Preliminary evidence that blocking the uptake of placenta-derived preeclamptic extracellular vesicles protects the vascular endothelium and prevents vasoconstriction.

Preeclampsia (PE) is a pregnancy syndrome characterized by hypertension and organ damage manifesting after 20 gestational weeks. The etiology is of multifactorial origin, where placental stress causes increased levels of placenta-derived extracellular vesicles (STBEVs) in the maternal circulation, shown to cause inflammation, endothelial activation, vasoconstriction, and anti-angiogenic activity. General endothelial dysfunction is believed to be initiated by endothelial insult during pregnancy that alters vascular function resulting in increased arterial stiffness, cardiac dysfunction, and increased risk of cardiovascular disease later in life. We compared the effect of normal and PE derived STBEVs in vitro on vascular contractility of human subcutaneous arteries using wire myography. Cellular structures of exposed vessels were investigated by transmission electron microscopy. We explored strategies to pharmacologically block the effects of the STBEVs on human vessels. The PE STBEVs caused significantly stronger angiotensin II-mediated contractions and extended structural damage to human subcutaneous arteries compared to normal STBEVs. These negative effects could be reduced by blocking vesicle uptake by endothelial cells, using chlorpromazine or specific antibodies towards the LOX-1 receptor. The therapeutic potential of blocking vesicle uptake should be further explored, to reduce the permanent damage caused on the vasculature during PE pregnancy to prevent future cardiovascular risk.

Moderate-to-vigorous group aerobic exercise versus group leisure activities for mild-to-moderate depression in adolescents: study protocol for a multicentre randomised controlled trial.

INTRODUCTION: Depression is common, increasing among adolescents and carries risk of disability, lower educational achievements, cardiovascular disease, substance abuse, self-harm and suicide. The effects of evidence-based treatments with medication or psychotherapy are modest. Aerobic exercise is a promising intervention for adolescents with depression, but available studies are hampered by methodological shortcomings. This study aims to evaluate aerobic group exercise versus an active comparator of leisure group activities in adolescents from clinical services with mild-to-moderate depression. METHODS AND ANALYSIS: This study is a multicentre randomised controlled trial at four psychiatric clinics in Sweden. Participants (n=122) will be randomised 1:1 to group exercise delivered by exercise professionals and supported by mental health (MH) workers or leisure activities lead by the same MH workers for 1 hour three times a week for 12 weeks. Participants will be assessed at baseline, single blind after 13 weeks and 26 weeks and openly after 1 year. Participants randomised to the leisure group will be offered exercise in the open phase. The primary outcome is clinician-rated Children's Depression Rating Scale-Revised. Secondary outcomes are self-rated Quick Inventory of Depressive Symptomatology, self-rated functioning; clinician-rated improvement and functioning; objectively measured aerobic capacity, muscular strength, muscular endurance, body composition and presence or activity of selected biological markers of neuroprotection and neuroinflammation in blood samples. Further outcomes are cost-effectiveness and adolescents', parents' and coaches' experiences of the interventions and an exploration of how the adolescents' health and lifestyle are influenced by the interventions through qualitative interviews. ETHICS AND DISSEMINATION: The study is approved by the Swedish Ethical Review Authority (Ref. 2021-05307-01). Informed consent in writing will be provided from patients and parents of participants below 15 years of age. The results of this study will be communicated to the included participants and healthcare providers and also submitted for publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT05076214.

Placental syncytiotrophoblast extracellular vesicles enter primary endothelial cells through clathrin-mediated endocytosis.

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.

Syncytiotrophoblast derived extracellular vesicles transfer functional placental miRNAs to primary human endothelial cells.

During the pregnancy associated syndrome preeclampsia (PE), there is increased release of placental syncytiotrophoblast extracellular vesicles (STBEVs) and free foetal haemoglobin (HbF) into the maternal circulation. In the present study we investigated the uptake of normal and PE STBEVs by primary human coronary artery endothelial cells (HCAEC) and the effects of free HbF on this uptake. Our results show internalization of STBEVs into primary HCAEC, and transfer of placenta specific miRNAs from STBEVs into the endoplasmic reticulum and mitochondria of these recipient cells. Further, the transferred miRNAs were functional, causing a down regulation of specific target genes, including the PE associated gene fms related tyrosine kinase 1 (FLT1). When co-treating normal STBEVs with HbF, the miRNA deposition is altered from the mitochondria to the ER and the cell membrane becomes ruffled, as was also seen with PE STBEVs. These findings suggest that STBEVs may cause endothelial damage and contribute to the endothelial dysfunction typical for PE. The miRNA mediated effects on gene expression may contribute to the oxidative and endoplasmic reticulum stress described in PE, as well as endothelial reprogramming that may underlay the increased risk of cardiovascular disease reported for women with PE later in life.

Placenta-derived extracellular vesicles: their cargo and possible functions.

The literature on extracellular vesicles consists of rapidly expanding and often contradictory information. In this paper we attempt to review what is currently known regarding extracellular vesicles released specifically from human placental syncytiotrophoblast cells with a focus on the common but complex pregnancy-associated syndrome pre-eclampsia, where the level of syncytiotrophoblast extracellular vesicle release is significantly increased. We review common methods for syncytiotrophoblast extracellular vesicle derivation and isolation and we discuss the cargo of syncytiotrophoblast extracellular vesicles including proteins, RNA and lipids and their possible functions. A meta-analysis of available trophoblast-derived extracellular vesicle proteomic datasets revealed only three proteins in common: albumin, fibronectin-1 and plasminogen activator inhibitor-1, suggesting some variability in vesicle cargo, most likely reflecting stage and cell type of origin. We discuss the possible sources of variability that may have led to the low number of common markers, which has led us to speculate that markers and density in common use may not be strict criteria for identifying and isolating placenta-derived exosomes.

Syncytiotrophoblast vesicles show altered micro-RNA and haemoglobin content after ex-vivo perfusion of placentas with haemoglobin to mimic preeclampsia.

BACKGROUND: Cell-free foetal haemoglobin (HbF) has been shown to play a role in the pathology of preeclampsia (PE). In the present study, we aimed to further characterize the harmful effects of extracellular free haemoglobin (Hb) on the placenta. In particular, we investigated whether cell-free Hb affects the release of placental syncytiotrophoblast vesicles (STBMs) and their micro-RNA content. METHODS: The dual ex-vivo perfusion system was used to perfuse isolated cotyledons from human placenta, with medium alone (control) or supplemented with cell-free Hb. Perfusion medium from the maternal side of the placenta was collected at the end of all perfusion phases. The STBMs were isolated using ultra-centrifugation, at 10,000×g and 150,000×g (referred to as 10K and 150K STBMs). The STBMs were characterized using the nanoparticle tracking analysis, identification of surface markers and transmission electron microscopy. RNA was extracted and nine different micro-RNAs, related to hypoxia, PE and Hb synthesis, were selected for analysis by quantitative PCR. RESULTS: All micro-RNAs investigated were present in the STBMs. Mir-517a, mir-141 and mir-517b were down regulated after Hb perfusion in the 10K STBMs. Furthermore, Hb was shown to be carried by the STBMs. CONCLUSION: This study showed that Hb perfusion can alter the micro-RNA content of released STBMs. Of particular interest is the alteration of two placenta specific micro-RNAs; mir-517a and mir-517b. We have also seen that STBMs may function as carriers of Hb into the maternal circulation.

The unique expression and function of miR-424 in human placental trophoblasts.

Placental hypoperfusion causes cellular hypoxia and is associated with fetal growth restriction and preeclampsia. In response to hypoxia, the repertoire of genes expressed in placental trophoblasts changes, which influences key cellular processes such as differentiation and fusion. Diverse miRNAs were recently found to modulate the cellular response to hypoxia. Here we show that miR-424, which was previously shown to be upregulated by hypoxia in nontrophoblastic cell types, is uniquely downregulated in primary human trophoblasts by hypoxia or chemicals known to hinder cell differentiation. We also identify FGFR1 as a direct target of miR-424 in human trophoblasts. This effect is unique to miR-424 and is not seen with other members of this miRNA family that are expressed in trophoblasts, such as miR-15 and miR-16. Our findings establish a unique role for miR-424 during differentiation of human trophoblasts.