scholarly journals A Comprehensive Proteomic SWATH-MS Workflow for Profiling Blood Extracellular Vesicles: A New Avenue for Glioma Tumour Surveillance

2020 ◽  
Vol 21 (13) ◽  
pp. 4754 ◽  
Author(s):  
Susannah Hallal ◽  
Ali Azimi ◽  
Heng Wei ◽  
Nicholas Ho ◽  
Maggie Yuk Ting Lee ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Biomarker Discovery ◽  
Ex Vivo ◽  
Tumour Progression ◽  
Principal Component ◽  
P Value ◽  
Diffuse Glioma ◽  
Blood Proteins ◽  
Treatment Protocols

Improving outcomes for diffuse glioma patients requires methods that can accurately and sensitively monitor tumour activity and treatment response. Extracellular vesicles (EV) are membranous nanoparticles that can traverse the blood–brain-barrier, carrying oncogenic molecules into the circulation. Measuring clinically relevant glioma biomarkers cargoed in circulating EVs could revolutionise how glioma patients are managed. Despite their suitability for biomarker discovery, the co-isolation of highly abundant complex blood proteins has hindered comprehensive proteomic studies of circulating-EVs. Plasma-EVs isolated from pre-operative glioma grade II–IV patients (n = 41) and controls (n = 11) were sequenced by Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) and data extraction was performed by aligning against a custom 8662-protein library. Overall, 4054 proteins were measured in plasma-EVs. Differentially expressed proteins and putative circulating-EV markers were identified (adj. p-value < 0.05), including those reported in previous in-vitro and ex-vivo glioma-EV studies. Principal component analysis showed that plasma-EV protein profiles clustered according to glioma histological-subtype and grade, and plasma-EVs resampled from patients with recurrent tumour progression grouped with more aggressive glioma samples. The extensive plasma-EV proteome profiles achieved here highlight the potential for SWATH-MS to define circulating-EV biomarkers for objective blood-based measurements of glioma activity that could serve as ideal surrogate endpoints to assess tumour progression and allow more dynamic, patient-centred treatment protocols.

scholarly journals A comprehensive proteomic SWATH-MS workflow for profiling blood extracellular vesicles: a new avenue for glioma tumour surveillance

2020 ◽  
Author(s):  
Susannah Hallal ◽  
Ali Azimi ◽  
Heng Wei ◽  
Nicholas Ho ◽  
Maggie Lee ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Biomarker Discovery ◽  
Data Extraction ◽  
Molecular State ◽  
Recurrent Glioblastoma ◽  
P Value ◽  
Cell Of Origin ◽  
Monitoring Method ◽  
Who Grade

AbstractThere is a real need for biomarkers that can indicate glioma disease burden and inform clinical management, particularly in the recurrent glioblastoma (GBM; grade IV glioma) setting where treatment-associated brain changes can confound current and expensive tumour surveillance methods. In this regard, extracellular vesicles (EVs; 30-1000 nm membranous particles) hold major promise as robust tumour biomarkers. GBM-EVs encapsulate molecules that reflect the identity and molecular state of their cell-of-origin and cross the blood-brain-barrier into the periphery where they are readily accessible. Despite the suitability of circulating-EVs for GBM biomarker discovery, sample complexity has hindered comprehensive quantitative proteomic studies. Here, sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) was used in conjunction with a targeted data extraction strategy to comprehensively profile circulating-EVs isolated from plasma. Plasma-EVs sourced from pre-operative glioma II-IV patients (n=41) and controls (n=11) were sequenced by SWATH-MS, and the identities and absolute quantities of the proteins were extracted by aligning the SWATH-MS data against a custom glioma spectral library comprised of 8662 high confidence protein species. Overall, 4054 plasma-EV proteins were quantified across the cohorts, and putative circulating-EV biomarker proteins identified (adjusted p-value<0.05) included previously reported GBM-EV proteins identified in vitro and in neurosurgical aspirates. Principle component analyses showed that plasma-EV protein profiles clustered according to glioma subtype and WHO-grade, and plasma-EV proteins reflected the extent of glioma aggression. Using SWATH-MS, we describe the most comprehensive proteomic plasma-EV profiles for glioma and highlight the promise of this approach as an accurate and sensitive tumour monitoring method. Objective blood-based measurements of glioma tumour activity will support the implementation of next-generation, patient-centred therapies and are ideal surrogate endpoints for recurrent progression that would allow clinical trial protocols to be more dynamic and adapt to the individual patient and their cancer.

scholarly journals Unraveling the In Vivo Protein Corona

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 132
Author(s):  
Johanna Simon ◽  
Gabor Kuhn ◽  
Michael Fichter ◽  
Stephan Gehring ◽  
Katharina Landfester ◽  
...  
Keyword(s):  
Plasma Proteins ◽  
Ex Vivo ◽  
Protein Corona ◽  
Blood Proteins ◽  
Therapeutic Application ◽  
Complex Situation ◽  
Ex Vivo Approach ◽  
In Vivo Administration

Understanding the behavior of nanoparticles upon contact with a physiological environment is of urgent need in order to improve their properties for a successful therapeutic application. Most commonly, the interaction of nanoparticles with plasma proteins are studied under in vitro conditions. However, this has been shown to not reflect the complex situation after in vivo administration. Therefore, here we focused on the investigation of magnetic nanoparticles with blood proteins under in vivo conditions. Importantly, we observed a radically different proteome in vivo in comparison to the in vitro situation underlining the significance of in vivo protein corona studies. Next to this, we found that the in vivo corona profile does not significantly change over time. To mimic the in vivo situation, we established an approach, which we termed “ex vivo” as it uses whole blood freshly prepared from an animal. Overall, we present a comprehensive analysis focusing on the interaction between nanoparticles and blood proteins under in vivo conditions and how to mimic this situation with our ex vivo approach. This knowledge is needed to characterize the true biological identity of nanoparticles.

scholarly journals Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 730
Author(s):  
Biji Mathew ◽  
Leianne A. Torres ◽  
Lorea Gamboa Gamboa Acha ◽  
Sophie Tran ◽  
Alice Liu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Extracellular Vesicles ◽  
Time Course ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Paracrine Effects

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC’s paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

scholarly journals Extracellular vesicles engineered with valency-controlled DNA nanostructures deliver CRISPR/Cas9 system for gene therapy

2020 ◽  
Vol 48 (16) ◽  
pp. 8870-8882 ◽  
Author(s):  
Jialang Zhuang ◽  
Jizhou Tan ◽  
Chenglin Wu ◽  
Jie Zhang ◽  
Ting Liu ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Primary Liver Cancer ◽  
The Body ◽  
Dna Aptamer ◽  
Great Promise ◽  
Tumor Growth Inhibition ◽  
Specific Cell ◽  
Dna Nanostructures

Abstract Extracellular vesicles (EVs) hold great promise for transporting CRISPR–Cas9 RNA-guided endonucleases (RNP) throughout the body. However, the cell-selective delivery of EVs is still a challenge. Here, we designed valency-controlled tetrahedral DNA nanostructures (TDNs) conjugated with DNA aptamer, and loaded the valency-controlled TDNs on EV surface via cholesterol anchoring for specific cell targeting. The targeting efficacy of different ratios of aptamer/cholesterol from 1:3 to 3:1 in TDNs on decorating EVs was investigated. TDNs with one aptamer and three cholesterol anchors (TDN1) efficiently facilitated the tumor-specific accumulation of the EVs in cultured HepG2 cells and human primary liver cancer-derived organoids, as well as xenograft tumor models. The intracellular delivery of RNP by TDN1-EVs successfully realized its subsequent genome editing, leading to the downregulation of GFP or WNT10B in specific cells. This system was ultimately applied to reduce the protein expression of WNT10B, which presented remarkable tumor growth inhibition in vitro, ex vivo and in vivo, and could be extended to other therapeutic targets. The present study provides a platform for the directional display of aptamer on surface labeling and the EVs-based Cas9 delivery, which provides a meaningful idea for future cell-selective gene editing.

scholarly journals microRNA-155 Is Decreased During Atherosclerosis Regression and Is Increased in Urinary Extracellular Vesicles During Atherosclerosis Progression

2020 ◽  
Vol 11 ◽  
Author(s):  
Stephen Fitzsimons ◽  
Silvia Oggero ◽  
Robyn Bruen ◽  
Cathal McCarthy ◽  
Moritz J. Strowitzki ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Macrophage Polarization ◽  
Chronic Inflammatory Disease ◽  
Cholesterol Diet ◽  
Human Coronary Artery ◽  
Inflammatory Signaling ◽  
Anti Inflammatory

BackgroundAtherosclerosis is a chronic inflammatory disease driven by macrophage accumulation in medium and large sized arteries. Macrophage polarization and inflammation are governed by microRNAs (miR) that regulate the expression of inflammatory proteins and cholesterol trafficking. Previous transcriptomic analysis led us to hypothesize that miR-155-5p (miR-155) is regulated by conjugated linoleic acid (CLA), a pro-resolving mediator which induces regression of atherosclerosis in vivo. In parallel, as extracellular vesicles (EVs) and their miR content have potential as biomarkers, we investigated alterations in urinary-derived EVs (uEVs) during the progression of human coronary artery disease (CAD).MethodsmiR-155 expression was quantified in aortae from ApoE−/− mice fed a 1% cholesterol diet supplemented with CLA blend (80:20, cis-9,trans-11:trans-10,cis-12 respectively) which had been previously been shown to induce atherosclerosis regression. In parallel, human polarized THP-1 macrophages were used to investigate the effects of CLA blend on miR-155 expression. A miR-155 mimic was used to investigate its inflammatory effects on macrophages and on ex vivo human carotid endarterectomy (CEA) plaque specimens (n = 5). Surface marker expression and miR content were analyzed in urinary extracellular vesicles (uEVs) obtained from patients diagnosed with unstable (n = 12) and stable (n = 12) CAD.ResultsHere, we report that the 1% cholesterol diet increased miR-155 expression while CLA blend supplementation decreased miR-155 expression in the aorta during atherosclerosis regression in vivo. CLA blend also decreased miR-155 expression in vitro in human THP-1 polarized macrophages. Furthermore, in THP-1 macrophages, miR-155 mimic decreased the anti-inflammatory signaling proteins, BCL-6 and phosphorylated-STAT-3. In addition, miR-155 mimic downregulated BCL-6 in CEA plaque specimens. uEVs from patients with unstable CAD had increased expression of miR-155 in comparison to patients with stable CAD. While the overall concentration of uEVs was decreased in patients with unstable CAD, levels of CD45+ uEVs were increased. Additionally, patients with unstable CAD had increased CD11b+ uEVs and decreased CD16+ uEVs.ConclusionmiR-155 suppresses anti-inflammatory signaling in macrophages, is decreased during regression of atherosclerosis in vivo and is increased in uEVs from patients with unstable CAD suggesting miR-155 has potential as a prognostic indicator and a therapeutic target.

Extracellular Vesicles Isolated from Mesenchymal Stromal Cells Primed with Hypoxia: Novel strategy in Regenerative Medicine

2020 ◽  
Vol 15 ◽  
Author(s):  
Shalmali Pendse ◽  
Vaijayanti Kale ◽  
Anuradha Vaidya
Keyword(s):  
Extracellular Vesicles ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Cell Types ◽  
Molecular Profile ◽  
Cell Contact ◽  
Hematopoietic Stem

: Mesenchymal stromal cells (MSCs) regulate other cell types through a strong paracrine component called the secretome, comprising of several bioactive entities. The composition of the MSCs’ secretome is dependent upon the microenvironment in which they thrive, and hence, it could be altered by pre-conditioning the MSCs during in vitro culture. The primary aim of this review is to discuss various strategies that are being used for pre-conditioning of MSCs, also known as “priming of MSCs”, in the context of improving their therapeutic potential. Several studies have underscored the importance of extracellular vesicles (EVs) derived from primed MSCs in improving their efficacy in the treatment of various diseases. We have previously shown that co-culturing hematopoietic stem cells (HSCs) with hypoxiaprimed MSCs improves their engraftment potential. Now the question we pose is would priming of MSCs with hypoxiafavorably alter theirsecretome and would this altered secretome work as effectively as the cell to cell contact did? Here we review the current strategies of using the secretome, specifically the EVs (microvesicles and exosomes), collected from the primed MSCs with the intention of expanding HSCs ex vivo. We speculate that an effective priming of MSCs in vitrocould modulate the molecular profile of their secretome, which could eventually be used as a cell-free biologic in clinical settings.

scholarly journals Extracellular Vesicles from Airway Secretions: New Insights in Lung Diseases

2021 ◽  
Vol 22 (2) ◽  
pp. 583
Author(s):  
Laura Pastor ◽  
Elisabeth Vera ◽  
Jose M. Marin ◽  
David Sanz-Rubio
Keyword(s):  
Extracellular Vesicles ◽  
Lung Diseases ◽  
Biomarker Discovery ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Lavage Fluid ◽  
In Vitro Models ◽  
Chronic Obstructive ◽  
Obstructive Pulmonary Disease

Lung diseases (LD) are one of the most common causes of death worldwide. Although it is known that chronic airway inflammation and excessive tissue repair are processes associated with LD such as asthma, chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF), their specific pathways remain unclear. Extracellular vesicles (EVs) are heterogeneous nanoscale membrane vesicles with an important role in cell-to-cell communication. EVs are present in general biofluids as plasma or urine but also in secretions of the airway as bronchoalveolar lavage fluid (BALF), induced sputum (IS), nasal lavage (NL) or pharyngeal lavage. Alterations of airway EV cargo could be crucial for understanding LD. Airway EVs have shown a role in the pathogenesis of some LD such as eosinophil increase in asthma, the promotion of lung cancer in vitro models in COPD and as biomarkers to distinguishing IPF in patients with diffuse lung diseases. In addition, they also have a promising future as therapeutics for LD. In this review, we focus on the importance of airway secretions in LD, the pivotal role of EVs from those secretions on their pathophysiology and their potential for biomarker discovery.

scholarly journals Percutaneous Coronary Intervention (PCI) Reprograms Circulating Extracellular Vesicles from ACS Patients Impairing Their Cardio-Protective Properties

2021 ◽  
Vol 22 (19) ◽  
pp. 10270
Author(s):  
Saveria Femminò ◽  
Fabrizio D’Ascenzo ◽  
Francesco Ravera ◽  
Stefano Comità ◽  
Filippo Angelini ◽  
...  
Keyword(s):  
Percutaneous Coronary Intervention ◽  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Coronary Intervention ◽  
Protective Properties ◽  
Coronary Syndrome ◽  
Percutaneous Coronary ◽  
Mrna Content ◽  
The Impact

Extracellular vesicles (EVs) are promising therapeutic tools in the treatment of cardiovascular disorders. We have recently shown that EVs from patients with Acute Coronary Syndrome (ACS) undergoing sham pre-conditioning, before percutaneous coronary intervention (PCI) were cardio-protective, while EVs from patients experiencing remote ischemic pre-conditioning (RIPC) failed to induce protection against ischemia/reperfusion Injury (IRI). No data on EVs from ACS patients recovered after PCI are currently available. Therefore, we herein investigated the cardio-protective properties of EVs, collected after PCI from the same patients. EVs recovered from 30 patients randomly assigned (1:1) to RIPC (EV-RIPC) or sham procedures (EV-naive) (NCT02195726) were characterized by TEM, FACS and Western blot analysis and evaluated for their mRNA content. The impact of EVs on hypoxia/reoxygenation damage and IRI, as well as the cardio-protective signaling pathways, were investigated in vitro (HMEC-1 + H9c2 co-culture) and ex vivo (isolated rat heart). Both EV-naive and EV-RIPC failed to drive cardio-protection both in vitro and ex vivo. Consistently, EV treatment failed to activate the canonical cardio-protective pathways. Specifically, PCI reduced the EV-naive Dusp6 mRNA content, found to be crucial for their cardio-protective action, and upregulated some stress- and cell-cycle-related genes in EV-RIPC. We provide the first evidence that in ACS patients, PCI reprograms the EV cargo, impairing EV-naive cardio-protective properties without improving EV-RIPC functional capability.

scholarly journals Λειτουργική ικανότητα υπερπληθυσμών λεμφoκυττάρων ασκιτικού υγρού ασθενών με καρκίνο των ωοθηκών

2013 ◽  
Author(s):  
Νικόλαος Πισταμαλτζιάν
Keyword(s):  
Ex Vivo ◽  
T Test ◽  
P Value ◽  
Paired T Test

Η προθυμοσίνη α (προΤα) είναι ένα ισχυρά όξινο πολυπεπτίδιο, 109 αμινοξικών καταλοίπων, με ανοσοενισχυτική δράση in vitro και in vivo. Οι ανοσοδραστικές της ιδιότητες, οφείλονται στο καρβοξυτελικό της δεκαπεπτίδιο, προΤα(100-109).Στην διατριβή, διερευνήθηκε η in vitro επίδραση των προΤα/προΤα(100-109), συνεργιστικά με ιντερλευκίνη–2, στις ανοσολογικές απαντήσεις λεμφοκυττάρων απομονωμένων από ασκιτικό υγρό ασθενών με καρκίνο ωοθηκών. Απομονωθέντα λεμφοκύτταρα, από 33 ασθενείς με καρκίνο ωοθηκών σταδίου ΙΙΙC (FIGO), χωρίς προηγούμενη έκθεση σε χημειοθεραπεία, επωάστηκαν ex vivo με προΤα, και προΤα(100–109), συνεργιστικά με χαμηλές συγκεντρώσεις ιντερλευκίνης–2, στα πλαίσια μικτής καλλιέργειας λεμφοκυττάρων/καρκινικών κυττάρων. Ως μέτρο σύγκρισης υπήρξαν κύτταρα που επωάστηκαν παρουσία ιντερλευκίνης–2 μόνο. Τα λεμφοκύτταρα ενεργοποιούνταν εβδομαδιαία, επί 3 εβδομάδες, με εγχύσεις αυτόλογων καρκινικών κυττάρων. Στις ημέρες 7, 14, 21 γίνονταν έλεγχος του ανοσοφαινότυπου των σχετιζόμενων με τον όγκο λεμφοκυττάρων (TALs) με τη μέθοδο της κυτταρομετρίας ροής, αλλά και της δραστικότητας τους με τη δοκιμασία κυτταροτοξικότητας. Για τη δοκιμασία της κυτταροτοξικότητας, χρησιμοποιήθηκαν ως στόχοι αυτόλογα καρκινικά κύτταρα και K562 κύτταρα. Οι στόχοι συνδέονταν με 51Cr (18 ώρες συνεπώασης, σε μια αναλογία εκτελεστών : στόχων 40:1), και γίνονταν μέτρηση της εκλυόμενης ραδιενέργειας από μετρητή γ-ακτινοβολίας. Για τον προσδιορισμό του ανοσοφαινότυπου χρησιμοποιήθηκαν anti-CD45, anti-CD56, anti-CD3, anti-CD4 και anti-CD8 μονοκλωνικά αντισώματα συνδεδεμένα με τις χρωστικές φλουορεσκεΐνη, R-φυκοερυθρίνη και R–φυκοερυθρίνη/Cychrome5. Για την κυτταρομετρία ροής, χρησιμοποιήθηκε κυτταρόμετρο BD FACS Calibur. Τα δεδομένα αναλύθηκαν με λογισμικό CellQuest. Για τη στατιστική επεξεργασία χρησιμοποιήθηκε το paired t–test, με αμφίπλευρα επίπεδα σημαντικότητας και p-value = 0,05. Για την ανάλυση χρησιμοποιήθηκε το SPSS v13.0. Συνολικά, τα πειραματικά δεδομένα υποδεικνύουν πως η προΤα και το προΤα(100-109) ενισχύουν σημαντικά την ειδική – για – το αντιγόνο κυτταροτοξικότητα των TALs, έναντι των αυτόλογων καρκινικών κυττάρων in vitro ( ~20% έναντι 12.5%, p = 0.001 για την ημέρα 7 και ~18% έναντι 11%, p=0.003 για την ημέρα 14), εμφανίζοντας μια λιγότερο ισχυρή επίδραση στην ικανότητα των ΝΚ κυττάρων, να λύουν ευαίσθητους σε αυτά στόχους (p=0.073). Η επίδραση της προθυμοσίνης α και του προΤα(100-109) είναι πιο ισχυρή τις ημέρες 7-14, και μειώνεται σημαντικά την ημέρα 21. Η δράση της προθυμοσίνης α είναι ταυτόσημη με αυτή του προΤα(100-109). Η χρήση δεκαπεπτιδίου με αναδιαταγμένη αλληλουχία αμινοξέων δεν προκάλεσε ανοσοενίσχυση, αποδεικνύοντας έτσι την ειδική για την αλληλουχία δράση του προΤα(100-109). Η συγκεκριμένη μελέτη παρουσιάζει για πρώτη φορά, την ανοσοενισχυτική δράση της προΤα και του προΤα(100-109) σε λεμφοκύτταρα ασκιτικού υγρού ασθενών με καρκίνο των ωοθηκών. Δεν υπήρξε κάποια συσχέτιση με την ηλικία, τον ιστολογικό τύπο ή την θεραπευτική ανταπόκριση.Συμπερασματικά, η προθυμοσίνη α και το ανοσοδραστικό καρβοξυτελικό δεκαπεπτίδιο προΤα(100-109), ενισχύουν σημαντικά την λυτική ικανότητα των TALs. Παρουσία καρκινικών αντιγόνων, ενισχύουν την μειωμένη κυτταροτοξικότητα των TALs έναντι των αυτόλογων καρκινικών κυττάρων, αναστρέφοντας την ισχυρή ανοσοκατασταλτική επίδραση που ασκεί σε αυτά, το περιβάλλον του ασκίτη ασθενών με καρκίνο των ωοθηκών.

scholarly journals Extracellular Vesicles Derived From Human Corneal Endothelial Cells Inhibit Proliferation of Human Corneal Endothelial Cells

2021 ◽  
Author(s):  
Mohit Parekh ◽  
Hefin Rhys ◽  
Tiago Ramos ◽  
Stefano Ferrari ◽  
Sajjad Ahmad
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Treatment Options ◽  
Proliferative Capacity ◽  
Corneal Endothelial Cells ◽  
Human Corneal Endothelial Cells ◽  
The Media

Abstract Corneal endothelial cells (CEnCs) are a monolayer of hexagonal cells that are responsible for maintaining the function and transparency of the cornea. Damage or dysfunction of CEnCs could lead to blindness. Human CEnCs (HCEnCs) have shown limited proliferative capacity in vivo hence, their maintenance is crucial. Extracellular vesicles (EVs), are responsible for inter- and intra-cellular communication, proliferation, cell-differentiation, migration, and many other complex biological processes. Therefore, we investigated the effect of EVs (derived from human corneal endothelial cell line – HCEC-12) on corneal endothelial cells. HCEC-12 cells were starved with serum-depleted media for 72 hours. The media was ultracentrifuged at 100,000xg to isolate the EVs. EV counting, characterization, internalization and localization were performed using NanoSight, flow cytometry, Dil labelling and confocal microscopy respectively. HCEC-12 and HCEnCs were cultured with media supplemented with EVs. Extracted EVs showed a homogeneous mixture of exosomes and microvesicles. Cells with EVs decreased the proliferation rate; increased apoptosis and cell size; showed poor wound healing response in vitro and on ex vivo human, porcine, and rabbit CECs. Thirteen miRNAs were found in the EV sample using next generation sequencing. We observed that increased cellular uptake of EVs by CECs limit the proliferative capacity of HCEnCs. These preliminary data may help in understanding the pathology of corneal endothelial dysfunction and provide further insights in the development of future therapeutic treatment options.

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