Circulating extracellular vesicles from patients with acute chest syndrome disrupt adherens junctions between endothelial cells

Intercellular junctions maintain the integrity of the endothelium. We previously found that the adherens and tight junctions between endothelial cells are disrupted by plasma extracellular vesicles from patients with sickle cell disease (especially those with Acute Chest Syndrome). In the current study, we evaluated the effects of these vesicles on endothelial gap junctions. The vesicles from sickle cell patients (isolated during episodes of Acute Chest Syndrome) disrupted gap junction structures earlier and more severely than the other classes of intercellular junctions (as detected by immunofluorescence). These vesicles were much more potent than those isolated at baseline from the same subject. The treatment of endothelial cells with these vesicles led to reduced levels of connexin43 mRNA and protein. These vesicles severely reduced intercellular communication (transfer of microinjected Neurobiotin). Our data suggest a hierarchy of progressive disruption of different intercellular connections between endothelial cells by circulating extracellular vesicles that may contribute to the pathophysiology of the endothelial disturbances in sickle cell disease.

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Extracellular vesicles from human airway basal cells respond to cigarette smoke extract and affect vascular endothelial cells

Scientific Reports ◽

10.1038/s41598-021-85534-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ashish Saxena ◽

Matthew S. Walters ◽

Jae-Hung Shieh ◽

Ling-Bo Shen ◽

Kazunori Gomi ◽

...

Keyword(s):

Endothelial Cells ◽

Cigarette Smoke ◽

Extracellular Vesicles ◽

Basal Cell ◽

Mapk Signaling ◽

Cigarette Smoke Extract ◽

Vegf Receptor ◽

Vascular Endothelial ◽

Basal Cells ◽

Human Airway

AbstractThe human airway epithelium lining the bronchial tree contains basal cells that proliferate, differentiate, and communicate with other components of their microenvironment. One method that cells use for intercellular communication involves the secretion of exosomes and other extracellular vesicles (EVs). We isolated exosome-enriched EVs that were produced from an immortalized human airway basal cell line (BCi-NS1.1) and found that their secretion is increased by exposure to cigarette smoke extract, suggesting that this stress stimulates release of EVs which could affect signaling to other cells. We have previously shown that primary human airway basal cells secrete vascular endothelial growth factor A (VEGFA) which can activate MAPK signaling cascades in endothelial cells via VEGF receptor–2 (VEGFR2). Here, we show that exposure of endothelial cells to exosome-enriched airway basal cell EVs promotes the survival of these cells and that this effect also involves VEGFR2 activation and is, at least in part, mediated by VEGFA present in the EVs. These observations demonstrate that EVs are involved in the intercellular signaling between airway basal cells and the endothelium which we previously reported. The downstream signaling pathways involved may be distinct and specific to the EVs, however, as increased phosphorylation of Akt, STAT3, p44/42 MAPK, and p38 MAPK was not seen following exposure of endothelial cells to airway basal cell EVs.

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Ubiquinone Metabolism and Transcription HIF-1 Targets Pathway Are Toxicity Signature Pathways Present in Extracellular Vesicles of Paraquat-Exposed Human Brain Microvascular Endothelial Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22105065 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5065

Author(s):

Tatjana Vujić ◽

Domitille Schvartz ◽

Anton Iliuk ◽

Jean-Charles Sanchez

Keyword(s):

Endothelial Cells ◽

Human Brain ◽

Extracellular Vesicles ◽

Physiological State ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

Cell Of Origin ◽

Functional Changes ◽

Data Independent Acquisition ◽

Microvascular Endothelial

Over the last decade, the knowledge in extracellular vesicles (EVs) biogenesis and modulation has increasingly grown. As their content reflects the physiological state of their donor cells, these “intercellular messengers” progressively became a potential source of biomarker reflecting the host cell state. However, little is known about EVs released from the human brain microvascular endothelial cells (HBMECs). The current study aimed to isolate and characterize EVs from HBMECs and to analyze their EVs proteome modulation after paraquat (PQ) stimulation, a widely used herbicide known for its neurotoxic effect. Size distribution, concentration and presence of well-known EV markers were assessed. Identification and quantification of PQ-exposed EV proteins was conducted by data-independent acquisition mass spectrometry (DIA-MS). Signature pathways of PQ-treated EVs were analyzed by gene ontology terms and pathway enrichment. Results highlighted that EVs exposed to PQ have modulated pathways, namely the ubiquinone metabolism and the transcription HIF-1 targets. These pathways may be potential molecular signatures of the PQ-induced toxicity carried by EVs that are reflecting their cell of origin by transporting with them irreversible functional changes.

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Delivery of Therapeutic Agents to the Central Nervous System and the Promise of Extracellular Vesicles

Pharmaceutics ◽

10.3390/pharmaceutics13040492 ◽

2021 ◽

Vol 13 (4) ◽

pp. 492

Author(s):

Charlotte A. René ◽

Robin J. Parks

Keyword(s):

Central Nervous System ◽

Endothelial Cells ◽

Nervous System ◽

Blood Brain Barrier ◽

Extracellular Vesicles ◽

Therapeutic Agents ◽

Target Cells ◽

Significant Barrier ◽

The Central Nervous System ◽

Delivery Vehicles

The central nervous system (CNS) is surrounded by the blood–brain barrier (BBB), a semipermeable border of endothelial cells that prevents pathogens, solutes and most molecules from non-selectively crossing into the CNS. Thus, the BBB acts to protect the CNS from potentially deleterious insults. Unfortunately, the BBB also frequently presents a significant barrier to therapies, impeding passage of drugs and biologicals to target cells within the CNS. This review provides an overview of different approaches to deliver therapeutics across the BBB, with an emphasis in extracellular vesicles as delivery vehicles to the CNS.

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Extracellular vesicles from arterial but not venous endothelial cells suppress endothelial-to-mesenchymal transition and endothelial inflammation

Atherosclerosis ◽

10.1016/j.atherosclerosis.2021.06.176 ◽

2021 ◽

Vol 331 ◽

pp. e61

Author(s):

D. Shishkova ◽

M. Sinitsky ◽

V. Markova ◽

A. Tsepokina ◽

A. Frolov ◽

...

Keyword(s):

Endothelial Cells ◽

Extracellular Vesicles ◽

Mesenchymal Transition ◽

Endothelial Inflammation ◽

Endothelial To Mesenchymal Transition

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MicroRNAs and Extracellular Vesicles as Distinctive Biomarkers of Precocious and Advanced Stages of Breast Cancer Brain Metastases Development

International Journal of Molecular Sciences ◽

10.3390/ijms22105214 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5214

Author(s):

Inês Figueira ◽

Joana Godinho-Pereira ◽

Sofia Galego ◽

Joana Maia ◽

János Haskó ◽

...

Keyword(s):

Breast Cancer ◽

Endothelial Cells ◽

Brain Metastases ◽

Extracellular Vesicles ◽

Triple Negative ◽

Survival Rates ◽

Brain Parenchyma ◽

Advanced Stages ◽

Breast Cancer Brain Metastases

Triple negative breast cancer presents higher mortality and poorer survival rates than other breast cancer (BC) types, due to the proneness to brain metastases formation, which are usually diagnosed at advanced stages. Therefore, the discovery of BC brain metastases (BCBM) biomarkers appears pivotal for a timely intervention. With this work, we aimed to disclose microRNAs (miRNAs) and extracellular vesicles (EVs) in the circulation as biomarkers of BCBM formation. Using a BCBM animal model, we analyzed EVs in plasma by nanoparticle tracking analysis and ascertained their blood-brain barrier (BBB) origin by flow cytometry. We further evaluated circulating miRNAs by RT-qPCR and their brain expression by in situ hybridization. In parallel, a cellular model of BCBM formation, combining triple negative BC cells and BBB endothelial cells, was used to differentiate the origin of biomarkers. Established metastases were associated with an increased content of circulating EVs, particularly of BBB origin. Interestingly, deregulated miRNAs in the circulation were observed prior to BCBM detection, and their brain origin was suggested by matching alterations in brain parenchyma. In vitro studies indicated that miR-194-5p and miR-205-5p are expressed and released by BC cells, endothelial cells and during their interaction. These results highlight miRNAs and EVs as biomarkers of BCBM in early and advanced stages, respectively.

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Spatial and temporal relationships between cadherins and PECAM-1 in cell-cell junctions of human endothelial cells.

The Journal of Cell Biology ◽

10.1083/jcb.126.1.247 ◽

1994 ◽

Vol 126 (1) ◽

pp. 247-258 ◽

Cited By ~ 132

Author(s):

O Ayalon ◽

H Sabanai ◽

M G Lampugnani ◽

E Dejana ◽

B Geiger

Keyword(s):

Endothelial Cells ◽

Plasma Membrane ◽

Vascular System ◽

Adherens Junctions ◽

Microscopic Analysis ◽

Membrane Receptors ◽

Temporal Organization ◽

Cell Junctions ◽

Short Treatment ◽

Cell Cell

The integrity of the endothelial layer, which lines the entire cavity of the vascular system, depends on tight adhesion of the cells to the underlying basement membrane as well as to each other. It has been previously shown that such interactions occur via membrane receptors that determine the specificity, topology, and mechanical properties of the surface adhesion. Cell-cell junctions between endothelial cells, in culture and in situ, involve both Ca(2+)-dependent and -independent mechanisms that are mediated by distinct adhesion molecules. Ca(2+)-dependent cell-cell adhesion occurs mostly via members of the cadherin family, which locally anchor the microfilament system to the plasma membrane, in adherens junctions. Ca(2+)-independent adhesions were reported to mainly involve members of the Ig superfamily. In this study, we performed three-dimensional microscopic analysis of the relative subcellular distributions of these two endothelial intercellular adhesion systems. We show that cadherins are located at adjacent (usually more apical), yet clearly distinct domains of the lateral plasma membrane, compared to PECAM-1. Moreover, cadherins were first organized in adherens junctions within 2 h after seeding of endothelial cells, forming multiple lateral patches which developed into an extensive belt-like structure over a period of 24 h. PECAM-1 became associated with surface adhesions significantly later and became progressively associated with the cadherin-containing adhesions. Cadherins and PECAM-1 also differed in their detergent extractability, reflecting differences in their mode of association with the cytoskeleton. Moreover, the two adhesion systems could be differentially modulated since short treatment with the Ca2+ chelator EGTA, disrupted the cadherin junctions leaving PECAM-1 apparently intact. These results confirm that endothelial cells possess distinct intercellular contact mechanisms that differ in their spatial and temporal organization as well as in their functional properties.

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Prolonged Detection of SARS CoV2 RNA in Extracellular Vesicles in Nasal Swab RT-PCR Negative Patients

10.1101/2021.12.16.21267912 ◽

2021 ◽

Author(s):

P Debishree Subudhi ◽

Sheetalnath Rooge ◽

Swati Thangriyal ◽

Reshu Aggarwal ◽

Ekta Gupta ◽

...

Keyword(s):

Endothelial Cells ◽

Viral Load ◽

Liver Disease ◽

Chronic Liver Disease ◽

Extracellular Vesicles ◽

Nasal Swab ◽

High Viral Load ◽

Chronic Liver ◽

Rt Pcr ◽

Route Of Transmission

Background: There is a prolonged RT PCR positivity seen in COVID-19 infected patients up to 2 to 3 months. It is assumed that this virus is usually non-infective but there are hardly any study on the reactivation of this virus within the respiratory tract. We aim to investigate the presence of viral particles inside Extracellular vesicles (EV) and its role in underlying liver disease patients. Methods: SARS CoV2 nasal and throat swab RT-PCR positive n=78 {n=24(66.6%) chronic liver disease (CLD); n=52 (81.3%) non liver disease} n=5 RT PCR negative subjects (HC) were studied. SARS CoV2 patients were also followed up for day (d) 7, 14 and 28. Nasal swab [collected in viral transport media (VTM)] and plasma samples were investigated at each time point. Extracellular vesicles were isolated using differential ultracentrifugation. SARS CoV2 RNA was measured using qRT-PCR by Altona Real Star kit. Cellular origin of EV was confirmed using epithelial cells (Epcam+ CK19+ CDh1+), endothelial cells (CD31+CD45-), and hepatocytes (ASGPR+) surface markers by Flow cytometry. Results: The COVID19 patients {Mean age 54±23 years; 41 males} were having severity between moderate to severe. In patients with cirrhosis, the most common aetiology of liver disease was alcohol (MELD 22±8). In baseline RT-PCR positive patients, SARS-CoV2 RNA inside the EV was present in 64/74 (82%) patients with comparable viral load between VTM and EV (mean 1/CT 0.033±0.005 vs. 1/CT 0.029±0.014, p=ns). On follow-up at day 7, of the 24 patients negative for COVID19, 10 (41%) had persistence of virus in the EV (1/CT 0.028±0.004) and on day 14, 14 of 40 (35%) negative RT-PCR had EVs with SARS CoV2 RNA (1/CT 0.028±0.06). The mean viral load decreased at day7 and day14 in nasal swab from baseline (p=0.001) but not in EV. SARS-CoV2 RNA otherwise undetectable in plasma, was found to be positive in EV in 12.5% of COVID19 positive patients. Interestingly, significantly prolonged and high viral load was found in EV at day 14 in CLD COVID19 patients compared to COVID19 alone (p=0.002). The high cellular injury was seen in CLD COVID19 infected patients with significant high levels of EV associated with endothelial cells and hepatocytes than COVID19 alone (p=0.004; 0.001). Conclusion: Identification of SARS-CoV2 RNA in EV, in RT-PCR negative patients indicates persistence of infection for and likely recurrence of the infection. It is suggestive of another route of transmission as EV harbour SARS CoV2 RNA. EV associated RNA may determine the ongoing inflammation and clinical course of subjects with undetectable SARS-CoV2 virus and this may also have relevance in management of chronic liver disease patients.

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Isolation of Primary Mouse Pulmonary Microvascular Endothelial Cells and Generation of an Immortalized Cell Line to Obtain Sufficient Extracellular Vesicles

Frontiers in Immunology ◽

10.3389/fimmu.2021.759176 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xu Liu ◽

Feiping Xia ◽

Xiao Wu ◽

Ying Tang ◽

Lu Wang ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Line ◽

Extracellular Vesicles ◽

Phenotypic Expression ◽

Microvascular Endothelial Cells ◽

Pulmonary Microvascular Endothelial Cells ◽

Immortalized Cell Line ◽

Primary Mouse ◽

Microvascular Endothelial ◽

Immortalized Cell

Pulmonary microvascular endothelial cells (PMECs) and the extracellular vesicles (EVs) derived from PMECs participate in maintaining pulmonary homeostasis and mediating the inflammatory response. However, obtaining a high-purity population of PMECs and their EVs from mouse is still notoriously difficult. Herein we provide a method to isolate primary mouse PMECs (pMPMECs) and to transduce SV40 lentivirus into pMPMECs to establish an immortalized cell line (iMPMECs), which provides sufficient quantities of EVs for further studies. pMPMECs and iMPMECs can be identified using morphologic criteria, a phenotypic expression profile (e.g., CD31, CD144, G. simplicifolia lectin binding), and functional properties (e.g., Dil-acetylated low-density protein uptake, Matrigel angiogenesis). Furthermore, pMPMEC–EVs and iMPMEC–EVs can be identified and compared. The characteristics of pMPMEC–EVs and iMPMEC–EVs are ascertained by transmission electron microscopy, nanoparticle tracking analysis, and specific protein markers. iMPMECs produce far more EVs than pMPMECs, while their particle size distribution is similar. Our detailed protocol to isolate and immortalize MPMECs will provide researchers with an in vitro model to investigate the specific roles of EVs in pulmonary physiology and diseases.

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