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The threats to chemical warfare-associated agents (CWA), including nitrogen mustard, are increasing, and no direct antidote is currently available to mitigate the deleterious cutaneous and systemic responses to prevent mortality. Though most of these agents act as alkylating agents, a significant knowledge gap exists in the molecular mechanisms of how these vesicants cause toxic effects. Studies, including ours, have shown that exposure to reactive oxygen species (ROS)-generating stimuli, including alkylating chemotherapeutic agents, and thermal burn injuries with ethanol produce the potent family of lipid mediators, Platelet-activating factor (PAF) agonists that induce local inflammation, and multi-system organ dysfunction (MOD). Notably, nano-sized microvesicle particles (MVPs), released from cells in response to stimuli, carry PAF-agonists and act as potent signaling agents to induce the local (cutaneous) and systemic responses. The current review highlights mechanistic insights and applicable approaches to mitigate CWA-induced local and systemic toxic responses with implications in cellular senescence and aging., Competing Interests: Declaration of competing interest None of the authors have a competing interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Traditional chemotherapy often encounters failure attributed to drug resistance mediated by tumor-repopulating cells (TRCs) and chemotherapy-triggered immune suppression. The effective inhibition of TRCs and the mitigation of drug-induced immune suppression are pivotal for the successful chemotherapy. Here, TRC-derived microparticles (3D-MPs), characterized by excellent tumor-targeting and high TRC uptake properties, are utilized to deliver metformin and the chemotherapeutic drug doxorubicin ((DOX+Met)@3D-MPs). (DOX+Met)@3D-MPs efficiently enhance tumor accumulation and are highly internalized in tumor cells and TRCs. Additionally, (DOX+Met)@3D-MPs significantly decrease the chemotherapy-triggered upregulation in P-glycoprotein expression to enhance intracellular doxorubicin retention, resulting in increased chemotherapy sensitivity and immunogenic cell death in tumor cells and TRCs for improved antitumor immunity. Importantly, (DOX+Met)@3D-MPs also remarkably reduce chemotherapy-induced PD-L1 expression, efficiently alleviating immune suppression facilitated by the PD-L1/PD-1 axis to further enhance immunological response against malignancy. These results underscore the (DOX+Met)@3D-MPs' potential as a viable platform for augmenting the efficacy of antitumor therapies.

Loss of plasma gelsolin (pGSN), a protein that lyses actin filaments, is implicated in the pathology of inflammatory and neurodegenerative diseases. We hypothesized that because pGSN is depleted in a murine model of decompression sickness (DCS), supplementation by administration of human recombinant (rhu-) pGSN would ameliorate inflammatory events. We observed that pGSN levels were persistently decreased in mice for at least 12 days postexposure to 790 kPa of air for 2 h. This decline was associated with elevated levels of inflammatory microparticles (MPs) in the blood and cervical lymph nodes, which previously were shown to cause neuroinflammation. In addition, these mice exhibited reduced expression of synaptic proteins, impaired neurogenesis and impaired cognitive and motor functions. Rhu-pGSN ameliorated the inflammatory changes and resulted in restored synaptic protein expression, neurogenesis, and neurological function. These findings demonstrate that neuronal dysfunction in our murine model of DCS is mediated by MPs and that rhu-pGSN can ameliorate injury even when administered in a delayed fashion. NEW & NOTEWORTHY A decrease in plasma gelsolin levels and the release of inflammatory microparticles (MPs) occur in response to high pressure followed by decompression, with expression of filamentous (F)-actin leading to persistent neuroinflammation and functional deficits lasting at least 12 days. The infusion of recombinant human plasma gelsolin lyses these MPs in decompressed mice, thereby alleviating particle-associated neuronal dysfunction. Rhu-gelsolin infusion may be beneficial as a prophylactic or treatment for decompression sickness.

Introduction: Large-volume packed red blood cell (pRBC) transfusion is associated with lung injury and worsened outcomes. Amitriptyline reduces lung injury and inflammation in a murine sepsis model. We hypothesized that red cell microparticles (MP) activate endothelial cells, leading to lung injury and that treatment with amitriptyline would blunt the inflammatory response MPs through inhibition of acid sphingomyelinase (ASM)., Methods: Murine pRBCs were obtained from C57Bl/6 mice and stored in AS3 for 14 d. The MPs were isolated from pRBCs by serial centrifugation. Mouse lung endothelial cells (MLECs) were pretreated with amitriptyline (0, 2.5, 25, 27 μM, n = 5) for 30 min prior to MP treatment. Chemokine secretion and adhesion molecule shedding was assessed. ASM activity was measured from cell lysates., Results: MPs increased the secretion of chemokines and shedding of adhesion molecules in MLECs at both four and 24 h. Amitriptyline treatment of MLECs decreased ASM activity in the setting of MPs. Amitriptyline pretreatment decreased the secretion of chemokines and shedding of adhesion molecules in response to MPs at 4 h but did not decrease adhesion molecule shedding at 24 h CONCLUSIONS: Endothelial cell treatment with MPs induces secretion of chemokines responsible for chemotaxis (keratinocyte chemoattractant, regulated upon activation normal T cell expressed and presumably secreted, and G-granulocyte colony-stimulating factor) as well as many downstream proinflammatory effects (interleukin-6). Additionally, MPs induce adhesion molecule shedding (vascular cell adhesion molecule-1, intracellular adhesion molecule-1, P-selectin, and E-selectin), which has been shown to be associated with endothelial cell activation. Amitriptyline pretreatment decreases MLEC inflammatory response and ASM activity is decreased. These data suggest that ASM inhibition in MLECs is a potential strategy to blunt the inflammatory response to the red blood cell storage lesion., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Endothelial dysfunction exacerbates hypertension and other vascular complications in diabetes mellitus (DM). Circulating microparticles (MPs) and extracellular vesicles released in patients with DM have emerged as novel regulators of endothelial dysfunction. The obstruction of mineralocorticoid receptors (MRs) is a potential therapeutic approach to reduce cardiovascular complications. Their impact on the obstruction of MRs on circulating MPs and endothelial dysfunction in DM remains unclear. DM was induced in mice through a single intravenous dose of streptozotocin (STZ; 200 mg/kg). Esaxerenone (ESAX; 3 mg/kg/day), a MR blocker was administered via diet for 8 weeks. In this study, the aortas of the DM group showed the endothelial dysfunction and the administration of ESAX ameliorated the endothelial-dependent responses. Moreover, ESAX influences the impaired endothelial-dependent responses of DM-derived MPs. Interestingly, MP levels increased in DM whereas decreased after ESAX administration. In the aorta, the DM-derived MPs increased the expression of intercellular adhesion molecule-1 (ICAM-1). ESAX inhibited the adhesion of DM-derived MPs. Moreover, the ICAM-1 inhibitor A205804 shows similar effects as ESAX. These results indicate that the release and adhesion properties of MPs can be partially obstructed by ESAX via the ICAM-1 signaling pathway, which clarifies the other functions beyond the anti-hypertensive effects of ESAX., (© 2024. The Author(s).)

Clodronate (Clod), a first-generation bisphosphonate, acts as a natural analgesic inhibiting vesicular storage of the nociception mediator ATP by vesicular nucleotide transporter (VNUT). Epidermal keratinocytes participate in cutaneous nociception, accumulating ATP within vesicles, which are released following different stimulations. Under stress conditions, keratinocytes produce microvesicles (MVs) by shedding from plasma membrane evagination. MV secretion has been identified as a novel and universal mode of intercellular communication between cells. The aim of this project was to evaluate if two nociceptive stimuli, Capsaicin and Potassium Hydroxide (KOH), could stimulate MV shedding from human keratinocytes, if these MVs could contain ATP, and if Clod could inhibit this phenomenon. In our cellular model, the HaCaT keratinocyte monolayer, both Capsaicin and KOH stimulated MV release after 3 h incubation, and the released MVs contained ATP. Moreover, Clod (5 µM) was able to reduce Caps-induced MV release and abolish the one KOH induced, while the Dansylcadaverine, an endocytosis inhibitor of Clod uptake, partially failed to block the bisphosphonate activity. Based on these new data and given the role of the activation of ATP release by keratinocytes as a vehicle for nociception and pain, the "old" bisphosphonate Clodronate could provide the pharmacological basis to develop new local analgesic drugs.

Extracellular vesicles are essential for intercellular communication and are involved in tumor progression. Inhibiting the direct release of extracellular vesicles seems to be an effective strategy in inhibiting tumor progression, but lacks of investigation. Here, we report a natural flavonoid compound, apigenin, could significantly inhibit the growth of hepatocellular carcinoma by preventing microvesicle secretion. Mechanistically, apigenin primarily targets the guanine nucleotide exchange factor ARHGEF1, inhibiting the activity of small G protein Cdc42, which is essential in regulating the release of microvesicles from tumor cells. In turn, this inhibits tumor angiogenesis related to VEGF 90K transported on microvesicles, ultimately impeding tumor progression. Collectively, these findings highlight the therapeutic potential of apigenin and shed light on its anticancer mechanisms through inhibiting microvesicle biogenesis, providing a solid foundation for the refinement and practical application of apigenin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Breast cancer, a pervasive malignancy affecting women, demands a diverse treatment approach including chemotherapy, radiotherapy, and surgical interventions. However, the effectiveness of doxorubicin (DOX), a cornerstone in breast cancer therapy, is limited when used as a monotherapy, and concerns about cardiotoxicity persist. Ginsenoside Rg3, a classic compound of traditional Chinese medicine found in Panax ginseng C. A. Mey., possesses diverse pharmacological properties, including cardiovascular protection, immune modulation, and anticancer effects. Ginsenoside Rg3 is considered a promising candidate for enhancing cancer treatment when combined with chemotherapy agents. Nevertheless, the intrinsic challenges of Rg3, such as its poor water solubility and low oral bioavailability, necessitate innovative solutions. Herein, we developed Rg3-PLGA@TMVs by encapsulating Rg3 within PLGA nanoparticles (Rg3-PLGA) and coating them with membranes derived from tumor cell-derived microvesicles (TMVs). Rg3-PLGA@TMVs displayed an array of favorable advantages, including controlled release, prolonged storage stability, high drug loading efficiency and a remarkable ability to activate dendritic cells in vitro . This activation is evident through the augmentation of CD86 + CD80 + dendritic cells, along with a reduction in phagocytic activity and acid phosphatase levels. When combined with DOX, the synergistic effect of Rg3-PLGA@TMVs significantly inhibits 4T1 tumor growth and fosters the development of antitumor immunity in tumor-bearing mice. Most notably, this delivery system effectively mitigates the toxic side effects of DOX, particularly those affecting the heart. Overall, Rg3-PLGA@TMVs provide a novel strategy to enhance the efficacy of DOX while simultaneously mitigating its associated toxicities and demonstrate promising potential for the combined chemo-immunotherapy of breast cancer.

Rationale : Caloric restriction improves the efficacy of anti-cancer therapy. This effect is largely dependent on the increase of the extracellular ATP concentration in the tumor microenvironment (TME). Pathways for ATP release triggered by nutrient deprivation are largely unknown. Methods: The extracellular ATP (eATP) concentration was in vivo measured in the tumor microenvironment of B16F10-inoculated C57Bl/6 mice with the pmeLuc probe. Alternatively, the pmeLuc-TG-mouse was used. Caloric restriction was in vivo induced with hydroxycitrate (HC). B16F10 melanoma cells or CT26 colon carcinoma cells were in vitro exposed to serum starvation to mimic nutrient deprivation. Energy metabolism was monitored by Seahorse. Microparticle release was measured by ultracentrifugation and by Nanosight. Results : Nutrient deprivation increases eATP release despite the dramatic inhibition of intracellular energy synthesis. Under these conditions oxidative phosphorylation was dramatically impaired, mitochondria fragmented and glycolysis and lactic acid release were enhanced. Nutrient deprivation stimulated a P2X7-dependent release of ATP-loaded, mitochondria-containing, microparticles as well as of naked mitochondria. Conclusions : Nutrient deprivation promotes a striking accumulation of eATP paralleled by a large release of ATP-laden microparticles and of naked mitochondria. This is likely to be a main mechanism driving the accumulation of eATP into the TME., Competing Interests: Competing Interests: FDV is a member of the Scientific Advisory Board of Biosceptre Ltd, a biotech Company involved in the development of anti-P2X7 antibodies, and a consultant with Axxam SpA. The other authors declare no conflict of interest., (© The author(s).)

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl 3 ) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl 3 and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases., Competing Interests: None declared., (Thieme. All rights reserved.)

Endothelial dysfunction contributes to the development of diabetic complications and the production of circulating microparticles (MPs). Our previous study showed that diabetic mice-derived MPs (DM MPs) had increased levels of extracellular regulated protein kinase 1/2 (ERK1/2) and impaired endothelial-dependent relaxation in aortas when compared with control mice-derived MPs. This study was designed to investigate whether PD98059, an ERK1/2 inhibitor, affects the function of aortas and DM MPs. MPs were obtained from streptozotocin-induced DM, DM after PD98059 treatment, and ICR mice as control. The mice and MPs were then analyzed on the basis of their vascular function and enzyme expressions. Compared with the controls, platelet-derived MPs and ERK1/2 levels in the MPs were significantly elevated in the DM but showed little change in PD98059-treated DM. PD98059 mainly decreased ERK1/2 phosphorylation in the MPs. In the aortas of DM and DM MPs the endothelium-dependent vascular function was impaired, and there was a significantly greater improvement in the vascular function in the PD98059-treated DM aortas and the aortas treated with PD98059-treated DM MPs than in DM aortas and the aortas treated with DM MPs. Furthermore, DM MPs increased ERK1/2 and intracellular adhesion molecule-1 (ICAM-1) expressions in the aortas, but PD98059-treated DM MPs did not show these effects. For the first time, these results indicate that PD98059 treatment improves endothelial dysfunction in DM, and adhesion properties of DM MPs can be partly blocked by PD98059 via ERK and ICAM-1. These effects may explain some of the vascular complications in diabetes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background & Aims: Circulating microvesicles (cMV) are both effectors and biomarkers of cardiovascular disease (CVD), and the effects of omega 3 polyunsaturated fatty acids (n3 PUFA) in MV shedding are not yet well known. Therefore, we aimed to investigate the effects of long-term n3 PUFA supplementation on cMV release from cells of the vascular compartment in elderly subjects at very high risk of CVD., Methods: We included 156 elderly patients 2-8 weeks after suffering an acute myocardial infarction from the OMEMI cohort. Subjects were randomly allocated to receive 930 mg EPA + 660 mg DHA (n3 PUFA intervention) or corn oil (56% linoleic acid, 32% oleic acid, 10% palmitic acid) used as placebo daily for two years. At inclusion and after one-year follow-up, prothrombotic [annexin V (AV) + ] cMV derived from blood and vascular cells were phenotyped by flow cytometry., Results: No differences were observed in the levels of cMV between the randomized groups at inclusion in the study. After one-year follow-up, total AV + , platelet-derived CD61 + /AV + , and endothelial-derived CD31 + /AV + and CD31 + /CD42b - /AV + cMV increased significantly in both groups. In the n3 PUFA supplemented group, platelet-derived CD62P + /AV + , CD42b + /AV + and CD31 + /CD42b + /AV + ; leukocyte-derived CD62L + /AV + , CD45 + /AV + , and CD11b + /AV + , as well as endothelial derived CD146 + /AV + , CD62E + /AV + , and CD309 + /AV + cMV also increased significantly. No significant differences were however, observed in the changes of cMV levels between groups., Conclusion: In elderly Norwegians who have suffered a recent acute myocardial infarction and treated as per guidelines, long-term supplementation with 1.8 g/day n3 PUFA does not modulate prothrombotic MV release from blood and vascular cells., Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01841944., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Benzene is a ubiquitous environmental pollutant. Recent population-based studies suggest that benzene exposure is associated with an increased risk for cardiovascular disease. However, it is unclear whether benzene exposure by itself is sufficient to induce cardiovascular toxicity. We examined the effects of benzene inhalation (50 ppm, 6 h/day, 5 days/week, 6 weeks) or HEPA-filtered air exposure on the biomarkers of cardiovascular toxicity in male C57BL/6J mice. Benzene inhalation significantly increased the biomarkers of endothelial activation and injury including endothelial microparticles, activated endothelial microparticles, endothelial progenitor cell microparticles, lung endothelial microparticles, and activated lung and endothelial microparticles while having no effect on circulating levels of endothelial adhesion molecules, endothelial selectins, and biomarkers of angiogenesis. To understand how benzene may induce endothelial injury, we exposed human aortic endothelial cells to benzene metabolites. Of the metabolites tested, trans,trans-mucondialdehyde (10 μM, 18h) was the most toxic. It induced caspases-3, -7 and -9 (intrinsic pathway) activation and enhanced microparticle formation by 2.4-fold. Levels of platelet-leukocyte aggregates, platelet macroparticles, and a proportion of CD4 + and CD8 + T-cells were also significantly elevated in the blood of the benzene-exposed mice. We also found that benzene exposure increased the transcription of genes associated with endothelial cell and platelet activation in the liver; and induced inflammatory genes and suppressed cytochrome P450s in the lungs and the liver. Together, these data suggest that benzene exposure induces endothelial injury, enhances platelet activation and inflammatory processes; and circulatory levels of endothelial cell and platelet-derived microparticles and platelet-leukocyte aggregates are excellent biomarkers of cardiovascular toxicity of benzene., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Sex steroid hormones like estrogens have a key role in the regulation of energy homeostasis and metabolism. In transwomen, gender-affirming hormone therapy like estradiol (in combination with antiandrogenic compounds) could affect metabolism as well. Given that the underlying pathophysiological mechanisms are not fully understood, this study assessed circulating estradiol-driven microRNAs (miRs) in transwomen and their regulation of genes involved in metabolism in mice., Methods: Following plasma miR-sequencing (seq) in a transwomen discovery (n = 20) and validation cohort (n = 30), we identified miR-224 and miR-452. Subsequent systemic silencing of these miRs in male C57Bl/6 J mice (n = 10) was followed by RNA-seq-based gene expression analysis of brown and white adipose tissue in conjunction with mechanistic studies in cultured adipocytes., Results: Estradiol in transwomen lowered plasma miR-224 and -452 carried in extracellular vesicles (EVs) while their systemic silencing in mice and cultured adipocytes increased lipogenesis (white adipose) but reduced glucose uptake and mitochondrial respiration (brown adipose). In white and brown adipose tissue, differentially expressed (miR target) genes are associated with lipogenesis (white adipose) and mitochondrial respiration and glucose uptake (brown adipose)., Conclusion: This study identified an estradiol-drive post-transcriptional network that could potentially offer a mechanistic understanding of metabolism following gender-affirming estradiol therapy.

Background: Endothelial microparticles (EMPs) carrying the protein disulfide isomerase (PDI) might play a key role in promoting platelet activation in diabetes. This study aimed to examine the activation of platelets, the amounts of MPs, PMPs, and EMPs, and the concentration and activity of PDI in patients with diabetic coronary heart disease (CHD) and non-diabetic CHD., Methods: Patients with CHD (n=223) were divided as non-diabetic CHD (n=121) and diabetic CHD (n=102). Platelet activation biomarkers, circulating microparticles (MPs), the concentration of protein disulfide isomerase (PDI), and MP-PDI activity were determined. The effect of EMPs on platelet activation was investigated in vitro . Allosteric GIIb/IIIa receptors that bind to PDI were detected by a proximity ligation assay (PLA)., Results: Platelet activation, platelet-leukocyte aggregates, circulating MPs, EMPs, PDI, and MP-PDI activity in the diabetic CHD group were significantly higher than in the non-diabetic CHD group ( P <0.05). Diabetes ( P =0.006) and heart rate <60 bpm ( P =0.047) were associated with elevated EMPs. EMPs from diabetes increased CD62p on the surface of the platelets compared with the controls ( P< 0.01), which could be inhibited by the PDI inhibitor RL90 ( P< 0.05). PLA detected the allosteric GIIb/IIIa receptors caused by EMP-PDI, which was also inhibited by RL90., Conclusions: In diabetic patients with CHD, platelet activation was significantly high. Diabetes and heart rate <60 bpm were associated with elevated EMPs and simultaneously increased PDI activity on EMP, activating platelets through the allosteric GPIIb/IIIa receptors.

Platelets can modulate cancer through budding of platelet microparticles (PMPs) that can transfer a plethora of bioactive molecules to cancer cells upon internalization. In acute myelogenous leukemia (AML) this can induce chemoresistance, partially through a decrease in cell activity. Here we investigated if the internalization of PMPs protected the monocytic AML cell line, THP-1, from apoptosis by decreasing the initial cellular damage inflicted by treatment with daunorubicin, or via direct modulation of the apoptotic response. We examined whether PMPs could protect against apoptosis after treatment with a selection of inducers, primarily associated with either the intrinsic or the extrinsic apoptotic pathway, and protection was restricted to the agents targeting intrinsic apoptosis. Furthermore, levels of daunorubicin-induced DNA damage, assessed by measuring gH2AX, were reduced in both 2N and 4N cells after PMP co-incubation. Measuring different BCL2-family proteins before and after treatment with daunorubicin revealed that PMPs downregulated the pro-apoptotic PUMA protein. Thus, our findings indicated that PMPs may protect AML cells against apoptosis by reducing DNA damage both dependent and independent of cell cycle phase, and via direct modulation of the intrinsic apoptotic pathway by downregulating PUMA. These findings further support the clinical relevance of platelets and PMPs in AML.

Acute promyelocytic leukemia (APL) cells constitutively express a large amount of tissue factor (TF) antigen, most of which is present in the cytoplasm. Coagulopathy may persist after induction therapy. We evaluated the overall role of circulating microparticles (MPs) in coagulation activation in APL-associated coagulopathy before and during induction therapy. Eleven adult patients with ≥ World Health Organization's (WHO) grade 2 bleeding events and 11 sex- and age-matched healthy controls were selected. All patients received arsenic trioxide alone as induction therapy. MP-associated TF (MP-TF) activity and MP procoagulant activity (MP-PCA) and 12 coagulation- and anticoagulation-associated indexes were measured before, during, and after induction therapy. Correlation between MP-associated indexes and the other 12 indexes was analyzed in patients. The MP-TF activity was negligible in controls, whereas it markedly increased in patients, dropped rapidly after treatment, and returned to normal at the end of induction therapy. The MP-PCA was similar between patients and controls. The correlation analysis revealed that TF-bearing MPs in patients mainly originated from APL cells. Partially differentiated APL cells could also release TF-bearing MPs, and the higher the degree of APL cell differentiation, the lower the ability of APL cells to release TF-bearing MPs. MP-TF was the main source of active TF in plasma and an important contributor for the coagulation activation in APL-associated coagulopathy. It was MPs released by APL cells/partially differentiated APL cells that served as the vehicle to transfer the large amount of TF to plasma to activate coagulation.

Background: Patients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition. Thus, we investigate, in vivo, in T2DM either with good (HbA1c ≤ 7.0%; GGC) or poor (HbA1c > 7.0%; PGC) glycemic control, the circulating levels of MPs, and in vitro, the molecular pathways involved in the release of MPs from platelets (PMP) and tested their pro-inflammatory effects on THP-1 transformed macrophages., Methods: In 59 T2DM, and 23 control subjects with normal glucose tolerance (NGT), circulating levels of CD62E+, CD62P+, CD142+, CD45+ MPs were determined by flow cytometry, while plasma levels of ICAM-1, VCAM-1, IL-6 by ELISA. In vitro, PMP release and activation of isolated platelets from GGC and PGC were investigated, along with their effect on IL-6 secretion in THP-1 transformed macrophages., Results: We found that MPs CD62P + (PMP) and CD142 + (tissue factor-bearing MP) were significantly higher in PGC T2DM than GGC T2DM and NGT. Among MPs, PMP were also correlated with HbA1c and IL-6. In vitro, we showed that acute thrombin exposure stimulated a significantly higher PMP release in PGC T2DM than GGC T2DM through a more robust activation of PAR-4 receptor than PAR-1 receptor. Treatment with PAR-4 agonist induced an increased release of PMP in PGC with a Ca 2+ -calpain dependent mechanism since this effect was blunted by calpain inhibitor. Finally, the uptake of PMP derived from PAR-4 treated PGC platelets into THP-1 transformed macrophages promoted a marked increase of IL-6 release compared to PMP derived from GGC through the activation of the NF-kB pathway., Conclusions: These results identify PAR-4 as a mediator of platelet activation, microparticle release, and inflammation, in poorly controlled T2DM.

[Figure: see text].

Recently, artificial blood vessels modified by integrin α4β1 ligand, such as REDV, showed endothelialization improvement and antithrombotic properties have been reported. Early endothelialization was affected by the type of circulating cells captured by the peptide in the initial transplantation state, however, it is still not clarified. In this study, we identified in vitro circulating cells bound with the peptides arginine-glutamic acid-aspartic acid-valine (REDV) or histidine-glycine-glycine-valine-arginine-leucine-tyrosine (HGGVRLY). The effect of free C- or N-terminal of HGGVRLY on the type of peptide-binding cells was also studied. The rat circulating cells were isolated from blood and incubated with 5(6)-carboxyfluorescein (5/6-FAM, F) labeled F-REDV (C-terminal free), F-HGGVRLY (C-terminal free), or HGGVRLY-F (N-terminal free). Furthermore, peptide-binding cells were identified by co-staining with various antibodies labeled with PE, PerCP/Cy5.5, or APC. N-terminal free HGGVRLY-F was found to bind to more circulating cells than C-terminal free F-REDV and F-HGGVRLY. The ratio of integrin α4β1 positive cell bound with F-REDV, F-HGGVRLY, or HGGVRLY-F reached over 90 %, demonstrating that HGGVRLY is also a ligand of integrin α4β1. Among identified cell types, we found that F-REDV mainly bounds with EPC and BMSC, while F-HGGVRLY with BMSC. HGGVRLY-F bounds with EPC and BMSC, exhibiting a higher EPC binding ratio than F-REDV and F-HGGVRLY., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: TF (Tissue factor) plays a key role in hemostasis, but an aberrant expression of TF leads to thrombosis. The objective of the present study is to investigate the effect of 4-hydroxy-2-nonenal (HNE), the most stable and major oxidant produced in various disease conditions, on the release of TF + microvesicles into the circulation, identify the source of TF + microvesicles origin, and assess their effect on intravascular coagulation and inflammation. Approach and Results: C57BL/6J mice were administered with HNE intraperitoneally, and the release of TF + microvesicles into circulation was evaluated using coagulation assays and nanoparticle tracking analysis. Various cell-specific markers were used to identify the cellular source of TF + microvesicles. Vascular permeability was analyzed by the extravasation of Evans blue dye or fluorescein dextran. HNE administration to mice markedly increased the levels of TF + microvesicles and thrombin generation in the circulation. HNE administration also increased the number of neutrophils in the lungs and elevated the levels of inflammatory cytokines in plasma. Administration of an anti-TF antibody blocked not only HNE-induced thrombin generation but also HNE-induced inflammation. Confocal microscopy and immunoblotting studies showed that HNE does not induce TF expression either in vascular endothelium or circulating monocytes. Microvesicles harvested from HNE-administered mice stained positively with CD248 and α-smooth muscle actin, the markers that are specific to perivascular cells. HNE was found to destabilize endothelial cell barrier integrity., Conclusions: HNE promotes the release of TF + microvesicles from perivascular cells into the circulation. HNE-induced increased TF activity contributes to intravascular coagulation and inflammation.

Purpose: Mesenchymal stem cells (MSCs) have appeared as a promising regenerative cell-based therapeutic, for degenerative conditions, such as OA, while the beneficial results from the application of MSCs have been attributed to the MSCs-derived secretome, which is the sum of cytoprotective factors produced by the MSCs. Aim of this study was to systematically review the literature in order to assess whether stem cell secretome (conditioned medium-CM, exosome-Exos or microvesicles-MV)(CM/Exos/MVs) treatment reduces inflammation and enhances cartilage regeneration in preclinical studies of experimental arthritis., Materials and Methods: An extensive electronic search was conducted by 2 independent reviewers by using the PubMed, Cochrane Library, Web of Science, and Scopus database, as well as Google Scholar, in order to identify the studies that met our inclusion criteria until August 2019. Included studies were assessed for quality and Risk of Bias (RoB) using the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines and a modification of Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) RoB tool for animal studies, respectively., Results: The initial search provided 525 records, with 28 fulfilling the inclusion criteria. The included studies presented great heterogeneity regarding the stem cells used, the preparation of therapeutic agent as well as the animal models used for testing. In addition, most studies presented with an unclear or high risk bias., Conclusion: In summary, the positive results of CM/Exos/MVs application in preclinical models of experimentally induced OA in terms of resolution of inflammation and cartilage regeneration are highlighted in this review, presenting a promising therapeutic solution for OA.

Purpose: Platelet microparticles (PMPs), which are microvesicles shed from platelets, participate in inflammation, vascular homeostasis, and thrombosis. PMPs are increased in obese women with polycystic ovary syndrome (PCOS). Agents that modulate hormonal aspects of PCOS could affect the levels of PMPs. The aim of the present study was to evaluate the effects of oral contraceptives (OCPs), antiandrogen, and metformin use for 6 and 12 months on PMPs in normal-weight women with PCOS., Methods: Forty-five women with PCOS and 13 healthy women were recruited. Biochemical, hormonal, and clinical parameters were recorded. Women with PCOS received treatment with OCPs, OCPs+antiandrogens, or metformin, depending on their main complaint or clinical/biochemical findings. PMPs were measured at baseline and after 6 and 12 months., Results: At baseline, patients with PCOS had higher levels of PMPs than controls (p = 0.017), which increased after 6-month treatment with OCPs (p = 0.006). Subsequently, they decreased after 12-month treatment (p = 0.046). Metformin had no effect on PMP levels., Conclusion: In conclusion, PMP levels are increased in PCOS and further increase with OCP use. This effect could possibly contribute to the increased risk of venous thromboembolism associated with OCP use. However, further studies are needed to elucidate the exact role of PMPs in PCOS.

Background: Vascular leakage is an important pathophysiological process of critical conditions such as shock and ischemia-reperfusion (I/R)-induced lung injury. Microparticles (MPs), including endothelial cell-derived microparticles (EMPs), platelet-derived microparticles (PMPs) and leukocyte-derived microparticles (LMPs), have been shown to participate in many diseases. Whether and which of these MPs take part in pulmonary vascular leakage and lung injury after I/R and whether these MPs have synergistic effect and the underlying mechanism are not known., Methods: Using hemorrhage/transfusion (Hemo/Trans) and aorta abdominalis occlusion-induced I/R rat models, the role of EMPs, PMPs and LMPs and the mechanisms in pulmonary vascular leakage and lung injury were observed., Results: The concentrations of EMPs, PMPs and LMPs were significantly increased after I/R. Intravenous administration of EMPs and PMPs but not LMPs induced pulmonary vascular leakage and lung injury. Furthermore, EMPs induced pulmonary sequestration of platelets and promoted more PMPs production, and played a synergistic effect on pulmonary vascular leakage. MiR-1, miR-155 and miR-542 in EMPs, and miR-126 and miR-29 in PMPs, were significantly increased after hypoxia/reoxygenation (H/R). Of which, inhibition of miR-155 in EMPs and miR-126 in PMPs alleviated the detrimental effects of EMPs and PMPs on vascular barrier function and lung injury. Overexpression of miR-155 in EMPs down-regulated the expression of tight junction related proteins such as ZO-1 and claudin-5, while overexpression of miR-126 up-regulated the expression of caveolin-1 (Cav-1), the trans-cellular transportation related protein such as caveolin-1 (Cav-1). Inhibiting EMPs and PMPs production with blebbistatin (BLE) and amitriptyline (AMI) alleviated I/R induced pulmonary vascular leakage and lung injury., Conclusions: EMPs and PMPs contribute to the pulmonary vascular leakage and lung injury after I/R. EMPs mediate pulmonary sequestration of platelets, producing more PMPs to play synergistic effect. Mechanically, EMPs carrying miR-155 that down-regulates ZO-1 and claudin-5 and PMPs carrying miR-126 that up-regulates Cav-1, synergistically mediate pulmonary vascular leakage and lung injury after I/R. Video Abstract.

Microvesicle particles (MVP) secreted by a variety of cell types in response to reactive oxygen species (ROS)-generating pro-oxidative stressors have been implicated in modifying the cellular responses including the sensitivity to therapeutic agents. Our previous studies have shown that expression of a G-protein coupled, platelet-activating factor-receptor (PAFR) pathway plays critical roles in pro-oxidative stressors-mediated cancer growth and MVP release. As most therapeutic agents act as pro-oxidative stressors, the current studies were designed to determine the role of the PAFR signaling in targeted therapies (i.e., gefitinib and erlotinib)-mediated MVP release and underlying mechanisms using PAFR-expressing human A549 and H1299 non-small cell lung cancer (NSCLC) cell lines. Our studies demonstrate that both gefitinib and erlotinib generate ROS in a dose-dependent manner in a process blocked by antioxidant and PAFR antagonist, verifying their pro-oxidative stressor's ability, and the role of the PAFR in this effect. We observed that these targeted therapies induce MVP release in a dose- and time-dependent manner, similar to a PAFR-agonist, carbamoyl-PAF (CPAF), and PAFR-independent agonist, phorbol myristate acetate (PMA), used as positive controls. To confirm the PAFR dependency, we demonstrate that siRNA-mediated PAFR knockdown or PAFR antagonist significantly blocked only targeted therapies- and CPAF-mediated but not PMA-induced MVP release. The use of pharmacologic inhibitor strategy suggested the involvement of the lipid ceramide-generating enzyme, acid sphingomyelinase (aSMase) in MVP biogenesis, and observed that regardless of the stimuli used, aSMase inhibition significantly blocked MVP release. As mitogen-activated protein kinase (MAPK; ERK1/2 and p38) pathways crosstalk with PAFR, their inhibition also significantly attenuated targeted therapies-mediated MVP release. These findings indicate that PAFR signaling could be targeted to modify cellular responses of targeted therapies in lung cancer cells.

Rationale: People living with HIV on effective antiretroviral therapy are at increased risk of cardiovascular complications, possibly due to off-target drug effects. Some studies have associated antiretroviral therapy with increased risk of myocardial infarction and endothelial dysfunction, but a link between endothelial function and antiretrovirals has not been established., Objective: To determine the effects of antiretrovirals in common clinical use upon in vitro endothelial function to better understand cardiovascular risk in people living with HIV., Methods and Results: Human umbilical cord vein endothelial cells or human coronary artery endothelial cells were pretreated with the antiretrovirals abacavir sulphate (ABC), tenofovir disoproxil fumarate, or tenofovir alafenamide. Expression of adhesion molecules, ectonucleotidases (CD39 and CD73), tissue factor (TF), endothelial-derived microparticle (EMP) numbers and phenotype, and platelet activation were evaluated by flow cytometry. TF and ectonucleotidase activities were measured using colourimetric plate-based assays. ABC-treated endothelial cells had higher levels of ICAM (intercellular adhesion molecule)-1 and TF expression following TNF (tumor necrosis factor)-α stimulation. In contrast, tenofovir disoproxil fumarate and tenofovir alafenamide treatment gave rise to greater populations of CD39 + CD73 + cells. These cell surface differences were also observed within EMP repertoires. ABC-treated cells and EMP had greater TF activity, while tenofovir disoproxil fumarate- and tenofovir alafenamide-treated cells and EMP displayed higher ectonucleotidase activity. Finally, EMP isolated from ABC-treated cells enhanced collagen-evoked platelet integrin activation and α-granule release., Conclusions: We report differential effects of antiretrovirals used in the treatment of HIV upon endothelial function. ABC treatment led to an inflammatory, prothrombotic endothelial phenotype that promoted platelet activation. In contrast, tenofovir disoproxil fumarate and tenofovir alafenamide conferred potentially cardioprotective properties associated with ectonucleotidase activity. These observations establish a link between antiretrovirals and specific functional effects that provide insight into cardiovascular disease in people living with HIV.

Scope: Diet rich in bilberries is considered cardioprotective, but the mechanisms of action are poorly understood. Cardiovascular disease is characterized by increased proatherogenic status and high levels of circulating microvesicles (MVs). In an open-label study patients with myocardial infarction receive an 8 week dietary supplementation with bilberry extract (BE). The effect of BE on patient MV levels and its influence on endothelial vesiculation in vitro is investigated., Methods and Results: MVs are captured with acoustic trapping and platelet-derived MVs (PMVs), as well as endothelial-derived MVs (EMVs) are quantified with flow cytometry. The in vitro effect of BE on endothelial extracellular vesicle (EV) release is examined using endothelial cells and calcein staining. The mechanisms of BE influence on vesiculation pathways are studied by Western blot and qRT-PCR. Supplementation with BE decreased both PMVs and EMVs. Furthermore, BE reduced endothelial EV release, Akt phosphorylation, and vesiculation-related gene transcription. It also protects the cells from P2X 7 -induced EV release and increase in vesiculation-related gene expression., Conclusion: BE supplementation improves the MV profile in patient blood and reduces endothelial vesiculation through several molecular mechanisms related to the P2X 7 receptor. The findings provide new insight into the cardioprotective effects of bilberries., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Background: Treatment of patients with hemophilia with an inhibitor is generally done using bypassing agents (BPA), wherein variability in response is observed. Due to lack of validated laboratory assays, monitoring is being carried out by clinical response only. Emerging biomarkers like procoagulant microparticles (MPs) may prove to be promising., Aim: To analyze whether procoagulant MP levels correlate with clinical response to FEIBA therapy., Materials and Methods: Total phosphatidylserine (PS) expressing MPs along with different cell derived MPs were measured in blood samples obtained prior and 2 hour post-FEIBA infusion in 64 bleeding episodes associated with 43 severe hemophilia patients., Results and Discussion: Patients with excellent response showed statistically significant increase in %MP of PS-MPs (p < 0.0001; 95.0% CI Range: -64.33 to -24.42) when compared to those with moderate response; platelet %MP change was also found significantly associated (p < 0.05) with clinical response. In search of an assay for monitoring FEIBA, results though preliminary seem to be promising with increase in %PS-MP correlating well with the clinical response. Coagulation being multifactorial process involves multiple factors for balanced hemostasis, which needs to be accounted. Larger studies in this line may provide indications for usage of MPs as monitoring and dose adjustment tool of FEIBA therapy., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aim: Tumor cell-derived microparticles (MP) can function as a targeted delivery carrier for anti-tumor drugs. Here, we aimed to generate paclitaxel-loaded microparticles (MP-PTX) from HeLa cells and examined its therapeutic potential on human cervical carcinoma., Methods: MP-PTX was generated from HeLa cells by ultraviolet radiation and subsequent centrifugation. The particle size, drug loading rate, and stability of MP-PTX were examined in vitro. Flow cytometry and the MTT assay were performed to test the inhibitory effect of MP-PTX using different cell lines. Immunodeficient mice bearing HeLa cervical carcinoma were treated with 0.9% normal saline, MP, paclitaxel (PTX) (2.5 mg/kg), or MP-PTX (PTX content identical to PTX group) every day for 6 consecutive days. Tumor volume and animal survival were observed. Micro 18 F-FDG PET/CT was performed to monitor the therapeutic efficacy. The proliferation activity of cells and microvessel density in tumor tissues were determined by immunohistochemical staining using Ki-67 and CD31, respectively., Results: Dynamic laser scattering measurements showed that the particle size of MP-PTX was 285.58 ± 2.95 nm and the polydispersity index was 0.104 ± 0.106. And the particle size of MP-PTX was not change at 4°C for at least one week. More than 1% of PTX in the medium could be successfully encapsulated into HeLa cell-derived MP. When compared with PTX, MP-PTX treatment significantly increased apoptosis of tumor cells and reduced their proliferation. In addition, MP-PTX showed lower toxicity to normal human umbilical vein endothelial cells (HUVEC) than PTX. In vivo studies further demonstrated that MP-PTX treatment significantly inhibited the growth of cervical carcinoma, prolonged the survival of tumor-bearing mice, and reduced the toxicity of PTX. Immunohistochemical staining revealed that MP-PTX treatment led to decreased Ki-67 positive tumor cells and decreased microvessel density in tumor tissues., Conclusion: Our results demonstrated that HeLa-derived MP-PTX significantly enhanced the anti-cancer effects of PTX with reduced toxicity, which may provide a novel strategy for the treatment of cervical carcinoma., Competing Interests: The authors report no conflicts of interest for this work., (© 2020 Peng et al.)

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 β-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., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Extracellular vesicles derived from mesenchymal stem cells (MSCs) represent a novel approach for regenerative and immunosuppressive therapy. Recently, cytochalasin B-induced microvesicles (CIMVs) were shown to be effective drug delivery mediators. However, little is known about their immunological properties. We propose that the immunophenotype and molecular composition of these vesicles could contribute to the therapeutic efficacy of CIMVs. To address this issue, CIMVs were generated from murine MSC (CIMVs-MSCs) and their cytokine content and surface marker expression determined. For the first time, we show that CIMVs-MSCs retain parental MSCs phenotype (Sca-1 + , CD49e + , CD44 + , CD45 - ). Also, CIMVs-MSCs contained a cytokine repertoire reflective of the parental MSCs, including IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-13, IL-17, CCL2, CCL3, CCL4, CCL5, CCL11, G-CSF, GM-CSF and TNF-α. Next, we evaluated the immune-modulating properties of CIMVs-MSCs in vivo using standard preclinical tests. MSCs and CIMVs-MSCs reduced serum levels of anti-sheep red blood cell antibody and have limited effects on neutrophil and peritoneal macrophage activity. We compared the immunomodulatory effect of MSCs, CIMVs and EVs. We observed no immunosuppression in mice pretreated with natural EVs, whereas MSCs and CIMVs-MSCs suppressed antibody production in vivo. Additionally, we have investigated the biodistribution of CIMVs-MSCs in vivo and demonstrated that CIMVs-MSCs localized in liver, lung, brain, heart, spleen and kidneys 48 h after intravenous injection and can be detected 14 days after subcutaneous and intramuscular injection. Collectively our data demonstrates immunomodulatory efficacy of CIMVs and supports their further preclinical testing as an effective therapeutic delivery modality.

Protein bound uremic toxins, such as p-cresol, cannot be effectively removed by conventional dialysis techniques and are accumulated in plasma, thus contributing to progression of both chronic kidney disease (CKD) and cardiovascular disease (CVD). Pathological effects of uremic toxins include activation of inflammatory response, endothelial dysfunction and release of endothelial microvesicles. To date, the role of p-cresol in endothelial microvesicles formation has not been analyzed. The aim of the present study was evaluate the effects of endothelial microvesicles released by p-cresol (PcEMV) on endothelial dysfunction. An in vitro model of endothelial damage mediated by p-cresol was proposed to evaluate the functional effect of PcEMV on the endothelial repair process carried out by endothelial cells and microRNA (miRNA) that could be involved in this process. We observed that p-cresol induced a greater release of microvesicles in endothelial cells. These microvesicles altered regenerative capacity of endothelial cells, decreasing their capacity for cell migration and their potential to form vascular structures in vitro. Moreover, we observed increased cellular senescence and a deregulation of miRNA-146b-5p and miRNA-223-3p expression in endothelial cells treated with endothelial microvesicles released by p-cresol. In summary our data show that microvesicles generated in endothelial cells treated with p-cresol (PcEMV) interfere with the endothelial repair process by decreasing the migratory capacity, the ability to form new vessels and increasing the senescence of mature endothelial cells. These alterations could be mediated by the upregulation of miRNA-146b-5p and miRNA-223-3p.

Platelet hyper-activation and platelet microparticles (PMPs) play a key role in the pathogenesis of cardiovascular diseases. Dietary polyphenols are believed to mimic antiplatelet agents by blunting platelet activation receptors via its antioxidant phenomenon. However, there is limited information on the anti-platelet activity of grain-derived polyphenols. The aim of the study is to evaluate the effects of sorghum extract (Shawaya short black 1 variety), an extract previously characterised for its high antioxidant activity and reduction of oxidative stress-related endothelial dysfunction, on platelet aggregation, platelet activation and PMP release. Whole blood samples collected from 18 healthy volunteers were treated with varying non-cytotoxic concentrations of polyphenol-rich black sorghum extract (BSE). Platelet aggregation study utilised 5 µg/mL collagen to target the GPVI pathway of thrombus formation whereas adenine phosphate (ADP) was used to stimulate the P2Y1/P2Y12 pathway of platelet activation assessed by flow cytometry. Procaspase-activating compound 1 (PAC-1) and P-selectin/CD62P were used to evaluate platelet activation- related conformational changes and degranulation respectively. PMPs were isolated from unstimulated platelets and quantified by size distribution and binding to CD42b. BSE treatment significantly reduced both collagen-induced platelet aggregation and circulatory PMP release at 40 µg/mL ( p < 0.001) when compared to control. However, there was no significant impact of BSE on ADP-induced activation-dependent conformational change and degranulation of platelets. Results of this study suggest that phenolic rich BSE may confer cardio-protection by modulating specific signalling pathways involved in platelet activation and PMP release.

Microvesicles (MVs) derived from human umbilical cord mesenchymal stem cells (hUC-MSCs-MVs) and miR-21 were demonstrated to ameliorate renal ischemia-reperfusion injury (IRI). Since hUC-MSC-MVs contained a substantial quantity of miR-21, we speculated that miR-21 might account for a part of the therapeutic effects of hUC-MSCs-MVs. The human tubule epithelial (HK-2) cells were cultured under low oxygen (LO) condition to mimic a cellular IRI model. A rat model of unilateral renal IRI was established. A co-culture model of HK-2 cells and MSC-MVs was utilized to examine the therapeutic role of MSC-MVs in HK-2 cell apoptosis and mechanism. The results showed that hUC-MSCs-MVs inhibited LO-induced HK-2 cell apoptosis through transferring miR-21 to HK-2 cells. Mechanistically, miR-21 directly targeted and negatively regulated programmed cell death protein 4 (PDCD4) in HK-2 cells. Moreover, PDCD4 overexpression in HK-2 cells abrogated the hUC-MSCs-MVs-inhibited HK-2 cell apoptosis under LO condition. Additionally, the beneficial effect of MSC-MVs on rat renal IRI was partly eliminated when miR-21 was knocked down in MSCs. Taken together, MSC-MVs inhibit tubular epithelial cell apoptosis and ameliorate renal IRI, at least partially, via delivery of miR-21.

Background and Objective: For many pathological states, microparticles are supposed to be one of the causes of hypercoagulation. Although there are some indirect data about microparticles participation in coagulation activation and propagation, the integral hemostasis test Thrombodynamics allows to measure micropaticles participation in these two coagulation phases directly. Demonstrates microparticles participation in coagulation activation by influence on the appearance of coagulation centres in the plasma volume and the rate of clot growth from the surface with immobilized tissue factor.Methods: Microparticles were obtained from platelets and erythrocytes by stimulation with thrombin receptor-activating peptide (SFLLRN) and calcium ionophore (A23187), respectively, from monocytes, endothelial HUVEC culture and monocytic THP cell culture by stimulation with lipopolysaccharides. Microparticles were counted by flow cytometry and titrated in microparticle-depleted normal plasma in the Thrombodynamics test., Results: Monocyte microparticles induced the appearance of clotting centres through the TF pathway at concentrations approximately 100-fold lower than platelet and erythrocyte microparticles, which activated plasma by the contact pathway. For endothelial microparticles, both activation pathways were essential, and their activity was intermediate. Monocyte microparticles induced plasma clotting by the appearance of hundreds of clots with an extremely slow growth rate, while erythrocyte microparticles induced the appearance of a few clots with a growth rate similar to that from surface covered with high-density tissue factor. Patterns of clotting induced by platelet and endothelial microparticles were intermediate. Platelet, erythrocyte and endothelial microparticles impacts on the rate of clot growth from the surface with tissue factor did not differ significantly within the 0-200·103/ul range of microparticles concentrations. However, at concentrations greater than 500·103/ul, erythrocyte microparticles increased the stationary clot growth rate to significantly higher levels than do platelet microparticles or artificial phospholipid vesicles consisting of phosphatidylcholine and phosphatidylserine., Conclusion: Microparticles of different origins demonstrated qualitatively different characteristics related to coagulation activation and propagation., Competing Interests: The authors have declared that no competing interests exist.

Curcumin, a hydrophobic polyphenol found in the rhizome of Curcuma longa, has been shown to reduce intracellular lipid accumulation in mouse models of lysosomal storage diseases such as Niemann-Pick type C. Exosomes are small extracellular vesicles secreted by cells in response to changes in intracellular ceramide composition. Curcumin can induce exosome/microvesicle release in cellular models of lipid deposition; however, the mechanism by which curcumin stimulates this release is unknown. In a model of lipid trafficking impairment in C6 glia cells, we show that curcumin stimulated ceramide synthesis by increasing the intracellular concentration of ceramide-dihydroceramide. Ceramide overload increased exosome/microvesicle secretion 10-fold, thereby reducing the concentration of lipids in the endolysosomal compartment. These effects were blocked by inhibitors of serine palmitoyltransferase (myriocin) and ceramide synthase (fumonisin B1). It is concluded that the decrease in intracellular lipid deposition induced by curcumin is mediated by increased ceramide synthesis and exosome/microvesicle release. This action may represent an additional health benefit of curcumin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Multiple myeloma (MM) cells accumulate in the bone marrow (BM) where their interactions impede disease therapy. We have shown that microvesicles (MVs) derived from BM mesenchymal stem cells (MSCs) of MM patients promote the malignant traits via modulation of translation initiation (TI), whereas MVs from normal donors (ND) do not. Here, we observed that this phenomenon is contingent on a MVs' protein constituent, and determined correlations between the MVs from the tumor microenvironment, for example, MM BM-MSCs and patients' clinical characteristics. BM-MSCs' MVs (ND/MM) proteomes were assayed (mass spectrometry) and compared. Elevated integrin CD49d (X80) and CD29 (X2) was determined in MM-MSCs' MVs and correlated with patients' staging and treatment response (free light chain, BM plasma cells count, stage, response to treatment). BM-MSCs' MVs uptake into MM cell lines was assayed (flow cytometry) with/without integrin inhibitors (RGD, natalizumab, and anti-CD29 monoclonal antibody) and recipient cells were analyzed for cell count, migration, MAPKs, TI, and drug response (doxorubicin, Velcade). Their inhibition, particularly together, attenuated the uptake of MM-MSCs MVs (but not ND-MSCs MVs) into MM cells and reduced MM cells' signaling, phenotype, and increased drug response. This study exposed a critical novel role for CD49d/CD29 on MM-MSCs MVs and presented a discriminate method to inhibit cancer promoting action of MM-MSCs MVs while retaining the anticancer function of ND-MSCs-MVs. Moreover, these findings demonstrate yet again the intricacy of the microenvironment involvement in the malignant process and highlight new therapeutic avenues to be explored., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Tanshinone IIA (TS IIA), a multi-pharmaceutical compound from traditional Chinese herb, is effective for treatment of atherothrombosis. However, the underlying mechanisms of TS IIA-mediated anti-platelet activation effect are still poorly understood. As shown in our previous study, platelet-derived microvesicles (PMVs) generated in response to oxidant insult could activate CD36/mitogen-activated protein kinase kinase 4/Jun N-terminal kinase 2 (CD36/MKK4/JNK2) signals and lead to platelet activation. The present study aims to investigate the effect of TS IIA on platelet activation and the possible mechanisms., Methods: The production of PMVs induced by Interleukin 6 (IL-6) was detected by flow cytometry. We performed activating studies of platelets with PMVs derived from IL-6-treated platelets (IL-6-PMVs) in vitro. Sometimes, platelet suspensions were incubated with serial concentrations of TS IIA for 15 min before being stimulated with IL-6-PMVs. Expression of platelet integrin α IIb β 3 and CD36 was detected by flow cytometry. Phosphorylation of MKK4 and JNK were detected by immunoblotting., Results: Here we demonstrated firstly that TS IIA could prevent platelet activation induced by PMVs and down-regulates CD36 and MKK4/JNK2 signaling pathway. CD36 may be the target of atherosclerosis (AS)-related thrombosis., Conclusions: This study showed the possible mechanisms of TS IIA-mediated anti-platelet activation and may provide a new strategy for the treatment of AS-related thrombosis by targeting platelet CD36.

Background and Aims: Circulating platelet microparticles (PMP) are the most abundant in bloodstream, are highly procoagulant and contribute to cross-talk with inflammatory cells. The aim of the present study was to investigate the interactions of PMP with platelets and explore the involvement of toll-like receptor 4 (TLR-4)., Methods: PMP were separated by ultracentrifugation of expired platelet concentrates and added to: i) washed platelets, to confirm uptake, by flow cytometry and confocal and transmission electron microscopy, ii) platelet rich plasma (PRP), to assess changes in platelet function due to uptake by aggregometry in response to ADP; and iii) whole blood, to evaluate heterotypic aggregate (HA) formation by flow cytometry. Moreover, whole blood previously enriched with platelets with internalized PMP was used to explore modifications in thromboelastometry parameters (ROTEM). The inhibitory action of anti-TLR-4 was investigated., Results: Confocal and ultrastructural microscopy studies revealed PMP internalization by platelets. Flow cytometry showed PMP-platelet association (p < 0.01 vs controls, at different PMP dilutions). PMP, at 1/20 dilution, increased HA (p < 0.05 vs controls), the percentage of maximal platelet aggregation to ADP (p < 0.05 vs controls), and accelerated clotting and clot formation times (p < 0.05 vs controls). Incubation of platelets with anti-TLR-4 prior to exposure to PMP reduced PMP-platelet association (p < 0.05 vs absence of the antibody), prevented HA formation, reduced maximal platelet aggregation and normalized ROTEM parameters., Conclusions: Platelets exhibit internalization ability towards their own PMP, a process that potentiates their thrombogenicity and is partially mediated by the innate immunity receptor TLR-4., Competing Interests: Declaration of competing interest The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Platelet-derived microvesicles (pMVs) are released from platelets in physiological and pathological conditions and exhibit a wide range of prothrombotic, antithrombotic, proatherogenic, and pro-inflammatory properties. Antiplatelet agents, such as acetylsalicylic acid (ASA), are widely used for the prevention and treatment of vascular diseases, but their impact on pMV release remains poorly understood and contradictory mainly because of discrepancies in the methodology and lack of well-standardized MV assessment protocols. The present study investigated the effects of ASA not only on total pMV release but also on their phenotypes defined using the surface expression of pro-inflammatory (CD40L, CD62P, CD31) and procoagulant (PS, PAC-1) markers in healthy subjects. Fifty healthy volunteers were enrolled in the study and received a daily dose of 150 mg ASA for 3 consecutive days. Circulating pMVs were characterized and quantified before and after the intervention period using flow cytometry. Serum levels of thromboxane B2 (TXB2) and whole blood impedance platelet aggregation under arachidonic acid (AA) stimulation were also investigated to assess ASA compliance. In general, ASA did not effect pMV numbers in healthy subjects despite its effective inhibition of platelet aggregation Moreover, in premenopausal women, we noticed an increase in the number of pMVs. Further studies are needed to assess whether dose modification of ASA or combinations or changes in antiplatelet therapy would reduce pMV formation, especially in patients with cardiovascular risk factors.

The prothrombotic state in patients with atrial fibrillation (AF) is related to endothelial injury, the activation of platelets and the coagulation cascade. We evaluated the levels of platelet- (CD42b) and endothelial-derived (CD144) microparticles in the plasma patients with non-valvular AF treated with dabigatran at the time of expected minimum and maximum drug plasma concentrations. Following that, we determined the peak dabigatran plasma concentration (c peak ). CD42b increased after taking dabigatran (median [IQR] 36.7 [29.4-53.3] vs. 45.6 [32.3-59.5] cells/µL; p = 0.025). The concentration of dabigatran correlated negatively with the post-dabigatran change in CD42b (ΔCD42b, r = -0.47, p = 0.021). In the multivariate model, the independent predictors of ΔCD42b were: c peak (HR -0.55; with a 95% confidence interval, CI [-0.93, -0.16]; p = 0.007), coronary artery disease (CAD) (HR -0.41; 95% CI [-0.79, -0.02]; p = 0.037) and peripheral artery disease (PAD) (HR 0.42; 95% CI [0.07, 0.74]; p = 0.019). CD144 did not increase after dabigatran administration. These data suggest that low concentrations of dabigatran may be associated with platelet activation. PAD and CAD have distinct effects on CD42b levels during dabigatran treatment.

Alpha hemolysin (HlyA) is the major virulence factor of uropathogenic Escherichia coli (UPEC) strains. Once in circulation, a low concentration of the toxin induces an increase in intracellular calcium that activates calpains - which proteolyse cytoskeleton proteins - and also favours the exposure of phosphatidylserine (PS) in the outer leaflet of erythrocyte membranes. All these events are considered part of eryptosis, as well as the delivery of microvesicles (MVs). Within this context, we studied the delivery of MVs by erythrocytes treated with sublytic concentrations of HlyA and demonstrated that HlyA-treated erythrocytes secrete MVs of diameter ∼200 nm containing HlyA and PS by a mechanism involving an increment of intracellular calcium concentration and purinergic receptor activation. Despite the presence of toxin in their membrane, HlyA-MVs are not hemolytically active and do not induce ATP release in untreated erythrocytes, thus suggesting that the delivery of HlyA-MVs might act as a protective mechanism on the part of erythrocytes that removes the toxin from the membrane to prevent the spread of infection. Although erythrocytes have been found to eliminate denatured hemoglobin and several membrane proteins by shedding MVs, the present work has revealed for the first time that an exogenous protein, such as a toxin, is eliminated by this process. This finding sheds light on the mechanism of action of the toxin and serves to further elucidate the consequences of UPEC infection in patients exhibiting HlyA-related diseases., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Objectives: This prospective, randomized, open-label study aimed to compare the effects of antihypertensive treatment based on amlodipine or hydrochlorothiazide on the circulating microparticles and central blood pressure values of hypertensive patients., Methods: The effects of treatments on circulating microparticles were assessed during monotherapy and after the consecutive addition of valsartan and rosuvastatin followed by the withdrawal of rosuvastatin. Each treatment period lasted for 30 days. Central blood pressure and pulse wave velocity were measured at the end of each period. Endothelial, monocyte, and platelet circulating microparticles were determined by flow cytometry. Central blood pressure values and pulse wave velocity were recorded at the end of each treatment period., Results: No differences in brachial blood pressure were observed between the treatment groups throughout the study. Although similar central blood pressure values were observed during monotherapy, lower systolic and diastolic central blood pressure values and early and late blood pressure peaks were observed in the amlodipine arm after the addition of valsartan alone or combined with rosuvastatin. Hydrochlorothiazide-based therapy was associated with a lower number of endothelial microparticles throughout the study, whereas a higher number of platelet microparticles was observed after rosuvastatin withdrawal in the amlodipine arm., Conclusions: Despite similar brachial blood pressure values between groups throughout the study, exposure to amlodipine was associated with lower central blood pressure values after combination with valsartan, indicating a beneficial interaction. Differences between circulating microparticles were modest and were mainly influenced by rosuvastatin withdrawal in the amlodipine arm.

Extracellular vesicles (EVs) are emerging as key players in intercellular communication. EVs can transfer biological macromolecules to recipient cells, modulating various physiological and pathological processes. It has been shown that tumor cells secrete large amounts of EVs that can be taken up by malignant and stromal cells, dictating tumor progression. In this study, we investigated whether EVs secreted by melanoma cells in response to chemotherapy modulate tumor response to alkylating drugs. Our findings showed that human and murine melanoma cells secrete more EVs after treatment with temozolomide and cisplatin. We observed that EVs shed by melanoma cells after temozolomide treatment modify macrophage phenotype by skewing macrophage activation towards the M2 phenotype through upregulation of M2-marker genes. Moreover, these EVs were able to favor melanoma re-growth in vivo, which was accompanied by an increase in Arginase 1 and IL10 gene expression levels by stromal cells and an increase in genes related to DNA repair, cell survival and stemness in tumor cells. Taken together, this study suggests that EVs shed by tumor cells in response to chemotherapy promote tumor repopulation and treatment failure through cellular reprogramming in melanoma cells.

Biological functions of extracellular vesicles (EVs) are being discovered to be critical in neurodegenerative disorders, including Parkinson's disease (PD). A previous study using cellular models of PD has suggested that EVs derived from microglia exposed to aggregated α-synuclein (α-Syn) leads to enhanced neurotoxicity. However, the function of EVs derived from microglia not treated with aggregated a-Syn or treated with monomeric α-Syn are unclear. Here, employing a widely used cellular model of PD, i.e. SH-SY5Y cells treated with MPP + , a well-established parkinsonian toxicant, we revealed that microglial EVs, when not stimulated by aggregated α-Syn, appeared to be protective, and the mechanisms, though remain to be defined further, appeared to involve mitochondrial dynamics, especially mitochondrial fission., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The purpose of the present study was to test the hypothesis that doxorubicin (DX) and cyclophosphamide (CY) adjuvant chemotherapy (CHT) acutely impairs neurovascular and hemodynamic responses in women with breast cancer. Sixteen women (age: 47.0 ± 2.0 yr; body mass index: 24.2 ± 1.5 kg/m) with stage II-III breast cancer and indication for adjuvant CHT underwent two experimental sessions, saline (SL) and CHT. In the CHT session, DX (60 mg/m 2 ) and CY (600 mg/m 2 ) were administered over 45 min. In the SL session, a matching SL volume was infused in 45 min. Muscle sympathetic nerve activity (MSNA) from peroneal nerve (microneurography), calf blood flow (CBF; plethysmography) and calf vascular conductance (CVC), heart rate (HR; electrocardiography), and beat-to-beat blood pressure (BP; finger plethysmography) were measured at rest before, during, and after each session. Venous blood samples (5 ml) were collected before and after both sessions for assessment of circulating endothelial microparticles (EMPs; flow cytometry), a surrogate marker for endothelial damage. MSNA and BP responses were increased ( P < 0.001), whereas CBF and CVC responses were decreased ( P < 0.001), during and after CHT session when compared with SL session. Interestingly, the vascular alterations were also observed at the molecular level through an increased EMP response to CHT ( P = 0.03, CHT vs. SL session). No difference in HR response was observed ( P > 0.05). Adjuvant CHT with DX and CY in patients treated for breast cancer increases sympathetic nerve activity and circulating EMP levels and, in addition, reduces muscle vascular conductance and elevates systemic BP. These responses may be early signs of CHT-induced cardiovascular alterations and may represent potential targets for preventive interventions. NEW & NOTEWORTHY It is known that chemotherapy regimens increase the risk of cardiovascular events in patients treated for cancer. Here, we identified that a single cycle of adjuvant chemotherapy with doxorubicin and cyclophosphamide in women treated for breast cancer dramatically increases sympathetic nerve activity and circulating endothelial microparticle levels, reduces the muscle vascular conductance, and elevates systemic blood pressure.

Medical devices, such as ventricular assist devices (VADs), introduce both foreign materials and artificial shear stress to the circulatory system. The effects these have on leukocytes and the immune response are not well understood. Understanding how these two elements combine to affect leukocytes may reveal why some patients are susceptible to recurrent device-related infections and provide insight into the development of pump thrombosis. Biomaterials-DLC: diamond-like carbon-coated stainless steel; Sap: single-crystal sapphire; and Ti: titanium alloy (Ti 6 Al 4 V) were attached to the parallel plates of a rheometer. Whole human blood was left between the two discs for 5 minutes at +37°C with or without the application of shear stress (0 s -1 or 1000 s -1 ). Blood was removed and used for complete blood cell counts, flow cytometry (leukocyte activation, cell death, microparticle generation, phagocytic ability, and reactive oxygen species [ROS] production), and the production of pro-inflammatory cytokines. L-selectin expression on monocytes was decreased when blood was exposed to the biomaterials both with and without shear. Applying shear stress to blood on a Sap and Ti surface led to activation of neutrophils shown as decreased L-selectin expression. Sap and Ti blunted the LPS-stimulated macrophage migration inhibitory factor (MIF) production, most notably when sheared on Ti. The biomaterials used here have been shown to activate leukocytes in a static environment. The introduction of shear appears to exacerbate this activation. Interestingly, a widely accepted biocompatible material (Ti) utilized in many different types of devices has the capacity for immune cell activation and inhibition of MIF secretion when combined with shear stress. These findings contribute to our understanding of the contribution of biomaterials and shear stress to recurrent infections and vulnerability to sepsis in some VAD patients as well as pump thrombosis., (© 2019 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Microvesicles (MVs) have been extensively identified in various biological fluids including bronchoalveolar lavage fluid (BALF), peripheral blood and ascitic fluids. Our previous study showed that MVs are responsible for acute lung injury, but the exact mechanism underlying MVs formation remains poorly understood. In the present study, we investigate the potential role of RhoA/Rock signaling in MVs generation and the biological activity of MVs in ventilator-induced lung injury (VILI). Our results revealed that high tide ventilation induced super MVs releasing into the lung and subsequently caused lung inflammation. Strikingly, intratracheal instillation of MVs that isolated from highly ventilated mice triggered significant lung inflammation in naive mice. The MVs production is strongly correlated with lung inflammation and the upregulation of RhoA, Rock and phospho-Limk (phosphorylation of Limk is the activated form). RhoA inhibitor decreased the expression of Rock and the phosphorylation of Limk, decreased MVs production and alleviated lung inflammation. Rock inhibitor also decreased the phosphorylation of Limk, decreased MVs production and alleviated lung inflammation. Our data demonstrated that the production of MVs requires RhoA/Rock signaling, and VILI might be potentially prevented by targeting RhoA/Rock signaling pathway., (Copyright © 2019. Published by Elsevier B.V.)