POSTER 21Placenta-brain communication via extracellular vesicles in preeclampsia

Hypertension and inflammation are the main characteristics of preeclampsia (PE), a pregnancy-driven disorder. PE serves as a predisposing factor for cardiovascular conditions later in life. The brain is one of the early victims of hypertension-induced organ damage characterized by cerebrovascular malfunction. Therefore, PE patients present anomalies such as brain edema and blood-brain barrier (BBB) failure. We have previously reported that exogenous EVs can reach the BBB, be internalized by endothelial cells, cross through and induce microglia activation. The placenta releases extracellular vesicles (EVs) to the maternal peripheral blood. Placenta-derived EVs are important carriers of bioactive mediators such as proteins, RNAs, lipids, and miRNAs. Furthermore, PE-EVs may trigger inflammatory responses and induce endothelial injury in the maternal vasculature. However, whether PE-EVs can reach the BBB and their possible effects on brain microvasculature are still to be explored. Plasma was obtained from patients with normal pregnancy (NP) and diagnosed with PE. EVs from plasma were enriched by differential ultracentrifugation and characterized by nanotracking analysis (NTA), Western blotting and transmission electron microscopy (TEM). The BBB representing endothelial hCMEC/D3 cell line was cultured in EndoGRO™-MV complete media in static and flow conditions. Cells were treated with unstained or PKH67-stained EVs from NP, PE or PBS as vehicle. The transendothelial electrical resistance (TEER) and permeability (FITC-dextran) were assessed in a trans-well BBB static model. Tight junction integrity was evaluated using a dynamic 3D on-chip BBB model. Localization of tight junction proteins and EV-uptake was imaged by confocal microscopy. In agreement with previous reports, we found elevated concentrations of sEVs and lEVs in plasma from PE patients compared to NP as measured by NTA and visualized by TEM. Enriched EVs contained placenta-specific markers including HLA-G, PLAP and miR-519d-3p, which differed between PE and NP samples. Using a trans-well BBB static model, uptake of EVs by hCMEC/D3 cells was demonstrated. Treatment with PE-EVs resulted in TEER reduction and increased FITC-dextran permeability. EV uptake was also confirmed under flow conditions using the BBB on-chip model. Increased paracellular permeability associated with decreased expression of tight junction proteins ZO-1 and β-catenin was observed upon treatment with PE-EVs. PE diagnosed pregnant women have higher plasma EV concentration in comparison with NP. The presence of placenta-specific markers confirms that plasma contains placenta-derived-EVs. PE-EVs can interact with endothelial cells in the BBB damaging its integrity and increasing paracellular transport. Altogether, our results suggest that PE-derived EVs may be associated with increased permeability of proinflammatory elements from the periphery into the brain increasing the risk for cerebrovascular disease.