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Introduction: The aim was to investigate syncytiotrophoblast extracellular vesicle (STBEV) uptake mechanisms by primary endothelial cells, the effects on gene expression, cell activation as well as the effect of aspirin., Methods: The STBEVs were derived using the placental perfusion system, from normal or preeclamptic placentas. Endothelial uptake was analysed with flow cytometry. To elucidate uptake, different inhibitors were tested; Cytochalasin D, Chlorpromazine hydrochloride, Methyl-B-cyclodextrin, Dynasore and Wortmannin. Endothelial gene expression was evaluated using an endothelial cell biology qPCR array. Cell activation was studied by ICAM-1 surface expression after STBEV exposure, with and without aspirin treatment., Results: Normal and preeclamptic STBEV uptake was blocked in similar ways. Chlorpromazine, Dynasore and Wortmannin almost completely blocked STBEV uptake. Methyl-B-cyclodextrin blocked 45-60% of the uptake while Cytochalasin D did not block uptake at all. Neither normal nor preeclamptic STBEVs had any significant effects on endothelial gene expression. Normal STBEVs down-regulated cell surface protein ICAM-1 expression, with and without aspirin treatment. Aspirin had no effect on STBEV uptake or cellular gene expression on its own, however it down regulated ICAM-1 protein expression in combination with preeclamptic STBEV exposure., Discussion: STBEV uptake primarily occurred through clathrin-mediated endocytosis. The STBEVs had no significant effect on gene expression but did have effects on ICAM-1 surface expression. The prophylactic mechanisms of aspirin may be by preventing the endothelium from being activated by the preeclamptic STBEVs., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Placental syncytiotrophoblast (STB) release extracellular vesicles (STB-EVs) that communicate physiological and pathological placental signals to the maternal organs. STB-EV release also increases in preeclampsia (PE). Here we explored the cargo of PP13 in STB-EVs from PE versus control placentas., Methods: Placentae were harvested following cesarean section deliveries, and dual placental lobe perfusion was used to harvest STB-EV. Maternal side perfusate was centrifuged at 10,000 × g to yield the STB microvesicles, and then at 150,000 × g to yield STB exosomes. Total STB-EVs (tSTB-EVs) were collected using a one step 150,000 × g centrifugation. Placental origin and size distribution were assessed by Western blotting and Nanoparticle Tracking Analysis, respectively. PP13 expression was determined by Western blot and ELISA., Results: Placental alkaline phosphatase (PLAP; a STB specific marker) was present in all preparations. Total tSTB-EVs and STB-EXs also expressed the exosome markers such as the Apoptosis-Linked Gene 2-Interacting Protein X (Alix) and the cluster differentiation protein 9 (CD9). PP13 was localized to the outer surface and intra-vesicular compartments of all fractions. Surface to total PP13 ratios were ∼1:1 for all STB-EV preparations. In contrast to the previously reported higher circulating concentrations of soluble PP13 in PE, significantly lower levels of PP13, normalized to total vesicular protein, were observed in PE samples. PP13 reduction in all STB-EVs' sub-populations may be attributed to differences in gestational age (GA). A simple correction for GA suggested that PE may be an important influence., Conclusions: PP13 is located in and on all types of STB-EVs. Circulating PP13 may therefore be either soluble or associated with extracellular vesicles with different pathophysiological effects in the maternal circulation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A variety of 'debris' is shed from the syncytial surface of the human placenta ranging from large deported multinuclear fragments to sub-cellular components. It is increasingly clear that at least some of this material has signalling functions. Many categories of circulating debris are increased in pre-eclampsia, and exhibit proteins that are pro-inflammatory and could contribute to the systemic inflammatory response in normal pregnancy, which is exaggerated in pre-eclampsia. It is now evident that there is a large 'hidden' population of microvesicles and nanovesicles (including exosomes) which are hard to investigate because of their size. We have used a new technology, nanoparticle tracking analysis, to measure the size and concentration of syncytiotrophoblast vesicles prepared by placental perfusion. The vesicles range in size from 50 nm to 1 μm with the majority being <500 nm (which includes both exosomes and microvesicles). We speculate whether changes not only in the numbers, but also in the size (beneficial syncytiotrophoblast exosomes and harmful microvesicles) might be important in the maternal syndrome of pre-eclampsia., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Preeclampsia can be lethal to both mother and baby. The prominent symptoms of this syndrome are hypertension, proteinuria and oedema, resulting from an exaggerated aseptic systemic inflammatory response, triggered by placental factors shed into the maternal circulation. Syncytiotrophoblast microparticles (STBM) are one possible factor, shed when the placenta is exposed to stressors such as hypoxia/reperfusion. These can disrupt mitochondria, triggering apoptosis and necrosis, placental pathologies which are increased in preeclampsia. We tested the effects of antioxidant vitamins C (50 microM) and E (50 microM) on trophoblast in culture, using term villous cytotrophoblast preparations. Following Percoll gradient centrifugation and MHC class I expressing cell depletion of placenta digests, syncytial fragments were removed using anti-placental alkaline phosphatase antibody. This yielded cytotrophoblasts of consistently high purity. EGF (10 ng/ml) stimulated syncytialisation and hCG and progesterone production. However, mitochondrial induced apoptosis (MIA) was evident 96h post-isolation, as mitochondrial membrane potential loss and caspase 9 and caspase 3 activation. ROCK-1 cleavage and syncytiotrophoblast particle shedding increased concurrently with apoptosis induction. Vitamins blocked MIA and syncytiotrophoblast particle shedding and significantly increased hCG (p<0.005) and progesterone (p<0.02) concentrations in culture supernatants, reflecting the increased survival rates. Although more cells survived in culture, syncytialisation rate (%) was significantly reduced (p<0.005). We conclude that vitamins C and E can significantly reduce mitochondrial damage generated following syncytialisation in vitro. However, further work is required to determine whether antioxidant vitamins interfere with normal fusion processes.

Rationale: Syncytiotrophoblast microparticles (STBM) are shed into the maternal circulation in higher amounts in pre-eclampsia compared to normal pregnancy and are believed to be the stimulus for the systemic inflammatory response and endothelial cell damage which characterises the maternal syndrome. The excess shedding of STBM may be caused by hypoxia as a result of poor placentation, which is often a feature of pre-eclampsia. Similar placental pathology occurs in some cases of normotensive intrauterine growth restriction (nIUGR), but in the absence of maternal disease., Objective: To examine whether the shedding of STBM in nIUGR occurs to the same extent as in pre-eclampsia., Methods: A prospective case-control study in a tertiary referral centre of: 1) women with early-onset pre-eclampsia (EOPET < 34 week), 2) women with late-onset pre-eclampsia (LOPET > or = 34 week), 3) women with nIUGR), 4) matched normal pregnant women (NPC), and 5) non-pregnant women. An ELISA using the antitrophoblast antibody NDOG2 was used to measure STBM levels in peripheral venous plasma. Non-parametric analyses were conducted with statistical significance set at p < 0.05., Results: STBM levels rise during normal pregnancy. EOPET was associated with increased STBM levels (EOPET (median): 41 ng/ml, n = 15) compared with matched normal pregnancy (16 ng/ml, n = 15; Wilcoxon p = 0.005). LOPET (50 ng/ml, n = 10) and nIUGR (18 ng/ml, n = 8) STBM levels did not differ from matched normal pregnancy (36 ng/ml, n = 15, and 36 ng/ml, n = 8, respectively). Background levels in non-pregnant plasma were 0.49 ng/ml, n = 10., Conclusions: Increased STBM levels were found in EOPET but not in nIUGR providing further evidence for their role in the pathogenesis of the maternal syndrome.