Exosome‐like Vesicles Facilitate Intercellular Communication between Uterine Artery Smooth Muscle Cells and Perivascular Adipose Tissue

Perivascular adipose tissue (PVAT) contributes to regulation of vascular tone. We recently reported that PVAT surrounding uterine arteries had anti‐dilatory effects on isolated uterine arteries and contributed to local regulation of uterine blood flow in pregnant rats. The objective of the current study was to investigate the mechanisms underlying the functional interaction between PVAT and uterine arterial wall. Adipose tissue passively secretes exosomes, which are membrane bound vesicles (30 – 150 nm in diameter). It is unknown whether PVAT, which has a different developmental origin from other adipose depots, secretes exosomes, and if exosomes facilitate intercellular communication between PVAT and vascular cells. We hypothesized that uterine PVAT sheds exosome‐like vesicles (Exo‐PVAT) that transfer their cargo into neighboring uterine vascular smooth muscle cells (USMCs), facilitating maternal uterine artery function. Methods Exo‐PVAT and primary USMCs were isolated from pregnant (gestational day 16, term=22–23 days) and non‐pregnant rats. Exosomes were isolated and purified with tissue culture, and ultrafiltration and ultracentrifugation techniques, and USMCs were isolated using enzymatic digestion. Exosome morphology was characterized via transmission electron microscopy, and exosome size distribution and molecular weight were determined via a Malvern Zetasizer and fast protein liquid chromatography (FPLC), respectively. Protein expression of various exosome markers was determined using Western blot analysis. To determine USMC uptake of Exo‐PVAT, uterine Exo‐PVAT were labelled with a membrane‐labeling dye (DiO, Invitrogen) and then co‐cultured with USMCs for 3 hours. Vascular reactivity to acetylcholine (ACh; 10 −9 – 3×10 −5 M) was measured in uterine arteries using wire myography after exposure to Exo‐PVAT or phosphate‐buffered saline (PBS, control) 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. Samples from pregnant rats had a relatively high expression of exosomes of 2000 kDa when compared to samples from non‐pregnant rats. Using immunocytochemistry, we demonstrated that USMCs were able to take up exosomes derived from their adjacent PVAT. Using a functional bioassay, we found that Exo‐PVAT reduced sensitivity to ACh in uterine arteries from pregnant rats ( p EC 50 , control: 7.46 vs. Exo‐PVAT PREG : 7.02) but had the opposite effect in non‐pregnant rats ( p EC 50 , control: 6.91 ± 0.04 vs. Exo‐PVAT NP : 7.03 ± 0.06, p<0.01). Conclusion Uterine PVAT sheds exosome‐like vesicles that transfer their cargo to the adjascent uterine arterial wall, where they are taken up by 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 local regulation of uterine blood flow in pregnancy. Support or Funding Information UNTHSC Seed Grant