Placental Vesicles Carry Active Endothelial Nitric Oxide Synthase and Their Activity is Reduced in Preeclampsia.

Abstract

Preeclampsia (PE), a multi-system hypertensive disorder of pregnancy, is associated 25 with increased systemic vascular resistance. Placentae from PE patients have 26 reduced levels of endothelial nitric oxide synthase (eNOS) and thus less nitric oxide 27 (NO). Syncytiotrophoblast extracellular vesicles (STBEV), comprised of microvesicles 28 (STBMV) and exosomes (STBEX), carry signals from the STB to the mother. We 29 hypothesized that STBEV bound eNOS (STBEV-eNOS), capable of producing NO, 30 are released into the maternal circulation. Dual-lobe ex vivo placental perfusion and 31 differential centrifugation was used to isolate STBEV from PE (n=8) and normal 32 pregnancies (NP) (n=11). Plasma samples of gestational age matched PE and NP 33 (n=6) were used to isolate circulating STBMV. STBEV expressed placental alkaline 34 phosphatase (PlAP), confirming placental origin. STBEV co-expressed eNOS, but not 35 iNOS, confirmed using Western blot, flow cytometry and immuno-depletion. STBEV-36 eNOS produced NO which was significantly inhibited by L-NAME (eNOS inhibitor, 37 *p<0.05), but not 1400W (iNOS inhibitor). STBEV-eNOS catalytic activity was 38 confirmed by visualising eNOS dimerization. STBEV-eNOS was more abundant in 39 uterine vein compared to peripheral blood, indicating placental origin. STBEV isolated 40 from PE perfused placentae had lower levels of STBEV-eNOS (STBMV; *p<0.05) and 41 overall lower NO activity (STBMV, ns; STBEX, *p<0.05) compared to NP. Circulating 42 plasma STBMV from PE women had lower STBEV-eNOS expression compared to NP 43 women (**p<0.01). This is the first observation of functional eNOS expressed on 44 STBEV from NP and PE placentae, as well as in plasma. The lower STBEV-eNOS 45 NO production seen in PE may contribute to the decreased NO bioavailability in this 46 disease.