Directional Endothelial Communication by Polarized Extracellular Vesicle Release.

BACKGROUND: Extracellular vesicles (EVs) contain bioactive cargo including miRNAs and proteins that are released by cells during cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels, interfacing with cells in the circulation and vascular wall. It is unknown whether ECs release EVs capable of governing recipient cells within these 2 separate compartments. Given their boundary location, we propose ECs use bidirectional release of distinct EV cargo in quiescent (healthy) and activated (atheroprone) states to communicate with cells within the circulation and blood vessel wall. METHODS: EVs were isolated from primary human aortic ECs (plate and transwell grown; ±IL [interleukin]-1ß activation), quantified, visualized, and analyzed by miRNA transcriptomics and proteomics. Apical and basolateral EC-EV release was determined by miRNA transfer, total internal reflection fluorescence and electron microscopy. Vascular reprogramming (RNA sequencing) and functional assays were performed on primary human monocytes or smooth muscle cells±EC-EVs. RESULTS: Activated ECs increased EV release, with miRNA and protein cargo related to atherosclerosis. EV-treated monocytes and smooth muscle cells revealed activated EC-EV altered pathways that were proinflammatory and atherogenic. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, activated basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis. In silico analysis determined compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and smooth muscle cells, respectively, with functional assays and in vivo imaging supporting this concept. CONCLUSIONS: Demonstrating that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance the design of endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated.

Circulating small extracellular vesicles mediate vascular hyperpermeability in diabetes.

AIMS/HYPOTHESIS: A hallmark chronic complication of type 2 diabetes mellitus is vascular hyperpermeability, which encompasses dysfunction of the cerebrovascular endothelium and the subsequent development of associated cognitive impairment. The present study tested the hypothesis that during type 2 diabetes circulating small extracellular vesicles (sEVs) exhibit phenotypic changes that facilitate pathogenic disruption of the vascular barrier. METHODS: sEVs isolated from the plasma of a mouse model of type 2 diabetes and from diabetic human individuals were characterised for their ability to disrupt the endothelial cell (EC) barrier. The contents of sEVs and their effect on recipient ECs were assessed by proteomics and identified pathways were functionally interrogated with small molecule inhibitors. RESULTS: Using intravital imaging, we found that diabetic mice (Leprdb/db) displayed hyperpermeability of the cerebrovasculature. Enhanced vascular leakiness was recapitulated following i.v. injection of sEVs from diabetic mice into non-diabetic recipient mice. Characterisation of circulating sEV populations from the plasma of diabetic mice and humans demonstrated increased quantity and size of sEVs compared with those isolated from non-diabetic counterparts. Functional experiments revealed that sEVs from diabetic mice or humans induced the rapid and sustained disruption of the EC barrier through enhanced paracellular and transcellular leak but did not induce inflammation. Subsequent sEV proteome and recipient EC phospho-proteome analysis suggested that extracellular vesicles (sEVs) from diabetic mice and humans modulate the MAPK/MAPK kinase (MEK) and Rho-associated protein kinase (ROCK) pathways, cell-cell junctions and actin dynamics. This was confirmed experimentally. Treatment of sEVs with proteinase K or pre-treatment of recipient cells with MEK or ROCK inhibitors reduced the hyperpermeability-inducing effects of circulating sEVs in the diabetic state. CONCLUSIONS/INTERPRETATION: Diabetes is associated with marked increases in the concentration and size of circulating sEVs. The modulation of sEV-associated proteins under diabetic conditions can induce vascular leak through activation of the MEK/ROCK pathway. These data identify a new paradigm by which diabetes can induce hyperpermeability and dysfunction of the cerebrovasculature and may implicate sEVs in the pathogenesis of cognitive decline during type 2 diabetes.

Multi-omic Landscape of Extracellular Vesicles in Human Carotid Atherosclerotic Plaque Reveals Endothelial Communication Networks.

Background: Carotid atherosclerosis is a multifaceted disease orchestrated by a myriad of cell-cell communication that drives progression along a clinical continuum (asymptomatic to symptomatic). Extracellular vesicles (EVs) are lipid bilayer membrane-enclosed cell-derived nanoparticles that represent a new paradigm in cellular communication. Little is known about their biological cargo, cellular origin/destination, and functional roles in human atherosclerotic plaque. Methods: EVs were enriched via size exclusion chromatography from human carotid endarterectomy samples dissected into plaque and marginal zones (n= 29 patients, paired plaque and marginal zone; symptomatic n=16, asymptomatic n=13), with further density gradient ultracentrifugation for proteomic analysis. EV cargoes were assessed via whole transcriptome miRNA sequencing and mass spectrometry-based proteomics. EV multi-omics were integrated with publicly available bulk and single cell RNA-sequencing (scRNA-seq) datasets to predict EV cellular origin and ligand-receptor interactions and multi-modal biological network integration of EV-cargo was completed. EV functional impact was assessed with endothelial angiogenesis assays. Results: Human carotid plaques contained greater quantities of EVs than adjacent marginal zones. EV-miRNA and protein content was different in diseased plaque versus adjacent marginal zones, with differential functions in key atherogenic pathways. EV cellular origin analysis suggested that tissue EV signatures originated from endothelial cells (EC), smooth muscle cells (SMC), and immune cells. Furthermore, EV signatures from SMCs and immune cells were most enriched in the marginal and plaque zones, respectively. Integrated tissue vesiculomics and scRNA-seq indicated complex EV-vascular cell communication strategies that changed with disease progression and plaque vulnerability (i.e., symptomatic disease). Plaques from symptomatic patients, but not asymptomatic patients, were characterized by increased involvement of endothelial pathways and more complex ligand-receptor interactions, relative to their marginal zones. Plaque-EVs were predicted to mediate communication with ECs. Pathway enrichment analysis delineated a strong endothelial signature with potential roles in angiogenesis and neovascularization - well-known indices of plaque instability. This was corroborated functionally, wherein human carotid symptomatic plaque EVs induced sprouting angiogenesis in comparison to their matched marginal zones. Conclusion: Our findings indicate that EVs may drive dynamic changes in plaques through EV-vascular cell communication and effector functions that typify vulnerability to rupture, precipitating symptomatic disease. The discovery of endothelial-directed processes mediated by EVs creates new avenues for novel therapeutics in atherosclerosis.