Proinflammatory Microparticles Research Articles

No relation between increases of spleen volume and reductions in circulating pro-inflammatory microparticles following fourteen days of apnea and hypoxia exposures

INTRODUCTION: Indigenous breath-hold diving populations have comparatively large spleen volumes, suggesting a functional benefit of a larger spleen in the context of diving. The spleen plays a role in clearing blood-borne microparticles (MPs). Pro-inflammatory MPs are increased with diving and have been associated with the etiology of decompression sickness. Thus, a potential benefit of a larger spleen may be enhanced splenic clearance of MPs. Repeated apneas and hypoxia exposure increases spleen volume in people without breath-hold diving ancestry. PURPOSE: Test the hypothesis that repeated apnea and hypoxia exposures reduce blood-borne pro-inflammatory MPs and the reduction is correlated with changes in spleen size. Methods: 20 healthy adults (10 women) completed 10 maximal apneas and 3 h of exposure to normobaric hypoxia (FiO2=0.14) for 14 days within a 16-day period. On Days 1 and 14, pre-exposure spleen volume was measured via ultrasound using the Pilström equation and venous blood was collected for blood-borne pro-inflammatory MPs analysis via flow cytometry. MPs data were log transformed for analyses. Data are presented as mean ± SD. Results: Average apnea duration increased from Day 1 (76±31 s) to Day 14 (91±30 s, p=0.004, dz=0.48). Pre-exposure spleen volume increased from Day 1 (169±36 ml) to Day 14 (182±39 ml, p=0.002, dz=0.33). Total MPs decreased from Day 1 (1407±814 #/μl) to Day 14 (1092±561#/μl, p=0.047, dz=0.45). MPs expressing CD66b+ (neutrophil origin; 194±89 vs. 156±72 #/μl, p=0.014, dz=0.47) and CD146 (endothelial origin; 337±197 vs. 250±116 #/μl, p=0.037, dz=0.53) were lower on Day 14, but MPs expressing CD41a (platelet origin) did not differ between Days 1 (82±78 #/μl) and 14 (55±38 #/μl, p=0.166, dz=0.45). Changes in MP counts from Day 1 to Day 14 were not correlated with changes in spleen volume for Total MPs (r=0.02, p=0.93) nor MPs expressing CD66b+ (r=0.30, p=0.23), CD146 (r=-0.04, p=0.86), or CD41a (r=0.31, p=0.20). CONCLUSION: Repeated apnea and hypoxia exposures increase spleen volume and reduce blood-borne pro-inflammatory MPs. However, there was no relation between exposure-induced changes in spleen size and MP counts. Thus, spleen size may not directly impact MP clearance. Rather, alterations in the MP phagocytic properties found within the spleen or another systemic change likely contribute to the observed reductions in MPs. Further work is needed to understand the role of splenic function in these observations. Support: Offce of Naval Research (N00014-20-1-2593). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Potential Prognostic, Diagnostic and Therapeutic Targets for Recurrent Arrhythmias in Patients with Coronary Restenosis and Reocclusions After Coronary Stenting.

The interplay of oxidative stress, proinflammatory microparticles, and proinflammatory cytokines in recurrent arrhythmias is unknown in elderly patients with coronary restenosis and reocclusions after coronary stenting. This research sought to investigate the potential diagnostic and therapeutic targets for recurrent arrhythmias in patients with coronary restenosis and reocclusions after coronary stenting. We examined whether oxidative stress, proinflammatory microparticles, and proinflammatory cytokines could have effects that lead to recurrent arrhythmias in elderly patients with coronary restenosis and reocclusions. We measured the levels of malondialdehyde (MDA), CD31 + endothelial microparticle (CD31 EMP), CD62E + endothelial microparticle (CD62E + EMP), high-sensitivity C-reactive protein (hs-CRP), interleukin- 1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α), as well as oxidized low-density lipoprotein (OX-LDL), and assessed the effects of relationship between oxidative stress, proinflammatory microparticles, and proinflammatory cytokines on recurrent atrial and ventricular arrhythmias in elderly patients with coronary restenosis and reocclusions after coronary stenting. The levels of CD31 + EMP, CD62E + EMP, MDA, hs-CRP, IL-1β, IL-6, IL-8, TNF-α and OX-LDL were found to be increased significantly in coronary restenosis + recurrent atrial arrhythmia group compared to without coronary restenosis and coronary restenosis + without recurrent atrial arrhythmia groups, respectively (P < 0.001). Patients in the coronary reocclusion + recurrent ventricular arrhythmia group also exhibited significantly increased levels of CD31 + EMP, CD62E + EMP, MDA, hs-CRP, IL-1β, IL-6, IL-8, TNF-α and OXLDL compared to without coronary reocclusion and coronary reocclusion + without recurrent ventricular arrhythmia groups, respectively (P < 0.001). Proinflammatory microparticles, proinflammatory cytokines, and oxidative stress might act as potential targets for recurrent arrhythmias in patients with coronary restenosis and reocclusions after coronary stenting.

Interplay of pro-inflammatory cytokines, pro-inflammatory microparticles and oxidative stress and recurrent ventricular arrhythmias in elderly patients after coronary stent implantations

BackgroundThe roles of pro-inflammatory microparticles, pro-inflammatory cytokines and oxidative stress were unknown in elderly patients with recurrent ventricular arrhythmias (VA). We evaluated whether cross talk between oxidative stress, pro-inflammatory microparticles, and pro-inflammatory cytokines play the roles in elderly patients with recurrent VA after coronary stenting. This research sought to investigate the effects of oxidative stress, pro-inflammatory microparticles, and pro-inflammatory cytokines on recurrent VA in elderly patients after coronary stenting. MethodsIn this study, we included 613 consecutive elderly patients with recurrent ventricular arrhythmias induced by coronary reocclusions after coronary stenting. We measured CD31+ endothelial microparticle (CD31+EMP), CD62E+ endothelial microparticle (CD62E+EMP), high-sensitivity C-reactive protein (hs-CRP), aldosterone (ALD), malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), soluble tumor necrosis factor receptor-1 (sTNFR-1) and soluble tumor necrosis factor receptor-2 (sTNFR-2) in elderly patients with recurrent VA and assessed impacts of pro-inflammatory microparticles, pro-inflammatory cytokines and oxidative stress on recurrent VA in elderly patients after coronary stenting. ResultsThe levels of CD31+EMP, CD62E+EMP, hs-CRP, ALD, MDA, TNF-α, sTNFR-1 and sTNFR-2 were increased in recurrent malignant ventricular arrhythmia, sustained ventricular tachycardia, multiple ventricular premature beat and left and right ventricular bundle branch block groups (P < 0.001) in elderly patients with coronary reocclusions after coronary stent implantation. Upregulation of pro-inflammatory microparticles, pro-inflammatory cytokines and oxidative stress markers induced recurrent VA in elderly patients after coronary stenting. ConclusionsHigh levels of pro-inflammatory microparticles, pro-inflammatory cytokines and oxidative stress markers were associated with recurrent VA in elderly patients after coronary stenting. Our results suggested that the pro-inflammatory microparticles, pro-inflammatory cytokines and oxidative stress may simultaneously induce and aggravate recurrent VA in elderly patients after coronary stenting.

Tiotropium inhibits proinflammatory microparticle generation by human bronchial and endothelial cells

Tiotropium is a muscarinic antagonist that reduces the risk of acute exacerbations of chronic obstructive pulmonary disease, possibly through an as yet incompletely characterized anti-inflammatory activity. We hypothesized that muscarinic activation of bronchial epithelial cells and endothelial cells causes the release of proinflammatory microparticles and that tiotropium inhibits the phenomenon. Microparticle generation was assessed by a functional assay, by flow cytometry and by NanoSight technology. Immortalized bronchial epithelial cells (16HBE) and umbilical vein endothelial cells were treated with acetylcholine in the presence of varying concentrations of tiotropium. Intracellular calcium concentration, extracellular regulated kinase phosphorylation and chemokine content in the conditioned media were assessed by commercial kits. Acetylcholine causes microparticle generation that is completely inhibited by tiotropium (50 pM). Microparticles generated by acetylcholine-stimulated cells increase the synthesis of proinflammatory mediators in an autocrine fashion. Acetylcholine-induced upregulation of microparticle generation is inhibited by an inhibitor of extracellular regulated kinase phosphorylation and by a phospholipase C inhibitor. Tiotropium blocks both extracellular regulated kinase phosphorylation and calcium mobilization, consistent with the hypothesis that the drug prevents microparticle generation through inhibition of these critical pathways. These results might contribute to explain the effect of tiotropium in reducing acute exacerbations of chronic obstructive pulmonary disease.

Platelets release proinflammatory microparticles in anti-neutrophil cytoplasmic antibody-associated vasculitis.

The biological functions of the platelets contributing to ANCA-associated vasculitis (AAV) are largely unclear. The current study aimed to investigate the potential role of platelet-derived microparticles (PMPs) in AAV. In the current study, microparticles in AAV patients were analysed by flow cytometry, and PMPs were probed for relative levels of 640 bioactive proteins secreted from patients' platelets using antibody microarrays. These data were then correlated with clinical and pathological parameters. PMPs were significantly increased in 69 AAV patients, predominantly MPO-ANCA positive patients in active stage compared with in remission [4406.8/μl (2135.4, 5485.0) vs 549.7/μl (350, 708.5), P < 0.0001], and 43% of microparticles in active AAV were PMPs. Compared with 15 patients in remission, highly expressed proinflammatory molecules in the microparticles from platelets in 15 AAV patients in active stage revealed that potential functions of PMPs were promotion of the effect of chemotaxis, adhesion, growth and apoptosis (all the patients for array analysis were MPO-ANCA positive). The level of PMPs had a significant association with disease activity, inflammation, and renal damage. PMPs may serve as inflammatory propagators through their wide production of proinflammatory cytokines in AAV, potentially providing a novel therapeutic target.

Downregulation of Microparticle Release and Pro-Inflammatory Properties of Activated Human Polymorphonuclear Neutrophils by LMW Fucoidan

Exposition of neutrophils (polymorphonuclear neutrophils, PMNs) to bacterial products triggers exacerbated activation of these cells, increasing their harmful effects on host tissues. We evaluated the possibility of interfering with the classic immune innate responses of human PMNs exposed to bacterial endotoxin (lipopolysaccharide, LPS), and further stimulated with bacterial formyl peptide (N-formyl-methionine-leucine-phenylalanine, fMLP). We showed that the low- molecular-weight fucoidan (LMW-Fuc), a polysaccharide extracted from brown algae, attenuated the exacerbated activation induced by fMLP on LPS-primed PMNs, in vitro, impairing chemotaxis, NET formation, and the pro-survival and pro-oxidative effects. LMW-Fuc also inhibited the activation of canonical signaling pathways, AKT, bad, p47phox and MLC, activated by the exposition of PMN to bacterial products. The activation of PMN by sequential exposure to LPS and fMLP induced the release of L-selectin⁺ microparticles, which were able to trigger extracellular reactive oxygen species production by fresh PMNs and macrophages. Furthermore, we observed that LMW-Fuc inhibited microparticle release from activated PMN. In vivo experiments showed that circulating PMN-derived microparticles could be detected in mice exposed to bacterial products (LPS/fMLP), being downregulated in animals treated with LMW-Fuc. The data highlight the autocrine and paracrine role of pro-inflammatory microparticles derived from activated PMN and demonstrate the anti-inflammatory effects of LMW-Fuc on these cells.

How to quantify microparticles in RBCs? A validated flow cytometry method allows the detection of an increase in microparticles during storage

The procoagulant and proinflammatory microparticles (MPs) released during storage of packed red blood cells (pRBCs) can potentially modify transfusion benefits. A robust method to quantify MPs in pRBCs is needed to evaluate their impact in clinical trials. The objective was to validate the preanalytic conditions required to prepare pRBC supernatant as well as a method to quantify and evaluate MP variations over 42 days of pRBC storage.A flow cytometry method with size-calibrated beads was developed and fully validated. Quantification of MPs in pRBCs (n = 109) was assessed during short-term (7 days) and long-term (42 days) storage at 4°C, during short-term storage (8 hours) at room temperature, and after 2 years frozen. Repeatability, reproducibility, and linearity of the quantification method were validated, and variations during conservation are presented. There was high variability in RBC (erythrocyte) MP (ERMP) and platelet MP (PMP) levels between RBC units, depending on the filter used for leukocyte reduction. During the 42 days of storage at 4°C, significant increases in ERMPs and PMPs occurred (from 58 to 138 ERMPs/µL from Day 2 to Day 42; p = 0.0002; and from 326 to 771 PMPs/µL from Day 2 to Day 42; p = 0.00026). We use a robust method to confirm that ERMPs and PMPs are present to various degrees in pRBCs and that storage for 42 days significantly increases their generation. This method is robust enough to allow MP quantification in pRBCs and is adapted to evaluate the clinical impact of transfused MPs in prospective clinical trials.

Endothelial, platelet, and macrophage microparticle levels do not change acutely following transcatheter aortic valve replacement.

BackgroundPatients with severe aortic stenosis have increased levels of prothrombotic and proinflammatory microparticles (MP), and MPs actively regulate pathological processes that lead to atherothrombotic cardiovascular events. Shear stress is a validated stimulus of MP production, and abnormal shear stress in aortic stenosis increases MP release in ex-vivo studies. We hypothesized that in patients with severe aortic stenosis, percutaneous replacement of the aortic valve (TAVR) would reduce abnormal shear stress and would decrease levels of circulating MPs.FindingsThe experimental protocol utilized flow cytometry (FC) and nanoparticle tracking analysis (NTA) to quantify circulating plasma MP levels in aortic stenosis patients at baseline and 5 days after TAVR. The baseline and 5 day MP counts measured by FC were 6.10⋅105 ± 1.21⋅105 MP/μL and 5.74⋅105 ± 9.54⋅104 MP/μL, respectively (p = 0.91). The baseline and 5 day MP counts measured by NTA were 9.29⋅1013 ± 1.66⋅1013 MP/μL and 3.95⋅1014 ± 3.11⋅1014 MP/μL, respectively (p = 0.91). When MPs were stratified by cell source, there was no difference in pre/post TAVR endothelial, platelet, or leukocyte MP levels.ConclusionLevels of circulating MPs do not change acutely following TAVR therapy for aortic stenosis.Trial registered at clinicaltrials.gov NCT02193035 on July 11, 2014.Electronic supplementary materialThe online version of this article (doi:10.1186/s12952-016-0051-2) contains supplementary material, which is available to authorized users.

Liraglutide protects Rin-m5f β 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 β cells were stimulated by H2 O2 or a combination of IL-1β 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.

Rapid shedding of proinflammatory microparticles by human mononuclear cells exposed to cigarette smoke is dependent on Ca2+ mobilization

Microparticles are membrane vesicles shed by cells upon activation and apoptosis. Agonists capable of inducing microparticle generation include cytokines, bacterial products, P-selectin, histamine. Cigarette smoke extract has also been recognized as an agonist involved in microparticle generation with an apoptosis-dependent mechanism. We investigated the possibility that cigarette smoke extract induces the rapid generation of proinflammatory microparticles by human mononuclear cells with a calcium-dependent mechanism. Human mononuclear cells were exposed to cigarette smoke extract. [Ca(2+)]i mobilization was assessed with the fluorescent probe Fluo-4 NW. Microparticles were quantified with a prothrombinase assay and by flow cytometry. Normal human bronchial epithelial cells and A549 alveolar cells were incubated with cigarette smoke extract-induced microparticles and the generation of ICAM-1, IL-8, and MCP-1 was assessed by ELISA. Exposure to cigarette smoke extract induced a rapid increase in [Ca(2+)]i mobilization. Microparticle generation was also increased. EGTA, verapamil and the calmodulin inhibitor, W-7, inhibited microparticle generation. Incubation of lung epithelial cells with cigarette smoke extract-induced microparticles increased the expression of proinflammatory mediators. Exposure of mononuclear cells to cigarette smoke extract causes a rapid shedding of microparticles with a proinflammatory potential that might add to the mechanisms of disease from tobacco use.

Role of tumour necrosis factor receptor-1 and nuclear factor-κB in production of TNF-α-induced pro-inflammatory microparticles in endothelial cells.

Tumour necrosis factor-α (TNF-α) is upregulated in many inflammatory diseases and is also a potent agent for microparticle (MP) generation. Here, we describe an essential role of TNF-α in the production of endothelial cell-derived microparticles (EMPs) in vivo and the function of TNF-α-induced EMPs in endothelial cells. We found that TNF-α rapidly increased blood levels of EMPs in mice. Treatment of human umbilical vein endothelial cells (HUVECs) with TNF-α also induced EMP formation in a time-dependent manner. Silencing of TNF receptor (TNFR)-1 or inhibition of the nuclear factor-κB (NF-κB) in HUVECs impaired the production of TNF-α-induced EMP. Incubation of HUVECs with PKH-67-stained EMPs showed that endothelial cells readily engulfed EMPs, and the engulfed TNF-α-induced EMPs promoted the expression of pro-apoptotic molecules and upregulated intercellular adhesion molecule-1 level on the cell surface, which led to monocyte adhesion. Collectively, our findings indicate that the generation of TNF-α-induced EMPs was mediated by TNFR1 or NF-κB and that EMPs can contribute to apoptosis and inflammation of endothelial cells.

Microparticle formation after co‐culture of human whole blood and umbilical artery in a novel in vitro model of flow

Cardiovascular disease (CVD) is now the largest killer in western society, and the importance of interactions between vascular endothelium and circulating blood components in disease pathogenesis is well established. Microparticles are a heterogeneous population of <1 μm blood borne particles that arise from blebbing or shedding of cell membranes. The microparticle population includes several classes of apoptotic bodies; however, increased numbers of procoagulant microparticles have been described in plasma from people with CVD. We have previously demonstrated that interactions of monocytes and platelets with isolated inflamed endothelial cells lead to production of pro-coagulant tissue factor bearing microparticles under laminar flow conditions. Here we have investigated microparticle production after perfusion of human whole blood through intact inflamed human umbilical artery. When blood was perfused through umbilical arteries which had been pre-stimulated with tumour necrosis factor (TNFα) for 18 h under flow conditions, there was significantly increased production of microparticles from both platelet and non-platelet sources, in particular from erythrocytes. To determine whether microparticles generated during interactions with inflamed endothelium could induce a pro-inflammatory response in trans, we isolated microparticles by centrifugation after co-culture and incubated with isolated quiescent endothelial cells followed by measurement of reactive oxygen species formation. Microparticles derived from co-culture with inflamed endothelium induced significantly enhanced levels of reactive oxygen species (ROS). These data suggest that presence of an inflamed endothelium causes release of pro-inflammatory microparticles from circulating blood cells, which could contribute to prolonged endothelial activation and subsequent atherosclerotic changes in blood vessels subjected to inflammatory insult.

Stimulation of blood mononuclear cells with bacterial virulence factors leads to the release of pro-coagulant and pro-inflammatory microparticles

Severe infectious diseases remain a major and life-threatening health problem. In serious cases a systemic activation of the coagulation cascade and hypovolemic shock are critical complications that are associated with high mortality rates. Here we report that blood mononuclear cells, stimulated with M1 protein of Streptococcus pyogenes or other bacterial virulence factors, produce not only pro-coagulant, but also pro-inflammatory microparticles (MPs). Our results also show that activation of the contact system on MPs contributes to these two effects. Phosphatidylserine (PS) plays an important role in these processes as its upregulation on MPs allows an assembly and activation of the contact system. This in turn results in stabilization of the tissue factor-induced clot and a processing of high-molecular-weight kininogen by plasma kallikrein followed by the release of bradykinin, a potent vascular mediator. Pro-coagulant monocyte-derived MPs were identified in plasma samples from septic patients and further analysis of MPs from these patients revealed that their pro-coagulant activity is dependent on the tissue factor- and contact system-driven pathway.