Neutrophil-derived extracellular vesicles induce endothelial inflammation and damage through the transfer of miRNAs

Extracellular Vesicles
/References

The critical role of neutrophils in pathological inflammation, notably in various autoimmune disorders, is currently the focus of renewed interest. Here, we demonstrate for the first time that activation of neutrophils with various inflammatory stimuli induces the release of extracellular vesicles (EVs) that are internalized by endothelial cells (ECs), thus leading to the transfer of miR-223, miR-142-3p and miR-451 and subsequent endothelial damage. Indeed, while miR-223 has little effect on EC responses, we show that the induced expression of miR-142-3p and miR-451 in ECs results in profound cell damage, especially in inflammatory conditions, characterized by a dramatic increase in cell apoptosis, impaired angiogenic repair responses, and the induction of IL-6, IL-8, CXCL10 and CXCL11 expression. We show that the strong deleterious effect of miR-142-3p may be due in part to its ability to block the activation of ERK1/2 and eNOS-mediated signals in ECs. miR-142-3p also inhibits the expression of RAC1, ROCK2 and CLIC4, three genes that are critical for EC migration and angiogenic responses. Importantly, miR-223, miR-142-3p and miR-451 are markedly increased in kidney biopsies from patients with active ANCA-associated vasculitis, a severe autoimmune disease that is prototypical of a neutrophil-induced microvascular damage. Taken together, our results suggest that miR-142-3p and miR-451 released in EVs by activated neutrophils can target EC to trigger an inflammatory cascade and induce direct vascular damage, and that therapeutic strategies based on the inhibition of these miRNAs in ECs will have implications for neutrophil-mediated inflammatory diseases.

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Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

2023
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