Platelet-derived extracellular vesicles promote endothelial dysfunction in sepsis by enhancing neutrophil extracellular traps

Abstract

Background The role of platelet-derived extracellular vesicles (PEVs) in the development of sepsis was investigated in this study. Methods After collection of blood samples from sepsis patients and normal volunteers, the extracellular vesicles (EVs) were separated, followed by the isolation of PEVs from the blood of rats. Next, a sepsis rat model was constructed by cecal ligation and puncture (CLP), and rats received tail vein injection of PEVs to explore the role of PEVs in sepsis. Subsequently, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were adopted to determine the diameter of EVs and observe the morphology of PEVs, respectively; flow cytometry to detect the percentage of CD41-and CD61-positive EVs in isolated EVs; and ELISA to assess neutrophil extracellular trap (NET) formation, endothelial function injury-related markers in clinical samples or rat blood and serum inflammatory factor level. Results Compared with normal volunteers, the percentage of CD41- and CD61-positive EVs and the number of EVs were significantly elevated in sepsis patients. Moreover, sepsis patients also presented notably increased histone H3, myeloperoxidase (MPO), angiopoietin-2 and endocan levels in the blood, and such increase was positively correlated with the number of EVs. Also, animal experiments demonstrated that PEVs significantly promoted NET formation, mainly manifested as up-regulation of histone H3, high mobility group protein B1 (HMGB1), and MPO; promoted endothelial dysfunction (up-regulation of angiopoietin-2, endocan, and syndecan-1); and stimulated inflammatory response (up-regulation of interleukin (IL) -1β, IL-6, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein (MCP) -1) in the blood of sepsis rats. Conclusion PEVs aggravate endothelial function injury and inflammatory response in sepsis by promoting NET formation.

The Role of Neutrophil Extracellular Traps in the Outcome of Malignant Epitheliomas: Significance of CA215 Involvement

“…These assays are functional in nature, involving the linkage of the tested molecule to extracellular DNA, which degrades a substrate and alters the final measured absorbance of the main biological product. The enzymatic activity is primarily measured using spectrophotometry [91][92][93][94].…”

“…Flow Cytometry: This method uses specific markers to evaluate NET formation at the single-cell level. The most commonly used markers for distinguishing between NETforming and non-NET-forming cells are citrullinated histones (H3, H4), neutrophil elastase, and myeloperoxidase, which can be identified using designated protocols [79,85,94,95]. Once NETs are formed, fluorescent markers are applied for staining.…”

“…A combination of DNA-binding dyes (such as Sytox Green or DAPI) and antibodies against specific NET components is commonly utilized. The flow cytometer allows for the differentiation between intact neutrophils, apoptotic cells, and NETs based on their size and fluorescence profile [79,85,94,95].…”

Communications between Neutrophil–Endothelial Interaction in Immune Defense against Bacterial Infection

“…NETs promote interleukin 8 (IL-8) release for the recruitment of other immune cells, such as M1-like macrophage [ 49 ]. However, research indicates that NETs amplify and propagate endothelial dysfunction [ 29 , 30 , 50 ]. On the other hand, prolonged infection and the death of neutrophils can also cause endothelial dysfunction and lead to inflammation.…”

Beyond Basic Characterization and Omics: Immunomodulatory Roles of Platelet-Derived Extracellular Vesicles Unveiled by Functional Testing

“…EVs containing the specific platelet and megakaryocyte integrin subunit CD41 (combined with CD61) constitute ~30 - 52% of all circulating EVs depending on the used analytical method 14,15 . Although some of these EVs may be of megakaryocyte-origin 16 , elevated levels of circulating PEVs have been associated with pathological conditions with immune-mediated inflammatory mechanisms, such as sepsis 17,18 , autoimmune conditions (lupus, rheumatoid arthritis) 19 , cardiovascular diseases 20 and cancer 21 . Regarding physiological functions, roles of PEVs have been assigned to coagulation and into recruitment and coordinating “homing” of innate immune cells to the site of vascular injury/infection, whereas their role in coordinating adaptive immune cell functions is less explored 22 .…”