Small extracellular vesicles from epicardial fat of patients with atrial fibrillation induce inflammation, fibrosis and re-entrant arrhythmias

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
O Shaihov-Teper ◽  
E Ram ◽  
R Brzezinski ◽  
D Volvovitch ◽  
E Zuroff ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Heart Surgery ◽  
Open Heart Surgery ◽  
Induced Pluripotent Stem Cell ◽  
Membrane Vesicles ◽  
Cell Types ◽  
Size Exclusion ◽  
Epicardial Fat ◽  
Atrial Remodeling

Abstract Background and aim Epicardial fat (eFat) has been linked to atrial remodeling and fibrillation (AF). Small extracellular vesicles (sEVs) are heterogeneous membrane vesicles released by all cell types. They can both protect and damage tissues by the delivery of multiple different messengers. Surprisingly, the role of sEVs in the pathogenesis of AF has not been studied. Thus, we aimed to determine whether sEVs derived from eFat play a role in the pathogenesis of AF. Methods and results We collected eFat specimens from patients with and without chronic or paroxysmal AF undergoing open-heart surgery. Isolated eFat specimens were cut into small pieces and incubated as organ cultures. We isolated sEVs from the medium of the explant by differential ultra-centrifugation, high-density gradient or size exclusion chromatography (SEC), and characterized vesicle size distribution, morphology, specific markers, histology and molecular cargo. Immunostaining for macrophage accumulation, fibrosis and apoptosis confirmed the pro-inflammatory and pro-fibrotic properties of eFat sEVs from patients with AF (Fig. 1). eFat sEVs labeled with PKH26 were massively up taken by endothelial cells (Fig. 2). Real-time PCR showed an increased level of oxidative stress genes in endothelial cells. eFat sEVs from patients with AF caused more fibrosis after injection into rat hearts than those without AF. (Fig. 3). Finally, while eFat sEVs from patients with and without AF induced shorter action potential duration, only eFat sEVs from patients with AF induced sustained re-entry (rotor) in human induced pluripotent stem cell (iPS)-derived cardiomyocytes (Fig. 4) Conclusion We show, for the first time, that sEVs from eFat of patients with AF demonstrate unique pro-inflammatory, pro-fibrotic and pro-arrhythmic properties. Our findings suggest that eFat sEVs can induce cellular, molecular and electrophysiological remodeling that can subsequently lead to the development of AF. Funding Acknowledgement Type of funding source: None

scholarly journals Extracellular Vesicles from Epicardial Fat Facilitate Atrial Fibrillation

Circulation ◽  
2021 ◽  
Author(s):  
Olga Shaihov-Teper ◽  
Eilon Ram ◽  
Nimer Ballan ◽  
Rafael Y. Brzezinski ◽  
Nili Naftali-Shani ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Extracellular Vesicles ◽  
Culture Medium ◽  
Heart Surgery ◽  
Biological Effects ◽  
Induced Pluripotent Stem Cell ◽  
Epicardial Fat ◽  
Human Mscs ◽  
And Migration

Background: The role of epicardial fat (eFat)-derived extracellular vesicles (EVs) in the pathogenesis of atrial fibrillation (AF) has never been studied. We tested the hypothesis that eFat-EVs transmit proinflammatory, profibrotic, and proarrhythmic molecules that induce atrial myopathy and fibrillation. Methods: We collected eFat specimens from patients with (n=32) and without AF (n=30) during elective heart surgery. eFat samples were grown as organ cultures, and the culture medium was collected every two days. We then isolated and purified eFat-EVs from the culture medium, and analyzed the EV number, size, morphology, specific markers, encapsulated cytokines, proteome, and miRNAs. Next, we evaluated the biological effects of unpurified and purified EVs on atrial mesenchymal stromal cells (MSCs) and endothelial cells (ECs) in vitro. To establish a causal association between eFat-EVs and vulnerability to AF, we modeled AF in vitro using induced pluripotent stem cell-derived cardiomyocytes (iCMs). Results: Microscopic examination revealed excessive inflammation, fibrosis, and apoptosis in fresh and cultured eFat tissues. Cultured explants from patients with AF secreted more EVs and harbored greater amounts of proinflammatory and profibrotic cytokines, as well as profibrotic miRNA, than those without AF. The proteomic analysis confirmed the distinctive profile of purified eFat-EVs from patients with AF. In vitro, purified and unpurified eFat-EVs from patients with AF had a greater effect on proliferation and migration of human MSCs and ECs, compared to eFat-EVs from patients without AF. Finally, while eFat-EVs from patients with and without AF shortened the action potential duration of iCMs, only eFat-EVs from patients with AF induced sustained reentry (rotor) in iCMs. Conclusions: We show, for the first time, a distinctive proinflammatory, profibrotic, and proarrhythmic signature of eFat-EVs from patients with AF. Our findings uncover another pathway by which eFat promotes the development of atrial myopathy and fibrillation.

scholarly journals Atrial Fibrillation Progression Is Associated with Cell Senescence Burden as Determined by p53 and p16 Expression

2019 ◽  
Vol 9 (1) ◽  
pp. 36
Author(s):  
Laurence Jesel ◽  
Malak Abbas ◽  
Sin-Hee Park ◽  
Kensuke Matsushita ◽  
Michel Kindo ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Heart Surgery ◽  
Open Heart Surgery ◽  
Right Atrial ◽  
Atrial Remodeling ◽  
Inflammatory Proteins ◽  
Underlying Mechanisms ◽  
The Right ◽  
New Evidence

Background: Whilst the link between aging and thrombogenicity in atrial fibrillation (AF) is well established, the cellular underlying mechanisms are unknown. In AF, the role of senescence in tissue remodeling and prothrombotic state remains unclear. Aims: We investigated the link between AF and senescence by comparing the expression of senescence markers (p53 and p16), with prothrombotic and inflammatory proteins in right atrial appendages from patients in AF and sinus rhythm (SR). Methods: The right atrial appendages of 147 patients undergoing open-heart surgery were harvested. Twenty-one non-valvular AF patients, including paroxysmal (PAF) or permanent AF (PmAF), were matched with 21 SR patients according to CHA2DS2-VASc score and treatment. Protein expression was assessed by tissue lysates Western blot analysis. Results: The expression of p53, p16, and tissue factor (TF) was significantly increased in AF compared to SR (0.91 ± 0.31 vs. 0.58 ± 0.31, p = 0.001; 0.76 ± 0.32 vs. 0.35 ± 0.18, p = 0.0001; 0.88 ± 0.32 vs. 0.68 ± 0.29, p = 0.045, respectively). Expression of endothelial NO synthase (eNOS) was lower in AF (0.25 ± 0.15 vs. 0.35 ± 0.12, p = 0.023). There was a stepwise increase of p53, p16, TF, matrix metalloproteinase-9, and an eNOS progressive decrease between SR, PAF, and PmAF. AF was the only predictive factor of p53 and p16 elevation in multivariate analysis. Conclusions: The study brought new evidence indicating that AF progression is strongly related to human atrial senescence burden and points at a link between senescence, thrombogenicity, endothelial dysfunction and atrial remodeling.

P2581Unique molecular profile of exosomes from epicardial fat of patients with atrial fibrillation

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
O Shaihov - Teper ◽  
E Ram ◽  
L Levin - Kotler ◽  
N Naftali - Shani ◽  
J Leor
Keyword(s):  
Heart Surgery ◽  
Micro Rna ◽  
Epicardial Fat ◽  
Molecular Profile ◽  
Atrial Remodeling ◽  
Intercellular Signaling ◽  
Intracellular Compartments ◽  
Novel Biomarkers ◽  
First Time

Abstract Background and aim Exosome, the smallest vesicles that originate from intracellular compartments, contains and delivers specific bioactive proteins, lipids, mRNA, and miRNA to facilitate intercellular signaling. However, the role and mechanism of exosomes in atrial remodeling and fibrillation (AF) has not been determined. While epicardial fat (eFat) and inflammation have been linked to atrial remodeling and fibrillation (AF), the role of eFat exosomes in the pathogenesis of AF remains unknown. Thus, we aimed to determine whether eFat exosomes play a role in the initiation and progression of AF. Methods and results We collected eFat specimens from patients with and without chronic or paroxysmal AF undergoing heart surgery. Isolated fat specimens were cut into small pieces and incubated as organ cultures. We isolated exosomes from the medium of the explant by differential ultra-centrifugation and characterized the vesicle size distributions, morphology, specific markers, and molecular cargo. Immunoblotting of CD63, CD81 and TSG101 confirmed the presence of exosomes (Fig.1 B). Significantly, eFat from patients with AF secreted higher amounts of exosomes compared with controls (p<0.001), which confirmed by Nanoparticle Tracking Analysis (NTA) (Fig.1 A). The levels of exosomal pro-inflammatory and pro-fibrotic cytokines were higher in exosomes from patients with, compared with those without, AF (Fig.1 C, D). Measurements of micro RNA (miRNA) by real-time PCR showed lower levels of anti-fibrotic miRNA-133a (p=0.2), and higher levels of pro-fibrotic miRNA-146b (p=0.2) in exosomes from patients with, compared with those without, AF (Fig.1 E, F) Conclusion We show, for the first time, that exosomes from eFat of patients with AF have a unique pro-inflammatory and pro-fibrotic profile. We suggest a novel mechanism that links eFat to AF and eFat exosomes as novel biomarkers and potential therapeutic targets for AF.

scholarly journals Extracellular Vesicles in Tumors: A Potential Mediator of Bone Metastasis

2021 ◽  
Vol 9 ◽  
Author(s):  
Shenglong Li ◽  
Wei Wang
Keyword(s):  
Bone Metastasis ◽  
Extracellular Vesicles ◽  
Lipid Membrane ◽  
Bone Cells ◽  
Membrane Vesicles ◽  
Metastatic Tumor ◽  
Cell Types ◽  
Biological Phenomena ◽  
Breast And Prostate Cancer ◽  
Metastatic Sites

As one of the most common metastatic sites, bone has a unique microenvironment for the growth and prosperity of metastatic tumor cells. Bone metastasis is a common complication for tumor patients and accounts for 15–20% of systemic metastasis, which is only secondary to lung and liver metastasis. Cancers prone to bone metastasis include lung, breast, and prostate cancer. Extracellular vesicles (EVs) are lipid membrane vesicles released from different cell types. It is clear that EVs are associated with multiple biological phenomena and are crucial for intracellular communication by transporting intracellular substances. Recent studies have implicated EVs in the development of cancer. However, the potential roles of EVs in the pathological exchange of bone cells between tumors and the bone microenvironment remain an emerging area. This review is focused on the role of tumor-derived EVs in bone metastasis and possible regulatory mechanisms.

Characterization and regulation of extracellular vesicles in the lumen of the ovine uterus†

2020 ◽  
Vol 102 (5) ◽  
pp. 1020-1032 ◽  
Author(s):  
Eleanore V O’Neil ◽  
Gregory W Burns ◽  
Christina R Ferreira ◽  
Thomas E Spencer
Keyword(s):  
Extracellular Vesicles ◽  
Cell Types ◽  
Size Exclusion ◽  
Rna Seq ◽  
Interferon Tau ◽  
Uterine Lumen ◽  
Pregnant Sheep ◽  
Exclusion Chromatography ◽  
Ovine Uterus

Abstract Secretions of the endometrium are vital for peri-implantation growth and development of the sheep conceptus. Extracellular vesicles (EVs) are present in the uterine lumen, emanate from both the endometrial epithelia of the uterus and trophectoderm of the conceptus, and hypothesized to mediate communication between those cell types during pregnancy establishment in sheep. Size-exclusion chromatography and nanoparticle tracking analysis determined that total EV number in the uterine lumen increased from days 10 to 14 of the cycle but was lower on days 12 and 14 of pregnancy in sheep. Intrauterine infusions of interferon tau (IFNT) did not affect total EV number in the uterine lumen. Quantitative mass spectrometric analyses defined proteins and lipids in EVs isolated from the uterine lumen of day 14 cyclic and pregnant sheep. In vitro analyses found that EVs decreased ovine trophectoderm cell proliferation and increased IFNT production without effects on gene expression as determined by RNA-seq. Collective results support the idea EVs impact conceptus growth during pregnancy establishment via effects on trophectoderm cell growth.

Soluble VCAM-1 is a very early marker of endothelial cell activation in cardiopulmonary bypass

Perfusion ◽  
2002 ◽  
Vol 17 (1) ◽  
pp. 15-21 ◽  
Author(s):  
Tonje Katrine Andresen ◽  
Jan L Svennevig ◽  
Vibeke Videm
Keyword(s):  
Endothelial Cells ◽  
Cardiopulmonary Bypass ◽  
Adhesion Molecules ◽  
Time Course ◽  
Heart Surgery ◽  
Culture Media ◽  
Open Heart Surgery ◽  
Endothelial Activation ◽  
Open Heart ◽  
Early Marker

Cardiopulmonary bypass causes a systemic inflammatory reaction, which leads to endothelial activation with increased expression of adhesion molecules. The study aim was to test whether activated endothelial cells secrete measurable amounts of soluble adhesion molecules during the time course of routine heart surgery, and whether these markers correlate with cellular activation responses. Endothelial cells from human umbilical cords were cultured by standard methods and stimulated with endotoxin. After 2 h, the expression of membrane-bound E-selectin on the cells had increased significantly ( p= 0.04), whereas soluble VCAM-1 (sVCAM-1) had increased significantly in the culture media ( p= 0.03). In agreement with these findings, sVCAM-1 increased from 399 ng/ml (median) to 581 ng/ml within 3 h postoperatively in sera from 11 patients undergoing open heart surgery ( p= 0.003). sVCAM-1, therefore, may be suitable as an early marker of endothelial activation related to the systemic inflammation after open heart surgery. The clinical significance of sVCAM-1 measurements must be further evaluated in future patient studies.

scholarly journals The Past, the Present, and the Future of the Size Exclusion Chromatography in Extracellular Vesicles Separation

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2272
Author(s):  
Hussein Kaddour ◽  
Malik Tranquille ◽  
Chioma M. Okeoma
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Cell Types ◽  
Size Exclusion ◽  
Target Cells ◽  
Purification And Characterization ◽  
Extracellular Milieu ◽  
Exclusion Chromatography ◽  
Single Vesicle

Extracellular vesicles (EVs) are cell-derived membranous particles secreted by all cell types (including virus infected and uninfected cells) into the extracellular milieu. EVs carry, protect, and transport a wide array of bioactive cargoes to recipient/target cells. EVs regulate physiological and pathophysiological processes in recipient cells and are important in therapeutics/drug delivery. Despite these great attributes of EVs, an efficient protocol for EV separation from biofluids is lacking. Numerous techniques have been adapted for the separation of EVs with size exclusion chromatography (SEC)-based methods being the most promising. Here, we review the SEC protocols used for EV separation, and discuss opportunities for significant improvements, such as the development of novel particle purification liquid chromatography (PPLC) system capable of tandem purification and characterization of biological and synthetic particles with near-single vesicle resolution. Finally, we identify future perspectives and current issues to make PPLC a tool capable of providing a unified, automated, adaptable, yet simple and affordable particle separation resource.

Abstract 432: Endothelial Cells Express miR-33a-5p and Release Extracellular Vesicles Containing miR-33a-5p

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Alexis Stamatikos ◽  
Lucia Vojtech ◽  
Nagadhara Dronadula ◽  
David Dichek
Keyword(s):  
Electron Microscopy ◽  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Small Rna ◽  
Cholesterol Efflux ◽  
Lipid Membrane ◽  
Cell Types ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Background: The microRNA miR-33a-5p increases cellular cholesterol by posttranscriptional silencing of genes involved in cholesterol efflux. Previous studies showed that miR-33a-5p inhibition protects against atherosclerosis. Atheroprotective effects of miR-33a-5p inhibition are attributed to actions in hepatocytes that raise plasma HDL and actions in macrophages that enhance cholesterol efflux. We hypothesized that miR-33a-5p is also expressed in primary arterial endothelial cells (EC) and that miR-33a-5p might contribute to atherosclerosis by promoting cholesterol accumulation in EC. We also hypothesized that EC could release miR-33a-5p in extracellular vesicles (EV; including exosomes and microvesicles) that transferred miR-33a-5p to neighboring vascular cells such as SMC and macrophages, inhibiting cholesterol efflux, promoting lipid accumulation in these cells, and accelerating atherosclerosis. Methods: We cultured bovine aortic endothelial cells (BAEC) in serum-free medium, collected the medium, and isolated EV by ultracentrifugation. To test whether we had isolated EV, we compared EV and BAEC lysates by SDS-PAGE, Coomassie blue staining, and immunoblotting. We measured EV size with nanoparticle tracking analysis and imaged the particles with transmission electron microscopy. We extracted total RNA from BAEC and EV, measured RNA size using BioAnalyzer, and detected miR-33a-5p expression using RT-PCR and restriction digestion as well as qRT-PCR. Results: Coomassie blue staining revealed large differences between BAEC and EV lysates. The exosome marker CD9 was present at higher levels in EV lysate compared to BAEC lysate. Most of the EV were in the size range of exosomes, and appeared as typical lipid membrane-bound spheres by electron microscopy. EV contained primarily small RNA (~25-200 nucleotides). miR-33a-5p was detected both in BAEC lysates and in detergent-soluble EV. Conclusions: Primary arterial EC express miR-33a-5p and release EV rich in small RNA, including miR-33a-5p. Manipulating miR-33a-5p expression in EC, for example with a miR-33a-5p antagomiR, may be an effective therapeutic approach for increasing cholesterol efflux from multiple vessel wall cell types and preventing/reversing atherosclerosis.

Abstract 224: Primary Human Cells Isolated From Atria and Bone Marrow Exhibit Comparable Anti-fibrotic Cell Phenotypes Upon Transfection With MicroRNA-301a

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Alison L Müller ◽  
Darren H Freed
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Heart Surgery ◽  
Cardiac Fibrosis ◽  
Open Heart Surgery ◽  
Cell Types ◽  
Scar Tissue ◽  
Open Heart ◽  
Cell Type

There are many cell types that can contribute to cardiac fibrosis including atrial fibroblasts (AFs) and bone marrow-derived progenitor cells (MPCs). We have previously shown that MPCs display a myofibroblast phenotype in vitro which is linked to altered microRNA(miR)-301a expression, a miR affiliated with maintaining proliferation in numerous cell types. We have also shown that miR-301a influences a dichotomous phenotype in primary human MPCs isolated from patients undergoing open heart surgery. As both MPCs and AFs display a dichotomous phenotype where each cell type displays a phenotype that pathologically contributes to fibrosis, we transfected both MPCs and AFs with miR-301a. AFs were also isolated from patients undergoing open heart surgery. We observed decreases in levels of both mRNA and protein of collagen I, non-muscle myosin IIA, and EDA-fibronectin. These proteins are expressed in myofibroblasts, the cell type predominantly responsible for causing cardiac fibrosis. In addition, transfection of miR301a caused both cell types to increase proliferation, which was analyzed using MTT proliferation assays. These results indicate that miR-301a could be influencing a non-fibrotic phenotype, which could prove useful in cell therapy trials where progenitor cells are injected into scar tissue in order to help heal patients who have suffered from a myocardial infarction. Over-expressing miR-301a in cells used could prevent them from differentiating into pro-fibrotic phenotypes and encourage their proliferation, thereby potentiating their efficacy.

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