Abstract
Objectives: Antiphospholipid antibodies (aPL) are autoantibodies that target the syncytiotrophoblast. Presence of aPL during pregnancy causes an increased risk of recurrent miscarriage, preeclampsia, preterm birth, stillbirth and fetal growth restriction. It is not clear how aPL contribute to these complications but aPL internalisation causes production and accumulation of ‘danger signals’ within the syncytiotrophoblast. Some of these ‘danger signals’ are packaged into extracellular vesicles (EVs) extruded into maternal blood. This study explores the extent of differential protein packaging into EVs after aPL treatment of placentae, providing insight into the functional effects these EVs may have on maternal physiology/pathology.
Methods: Term placental explants were cultured in Netwells with 50 μg/mL of aPL (n=5) or control IgG (n=5) in advanced DMEM/F12 with 2% FBS, 1% penicillin/streptomycin overnight. Media were centrifuged at 2000 xg for 5 minutes to remove debris, the supernatants centrifuged at 20,000 xg for 1 hour at 4°C, followed by a 100,000 xg spin for 1 hour at 4°C to obtain large- and small-EVs, respectively. EVs were enriched through a qEV legacy column. EV-protein profiles were compared by quantitative proteomics using SWATH-mass spectrometry followed by enrichment analyses of differentially abundant proteins using DAVID.
Results: aPL treatment of placentae led to extrusion of large-EVs with increased transcription factors and protein translation/splicing machinery. aPL treatment of placentae led to production of small-EVs with a larger number of increased protein production/splicing machinery and proteasome components, as well as decreased DNA damage sensing/DNA repair machinery and cytoskeletal components.
Conclusion: The differential changes in protein packaging into small- and large-EVs from aPL-treated placentae confirms that these EV subtypes carry cargos that are altered in placental pathology. Therefore, each EV subtype may have different effects on maternal physiology. Studying the effects of a single EV subtype on maternal physiology may have misleading results.