Exploring Differences in The Cargos of Placental Extracellular Vesicles in Physiological and Pathological Pregnancy

Abstract

Introduction: Placental extracellular vesicles (pEVs) are lipid bilayer particles that carry various types of cargos from the placenta to the maternal circulation. Recognized as essential messengers in fetal-maternal communication, the pEVs have been reported to regulate vascular tone during normal pregnancy. However, it remains unclear whether these regulatory functions differ between pEVs from 1st and 3rd-trimesters placentae. Meanwhile, pEVs from preeclamptic pEVs have been shown to induce or exacerbate hypertension, the key presentation of this condition, suggesting they may be involved in the pathogenesis of preeclampsia via an unknown mechanism. Aims: To investigate the differences of pEVs during different gestational stages in physiological pregnancy, as well as the differences of pEVs during preeclampsia and normotensive pregnancy. Methods: Wire myography was performed on the mesenteric resistant arteries isolated from pregnant CD-1mice injected with 1st and 3rd-trimester pEVs to compare the vascular effects of pEVs from different gestational ages in normal pregnancy. Small RNAs sequencing was conducted on pEVs collected from 1) 1st and 3rd-trimester placentae and 2) early-onset preeclampsia (EOPE), late-onset preeclampsia (LOPE) and normotensive (NT) placentae, with the pEVs isolated from an ex vivo placental explant culture model. In parallel, Sequential Window Acquisition of All Theoretical Mass Spectrometry was employed to profile the proteomic content of EOPE, LOPE, and NT pEVs derived from the same explant model. To investigate the functional relevance of the significantly differentially abundant miRNAs in preeclamptic versus normotensive pEVs, human microvascular endothelial cells were transfected with specific miRNA inhibitors, followed by monocyte adhesion assays to assess endothelial activation. Additionally, the biomarker potential of altered membrane-associated proteins on pEVs was evaluated using a magnetic affinity sorting assay. Findings: 1st-trimester pEVs reduced the vascular sensitivity to vasoconstrictors and enhanced the vascular sensitivity to vasodilators, in comparison to 3rd-trimester pEVs. These functional differences may be partially explained by the differences in the miRNA profiles between the 1st and 3rd trimester pEVs. In preeclamptic pEVs, miRNAs and proteins with different abundance compared to normotensive pEVs are involved in various cardiovascular functions. Notably, the lower abundance of miR-148b-3p in preeclamptic pEVs compared to normotensive pEVs may lead to upregulation of Integrin α5 on endothelial cells, thereby enhancing monocyte adhesion. Additionally, TPBG, a membrane protein found to be more abundant in EOPE pEVs than in NT pEVs, was unable to sort pEVs via magnetic affinity assay, underscoring the complexity of EV surface biology. Conclusion: This study provides novel insights into the potential roles of pEVs in maternal cardiovascular adaptation during both physiological and pathological pregnancy. Collectively, this study, when combined with others, offers promising avenues for the identification of biomarkers and therapeutic targets for the clinical management of preeclampsia