Endothelial Extracellular Vesicles Research Articles

Neurovascular coupling, functional connectivity, and cerebrovascular endothelial extracellular vesicles as biomarkers of mild cognitive impairment.

Mild cognitive impairment (MCI) is a prodromal stage of dementia. Understanding the mechanistic changes from healthy aging to MCI is critical for comprehending disease progression and enabling preventative intervention. Patients with MCI and age-matched controls (CN) were administered cognitive tasks during functional near-infrared spectroscopy (fNIRS) recording, and changes in plasma levels of extracellular vesicles (EVs) were assessed using small-particle flow cytometry. Neurovascular coupling (NVC) and functional connectivity (FC) were decreased in MCI compared to CN, prominently in the left-dorsolateral prefrontal cortex (LDLPFC). We observed an increased ratio of cerebrovascular endothelial EVs (CEEVs) to total endothelial EVs in patients with MCI compared to CN, correlating with structural MRI small vessel ischemic damage in MCI. LDLPFC NVC, CEEV ratio, and LDLPFC FC had the highest feature importance in the random Forest group classification. NVC, CEEVs, and FC predict MCI diagnosis, indicating their potential as markers for MCI cerebrovascular pathology. Neurovascular coupling (NVC) is impaired in mild cognitive impairment (MCI). Functional connectivity (FC) compensation mechanism is lost in MCI. Cerebrovascular endothelial extracellular vesicles (CEEVs) are increased in MCI. CEEV load strongly associates with cerebral small vessel ischemic lesions in MCI. NVC, CEEVs, and FC predict MCI diagnosis over demographic and comorbidity factors.

#1841 Trimethylamine N-oxide (TMAO) induces the formation of endothelial extracellular vesicles

Abstract Background and Aims Endothelial dysfunction caused by the accumulation of uremic toxins is an important contributor to the development of cardiovascular disease in patients with chronic kidney disease (CKD). The uremic toxin trimethylamine n-oxide (TMAO) is associated with oxidative stress, vascular calcification, and inflammation, triggering the release of several proinflammatory molecules and Extracellular Vesicles (EVs). Clinical studies have shown that patients with CKD have high levels of circulating EVs, however, their role is still poorly understood. In this dysfunctional scenario, EVs can be used as damage biomarkers for CKD, as they carry information from their cells of origin. Here, we aim to elucidate the formation of EVs in endothelial cells exposed to TMAO. Method Human endothelial cells (EA.hy926, ATCC CRL-2922) were exposed for 24 h with TMAO at uremic concentrations (7.49 mg/L). EVs were isolated by differential ultracentrifugation. Protein concentration was analyzed by Pierce™ BCA Protein Assay Kit, and particle size and particle concentration were analyzed by NanoParticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), and Scanning Electron Microscopy (SEM). EVs were stained with calcein and their internalization in endothelial cells was analyzed with Fluorescence Confocal Microscopy. Results TMAO treatment at uremic concentration induced the formation of EVs with a size of 202.8 ± 3.1 nm (image 1B) and 1.09 × 109 ± 2.68 × 107 particles/mL and a total protein concentration of 88.57 µg/mL. The morphological features of EVs are shown in images 1C-1F. TMAO-induced EVs were incorporated by endothelial cells, being dispersed in the cytoplasm or perinuclear regions (images 1G and end 1H). Conclusion TMAO was able to induce the formation of EVs in endothelial cells, with characteristics morphological features. When compared to control EVs (image 1A), TMAO-EVs have a more heterogeneous size, which indicate a change in the size pattern, and thus the nature of the EVs induced by TMAO. TMAO-EVs were able to be internalized by human endothelial cells. This uptake can be closely related to the effects of EVs in endothelial cells, but more experiments need to be performed. Taken together, these data could contribute to understanding how the formation and uptake of TMAO-EVs by the endothelium occurs, relating their effects to the cardiovascular complications of CKD.

A potential mechanism for the cytoprotective effects of activated protein C–released endothelial extracellular vesicles

AbstractActivated protein C (APC) was shown to release extracellular vesicles (EVs). APC bound to the EVs was thought to be responsible for cytoprotection. Our study demonstrates that the cytoprotective effects of APC-released EVs are independent of APC. APC-released EVs carry anti-inflammatory microRNAs in their cargo.

Endothelial Extracellular Vesicles Enriched in microRNA-34a Predict New-Onset Diabetes in Coronavirus Disease 2019 (COVID-19) Patients: Novel Insights for Long COVID Metabolic Sequelae.

Emerging evidence indicates that the relationship between coronavirus disease 2019 (COVID-19) and diabetes is 2-fold: 1) it is known that the presence of diabetes and other metabolic alterations poses a considerably high risk to develop a severe COVID-19; 2) patients who survived a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have an increased risk of developing new-onset diabetes. However, the mechanisms underlying this association are mostly unknown, and there are no reliable biomarkers to predict the development of new-onset diabetes. In the present study, we demonstrate that a specific microRNA (miR-34a) contained in circulating extracellular vesicles released by endothelial cells reliably predicts the risk of developing new-onset diabetes in COVID-19. This association was independent of age, sex, body mass index (BMI), hypertension, dyslipidemia, smoking status, and D-dimer. SIGNIFICANCE STATEMENT: We demonstrate for the first time that a specific microRNA (miR-34a) contained in circulating extracellular vesicles released by endothelial cells is able to reliably predict the risk of developing diabetes after having contracted coronavirus disease 2019 (COVID-19). This association was independent of age, sex, body mass index (BMI), hypertension, dyslipidemia, smoking status, and D-dimer. Our findings are also relevant when considering the emerging importance of post-acute sequelae of COVID-19, with systemic manifestations observed even months after viral negativization (long COVID).

Circulating endothelial extracellular vesicle signatures correspond with ICU requirement: an exploratory study in COVID-19 patients

Extracellular vesicles (EVs) represent nanometer-sized, subcellular spheres, that are released from almost any cell type and carry a wide variety of biologically relevant cargo. In severe cases of coronavirus disease 2019 (COVID-19) and other states of systemic pro-inflammatory activation, EVs, and their cargo can serve as conveyors and indicators for disease severity and progression. This information may help distinguish individuals with a less severe manifestation of the disease from patients who exhibit severe acute respiratory distress syndrome (ARDS) and require intensive care measures. Here, we investigated the potential of EVs and associated miRNAs to distinguish normal ward patients from intensive care unit (ICU) patients (N = 10/group), with 10 healthy donors serving as the control group. Blood samples from which plasma and subsequently EVs were harvested by differential ultracentrifugation (UC) were obtained at several points in time throughout treatment. EV-enriched fractions were characterized by flow cytometry (FC), nanoparticle tracking analysis (NTA), and qPCR to determine the presence of selected miRNAs. Circulating EVs showed specific protein signatures associated with endothelial and platelet origin over the course of the treatment. Additionally, significantly higher overall EV quantities corresponded with increased COVID-19 severity. MiR-223-3p, miR-191-5p, and miR-126-3p exhibited higher relative expression in the ICU group. Furthermore, EVs presenting endothelial-like protein signatures and the associated miR-126-3p showed the highest area under the curve in terms of receiver operating characteristics regarding the requirement for ICU treatment. In this exploratory investigation, we report that specific circulating EVs and miRNAs appear at higher levels in COVID-19 patients, especially when critical care measures are indicated. Our data suggest that endothelial-like EVs and associated miRNAs likely represent targets for future laboratory assays and may aid in clinical decision-making in COVID-19.

MicroRNA-10a enrichment in factor VIIa–released endothelial extracellular vesicles: potential mechanisms

BackgroundFactor VIIa induces the release of extracellular vesicles (EVs) from endothelial cells (EEVs). Factor VIIa–released EEVs are enriched with microRNA-10a (miR10a) and elicit miR10a-dependent cytoprotective responses. ObjectivesTo investigate mechanisms by which FVIIa induces miR10a expression in endothelial cells and sorts miR10a into the EVs. MethodsActivation of Elk-1 and TWIST1 expression was analyzed by immunofluorescence microscopy and immunoblot analysis. Small interfering RNA silencing approach was used to knock down the expression of specific genes in endothelial cells. EVs secreted from endothelial cells or released into circulation in mice were isolated by centrifugation and quantified by nanoparticle tracking analysis. Factor VIIa or EVs were injected into mice; mice were challenged with lipopolysaccharides to assess the cytoprotective effects of FVIIa or EVs. ResultsFVIIa activation of ERK1/2 triggered the activation of Elk-1, which led to the induction of TWIST1, a key transcription factor involved in miR10a expression. Factor VIIa also induced the expression of La, a small RNA-binding protein. Factor VIIa–driven acid sphingomyelinase (ASM) activation and the subsequent activation of the S1P receptor pathway were responsible for the induction of La. Silencing of ASM or La significantly reduced miR10a levels in FVIIa-released EEVs without affecting the cellular expression of miR10a. Factor VIIa–EEVs from ASM knocked-down cells failed to provide cytoprotective responses in cell and murine model systems. Administration of FVIIa protected wild-type but not ASM−/− mice against lipopolysaccharide-induced inflammation and vascular leakage. ConclusionOur data suggest that enhanced cellular expression of miR10a coupled with La-dependent sorting of miR10a is responsible for enriching FVIIa-released EVs with miR10a.

Markers of systemic endotheliosis in early-onset preeclampsia relapse

Aim: to assess the blood level of biological endothelial damage markers in women with previous early-onset preeclampsia (еPE) during both the preconception (PC) stage and the first trimester of pregnancy.Materials and Methods. A prospective single-center cohort study was conducted. The control group comprised 40 women with uncomplicated pregnancies. The observation group consisted of 97 patients with a history of ePE, stratified based on pregnancy outcome into the comparison group (n = 59) characterized by a favorable gestational course, and the main group (n = 38) with relapsed еPE (30 patients were excluded from the study). Markers of systemic endotheliosis were determined in women at the PC stage and at 11–13 weeks of gestation: endothelin-1 (ET-1) and endothelial extracellular vesicles (EVs; CD-144).Results. At the PC stage, women in the control group and patients from the comparison group had comparable median (Me) ET-1 levels – 0.39 and 0.40 pmol/ml (p = 0.5935), respectively; at the same time, patients with relapsed еPE vs. control group had it significantly elevated (Me = 0.55 pmol/ml; p = 0.0382). At gestational age of 11–13 weeks, ET-1 level was significantly higher in the group with relapsed еPE (Me = 0.93 pmol/ml) than in control group (Me = 0.29 pmol/ml; p < 0.0001) and comparison group (Me = 0.42 pmol/ml; p = 0.0003). No significant changes in E-EVs (CD-144) level at various PС stages were observed, whereas in the study groups such parameters remained differed.Conclusion. Biological markers evidencing about systemic endothelial dysfunction/destruction – ET-1 and E-EVs (CD-144) in patients with previous ePE can be considered as predictive tests of disease relapse with an accuracy of 63.5 % and 83.0 % at the PС stage and 85.7 % and 94.2 % at gestational age of 11–13 weeks, respectively.

Extracellular Vesicle-Serpine-1 Affects Neural Progenitor Cell Mitochondrial Networks and Synaptic Density: Modulation by Amyloid Beta and HIV-1

Brain endothelial extracellular vesicles carrying amyloid beta (EV-Aβ) can be transferred to neural progenitor cells (NPCs) leading to NPC dysfunction. However, the events involved in this EV-mediated Aβ pathology are unclear. EV-proteomics studies identified Serpine-1 (plasminogen activator inhibitor 1, PAI-1) as a major connecting “hub” on several protein–protein interaction maps. Serpine-1 was described as a key player in Aβ pathology and was linked to HIV-1 infection as well. Therefore, the aim of this work was to address the hypothesis that Serpine-1 can be transferred via EVs from brain endothelial cells (HBMEC) to NPCs and contribute to NPC dysfunction. HBMEC concentrated and released Serpine-1 via EVs, the effect that was potentiated by HIV-1 and Aβ. EVs loaded with Serpine-1 were readily taken up by NPCs, and HIV-1 enhanced this event. Interestingly, a highly specific Serpine-1 inhibitor PAI039 increased EV-Aβ transfer to NPCs in the presence of HIV-1. PAI039 also partially blocked mitochondrial network morphology alterations in the recipient NPCs, which developed mainly after HIV + Aβ-EV transfer. PAI039 partly attenuated HIV-EV-mediated decreased synaptic protein levels in NPCs, while increased synaptic protein levels in NPC projections. These findings contribute to a better understanding of the complex mechanisms underlying EV-Serpine-1 related Aβ pathology in the context of HIV infection. They are relevant to HIV-1 associated neurocognitive disorders (HAND) in an effort to elucidate the mechanisms of neuropathology in HIV infection.

Endothelial extracellular vesicles induce acute lung injury via follistatin-like protein 1.

Cardiopulmonary bypass has been speculated to elicit systemic inflammation to initiate acute lung injury (ALI), including acute respiratory distress syndrome (ARDS), in patients after cardiac surgery. We previously found that post-operative patients showed an increase in endothelial cell-derived extracellular vesicles (eEVs) with components of coagulation and acute inflammatory responses. However, the mechanism underlying the onset of ALI owing to the release of eEVs after cardiopulmonary bypass, remains unclear. Plasma plasminogen-activated inhibitor-1 (PAI-1) and eEV levels were measured in patients with cardiopulmonary bypass. Endothelial cells and mice (C57BL/6, Toll-like receptor 4 knockout (TLR4-/-) and inducible nitric oxide synthase knockout (iNOS-/-)) were challenged with eEVs isolated from PAI-1-stimulated endothelial cells. Plasma PAI-1 and eEVs were remarkably enhanced after cardiopulmonary bypass. Plasma PAI-1 elevation was positively correlated with the increase in eEVs. The increase in plasma PAI-1 and eEV levels was associated with post-operative ARDS. The eEVs derived from PAI-1-stimulated endothelial cells could recognize TLR4 to stimulate a downstream signaling cascade identified as the Janus kinase 2/3 (JAK2/3)-signal transducer and activator of transcription 3 (STAT3)-interferon regulatory factor 1 (IRF-1) pathway, along with iNOS induction, and cytokine/chemokine production in vascular endothelial cells and C57BL/6 mice, ultimately contributing to ALI. ALI could be attenuated by JAK2/3 or STAT3 inhibitors (AG490 or S3I-201, respectively), and was relieved in TLR4-/- and iNOS-/- mice. eEVs activate the TLR4/JAK3/STAT3/IRF-1 signaling pathway to induce ALI/ARDS by delivering follistatin-like protein 1 (FSTL1), and FSTL1 knockdown in eEVs alleviates eEV-induced ALI/ARDS. Our data thus demonstrate that cardiopulmonary bypass may increase plasma PAI-1 levels to induce FSTL1-enriched eEVs, which target the TLR4-mediated JAK2/3/STAT3/IRF-1 signaling cascade and form a positive feedback loop, leading to ALI/ARDS after cardiac surgery. Our findings provide new insight into the molecular mechanisms and therapeutic targets for ALI/ARDS after cardiac surgery.

PS-C17-6: MATERNAL ENDOTHELIAL MITOCHONDRIAL DYSFUNCTION IN GESTATIONAL DIABETES MELLITUS

Objective: Gestational diabetes mellitus (GDM) is associated with adverse pregnancy outcomes, and short and long-term maternal and neonatal cardiovascular risks. Our laboratory has shown that elevated levels of endothelial extracellular vesicles (EVs) in pregnant women is predictive of adverse pregnancy outcomes in type 1 diabetes. However, this matter has not been explored in GDM. We therefore investigated the impact of GDM on maternal vascular health, focusing on the mitochondria. Design and method: We determined the mitochondrial protein expression altered by GDM in rat kidneys by Western blot analysis. We examined the levels of circulating endothelial EVs and EV mitochondrial DNA (mtDNA) from rat plasma samples via flow cytometry and quantitative real-time PCR, respectively. We assessed the effects of high glucose exposure for 48 hours on endothelial mitochondrial function in human umbilical vein endothelial cells (HUVECs). Results: In a rat model of diet-induced GDM (Pereira, 2015 J Physiol), we observed significant decreases in expression of oxidative phosphorylation Complexes I (p < 0.05) and II (p < 0.01) in kidneys. We also observed a three-fold increase in late gestational levels of circulating endothelial EVs in GDM rats (p < 0.01) as well as increases in levels of EV-associated mtDNA, COX2 (p < 0.05) and ND2 (p < 0.01), suggesting mitochondrial dysfunction. In cultured HUVECs exposed to high glucose for 48 hours, we observed a significant decrease in Complex II expression (p < 0.01) and five-fold increases in EV mtDNA levels. This effect persisted even after removing high glucose exposure for 24 hours. Conclusion: These data show maternal endothelial dysfunction in GDM, accompanied by mitochondrial dysfunction and increased endothelial EV release. These findings suggest that endothelial mitochondrial health may represent a novel approach to reduce risks associated with GDM.

Endothelial Extracellular Vesicles Regulate Platelet Purinergic Signaling in Sickle Cell Disease

Introduction. Acute chest syndrome (ACS) is a type of acute lung injury and the leading cause of mortality among sickle cell disease (SCD) patients. ACS is often preceded by thrombocytopenia and involves massive thrombosis across pulmonary artery branches. Although, released during hemolysis, adenosine diphosphate (ADP) is known to activate platelets by stimulating their P2Y1 and P2Y12 purinergic receptors, antagonists of P2Y12 have not shown any benefit in ACS therapy, justifying the need for better understanding of purinergic signaling in SCD. CD39 maintains ADP homeostasis by degrading excessive ADP. Though CD39 inhibits ADP-dependent platelet activation, its role in the pathophysiology of SCD is still unidentified. Methods. To evaluate SCD platelet response to ADP in vivo we used a state-of-the-art intravital lung microscopy and a novel in vivo model of ADP-triggered thrombocytopenia in transgenic humanized SCD mice. Additionally, both mouse and human SCD platelet ADP-dependent aggregation was examined using in vitro turbidimetric aggregation assay. Hemin, a major host-derived damage associated molecular pattern associated with SCD, was incubated with Human Lung Micro-Vascular Endothelial Cells (HMVEC-L). Extracellular vesicles (EVs) were obtained from mouse and human plasma samples, and cell culture medium samples using size exclusion chromatography. Obtained EVs were subjected to nanoparticle tracking analysis. EVs CD39 levels and activity were determined using ELISA, western blot and malachite green phosphate assays, respectively. Results. Intravital lung microscopy and in vivo thrombocytopenia studies revealed that intravascular administration of ADP triggered acute pulmonary thrombosis in control but not in SCD mice. In vitro aggregation study confirmed our in vivo findings and demonstrated impaired SCD mouse and human platelet response to ADP, which was, further, significantly augmented by a specific CD39 inhibitor. Hemin triggered shedding of CD39-bearing EVs by HMVEC-L. Indeed, we discovered that isolated from mouse and human plasma endothelial cell-derived SCD EVs expressed higher CD39 levels and activity in comparison to control EVs. Conclusions. Our findings suggest that CD39-bearing EVs prevent ADP-mediated platelet aggregation and pulmonary thrombosis in SCD. Current study explains why P2Y12 blockers are not effective in SCD therapy.

MiR-1249 on Endothelial Extracellular Vesicles Mediates Cigarette Smoke-Induced Pulmonary Hypertension by Inhibiting HDAC10 (Histone Deacetylase 10)-NFκB (Nuclear Factor κB)-CaSR (Calcium-Sensing Receptor) Cascade.

Overproduction of endothelial extracellular vesicles (eEVs) is correlated with pulmonary hypertension progression, but the precise mechanism remains largely unclear. MicroRNA-chip and real-time polymerase chain reaction were conducted to screen and validate microRNA profiles in blood plasma eEVs of rats and human with or without cigarette smoking. Pulmonary artery smooth muscle cells were cultured to study signaling pathways. Pulmonary hypertension phenotypes were evaluated in wild-type and calcium-sensing receptor knockout rats to identify the pathophysiological significance of the microRNA pathway. MicroR-1249 was predominant highly expressed in eEVs from plasma of rats exposed to cigarette smoking, and confirmed in eEVs from plasma of human smokers as well as in eEVs from cigarette smoke extract-treated pulmonary artery endothelial cells, but not in cigarette smoke extract-treated pulmonary artery smooth muscle cells. In cultured pulmonary artery smooth muscle cells, microR-1249 downregulated the expression of histone deacetylase 10, which in turn enhanced the acetylated form of NFκB (nuclear factor κB) level and its nuclear translocation leading to increased expression of calcium-sensing receptor. In rats, the repression of microR-1249 in eEVs by microR-1249 inhibitor, histone deacetylase 10 overexpression, or calcium-sensing receptor knockout profoundly inhibited the proliferative capacities and diminished apoptosis-resistance of pulmonary artery smooth muscle cells and pulmonary hypertension development in rats intravenously administrated with eEVs preparation from cigarette smoke extract-treated pulmonary artery endothelial cells. Cigarette smoke-enriched microR-1249 in endothelial extracellular vesicles facilitates the hyperproliferative and antiapoptotic status of pulmonary artery smooth muscle cells promoting pulmonary hypertension evolution through the inhibition of histone deacetylase 10-NFκB-calcium-sensing receptor cascade.

Biogenesis and release of endothelial extracellular vesicles: Morphological aspects

BackgroundCell-cell communication through extracellular vesicles (EVs) including exosomes, microvesicles and apoptotic bodies has been shown to be important in physiological homoeostasis as well as pathological processes such as atherosclerosis. However, while the cellular machinery controlling EV formation and composition has been studied during the past decade, less is known about the morphological process of their formation and release. MethodsUsing different electron microscopic approaches including transmission-, scanning-, immun-, and serial block face electron microscopy we studied the morphogenetic events of EV formation and release. We analysed the different steps of EV formation and release in cultured myocardial endothelial (MyEnd) and aortic endothelial (AoEnd) cell lines under serum starvation and under inflammatory conditions. ResultsWe show that in a narrow time frame, the number of active cells and microvesicle (MV) producing cells increased in dependence of time spent in cultivation and additional stimulation by TNF-α. However, MV secretion was a highly heterogeneous process which couldn´t be seen in all cells cultivated under the same conditions. Release of MVs could be observed all over the cells’ surface with no preferred release site. While no single specific microscopic approach applied was sufficient to provide a comprehensive analysis of EV biogenesis, we show that the limitations of one technique could be compensated by the qualities of the respective other applied techniques, thus enabling us to provide a detailed ultrastructural analysis of MV and exosome biogenesis. Surprisingly, exosome release in endothelial cells occurred via a yet undescribed process indicating that MVBs were incorporated into a novel distinct cellular compartment covered by fenestrated endothelium before exosome release. Lastly, we could show that TNF-α stimulation of AoEnd cells leads not only to the upregulation of CD44 in parental cells, but also to incorporation of CD44 into the membranes of generated MVs and exosomes. ConclusionsTaken together, our data contribute to a better understanding of biogenesis and release of EVs. We conclude that under inflammatory conditions, EVs can mediate the transfer of CD44 from endothelial cells to target cells at distant sites including vessel wall cells and this could be a mechanism by which MVs may change the and thus contribute to the development and progression of atherosclerotic lesions.

Increased Circulating CD62E+ Endothelial Extracellular Vesicles Predict Severity and in- Hospital Mortality of COVID-19 Patients.

COVID-19 and infectious diseases have been included in strategic development goals (SDG) of United Nations (UN). Severe form of COVID-19 has been described as an endothelial disease. In order to better evaluate Covid-19 endotheliopathy, we characterized several subsets of circulating endothelial extracellular vesicles (EVs) at hospital admission among a cohort of 60 patients whose severity of COVID-19 was classified at the time of inclusion. Degree of COVID-19 severity was determined upon inclusion and categorized as moderate to severe in 40 patients and critical in 20 patients. We measured citrated plasma EVs expressing endothelial membrane markers. Endothelial EVs were defined as harboring VE-cadherin (CD144+), PECAM-1 (CD31 + CD41-) or E-selectin (CD62E+). An increase in CD62E + EV levels on admission to the hospital was significantly associated with critical disease. Moreover, Kaplan-Meier survival curves for CD62E + EV level showed that level ≥ 88,053 EVs/μL at admission was a significant predictor of in hospital mortality (p = 0.004). Moreover, CD62E + EV level ≥ 88,053 EV/μL was significantly associated with higher in-hospital mortality (OR 6.98, 95% CI 2.1–26.4, p = 0.002) in a univariate logistic regression model, while after adjustment to BMI CD62E + EV level ≥ 88,053 EV/μL was always significantly associated with higher in-hospital mortality (OR 5.1, 95% CI 1.4–20.0, p = 0.01). The present findings highlight the potential interest of detecting EVs expressing E-selectin (CD62) to discriminate Covid-19 patients at the time of hospital admission and identify individuals with higher risk of fatal outcome.Graphical abstract

Endothelial extracellular vesicles promote tumour growth by tumour-associated macrophage reprogramming.

Tumour‐derived extracellular vesicles (EVs) participate in tumour progression by deregulating various physiological processes including angiogenesis and inflammation. Here we report that EVs released by endothelial cells in a mammary tumour environment participate in the recruitment of macrophages within the tumour, leading to an immunomodulatory phenotype permissive for tumour growth. Using RNA‐Seq approaches, we identified several microRNAs (miRNAs) found in endothelial EVs sharing common targets involved in the regulation of the immune system. To further study the impact of these miRNAs in a mouse tumour model, we focused on three miRNAs that are conserved between humans and mouse, that is, miR‐142‐5p, miR‐183‐5p and miR‐222‐3p. These miRNAs are released from endothelial cells in a tumour microenvironment and are transferred via EVs to macrophages. In mouse mammary tumour models, treatment with EVs enriched in these miRNAs leads to a polarization of macrophages toward an M2‐like phenotype, which in turn promotes tumour growth.

Circulating extracellular vesicles during pregnancy in women with type 1 diabetes: a secondary analysis of the CONCEPTT trial

BackgroundExtracellular vesicles are membrane vesicles that are released into the extracellular environment and accumulate in the circulation in vascular disease. We aimed to quantify circulating extracellular vesicles in pregnant women with type 1 diabetes and to examine associations between extracellular vesicle levels, continuous glucose measures, and pregnancy outcomes.MethodsWe used plasma samples from the Continuous Glucose Monitoring in Women with Type 1 Diabetes in Pregnancy Trial study and quantified circulating extracellular vesicles by flow cytometry (n = 163). Relationships with clinical variables were assessed by repeated measures correlation. Logistic regression was used to assess associations between elevated extracellular vesicle levels and pregnancy outcomes.ResultsPlatelet extracellular vesicle levels were inversely associated with glucose time above range and glycaemic variability measures (P < 0.05). A weak positive association was observed between endothelial extracellular vesicles and mean amplitude of glycemic excursion (P < 0.05). In a univariate logistic regression model, high baseline endothelial extracellular vesicles was associated with increased risk of neonatal intensive care unit (NICU) admission (OR: 2.06, 1.03–4.10), and respiratory distress requiring ventilation (OR: 4.98, 1.04–23.92). After adjusting for HbA1c and blood pressure the relationship for NICU admission persisted and an association with hyperbilirubinemia was seen (OR: 2.56, 1.10–5.94). Elevated platelet extracellular vesicles were associated with an increased risk of NICU admission (OR: 2.18, 1.04–4.57), and hyperbilirubinemia (OR: 2.61, 1.11–6.12) after adjusting for HbA1c and blood pressure.ConclusionsHigh levels of extracellular vesicles in early pregnancy were associated with adverse neonatal outcomes. Assessment of extracellular vesicles may represent a novel approach to personalized care in type 1 diabetes pregnancy.

Factor VIIa suppresses inflammation and barrier disruption through the release of EEVs and transfer of microRNA 10a

AbstractCoagulation protease, factor VIIa (FVIIa), binds to endothelial cell protein C receptor (EPCR) and induces anti-inflammatory and endothelial barrier protective responses via protease-activated receptor-1 (PAR1)–mediated, biased signaling. Our recent studies had shown that the FVIIa-EPCR-PAR1 axis induces the release of extracellular vesicles (EVs) from endothelial cells. In the present study, we investigated the mechanism of FVIIa release of endothelial EVs (EEVs) and the contribution of FVIIa-released EEVs to anti-inflammatory and vascular barrier protective effects, in both in vitro and in vivo models. Multiple signaling pathways regulated FVIIa release of EVs from endothelial cells, but the ROCK-dependent pathway appeared to be a major mechanism. FVIIa-released EEVs were enriched with anti-inflammatory microRNAs (miRs), mostly miR10a. FVIIa-released EEVs were taken up readily by monocytes/macrophages and endothelial cells. The uptake of FVIIa-released EEVs by monocytes conferred anti-inflammatory phenotype to monocytes, whereas EEV uptake by endothelial cells resulted in barrier protection. In additional experiments, EEV-mediated delivery of miR10a to monocytes downregulated the expression of TAK1 and activation of the NF-κB–mediated inflammatory pathway. In in vivo experiments, administration of FVIIa-released EEVs to wild-type mice attenuated LPS-induced increased inflammatory cytokines in plasma and vascular leakage into vital tissues. The incorporation of anti-miR10a into FVIIa-released EEVs diminished the ability of FVIIa-released EEVs to confer cytoprotective effects. Administration of the ROCK inhibitor Y27632, which significantly inhibits FVIIa release of EEVs into the circulation, to mice attenuated the cytoprotective effects of FVIIa. Overall, our study revealed novel insights into how FVIIa induces cytoprotective effects and communicates with various cell types.

Large Platelet and Endothelial Extracellular Vesicles in Cord Blood of Preterm Newborns: Correlation with the Presence of Hemolysis.

Different biomarkers are investigated to detect the causes of severe complications in preterm infants. Extracellular vesicles (EVs) are recognized as an important part of cell-to-cell communication, and their increased levels were reported in numerous pathological states. We aimed to increase our knowledge about the incidence of platelet and endothelial EVs in cord blood of preterm newborns using conventional flow cytometry. The presence of platelet (CD36+CD41+), activated platelet (CD41+CD62+), and endothelial (CD31+CD105+) EVs was analyzed. Immune electron microscopy was used to confirm the presence of EVs and the specificity of their labeling. The size of detected extracellular vesicles was in the range 400–2000 nm. The differences in the counts of EVs between the preterm and control group were not significant and no correlation of EVs count with gestation age was recorded. Cord blood plasma samples with free hemoglobin level > 1 mg/mL had more than threefold higher counts of CD36+CD41+ and CD41+CD62+ EVs (p < 0.001), while the count of CD31+CD105+ EVs was only moderately increased (p < 0.05). Further studies utilizing cytometers with improved sensitivity are needed to confirm that the analysis of large platelet and endothelial EVs mirrors the quantitative situation of their whole plasma assemblage.

Different Effects of Endothelial Extracellular Vesicles and LPS-Induced Endothelial Extracellular Vesicles on Vascular Smooth Muscle Cells: Role of Curcumin and Its Derivatives.

Background: During the progression of atherosclerosis (AS), the vascular endothelial and smooth muscle cells are reciprocally regulated by extracellular vesicles (EVs). EVs have different effects on pathological and physiological processes due to the different cargoes contained in EVs.Purpose: To study the effects of endothelial cells-derived EVs on normal and inflammatory conditions. To investigate the effects of curcumin and curcumin derivatives (Nicotinic-curcumin) on endothelial EVs.Methods: EVs were isolated from human umbilical vein endothelial cells (HUVECs) by ultracentrifugation. To examined the effect of normal and LPS-induced endothelial cells-derived EVs on the proliferation of human aortic smooth muscle cells (HASMCs), the CCK-8 assay was performed. Transwell and wound healing assays were conducted to assess cell migration. The effects of EVs on lipid accumulation following treatment with oxidized low-density lipoprotein (Ox-LDL) were evaluated with the oil red O staining assay and HPLC. The number of EVs was calculated using the nanoparticle tracking analysis (NTA) and BCA. The expression levels of Rab27a and Rab27b that regulate the EVs secretion were measured by Western blotting assay. The differential expression of miRNAs in endothelial EVs and LPS-induced endothelial EVs was analyzed using miRNA-Sequencing (miRNA-Seq) and RT-PCR.Results: Treatment with endothelial EVs reduced the proliferation and migration of HASMCs as well as lipid accumulation in HASMCs. However, treatment with LPS-induced endothelial EVs did not inhibit the migration of HASMCs or lipid accumulation, instead it promoted the proliferation of HASMCs. Treatment with the two types of EVs induced differential expression of several miRNAs, including miR-92a-3p, miR-126-5p, miR-125a-3p, miR-143-3p, etc. Moreover, 1 μg/mL LPS induction greatly increased secretion of endothelial EVs. Treatment with curcumin and nicotinic-curcumin reduced endothelial EVs secretion, possibly by inhibiting inflammation.Conclusion: Endothelial EVs may confer beneficial effects on atherosclerosis by regulating vascular smooth muscle cell (VSMCs), whereas pro-inflammatory factors may disrupt this effect.

Uremic endothelial-derived extracellular vesicles: Mechanisms of formation and their role in cell adhesion, cell migration, inflammation, and oxidative stress

p-Cresyl sulfate (PCS), indoxyl sulfate (IS), and inorganic phosphate (Pi) are uremic toxins found in chronic kidney disease (CKD) that are closely related to endothelial extracellular vesicles (EVs) formation. The present study aimed to understand the role of EVs and their role in cell adhesion and migration, inflammation, and oxidative stress. Human endothelial cells were treated with PCS, IS, and Pi in pre-established uremic and kinetic recommendations. EVs were characterized using scanning electron microscopy, flow cytometry, and NanoSight assays. The concentrations of EVs were established using Alamar Blue and MTT assays. Cell adhesion to extracellular matrix proteins was analyzed using an adhesion assay. Inflammation and oxidative stress were assessed by vascular cell adhesion molecule-1 expression/monocyte migration and reactive oxygen species production, respectively. The capacity of EVs to stimulate endothelial cell migration was evaluated using a wound-healing assay. Our data showed that endothelial cells stimulated with uremic toxins can induce the formation of EVs of different sizes, quantities, and concentrations, depending on the uremic toxin used. Cell adhesion was significantly (P < 0.01) stimulated in cells exposed to PCS-induced extracellular vesicles (PCSEVs) and inorganic phosphate-induced extracellular vesicles (PiEVs). Cell migration was significantly (P < 0.05) stimulated by PCSEVs. VCAM-1 expression was evident in cells treated with PCSEVs and IS-induced extracellular vesicles (ISEVs). EVs are not able to stimulate monocyte migration or oxidative stress. In conclusion, EVs may be a biomarker of endothelial injury and the inflammatory process, playing an important role in cell-to-cell communication and pathophysiological processes, although more studies are needed to better understand the mechanisms of EVs in uremia.