Grafted dinuclear zinc complexes for selective recognition of phosphatidylserine: Application to the capture of extracellular membrane microvesicles.

Microvesicles (MVs) are key markers in human body fluids that reflect cellular activation related to diseases as thrombosis. These MVs display phosphatidylserine at the outer leaflet of their plasma membrane as specific recognition moieties. The work reported in this manuscript focuses on the development of an original method where MVs are captured by bimetallic zinc complexes. A set of ligands have been synthetized based on a phenol spacer bearing in para position an amine group appended to a short or a longer alkyl chain (for grafting on surface) and bis(dipicolylamine) arms in ortho position (for zinc coordination). The corresponding dibridged zinc phenoxido and hydroxido complexes have been prepared in acetronitrile in presence of triethylamine and characterized by several spectroscopic techniques. The pH-driven interconversion studies for both complexes in H2O:DMSO (70:30) evidence that at physiologic pH the main species are mono-bridged by the phenoxido spacer. An X-Ray structure obtained from complex 2 (based on the ligand with the amine group on the short chain) in aqueous medium confirms the presence of a mono-bridged complex. Then, the complexes have been used for interaction studies with short-chain phospholipids. Both have established the selective recognition of the anionic phosphatidylserine model versus zwitterionic phospholipids (in solution by 31P NMR and after immobilization on solid support by surface plasmon resonance (SPR)). Moreover, both complexes have also demonstrated their ability to capture MVs isolated from human plasma. These complexes are thus promising candidates for MVs probing by a new approach based on coordination chemistry.

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.

Epithelial-mesenchymal transition and membrane microparticles: Potential implications for bronchiolitis obliterans syndrome after lung transplantation.

Long term survival post lung transplantation (LTx) is limited by the occurrence of bronchiolitis obliterans syndrome (BOS). One mechanism involved is the epithelial-mesenchymal transition (EMT). Membrane microparticles (MPs) are known to be involved in some respiratory diseases and in other organs allograft rejection episodes. We hypothesized that leukocyte-derived MPs likely contribute to EMT. To emphasize this physiological concept, our objectives were to: (1) confirm the presence of EMT on explanted lungs from patients who underwent a second LTx for BOS; 2) characterize circulating MPs in transplanted patients, with or without BOS; (3) evaluate in vitro the effect of monocyte-derived MPs in EMT of human bronchial epithelial cells. Our IHC analysis on explanted graft lungs revealed significant pathological signs of EMT with an inhomogeneous destruction of the bronchial epithelium, with decreased expression of the epithelial protein E-cadherin and increased expression of the mesenchymal protein Vimentin. The immunophenotyping of MPs demonstrated that the concentration of MPs carrying E-cadherin was lower in patients affected by BOS (p = .007). In vitro, monocyte-derived MPs produced with LPS were associated with decreased E-cadherin expression (p < .05) along with significant morphological and functional cell modifications. MPs may play a role in EMT onset in bronchial epithelium following LTx.

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.

Assessment of plasma microvesicles to monitor pancreatic islet graft dysfunction: Beta cell- and leukocyte-derived microvesicles as specific features in a pilot longitudinal study.

Markers of early pancreatic islet graft dysfunction and its causes are lacking. We monitored 19 type 1 diabetes islet-transplanted patients for up to 36 months following last islet injection. Patients were categorized as Partial (PS) or complete (S) Success, or Graft Failure (F), using the ß-score as an indicator of graft function. F was the subset reference of maximum worsened graft outcome. To identify the immune, pancreatic, and liver contribution to the graft dysfunction, the cell origin and concentration of circulating microvesicles (MVs) were assessed, including MVs from insulin-secreting ß-cells typified by polysialic acid of neural cell adhesion molecule (PSA-NCAM), and data were compared with values of the ß-score. Similar ranges of PSA-NCAM+ -MVs were found in healthy volunteers and S patients, indicating minimal cell damage. In PS, a 2-fold elevation in PSA-NCAM+ -MVs preceded each ß-score drop along with a concomitant rise in insulin needs, suggesting ß-cell damage or altered function. Significant elevation of liver asialoglycoprotein receptor (ASGPR)+ -MVs, endothelial CD105+ -MVs, neutrophil CD66b+ -MVs, monocyte CD 14+ -MVs, and T4 lymphocyte CD4+ -MVs occurred before each ß-score drop, CD8+ -MVs increased only in F, and B lymphocyte CD19+ -MVs remained undetectable. In conclusion, PSA-NCAM+ -MVs are noninvasive early markers of transplant dysfunction, while ASGPR+ -MVs signal host tissue remodeling. Leukocyte MVs could identify the cause of graft dysfunction.