Syncytiotrophoblast Extracellular Vesicles from Late-Onset Preeclampsia Placentae suppress Pro-Inflammatory immune response in THP-1 Macrophages

© 2021 Awoyemi, Motta-Mejia, Zhang, Kouser, White, Kandzija, Alhamlan, Cribbs, Tannetta, Mazey, Redman, Kishore and Vatish. Syncytiotrophoblast derived Extracellular Vesicles (STBEV) from normal pregnancy (NP) have previously been shown to interact with circulating monocytes and B cells, and induce pro-inflammatory cytokine release. Early-onset preeclampsia (EOPE) is associated with an exacerbated inflammatory response, yet there is little data regarding late-onset PE (LOPE) and immune function. Here, using a macrophage/monocyte cell line THP-1, we investigated the inflammatory potential of STBEV, comprising medium/large-STBEV (>200nm) and small-STBEV (<200nm), isolated from LOPE (n=6) and normal (NP) (n=6) placentae via dual-lobe ex-vivo placental perfusion and differential centrifugation. THP-1 cells bound and internalised STBEV isolated from NP and LOPE placentae, as revealed by flow cytometry, confocal microscopy and ELISA. STBEV-treated THP-1 cells were examined for cytokine gene expression by RT-qPCR and the cell culture media examined for secreted cytokines/chemokines. As has been previously reported, NP medium/large-STBEV significantly upregulated the transcriptional expression of TNF-α, IL-10, IL-6, IL-12, IL-8 and TGF-β compared to PE medium/large-STBEV, however, there was no significant difference in the small STBEV population between the two groups though in general, NP small STBEVs slightly upregulated the same cytokines. In contrast, LOPE STBEV (medium and large) did not induce pro-inflammatory responses by differentiated THP-1 macrophages. This decreased effect of LOPE STBEV was echoed in cytokine/chemokine release. Our results appear to suggest that STBEV from LOPE placentae do not have a major immune-modulatory effect on macrophages. In contrast, NP STBEV caused THP-1 cells to release pro-inflammatory cytokines. Thus, syncytiotrophoblast extracellular vesicles from LOPE dampen immune functions of THP-1 macrophages, suggesting an alternative mechanism leading to the pro-inflammatory environment observed in LOPE

Placental capillary pericytes release excess extracellular vesicles under hypoxic conditions inducing a pro-angiogenic profile in term pregnancy

Supplementary data available online at: https://www.sciencedirect.com/science/article/pii/S0006291X2300181X#appsec1Copyright © 2023 The Authors. Pericytes are multifunctional cells wrapped around capillary endothelia, essential for vascular health, development, and blood flow regulation, although their role in human placental chorionic villi has not been fully explored. The second half of normal pregnancy is characterized by a progressive decline in placental and fetal oxygen levels which, by term, comprises a substantial degree of hypoxia. We hypothesized this hypoxia would stimulate pericyte regulation of chorionic villous capillary function. This study's objective was to investigate the role of hypoxia on normal term placental pericytes (PLVP) and their signaling to endothelial cells. First, we confirmed fetoplacental hypoxia at term by a new analysis of umbilical arterial blood oxygen tension of 3,010 healthy singleton neonates sampled at caesarean section and before labor. We then measured the release of cytokines, chemokines, and small extracellular vesicles (PLVPsv), from PLVP cultured at 20%, 8% and 1% O2. As O2 levels decreased, secreted cytokines and chemokines [interleukin-6 (IL-6), interleukin-1α (IL-1α) and vascular endothelial growth factor (VEGF)], and small extracellular vesicle markers, (Alix, Syntenin and CD9) increased significantly in the culture supernatants. When primary human umbilical vein endothelial cells (HUVEC) were cultured with PLVPsv, polygon formation, number, and tube formation length was significantly increased compared to cells not treated with PLVPsv, indicating PLVPsv stimulated angiogenesis. We conclude that adding PLVPsv stimulates angiogenesis and vessel stabilization on neighboring endothelial cells in response to hypoxia in term pregnancy compared to no addition of PLVPsv. Our finding that PLVP can release angiogenic molecules via extracellular vesicles in response to hypoxia may apply to other organ systems.MRC Program Grant (MR/J003360/1); rad Sutherland is supported by the National Health and Medical Research Council of Australia (APP1137776)

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

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

Syncytiotrophoblast extracellular vesicles: their role in gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is the most common metabolic complication of pregnancy. It affects more than 17 million women each year and causes adverse consequences for both mother and baby. Previous research has observed that the placenta plays an important role in the pathogenesis of GDM, since delivery of the placenta results in the immediate reversal of diabetic symptoms. The aim of this thesis is to investigate the nature of the placenta’s role in GDM, building on the knowledge that the organ releases extracellular vesicles (EVs) into the maternal circulation from six weeks gestation until delivery. EVs carry a variety of ligands, including both proteins and RNA species, between the cells. As such, they have an important role in cell-cell communication in both physiological and pathophysiological conditions, and may provide novel biomarkers of disease. We hypothesized that placental EVs might carry factors impacting insulin availability, a hallmark of pregnancy altered during the establishment of GDM. We identified two novel biomarkers (dipeptidyl peptidase 4 (DPPIV) and insulin receptor (IR)) on placental EVs. DPPIV is a glycoprotein that rapidly degrades incretin hormones, which are known to increase insulin secretion, and thus regulate glucose homeostasis. DPPIV inhibitors are established as therapeutic agents for type 2 diabetes mellitus, and we confirmed that they can be used to inhibit placental DPPIV-EV activity. IR is a key regulator of glucose uptake by skeletal muscles and adipose cells. We showed that placental IR-EVs can act as decoy receptors capable of neutralising maternal insulin. We demonstrated that GDM patients exhibited significantly higher levels of circulating placental DPPIV-EVs and IR-EVs. The final aim of this project was to characterise the protein cargo of placental EVs from GDM pregnancy using mass spectrometry. We identified 56 proteins as differentially expressed between GDM and normal pregnancy. In conclusion, this DPhil project shows that STB-EVs carry biologically active molecules that have the potential to regulate maternal insulin levels in GDM and could represent biologically plausible biomarkers for the disease

Circulating soluble fms-like tyrosine kinase-1 is placentally derived in normal pregnancy: first in vivo evidence

Circulating sFlt-1 increases significantly in pregnancy compared to the non-pregnant state and even more so in the event of preeclampsia. We set out to determine if circulating sFlt-1 is placentally derived in normal pregnancy. Paired uterine and peripheral vein samples were collected at time of caesarean section. A follow up peripheral sample was collected in the postpartum period. There was a significant sFlt-1 gradient between uterine and peripheral veins. sFlt-1 levels dropped significantly when the placenta has been removed, i.e. postnatally. This is in keeping with the placenta being the main site of sFlt-1 production in normal pregnancies

Circulating soluble fms-like tyrosine kinase-1 is placentally derived in normal pregnancy: first in vivo evidence

Circulating sFlt-1 increases significantly in pregnancy compared to the non-pregnant state and even more so in the event of preeclampsia. We set out to determine if circulating sFlt-1 is placentally derived in normal pregnancy. Paired uterine and peripheral vein samples were collected at time of caesarean section. A follow up peripheral sample was collected in the postpartum period. There was a significant sFlt-1 gradient between uterine and peripheral veins. sFlt-1 levels dropped significantly when the placenta has been removed, i.e. postnatally. This is in keeping with the placenta being the main site of sFlt-1 production in normal pregnancies

Placental syncytiotrophoblast-derived extracellular vesicles carry active NEP (neprilysin) and are increased in preeclampsia

NEP (neprilysin) is a widely expressed membrane-bound metalloprotease, which binds and cleaves a variety of peptides including vasodilators, natriuretics, and diuretics. Higher levels of NEP result in hypertension—a cardinal feature of the placental disease preeclampsia. Syncytiotrophoblast-derived extracellular vesicles (EVs), comprising microvesicles and exosomes, are released into the peripheral circulation in pregnancy and are postulated as a key mechanism coupling placental dysfunction and maternal phenotype in preeclampsia. We aimed to determine whether higher levels of active NEP are found in syncytiotrophoblast-derived EVs in preeclampsia compared with normal pregnancy. Using immunostaining and Western blotting, we first demonstrated that NEP levels are greater not only in preeclampsia placental tissue but also in syncytiotrophoblast-derived microvesicles and exosomes isolated from preeclampsia placentas (P&lt;0.05, n=5). We confirmed placental origin using antibody-coated magnetic beads to isolate NEP-bound vesicles, finding that they stain for placental alkaline phosphatase. NEP on syncytiotrophoblast-derived EVs is active and inhibited by thiorphan (P&lt;0.01, n=3; specific inhibitor). Syncytiotrophoblast-derived microvesicles, isolated from peripheral plasma, demonstrated higher NEP expression in preeclampsia using flow cytometry (P&lt;0.05, n=8). We isolated plasma exosomes using size-exclusion chromatography and showed greater NEP activity in preeclampsia (P&lt;0.05, n=8). These findings show that the placenta releases active NEP into the maternal circulation on syncytiotrophoblast-derived EVs, at significantly greater levels in preeclampsia. NEP has pathological roles in hypertension, heart failure, and amyloid deposition, all of which are features of preeclampsia. Circulating syncytiotrophoblast-derived EV-bound NEP thus may contribute to the pathogenesis of this disease

Circulating syncytiotrophoblast-derived extracellular vesicles exhibit variation in release between night and day

: Syncytiotrophoblast-derived extracellular vesicles (STBEV) are a key focus of placental research, proposed as a mediator of feto-maternal communication. Recent work has described differences in STBEV number and content in normal and disease states, with attention directed to their value as disease biomarkers in preeclampsia and gestational diabetes. Circadian rhythms are fundamental to human biology; three studies have shown diurnal variation in specific circulating extracellular vesicles outside pregnancy; no study has investigated diurnal variation of STBEV. We hypothesise that STBEV release is different between night and day

Placental syncytiotrophoblast-derived extracellular vesicles carry active NEP (neprilysin) and are increased in preeclampsia

NEP (neprilysin) is a widely expressed membrane-bound metalloprotease, which binds and cleaves a variety of peptides including vasodilators, natriuretics, and diuretics. Higher levels of NEP result in hypertension—a cardinal feature of the placental disease preeclampsia. Syncytiotrophoblast-derived extracellular vesicles (EVs), comprising microvesicles and exosomes, are released into the peripheral circulation in pregnancy and are postulated as a key mechanism coupling placental dysfunction and maternal phenotype in preeclampsia. We aimed to determine whether higher levels of active NEP are found in syncytiotrophoblast-derived EVs in preeclampsia compared with normal pregnancy. Using immunostaining and Western blotting, we first demonstrated that NEP levels are greater not only in preeclampsia placental tissue but also in syncytiotrophoblast-derived microvesicles and exosomes isolated from preeclampsia placentas (P<0.05, n=5). We confirmed placental origin using antibody-coated magnetic beads to isolate NEP-bound vesicles, finding that they stain for placental alkaline phosphatase. NEP on syncytiotrophoblast-derived EVs is active and inhibited by thiorphan (P<0.01, n=3; specific inhibitor). Syncytiotrophoblast-derived microvesicles, isolated from peripheral plasma, demonstrated higher NEP expression in preeclampsia using flow cytometry (P<0.05, n=8). We isolated plasma exosomes using size-exclusion chromatography and showed greater NEP activity in preeclampsia (P<0.05, n=8). These findings show that the placenta releases active NEP into the maternal circulation on syncytiotrophoblast-derived EVs, at significantly greater levels in preeclampsia. NEP has pathological roles in hypertension, heart failure, and amyloid deposition, all of which are features of preeclampsia. Circulating syncytiotrophoblast-derived EV-bound NEP thus may contribute to the pathogenesis of this disease