11:10-11:20 Uptake of preeclampsia-derived EVs activates microglia in vitro

Extracellular vesicles (EVs) are cell-derived particles that constitute a mechanism of intercellular communication transporting cell-type-specific cargos involved in multiple physiological and pathological processes. The concentration of EVs increases in plasma of normal pregnancy (NP) throughout gestation exhibiting particular molecular profiles and is higher in patients with preeclampsia (PE). PE is characterized by new-onset hypertension and associated with cerebrovascular and neurological impairment. EVs are related with neuroinflammation through activation of microglia and with neurodegenerative conditions. Despite few studies have suggested that EVs are involved in the relationship between PE and central nervous system injury, the underlying mechanisms are unknown. Here we investigated effects of plasma-derived EVs from PE on microfluidically supported and static models of the human blood-brain barrier (BBB) and microglia. EVs were isolated from NP and PE plasma through differential ultracentrifugation, and characterized by nanotracking analysis, cryogenic electron microscopy and Western blotting. Human brain endothelial cells (hCMEC/D3) were cultured with EndoGRO supplemented with exosome-depleted FBS in microfluidically supported biochips -that mimic cerebral blood flow and induce endothelial polarization, and stimulated with NP- and PE-derived EVs for 24 h. A co-culture of hCMEC/D3 cells and human microglial cells (HMC3) in a two-compartment static model of the BBB was also used. PKH67-labeled EVs, endothelial adhesion proteins and microglial activation markers were analyzed by confocal microscopy. Cytokines released in the cell culture supernatant were measured by flow cytometry. Enriched fractions of small (<150 nm) and large (>150 nm) EVs were obtained exhibiting polydisperse spherical morphology. Compared to NP, elevated concentrations of EVs were observed in PE. sEV harbored CD63, ALIX and TSG101, suggesting exosome-enrichment, and HLA-G and PLAP, demonstrating the presence of their placental origin. In assessing the effect of EVs on the integrity of the BBB under flow conditions, PE-EVs were taken up by hCMEC/D3 cells and triggered decrease of endothelial claudin-5 and occludin compared to NP-EVs. Furthermore, PE-EVs crossed the endothelial monolayer in the static co-culture set-up and were taken up by HMC3 microglial cells, inducing increase of the microglial activation markers CD11b, IBA1, and release of TNF-α, IL-1β and IFN-γ. Our results show that placenta derived EVs reach the maternal periphery. PE-EVs disrupt and cross the BBB, are taken up by endothelial and microglial cells and induce microglial activation in vitro. This study suggests that placenta-derived EVs play a role in the mechanism of inter-organ communication in the context of pregnancy disorders, and could be the link between PE and the cerebrovascular and neurological impairment observed in those patients.