(676f) Extracellular Vesicles As Potentiators of Stress Signals to Alter Placental and Fetal Development

Introduction: Maternal stress is linked to a variety of adverse fetal and pregnancy outcomes. In our well-established mouse model of early prenatal stress (EPS), we observe impaired cognitive function, alterations in metabolic programming, and heightened stress sensitivity, specifically in male but not female offspring. Transcriptomic analysis of the placenta demonstrated that EPS impacts endo- and exosomal processes in the placenta, supporting an involvement of extracellular vesicle signaling. Extracellular vesicles (EVs) are cell-derived nanoparticles that play an important role in intercellular communication, by transporting enzymes, immune signals, and nucleic acids between cells and tissues. During pregnancy, circulating EVs increase in number, in part due to the generation of EVs by the placenta. We hypothesize that, during pregnancy, EVs play an important role in communication between maternal and fetal tissues, and that EV contents and characteristics are altered in response to the maternal environment. Here, we utilize our established EPS murine model to examine circulating maternal EVs over the course of pregnancy, and to probe the mechanism by which maternal EVs impact fetal brain development.

Methods: We first addressed the role of stress in EV signaling by isolating circulating EVs from donor dams in control and EPS-exposed groups. We isolated EVs from plasma collected on embryonic days 12.5, 15.5, and 18.5 (E12.5, E15.5, E18.5) using size exclusion chromatography. Nanoparticle tracking analysis was used to measure the size, concentration, and surface charge of isolated EVs. Next, small RNA-sequencing and proteomics were used to analyze the content of EVs from control and EPS-exposed dams. Labeled EVs were injected into naïve dams, and an in vivo imaging system (IVIS) was used to detect EV trafficking in maternal and fetal compartments. A separate cohort was used to detect transcriptomic changes in placental and fetal tissues after exposure to isolated EVs from either control or EPS-exposed dams. EVs were administered to a cohort of naïve dams, and offspring were monitored throughout adulthood for growth and hypothalamic-pituitary-adrenal (HPA) stress axis reactivity.

Results: We observed that, while the size of circulating EVs remains the same over the course of gestation (~120 nm), the concentration of EVs tends to decrease as gestation progresses. While it was not significant, we observed a lower concentration of EVs isolated from E18.5 EPS dams, than from E18.5 control dams. We observed the protein content of EVs isolated from derived from EPS-exposed dams showed a significant decrease in proteins related to the immune response, and an increase in proteins related to metabolic processes. IVIS revealed that injected EVs traffic to the placenta and to the maternal brain, regardless of EPS exposure. RNA-sequencing revealed that control-derived EVs were able to significantly alter protein targeting and messenger RNA catabolic processes in male placentas from EPS-exposed dams.

Conclusions: We have demonstrated the potential for EVs to have a lasting impact on placental and fetal neurological development. Together, these studies provide insight into the role EVs play in promoting stress signals in both maternal and fetal circulation, their interaction at the level of the placenta, and the impact of prenatal stress on important signaling dynamics between maternal and fetal compartments during gestation. Future work will be aimed at engineering biomimetic lipid nanoparticles to target the placenta and fetal brain and rescue the EPS phenotype observed in male offspring.

Funding: NIEHS ES028202, NICHD HD097093, NIMH MH104184 & MH108286