Cushen, SpencerRaetz, MeganSaranya Conjeevaram Nagarajan, BhavaniRaut, SangramGoulopoulou, Styliani2020-12-102020-12-102020https://hdl.handle.net/20.500.12503/30108Introduction Perivascular adipose tissue (PVAT) regulates uterine artery tone during pregnancy. However, the mechanisms underlying its functional role in uterine arteries is unknown. Exosomes are cargo carrying membrane-bound extracellular vesicles used in intercellular communication. It is unknown whether PVAT secretes exosomes. We hypothesized that uterine PVAT sheds exosomes (Exo-PVAT) that are uptaken by neighboring uterine vascular smooth muscle cells (USMCs). Methods Exo-PVAT were isolated and purified with tissue culture and ultracentrifugation, and primary USMCs were isolated using enzymatic digestion from pregnant and non-pregnant rats. Exosome protein content, size and molecular weight were determined via western blot, Malvern Zetasizer and fast protein liquid chromatography (FPLC), respectively. To determine USMC uptake of Exo-PVAT, Exo-PVAT were labelled with a membrane-labeling dye and co-cultured with USMCs for 3 hours. Results Exo-PVAT expressed TSG101, Alix, and CD9. Pregnancy did not affect Exo-PVAT size [Median(IQR) (nm), Non-pregnant: 99.4 (80.5) vs. Pregnant: 46.7 (21.2), p=0.5]. Using FPLC, we identified exosomes of 40-200 kDa in samples from both pregnant and non-pregnant rats. PVAT from pregnant rats secreted a relatively high amount of exosomes of 2000 kDa compared to non-pregnat rats. Immunocytochemical assessments revealed that USMCs took up exosomes derived from their adjacent PVAT. Conclusion Uterine PVAT sheds exosome-like vesicles which are uptaken by adjacent USMCs. The interaction between Exo-PVAT and USMCs is a novel type of intercellular communication that may have important implications in uterine artery function and blood flow in pregnancy.enpresentation