Proteomic analyses of placental extracellular vesicles and glycoproteins on the surface of target immune cells

• Applicant: Dr. Diana Maria Morales Prieto
• Subject Area: Reproductive Medicine, Urology
• Term: from 2014 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 255955419

Successful pregnancy relies on an intricate communication between fetal trophoblast and maternal immune cells that allows remodeling of uterine vessels, establishment of maternal tolerance and protection of mother and fetus from pathogens. This communication can be carried out by release of extracellular vesicles (EVs) that harbor among other proteins and nucleic acids from the parental cell. The microRNA (miRNA) cargo of EVs received great attention because of the potential to modify gene expression post-transcriptionally in the recipient cells. We demonstrated previously that trophoblast cells express a singular miRNA profile which includes species that are almost uniquely expressed by the placenta tissue. These miRNAs regulate cellular processes of trophoblast cells in vitro such as cell proliferation and invasion, as well as their ability to interact with endothelial cells. In a second step, we investigated the miRNA profile of placenta tissue in normal and pathological pregnancies namely preeclampsia (PE), intrauterine growth restriction (IUGR) and placenta accreta syndrome (PAS). Altered miRNA species were identified and associated with intracellular signaling pathways and cellular functions. Thereafter, cell lines modeling singular immune cell populations were treated with trophoblast-derived EVs containing abnormal levels of placental miRNAs. We confirmed that placental miRNAs, including those dysregulated in pathological settings, are secreted via EVs by trophoblast cells and that after internalization by immune cells, affect gene expression therein. In vivo, variations in decidual immune cells facilitate the establishment of pregnancy, whilst hematological changes in the peripheral blood are needed to maintain surveillance and to recognize and respond to invading microorganisms. These immune responses are mediated by glycoproteins on the surface of cells, whose expression could be modified by uptake of vesicular proteins carried by EVs. We hypothesize that the proteome of placental-derived EVs (PDEVs) is transferred to immune cells and shapes their function specifically through modification of their glycoprotein expression. To prove this, PDEVs will be obtained by ex vivo placenta perfusion. Two populations of PDEVs will be isolated by ultracentrifugation and then characterized in terms of size, concentration and protein content by proteomic analysis. Peripheral mononuclear blood cells (PMBCs) will be isolated from whole blood samples and PDEV uptake will be assessed by flow cytometry and confocal microscopy. Finally, proteomic analyses of recipient cells will be carried out with special regard to glycoprotein identification followed by a bioinformatics analysis for identification of altered proteins and their associated pathways.

• DFG Programme: Research Grants