Evaluation of EV Storage Buffer for Efficient Preservation of Engineered Extracellular Vesicles.

Extracellular vesicles (EVs), detectable in all bodily fluids, mediate intercellular communication by transporting molecules between cells. The capacity of EVs to transport molecules between distant organs has drawn interest for clinical applications in diagnostics and therapeutics. Although EVs hold potential for nucleic-acid-based and other molecular therapeutics, the lack of standardized technologies, including isolation, characterization, and storage, leaves many challenges for clinical applications, potentially resulting in misinterpretation of crucial findings. Previously, several groups demonstrated the problems of commonly used storage methods that distort EV integrity. This work aims to evaluate the process to optimize the storage conditions of EVs and then characterize them according to the experimental conditions and the models used previously. Our study reports a highly efficient EV storage condition, focusing on EV capacity to protect their molecular cargo from biological, chemical, and mechanical damage. Compared with commonly used EV storage conditions, our EV storage buffer leads to less size and particle number variation at both 4 °C and -80 °C, enhancing the ability to protect EVs while maintaining targeting functionality.

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection.

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enables the endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model. P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 107 bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitoneal P. gingivalis infection.

Significance of neutrophil microparticles in ischaemia-reperfusion: Pro-inflammatory effectors of endothelial senescence and vascular dysfunction.

Endothelial senescence is an emerging cause of vascular dysfunction. Because microparticles are effectors of endothelial inflammation and vascular injury after ischaemia-reperfusion, we examined leucocyte-derived microparticles of spleen origin as possible contributors. Microparticles were generated from primary rat splenocytes by either lipopolysaccharide or phorbol-myristate-acetate/calcium ionophore, under conditions mimicking innate and adaptive immune responses. Incubation of primary porcine coronary endothelial cells with either type of microparticles, but not with those from unstimulated splenocytes, leads to a similar threefold raise in senescence-associated ß-galactosidase activity within 48 hours, indicating accelerated senescence, to endothelial oxidative stress, and a fivefold and threefold increase in p21 and p16 senescence markers after 24 hours. After 12-hour incubation, the endothelial-dependent relaxation of coronary artery rings was reduced by 50%, at distinct optimal microparticle concentration. In vitro, microparticles were pro-thrombotic by up-regulating the local angiotensin system, by prompting tissue factor activity and a secondary generation of pro-coagulant endothelial microparticles. They initiated an early pro-inflammatory response by inducing phosphorylation of NF-κB, MAP kinases and Akt after 1 hour, and up-regulated VCAM-1 and ICAM-1 at 24 hours. Accordingly, VCAM-1 and COX-2 were also up-regulated in the coronary artery endothelium and eNOS down-regulated. Lipopolysaccharide specifically favoured the shedding of neutrophil- and monocyte-derived microparticles. A 80% immuno-depletion of neutrophil microparticles reduced endothelial senescence by 55%, indicating a key role. Altogether, data suggest that microparticles from activated splenocytes prompt early pro-inflammatory, pro-coagulant and pro-senescent responses in endothelial cells through redox-sensitive pathways. The control of neutrophil shedding could preserve the endothelium at site of ischaemia-reperfusion-driven inflammation and delay its dysfunction.

Porphyromonas gingivalis triggers the shedding of inflammatory endothelial microvesicles that act as autocrine effectors of endothelial dysfunction.

A link between periodontitis and atherothrombosis has been highlighted. The aim of this study was to determine the influence of Porphyromonas gingivalis on endothelial microvesicles (EMVPg) shedding and their contribution to endothelial inflammation. Endothelial cells (EC) were infected with P. gingivalis (MOI = 100) for 24 h. EMVPg were isolated and their concentration was evaluated by prothrombinase assay. EMVPg were significantly increased in comparison with EMVCtrl shedded by unstimulated cells. While EMVCtrl from untreated EC had no effect, whereas, the proportion of apoptotic EC was increased by 30 nM EMVPg and viability was decreased down to 25%, a value elicited by P. gingivalis alone. Moreover, high concentration of EMVPg (30 nM) induced a pro-inflammatory and pro-oxidative cell response including up-regulation of TNF-α, IL-6 and IL-8 as well as an altered expression of iNOS and eNOS at both mRNA and protein level. An increase of VCAM-1 and ICAM-1 mRNA expression (4.5 folds and 3 folds respectively (p < 0.05 vs untreated) was also observed after EMVPg (30 nM) stimulation whereas P. gingivalis infection was less effective, suggesting a specific triggering by EMVPg. Kinasome analysis demonstrated the specific effect induced by EMVPg on main pro-inflammatory pathways including JNK/AKT and STAT. EMVPg are effective pro-inflammatory effectors that may have detrimental effect on vascular homeostasis and should be considered as potential autocrine and paracrine effectors involved in the link between periodontitis and atherothrombosis.