cells (HUVECs) were transfected with EVs from either nutrient complete or nutrient-restricted hTSCs for an additional 48 hours. Subsequently, HUVEC mRNA was sequenced. Genes with significantly altered levels of expression between transfection types were mapped using the machine learning tools PANTHER (Protein ANalysis THrough Evolutionary Relationships) and Ingenuity Pathway Analysis (IPA) to identify potential targets of placentation, specifically angiogenic molecular pathways.

Results: HUVECs transfected with EVs from nutrient-restricted hTSCs demonstrated altered gene expression profiles in pathways involving angiogenesis and extracellular matrix (ECM) remodeling. 13,405 total genes were sequenced, of which 479 were found to be significantly upregulated and 598 significantly downregulated (Figure 1). PANTHER analysis identified that nutrient-restricted EVs downregulated molecular pathways involving Vascular Endothelial Growth Factor (VEGF), Platelet-Derived Growth Factor (PDGF), and Integrin pathways, all essential for placental angiogenesis (Figure 2). IPA identified ECM organization as a top conical pathway with altered gene expression profiles showing significant downregulation of multiple genes involved with ECM remodeling. The three most significantly downregulated genes associated with angiogenic pathways were YAP1, MMP17, and COL6A1 (Figure 1).

Conclusion: Using an in vitro FGR model, we demonstrated that EVs from nutrient-restricted hTSCs altered gene expression in HUVECs, notably downregulating ECM remodeling and impairing angiogenic pathways. This study uncovers potentially significant new roles for EVs in placentation and the role of malnutrition in placental signaling. Further studies are necessary to determine the cargo within EVs that target such pathways and their role in placental development.