Mesenchymal stem cell-derived exosomes as cell free nanotherapeutics and nanocarriers.

Many strategies for regenerating the damaged tissues or degenerating cells are employed in regenerative medicine. Stem cell technology is a modern strategy of the recent approaches, particularly the use of mesenchymal stem cells (MCSs). The ability of MSCs to differentiate as well as their characteristic behaviour as paracrine effector has established them as key elements in tissue repair (Shaer et al., 20141). Recently, extracellular vesicles (EVs) shed by MSCs have emerged as a promising cell free therapy (Citation}Rani, S., Ryan, A. E., Griffin, M. D., and Ritter, T., 20152). This comprehensive review encompasses MSCs-derived exosomes and their therapeutic potential as nanotherapeutics. We also discuss their potency as drug delivery nano-carriers in comparison with liposomes. A better knowledge of EVs behaviour in vivo and of their mechanism of action are key to determine parameters of an optimal formulation in pilot studies and to establish industrial processes.

Distinguishing Plasmin-Generating Microvesicles: Tiny Messengers Involved in Fibrinolysis and Proteolysis.

A number of stressors and inflammatory mediators (cytokines, proteases, oxidative stress mediators) released during inflammation or ischemia stimulate and activate cells in blood, the vessel wall or tissues. The most well-known functional and phenotypic responses of activated cells are (1) the immediate expression and/or release of stored or newly synthesized bioactive molecules, and (2) membrane blebbing followed by release of microvesicles. An ultimate response, namely the formation of extracellular traps by neutrophils (NETs), is outside the scope of this work. The main objective of this article is to provide an overview on the mechanism of plasminogen reception and activation at the surface of cell-derived microvesicles, new actors in fibrinolysis and proteolysis. The role of microvesicle-bound plasmin in pathological settings involving inflammation, atherosclerosis, angiogenesis, and tumour growth, remains to be investigated. Further studies are necessary to determine if profibrinolytic microvesicles are involved in a finely regulated equilibrium with pro-coagulant microvesicles, which ensures a balanced haemostasis, leading to the maintenance of vascular patency.

Angiotensin II-induced upregulation of SGLT1 and 2 contributes to human microparticle-stimulated endothelial senescence and dysfunction: protective effect of gliflozins.

BACKGROUND: Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduced cardiovascular risk in type 2 diabetes patients independently of glycemic control. Although angiotensin II (Ang II) and blood-derived microparticles are major mediators of cardiovascular disease, their impact on SGLT1 and 2 expression and function in endothelial cells (ECs) and isolated arteries remains unclear. METHODS: ECs were isolated from porcine coronary arteries, and arterial segments from rats. The protein expression level was assessed by Western blot analysis and immunofluorescence staining, mRNA levels by RT-PCR, oxidative stress using dihydroethidium, nitric oxide using DAF-FM diacetate, senescence by senescence-associated beta-galactosidase activity, and platelet aggregation by aggregometer. Microparticles were collected from blood of patients with coronary artery disease (CAD-MPs). RESULTS: Ang II up-regulated SGLT1 and 2 protein levels in ECs, and caused a sustained extracellular glucose- and Na+-dependent pro-oxidant response that was inhibited by the NADPH oxidase inhibitor VAS-2780, the AT1R antagonist losartan, sotagliflozin (Sota, SGLT1 and SGLT2 inhibitor), and empagliflozin (Empa, SGLT2 inhibitor). Ang II increased senescence-associated beta-galactosidase activity and markers, VCAM-1, MCP-1, tissue factor, ACE, and AT1R, and down-regulated eNOS and NO formation, which were inhibited by Sota and Empa. Increased SGLT1 and SGLT2 protein levels were observed in the rat aortic arch, and Ang II- and eNOS inhibitor-treated thoracic aorta segments, and were associated with enhanced levels of oxidative stress and prevented by VAS-2780, losartan, Sota and Empa. CAD-MPs promoted increased levels of SGLT1, SGLT2 and VCAM-1, and decreased eNOS and NO formation in ECs, which were inhibited by VAS-2780, losartan, Sota and Empa. CONCLUSIONS: Ang II up-regulates SGLT1 and 2 protein expression in ECs and arterial segments to promote sustained oxidative stress, senescence and dysfunction. Such a sequence contributes to CAD-MPs-induced endothelial dysfunction. Since AT1R/NADPH oxidase/SGLT1 and 2 pathways promote endothelial dysfunction, inhibition of SGLT1 and/or 2 appears as an attractive strategy to enhance the protective endothelial function.

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.

Angiotensin II-induced redox-sensitive SGLT1 and 2 expression promotes high glucose-induced endothelial cell senescence.

High glucose (HG)-induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co-transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG-induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence-associated beta-galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT-PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2-NBD-glucose. HG increased ECs senescence markers and oxidative stress, down-regulated eNOS expression and NO formation, and induced the expression of VCAM-1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX-4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H2 O2 in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG-induced ECs senescence and SGLT1 and 2 expression. Thus, HG-induced ECs ageing is driven by the local angiotensin system via the redox-sensitive up-regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity.

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.

DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma.

Activation of platelets and neutrophils in septic shock results in the formation of microvascular clots containing an intricate scaffold of fibrin with neutrophil extracellular traps (NETs) DNA. NETs contain multiple components that might impact endogenous fibrinolysis, resulting in failure to lyse clots in the microcirculation and residual systemic microthrombosis. We propose herein that the reservoir of human neutrophil elastase (HNE) on NETs may directly interfere with the fibrinolytic mechanism via a plasminogen proteolytic pathway. To investigate this mechanism, we constructed fibrin-NETs matrices by seeding and activating neutrophils onto a fibrin surface and monitored plasminogen activation or degradation. We demonstrate that the elastase activity of HNE-DNA complexes is protected from inhibition by plasma antiproteases and sustains its ability to degrade plasminogen. Using mass spectrometry proteomic analysis, we identified plasminogen fragments composed of kringle (K) domains (K1+2+3, k1+2+3+4) and the serine protease (SP) region (K5-SP). We further demonstrate that patients with septic shock with disseminated intravascular coagulation have circulating HNE-DNA complexes, HNE-derived plasminogen fragments, a low plasminogen concentration, and a reduced capacity to generate plasmin onto fibrin. In conclusion, we show that NETs bearing active HNE-DNA complexes reduce plasminogen into fragments, thus impairing fibrinolysis by decreasing the local plasminogen concentration, plasminogen binding to fibrin, and localized plasmin formation.-Barbosa da Cruz, D., Helms, J., Aquino, L. R., Stiel, L., Cougourdan, L., Broussard, C., Chafey, P., Riès-Kautt, M., Meziani, F., Toti, F., Gaussem, P., Anglés-Cano, E. DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma.

Oral Intake of EPA:DHA 6:1 by Middle-Aged Rats for One Week Improves Age-Related Endothelial Dysfunction in Both the Femoral Artery and Vein: Role of Cyclooxygenases.

In humans, aging is associated with endothelial dysfunction and an increased risk of venous thromboembolism. Although intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) at a ratio of 6:1 by old rats improved the endothelial dysfunction in arteries, the impact on veins remains unclear. Eight-month-old male Wistar rats were either untreated or orally administered corn oil, EPA:DHA 1:1, or EPA:DHA 6:1 (500 mg/kg/d) for seven days. Vascular reactivity was studied by myography. In middle-aged femoral artery rings, acetylcholine caused a partial relaxation at low concentrations and a contractile response at high concentrations, whereas in the old femoral vein only a partial relaxation was observed. The EPA:DHA 6:1 treatment blunted the contractile response to acetylcholine in the middle-aged femoral artery and both EPA:DHA 6:1 and 1:1 increased the relaxation to acetylcholine in the old femoral vein. No such effects were observed with corn oil. Both the non-selective cyclooxygenase inhibitor indomethacin and the COX-1 inhibitor SC-560 increased the relaxation to acetylcholine in the middle-aged femoral artery whereas the COX-2 inhibitor NS-398 increased that in the middle-aged femoral vein. In conclusion, our results indicate that aging is associated with an endothelial dysfunction in the femoral artery and vein, which can be improved by EPA:DHA 6:1 treatment-most likely via a cyclooxygenase-dependent mechanism.

Endothelial Microparticles From Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity: Role of the Ang II/AT1 Receptor/NADPH Oxidase-Mediated Activation of MAPKs and PI3-Kinase Pathways.

BACKGROUND: Microparticles (MPs) have emerged as a surrogate marker of endothelial dysfunction and cardiovascular risk. This study examined the potential of MPs from senescent endothelial cells (ECs) or from patients with acute coronary syndrome (ACS) to promote premature EC aging and thrombogenicity. METHODS: Primary porcine coronary ECs were isolated from the left circumflex coronary artery. MPs were prepared from ECs and venous blood from patients with ACS (n=30) and from healthy volunteers (n=4) by sequential centrifugation. The level of endothelial senescence was assessed as senescence-associated ß-galactosidase activity using flow cytometry, oxidative stress using the redox-sensitive probe dihydroethidium, tissue factor activity using an enzymatic Tenase assay, the level of target protein expression by Western blot analysis, platelet aggregation using an aggregometer, and shear stress using a cone-and-plate viscometer. RESULTS: Senescence, as assessed by senescence-associated ß-galactosidase activity, was induced by the passaging of porcine coronary artery ECs from passage P1 to P4, and was associated with a progressive shedding of procoagulant MPs. Exposure of P1 ECs to MPs shed from senescent P3 cells or circulating MPs from ACS patients induced increased senescence-associated ß-galactosidase activity, oxidative stress, early phosphorylation of mitogen-activated protein kinases and Akt, and upregulation of p53, p21, and p16. Ex vivo, the prosenescent effect of circulating MPs from ACS patients was evidenced only under conditions of low shear stress. Depletion of endothelial-derived MPs from ACS patients reduced the induction of senescence. Prosenescent MPs promoted EC thrombogenicity through tissue factor upregulation, shedding of procoagulant MPs, endothelial nitric oxide synthase downregulation, and reduced nitric oxide-mediated inhibition of platelet aggregation. These MPs exhibited angiotensin-converting enzyme activity and upregulated AT1 receptors and angiotensin-converting enzyme in P1 ECs. Losartan, an AT1 receptor antagonist, and inhibitors of either mitogen-activated protein kinases or phosphoinositide 3-kinase prevented the MP-induced endothelial senescence. CONCLUSIONS: These findings indicate that endothelial-derived MPs from ACS patients induce premature endothelial senescence under atheroprone low shear stress and thrombogenicity through angiotensin II-induced redox-sensitive activation of mitogen-activated protein kinases and phosphoinositide 3-kinase/Akt. They further suggest that targeting endothelial-derived MP shedding and their bioactivity may be a promising therapeutic strategy to limit the development of an endothelial dysfunction post-ACS.

Endothelial microparticles released by activated protein C protect beta cells through EPCR/PAR1 and annexin A1/FPR2 pathways in islets.

Islet transplantation is associated with early ischaemia/reperfusion, localized coagulation and redox-sensitive endothelial dysfunction. In animal models, islet cytoprotection by activated protein C (aPC) restores islet vascularization and protects graft function, suggesting that aPC triggers various lineages. aPC also prompts the release of endothelial MP that bear EPCR, its specific receptor. Microparticles (MP) are plasma membrane procoagulant vesicles, surrogate markers of stress and cellular effectors. We measured the cytoprotective effects of aPC on endothelial and insulin-secreting Rin-m5f ß-cells and its role in autocrine and paracrine MP-mediated cell crosstalk under conditions of oxidative stress. MP from aPC-treated primary endothelial (EC) or ß-cells were applied to H2 O2 -treated Rin-m5f. aPC activity was measured by enzymatic assay and ROS species by dihydroethidium. The capture of PKH26-stained MP and the expression of EPCR were probed by fluorescence microscopy and apoptosis by flow cytometry. aPC treatment enhanced both annexin A1 (ANXA1) and PAR-1 expression in EC and to a lesser extent in ß-cells. MP from aPC-treated EC (eMaPC ) exhibited high EPCR and annexin A1 content, protected ß-cells, restored insulin secretion and were captured by 80% of ß cells in a phosphatidylserine and ANXA1-dependent mechanism. eMP activated EPCR/PAR-1 and ANXA1/FPR2-dependent pathways and up-regulated the expression of EPCR, and of FPR2/ALX, the ANXA1 receptor. Cytoprotection was confirmed in H2 O2 -treated rat islets with increased viability (62% versus 48% H2 O2 ), reduced apoptosis and preserved insulin secretion in response to glucose elevation (16 versus 5 ng/ml insulin per 10 islets). MP may prove a promising therapeutic tool in the protection of transplanted islets.

ß cell membrane remodelling and procoagulant events occur in inflammation-driven insulin impairment: a GLP-1 receptor dependent and independent control.

Inflammation and hyperglycaemia are associated with a prothrombotic state. Cell-derived microparticles (MPs) are the conveyors of active procoagulant tissue factor (TF) and circulate at high concentration in diabetic patients. Liraglutide, a glucagon-like peptide (GLP)-1 analogue, is known to promote insulin secretion and ß-cell preservation. In this in vitro study, we examined the link between insulin impairment, procoagulant activity and plasma membrane remodelling, under inflammatory conditions. Rin-m5f ß-cell function, TF activity mediated by MPs and their modulation by 1 µM liraglutide were examined in a cell cross-talk model. Methyl-ß-cyclodextrine (MCD), a cholesterol depletor, was used to evaluate the involvement of raft on TF activity, MP shedding and insulin secretion as well as Soluble N-éthylmaleimide-sensitive-factor Attachment protein Receptor (SNARE)-dependent exocytosis. Cytokines induced a two-fold increase in TF activity at MP surface that was counteracted by liraglutide. Microparticles prompted TF activity on the target cells and a two-fold decrease in insulin secretion via protein kinase A (PKA) and p38 signalling, that was also abolished by liraglutide. Large lipid raft clusters were formed in response to cytokines and liraglutide or MCD-treated cells showed similar patterns. Cells pre-treated by saturating concentration of the GLP-1r antagonist exendin (9-39), showed a partial abolishment of the liraglutide-driven insulin secretion and liraglutide-decreased TF activity. Measurement of caspase 3 cleavage and MP shedding confirmed the contribution of GLP-1r-dependent and -independent pathways. Our results confirm an integrative ß-cell response to GLP-1 that targets receptor-mediated signalling and membrane remodelling pointing at the coupling of insulin secretion and inflammation-driven procoagulant events.

Neutrophil Fluorescence: A New Indicator of Cell Activation During Septic Shock-Induced Disseminated Intravascular Coagulation.

OBJECTIVE: To investigate the contribution of neutrophil activation as innate immune cells during septic shock-induced disseminated intravascular coagulation. DESIGN: Prospective study. SETTING: One University Hospital ICU. PARTICIPANTS: Hundred patients with septic shock. Thirty-five patients had disseminated intravascular coagulation according to Japanese Association for Acute Medicine 2006 score. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Neutrophil chromatin decondensation was assessed by measuring neutrophil fluorescence (NEUT-side-fluorescence light) labeled by a fluorochrome-based polymethine reagent using a routine automated flow cytometer Sysmex XN20 (Sysmex, Kobe, Japan) and neutrophil-derived CD66b microparticles by prothrombinase assay. Measurements in disseminated intravascular coagulation and no disseminated intravascular coagulation patients showed that a mean value of NEUT-side-fluorescence light above 57.3 arbitrary units had a sensitivity of 90.91% and a specificity of 80.60% for disseminated intravascular coagulation diagnosis. NEUT-side-fluorescence light was correlated to the CD66b microparticles/neutrophil count, a surrogate of neutrophil activation associated with septic shock-induced disseminated intravascular coagulation. CONCLUSION: NEUT-side-fluorescence light, routinely available, could prove an accurate biomarker of neutrophil activation.

Early Detection of Disseminated Intravascular Coagulation During Septic Shock: A Multicenter Prospective Study.

OBJECTIVES: Inadequate stratification of septic shock patients may result in inappropriate treatment allocation in randomized clinical trials, especially regarding anticoagulant. We previously reported that endothelial-derived microparticles are relevant biomarkers of sepsis-induced disseminated intravascular coagulation. In this validation cohort, we assess microparticles as surrogates of cell activation to improve early disseminated intravascular coagulation diagnosis and patient stratification. DESIGN: Prospective observational study in septic shock patients. SETTINGS: Four medical ICUs in university hospitals. PATIENTS AND METHODS: Two hundred sixty-five patients with septic shock from four ICUs were consecutively enrolled. Disseminated intravascular coagulation was diagnosed according to Japanese Association for Acute Medicine 2006 score. Endothelial- and leukocyte-derived circulating procoagulant microparticles were isolated and quantified by prothrombinase assay at admission, day 3, and day 7. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Two hundred fifty-nine patients were analyzed. Sixty-one had disseminated intravascular coagulation at admission, and 32 developed disseminated intravascular coagulation during the first 24 hours after admission. Multiple logistic regression model confirmed that endothelial cell-derived microparticles were associated with disseminated intravascular coagulation: CD105-microparticles (odds ratio, 2.13) and CD31-microparticles (odds ratio, 0.65) (p < 0.05). Furthermore, CD11a-microparticles to leukocyte ratio evidenced leukocyte activation (odds ratio, 1.59; p < 0.05). Prediction of disseminated intravascular coagulation was also analyzed after exclusion of patients with disseminated intravascular coagulation at admission. A new multiple logistic regression analysis demonstrated the association of CD105-microparticles (> 0.60 nM eq. PhtdSer; odds ratio, 1.67; p < 0.01), platelets count (≤ 127 g/L; odds ratio, 0.99; p < 0.01), and prothrombin time (≤ 58%; odds ratio, 0.98; p < 0.05) with disseminated intravascular coagulation. A combining score at admission is predictive of the absence of disseminated intravascular coagulation (area under the curve, 72.9%; specificity, 71.2%; sensitivity, 71.0%, with a negative predictive value of 93.1% and a positive predictive value of 31.0%). CONCLUSIONS: Procoagulant microparticles from endothelial cells and leukocytes reflect a vascular injury during sepsis-induced disseminated intravascular coagulation that precedes obvious activation of coagulation. A combination of prothrombin time, endothelium-derived CD105-microparticles, and platelet count at admission could predict the absence of disseminated intravascular coagulation and allow a better stratification in future randomized clinical trials.

Differential influence of tacrolimus and sirolimus on mitochondrial-dependent signaling for apoptosis in pancreatic cells.

To examine and compare the mitochondria-related cellular mechanisms by which tacrolimus (TAC) or sirolimus (SIR) immunosuppressive drugs alter the pancreatic exocrine and endocrine ß-cell fate. Human exocrine PANC-1 and rat endocrine insulin-secreting RIN-m5F cells and isolated rat islets were submitted to 1-100 nM TAC or SIR. In cultures, insulin secretion was measured as endocrine cell function marker. Apoptosis was quantified by annexin 5 and propidium iodide staining. Cleaved caspase-3, Bax apoptosis indicators, and p53, p21 cell cycle regulators were detected by Western blot. Cell cycle and mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry and SA-beta-galactosidase (SA-ß-gal) activity by fluorescence microscopy. Only TAC reduced insulin secretion by RIN-m5F after 24 h. TAC and SIR promoted moderate apoptosis in both PANC-1 and RIN-m5F after 24 h. Apoptosis was associated with up-regulated Bax (threefold) and cleaved caspase-3 (fivefold) but only in PANC-1, while p53 and p21 were up-regulated (twofold) in both cell lines. ΔΨm was impaired only in PANC-1 by TAC and SIR. Only SIR prompted cell cycle arrest in both cell lines. The induction of a premature senescence-like phenotype was confirmed in isolated islets by SA-ß-gal activity. TAC and SIR are early inducers of pancreatic cell dysfunction and apoptosis but differentially alter endocrine and exocrine cells via mitochondrial-driven pathways. In rat islets, TAC and SIR prompt a senescence-like phenotype.

Do atrial differences in endothelial damage, leukocyte and platelet activation, or tissue factor activity contribute to chamber-specific thrombogenic status in patients with atrial fibrillation?

BACKGROUND: Thrombi form mainly in the left rather than the right atria of patients with atrial fibrillation (AF), the reason of this predilection being unknown. OBJECTIVE: The purpose of this study was to investigate whether atrial-specific differences in endothelial damage, leukocyte activation, platelet stimulation, and tissue factor activity occur in patients with AF. METHODS: Twenty-two patients (16 men, 6 women; age 56 ± 8 years; 16 paroxysmal AF, 6 persistent AF) with AF undergoing pulmonary vein isolation were investigated. Blood samples from the left and the right atrium were obtained at the start of the procedure. Microparticles (MPs) released by apoptotic/stimulated cells were measured by capture assays. Their procoagulant abilities were quantified by functional prothrombinase and tissue factor assays and their cellular origin were determined (endothelium, platelet, leukocyte). Platelet reactivity was evaluated by whole blood flow cytometry for expression of platelet P-selectin (CD62P), active glycoprotein IIb/IIIa receptor (PAC-1). Platelet aggregation was evaluated using ADP, TRAP and collagen-induced whole blood aggregometry. RESULTS: There were no atrial-specific differences in the levels of total procoagulant MPs, leukocyte-derived-MPs or platelet-derived MPs. Conversely, endothelial-derived MPs and tissue factor activity and collagen-induced platelet aggregation were slightly elevated in the right atrium (P < 0.05). CONCLUSIONS: Our data show no evidence for increased thrombogenic status in the left atrium that would account for its greater propensity for thrombus formation in patients with AF.

Liraglutide protects Rin-m5f ß cells by reducing procoagulant tissue factor activity and apoptosis prompted by microparticles under conditions mimicking Instant Blood-Mediated Inflammatory Reaction.

Instant Blood-Mediated Inflammatory Reaction (IBMIR) occurs at the vicinity of transplanted islets immediately after intraportal infusion and is characterized by cytokine secretion, tissue factor (TF) expression, and ß cell loss. Microparticles (MPs) are cellular effectors shed from the plasma membrane of apoptotic cells. Modulation of the properties of ß cell-derived MPs by liraglutide was assessed in a cellular model designed to mimic IBMIR oxidative and inflammatory conditions. Rin-m5f rat ß cells were stimulated by H2 O2 or a combination of IL-1ß and TNF-α. Cell-derived MPs were applied to naive Rin-m5f for 24 h. Apoptosis, MP release, TF activity, P-IκB expression, and MP-mediated apoptosis were measured in target cells. Direct protection by liraglutide was shown by a significant decrease in the oxidative stress-induced apoptosis (18.7% vs. 7.6%, P < 0.0001 at 1 µm liraglutide) and cellular TF activity (-40% at 100 nm liraglutide). Indirect cytoprotection led to 20% reduction in MP generation, thereby lowering MP-mediated apoptosis (6.3% vs. 3.7%, P = 0.022) and NF-κB activation (-50%) in target cells. New cytoprotective effects of liraglutide were evidenced, limiting the expression of TF activity by ß cells and the generation of noxious MPs. Altogether, these data suggest that liraglutide could target pro-apoptotic and pro-inflammatory MPs in transplanted islets.

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.

Cellular mechanisms underlying the formation of circulating microparticles.

Microparticles (MPs) derived from platelets, monocytes, endothelial cells, red blood cells, and granulocytes may be detected in low concentrations in normal plasma and at increased levels in atherothrombotic cardiovascular diseases. The elucidation of the cellular mechanisms underlying the generation of circulating MPs is crucial for improving our understanding of their pathophysiological role in health and disease. The flopping of phosphatidylserine (PS) to the outer leaflet of the plasma membrane is the key event that will ultimately lead to the shedding of procoagulant MPs from activated or apoptotic cells. Research over the last few years has revealed important roles for calcium-, mitochondrial-, and caspase-dependent mechanisms leading to PS exposure. The study of Scott cells has unraveled different molecular mechanisms that may contribute to fine-tuning of PS exposure and MP release in response to a variety of specific stimuli. The pharmacological modulation of MP release may have a substantial therapeutic impact in the management of atherothrombotic vascular disorders. Because PS exposure is a key feature in pathological processes different from hemostasis and thrombosis, the most important obstacle in the field of MP-modulating drugs seems to be carefully targeting MP release to relevant cell types at an optimal level, so as to achieve a beneficial action and limit possible adverse effects.

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.