Extracellular vesicles opsonized by monomeric C-reactive protein (CRP) are accessible as autoantigens in patients with systemic lupus erythematosus and associate with autoantibodies against CRP.

The pentraxin C-reactive protein (CRP) is a pentameric protein now known to be able to undergo dissociation into a monomeric, modified isoform, referred to as mCRP. In carefully assessing the bioactivities of each isoform, mCRP has strong pro-inflammatory activities while pCRP has mild anti-inflammatory activities. Systemic lupus erythematosus (SLE) is a disease characterized by a vast number of autoantibodies, including anti-CRP autoantibodies which have been associated with SLE disease activity and lupus nephritis. The origin of these autoantibodies is currently unknown. Extracellular vesicles (EVs) have been implicated in SLE pathogenesis as they can expose nuclear antigens on their outside surface, thereby being a potential adjuvant for the generation of autoantibodies. Herein, we studied exposure of both pCRP and mCRP on EVs in SLE plasma and the implications of each in disease activity, organ damage and clinical manifestations. We used flow cytometry to detect CRP isoforms on EV surfaces in 67 well-characterized SLE patients and 60 sex- and age-matched healthy controls. Autoantibodies against mCRP were measured using ELISA. We found an abundance of both pCRP and mCRP on SLE EVs compared to controls. Furthermore, mCRP+ but not pCRP+ EVs were elevated in patients with active disease and in anti-CRP positive patients. The proportions of mCRP+ EVs were lower in patients with acquired organ damage, especially in patients with lupus nephritis (LN), and displayed an inverse relationship with disease duration in LN and patients with active disease. Speculatively, these data suggest EV-bound mCRP as a relevant factor in SLE pathogenesis, which could contribute to development of anti-CRP autoantibodies by stimulating an immune response.

The binding of SLE autoantibodies to mitochondria.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by immune complexes. Because these complexes contain mitochondrial components, we assessed the presence of antibodies to whole mitochondria (wMITO) using an ELISA in which mitochondria from mouse liver are bound to microtiter plates pre-coated with poly-l-lysine. Studies with this ELISA demonstrated that SLE plasmas contain abundant anti-wMITO activity. While digestion with DNase 1 did not affect anti-wMITO activity, adsorption of plasma on DNA affinity columns could reduce binding activity. Assay for anti-mitochondrial antibodies (AMA) by immunofluorescence and an ELISA with the M2 antigen (2-oxo-acid dehydrogenase protein complex) showed a low frequency of positivity, indicating that AMA and anti-wMITO are distinct specificities. In the study of 204 patients with SLE, the levels of anti-wMITO were higher in active SLE and correlated with levels of anti-DNA. These findings suggest that anti-wMITO can form immune complexes with mitochondria which may drive pathogenesis.

Microparticles in the blood of patients with SLE: Size, content of mitochondria and role in circulating immune complexes.

OBJECTIVE: Microparticles (MPs) are small extracellular vesicles released from apoptotic or activated cells through a blebbing process. MPs express surface molecules from their parental cells and they bind IgG to form circulating immune complexes (MP-ICs) in patients with systemic lupus erythematosus (SLE). Through investigation of MP size, IgG expression, content of nucleic acids and mitochondrial molecules, we hypothesized that unrecognized particle populations can be identified in SLE. METHODS: We investigated 327 well-characterized SLE patients and 304 controls divided into two sets (280/280 and 47/24). We measured MPs by flow cytometry using a gating strategy to encompass small (0.2-0.7 µm) and large (0.7-3.0 µm) MPs. Nucleic acids were labeled with SYTO 13 and mitochondria with MitoTracker. Expression of mitochondria markers TOM-20 and Hexokinase 1 and the presence of IgG was investigated. RESULTS: MPs staining with SYTO 13 were more frequent in 280 SLE patients compared to 280 controls. In 47 SLE patients, levels of large MPs were elevated compared to 24 controls. The majority of large MPs contained mitochondria (mitoMPs). The number of mitoMPs associated positively with high disease activity, anti-dsDNA antibodies and pro-inflammatory cytokines. Patients with active lupus nephritis had higher levels of mitoMPs and IgG-positive mitoMPs. CONCLUSION: Blood of patients with SLE contain a previously unrecognized population of circulating large MPs with bound IgG and mitochondrial proteins. Levels of these particles are related to several measures of active SLE, suggesting that these structures may have a role in disease pathogenesis.

Meal intake increases circulating procoagulant microparticles in patients with type 1 and type 2 diabetes mellitus.

Diabetes mellitus (DM) is associated with prothrombotic alterations, and postprandial hyperglycemia is an independent risk factor for cardiovascular complications. We therefore investigated whether a standardized mixed meal alters circulating microparticles (MPs) and their procoagulant activity in DM patients. Patients with DM type 1 (T1DM, n = 11) and type 2 (T2DM; n = 9) were studied before and 90 min after a standardized meal (without premeal insulin). MPs in plasma derived from platelets (PMPs), endothelial cells (EMPs), or monocytes (MMPs) were measured by flow cytometry. MP-induced thrombin generation in plasma was assessed by a calibrated automated thrombogram. In the fasting state, MPs did not differ significantly between T1DM and T2DM. Meal intake increased the following microparticles: PMPs expressing phosphatidylserine (by 55%, on average), P-selectin (by 86%), and tissue factor (TF; by 112%); EMPs expressing E-selectin (by 96%) and MMPs expressing TF (by 164%), with no significant group differences between T1DM and T2DM. There were no increments in EMPs expressing phosphatidylserine or TF. Meal intake increased MP-induced thrombin generation similarly in T1DM and T2DM with increased endogenous thrombin potential (p = 0.02) and peak thrombin (p = 0.03) and shortened time to peak (p = 0.02). Phosphatidylserine inhibition by lactadherin completely abolished MP-induced thrombin generation, while an anti-TF antibody had no effect. In conclusion, meal intake increased several types of circulating MPs in patients with diabetes mellitus. These MPs have a procoagulant potential, which is related to phosphatidylserine expression and negatively charged MP surfaces rather than to TF.

Microvesicles from patients with acute coronary syndrome enhance platelet aggregation.

Microvesicles (MVs) released from leukocytes, platelets and endothelial cells are elevated in patients with acute coronary syndrome (ACS). In the present study, we assessed the potential pro-aggregatory properties of MVs obtained from ACS patients. Thus, we divided the patients into two groups based on clopidogrel-responsiveness, i.e. high on-treatment platelet reactivity (HPR; n = 16), and low or normal on-treatment platelet reactivity (non-HPR; n = 14), respectively. MVs from patients were obtained by high-speed centrifugation, and the pro-aggregatory effect of MVs added to fresh isolated platelets from healthy subjects were analyzed by 96-well microplate aggregometry. MVs from HPR patients significantly enhanced spontaneous platelet aggregation around two times more than MVs from non-HPR patients. The pro-aggregatory effect of three out of four MV phenotypes correlated to MV-concentrations as determined by flow cytometry. Furthermore, MVs from patients with diabetes mellitus (n = 9) had a stronger pro-aggregatory effect compared to MVs from those without diabetes (n = 21; p = .025 between groups). In conclusion, MVs from ACS patients with clopidogrel non-responsiveness enhance platelet aggregation, as do MVs from ACS patients with diabetes. Thus, MVs from patients with hyperreactive platelets boost platelet aggregation. Blocking MV-formation may reduce platelet hyperreactivity.

Changes in microparticle profiles by vitamin D receptor activation in chronic kidney disease - a randomized trial.

BACKGROUND: Microparticles (MPs) are biomarkers and mediators of disease through their expression of surface receptors, reflecting activation or stress in their parent cells. Endothelial markers, ICAM-1 and VCAM-1, are implicated in atherosclerosis and associated with cardiovascular risk. Chronic kidney disease (CKD) patients have endothelial dysfunction and high levels of endothelial derived MPs. Vitamin D treatment has been reported to ameliorate endothelial function in CKD patients. We aimed to examine cell specific MP profiles and concentrations of MPs expressing the atherosclerotic markers ICAM-1 and VCAM-1 after treatment with paricalcitol in patients with CKD stage 3-4. METHODS: Sub-study of the previously reported SOLID trial where 36 patients were randomly assigned to placebo, 1 or 2 µg paricalcitol, for 12 weeks. MPs were measured by flow cytometry after labelling with antibodies against endothelial (CD62E), platelet (CD62P, CD41, CD154) leukocyte (CD45) and vascular (CD54, CD106) markers. RESULTS: Patients had a mean age of 65 years with a mean eGFR of 40 mL/min/1.73m2. Concentrations of ICAM-1 positive MPs were significantly reduced by treatment (repeated measures ANOVA p = 0.04). Repeated measures MANOVA of concentrations of endothelial, platelet and leukocyte MPs showed sustained levels in the 2 µg treatment group (p = 0.85) but a decline in the 1 µg (p = 0.04) and placebo groups (p = 0.005). CONCLUSIONS: Treatment with paricalcitol reduces concentrations of ICAM-1 positive MPs. This is accompanied by sustained concentrations of all cell specific MPs in the 2 µg group, and decreasing concentrations in the other groups, possibly due to a more healthy and reactive endothelium with paricalcitol treatment.

Increased concentrations of platelet- and endothelial-derived microparticles in patients with myocardial infarction and reduced renal function- a descriptive study.

BACKGROUND: Patients with chronic kidney disease (CKD) have a high risk of recurring thrombotic events following acute myocardial infarction (AMI). Microparticles (MPs) are circulating small vesicles shed from various cells. Platelet microparticles (PMPs) reflect platelet activation and endothelial microparticles (EMPs) reflect endothelial activation or dysfunction. Both increase following AMI, and may mediate important biological effects. We hypothesized that AMI patients with CKD have further elevated PMPs and EMPs compared with non-CKD patients, despite concurrent antithrombotic treatment. METHODS: We performed a descriptive study of patients with AMI. Fasting blood samples were acquired from 47 patients on dual antiplatelet treatment. Patients were stratified by renal function: normal (H; n = 19) mean eGFR 88; moderate CKD (CKD3; n = 15) mean eGFR 47, and severe CKD (CKD4-5; n = 13) mean eGFR 20 mL/min/1.73 m2. MPs were measured by flow-cytometry and phenotyped according to size (< 1.0 µm) and expression of CD41 (GPIIb; PMPs) and CD62E (E-selectin; EMPs). In addition, expression of platelet activation markers P-selectin (CD62P) and CD40ligand (CD154) were also investigated. RESULTS: PMPs expressing CD40 ligand were higher in CKD4-5: 210 /µl (174-237); median and interquartile range; vs. group H; 101 /µl (71-134; p < 0.0001) and CKD 3: 142 /µl (125-187; p = 0.006). PMPs expressing P-selectin were higher in CKD4-5 compared with H, but not in CKD3. EMPs were higher in CKD4-5; 245 /µl (189-308) compared with H; 83 /µl (53-140; p < 0.0001) and CKD3; 197 /µl (120-245; p < 0.002). CONCLUSIONS: In AMI patients, PMPs and EMPs from activated platelets and endothelial cell are further elevated in CKD patients. This indicate impaired endothelial function and higher platelet activation in CKD patients, despite concurrent antiplatelet treatment.

Microparticles as autoantigens in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of antibodies to components of the cell nucleus (antinuclear antibodies or ANAs) and the formation of immune complexes with nuclear antigens. These complexes can drive pathogenesis by depositing in the tissue to incite inflammation or induce cytokine production by cells of the innate immune system. While ANAs can bind to purified nuclear molecules, nuclear autoantigens in vivo most likely exist attached to other molecules or embedded in larger structures. Among these structures, microparticles (MPs) are membrane bound vesicles that are released from dead and dying cells by a blebbing process; MPs can also be released during activation of platelets. The presence of MPs in the blood or tissue culture media can be assayed by flow cytometry on the basis of light scattering as well as binding of marker antibodies to identify the cell of origin. As shown by biochemical analyses, MPs contain an ensemble of intracellular components including nuclear, cytoplasmic and membrane molecules. Because of the display of these molecules on the particle surface or in an otherwise accessible form, ANAs, including anti-DNA, can bind to particles. Levels of MPs are increased in the blood of patients with SLE, with flow cytometry demonstrating the presence of IgG-containing particles. In addition to forming immune complexes, MPs can directly stimulate immune responses. Together, these findings suggest an important role of particles in the pathogenesis of SLE and their utility as biomarkers.

The expression of microvesicles in the blood of patients with Graves' disease and its relationship to treatment.

OBJECTIVE: Graves' disease (GD) is an autoimmune disease characterized by the presence of circulating autoantibodies against thyroid-stimulating hormone (TSH) receptor. Despite extensive research, the pathogenic mechanisms remain unclear. Immune responses associated with the disease may lead to cell activation/apoptosis and the release of microvesicles (MVs) into the circulation. MVs can display biological activities which may aggravate GD further. We studied immune mechanisms in GD by investigating the numbers and phenotype of circulating MVs in patients before and after antithyroid therapy with thiamazole. PATIENTS AND MEASUREMENTS: Samples were obtained from 15 patients with GD in the acute phase of hyperthyroidism and following 17-26 months treatment and 14 healthy controls. MVs from platelets, endothelial cells and monocytes exposing inflammation/activation markers (P-selectin, CD40 ligand, E-selectin and HMGB1) and MVs containing nuclear molecules were measured with flow cytometry. RESULTS: Patients had elevated baseline values of MVs (P < 0·001 for all types of MVs), while the levels decreased during thiamazole treatment (P < 0·05 for all types of MVs). The majority of MV populations remained, however, significantly higher in patients after treatment compared to levels in controls. CONCLUSIONS: GD patients have elevated levels of MVs that carry molecules with potential biological activities. MVs are significantly reduced after antithyroid treatment with thiamazole but still higher compared to levels in healthy controls. Assessment of MV levels and pattern may therefore provide additional information on underlying immune disturbances not obtained by measurements of hormone levels alone.

Deletion of mPGES-1 affects platelet functions in mice.

Microsomal prostaglandin E2 synthase-1 (mPGES-1) constitutes an essential player in inflammation and is involved in the pathogenesis of rheumatoid arthritis. Platelets participate in the regulation of inflammatory processes by the release of proinflammatory mediators and platelet-derived microparticles (PMPs). However, the role of the inducible mPGES-1/PGE2 pathway in platelet functions has not been investigated. In the present study we report a significant impact of mPGES-1 on platelet functions during inflammation. Wild-type (WT) and mPGES-1-/- knockout (KO) mice were stimulated with lipopolysaccharide (LPS) for 24 h. Platelet counts and activation were assessed by flow cytometry analysing CD62P-CD154 expression, PMP numbers, platelet-leukocyte aggregates and platelet aggregation. The accumulation of platelets and fibrinogen in the liver was analysed by immunofluorescent staining. In native platelets from WT and mPGES-1 KO mice, there were no differences among the investigated functions. After LPS treatment, the number of platelets was significantly decreased in WT, but not in KO mice. Platelet activation, platelet-leukocyte aggregates and PMP numbers were all significantly lower in KO mice compared with WT mice after LPS treatment. In addition, KO mice displayed a significant reduction in platelet aggregation ex vivo In the liver of LPS-stimulated WT and KO mice, there were no differences in platelet accumulation, although the percentage of total vessel area in the KO liver was significantly lower compared with the WT one. Our results demonstrate that systemic inhibition of mPGES-1 prevents platelet activation, which should have important implications with regard to the cardiovascular safety of mPGES-1 inhibitors.

Effects of lipid-lowering treatment on circulating microparticles in patients with diabetes mellitus and chronic kidney disease.

BACKGROUND: Elevated levels of circulating microparticles (MPs) may contribute to the high cardiovascular risk in diabetes mellitus (DM) and chronic kidney disease (CKD). Therefore, we investigated the effects of lipid-lowering treatment (LLT) with simvastatin alone (S) or with ezetimibe (S+E) on MPs in DM patients with or without CKD. METHODS: After a placebo run-in period, 18 DM patients with an estimated glomerular filtration rate (eGFR) of 15-59 mL/min (CKD stages 3-4) (DM-CKD) and 21 DM patients with eGFR >75 mL/min (DM-only) were treated with S and S+E in a randomized, double-blind, crossover study. MPs from platelets, monocytes and endothelial cells (PMPs, MMPs and EMPs), and their expression of phosphatidylserine (PS), P-selectin, CD40 ligand (CD40L) and tissue factor (TF) were measured by flow cytometry. RESULTS: At baseline, all types of MPs, except TF-positive MMPs, were elevated in DM-CKD compared with DM-only patients. All MPs, regardless of origin and phenotype, were inversely correlated with eGFR. S reduced the expression of P-selectin, TF and CD40L on PMPs and of TF on MMPs in both patient groups. S+E had no further effect. S also reduced total PS-positive procoagulant MPs, PMPs and MMPs in DM-CKD but not in DM-only patients. CONCLUSIONS: DM patients with CKD stages 3-4 had elevated PMPs, EMPs and MMPs compared with DM patients with normal GFR. Simvastatin reduced procoagulant MPs, MMPs and PMPs in DM-CKD patients, suggesting a beneficial reduction of hypercoagulability in this high-risk patient group. Differences between DM-CKD and DM-only patients were counteracted by LLT.

Association between platelet reactivity and circulating platelet-derived microvesicles in patients with acute coronary syndrome.

High on-treatment platelet reactivity (HPR) to clopidogrel has been shown to increase the risk of cardiovascular events. Platelet-derived microvesicles (PMVs) may be prothrombotic and contribute to the risk of recurrent events observed in patients with HPR. However, PMVs may also serve as biomarkers and be used to assess platelet function. We investigated the association between platelet responses to clopidogrel (measured by whole blood impedance aggregometry) and circulating PMVs in patients with acute coronary syndrome (ACS). Blood samples were obtained at discharge from 200 patients with ACS who had undergone percutaneous coronary intervention (PCI). All patients were loaded with aspirin and clopidogrel before PCI. ADP-induced whole blood impedance aggregometry and measurement of PMVs were performed. Cut-off values for HPR and other reactivity (i.e. normal on-treatment reactivity, NPR and low on-treatment reactivity, LPR) to clopidogrel were set according to data from large prospective studies. We measured PMVs as phosphatidylserine and CD42a positive vesicles, together with CD62P or CD40L, using flow cytometry. ADP-induced platelet aggregation revealed that approximately 20% of patients had HPR. Levels of PMVs were almost two-fold higher in the HPR group compared with patients without HPR (for both CD42a- and CD62P-positive PMVs, p < 0.01). Furthermore, patients with LPR to clopidogrel had significantly fewer PMVs exposing CD62P than patients with HPR or those with NPR to clopidogrel. Patients with HPR during clopidogrel treatment have elevated levels of circulating PMVs, indicating ongoing platelet activation despite clopidogrel treatment. Moreover, in patients with LPR to clopidogrel, circulating PMV numbers are decreased. Taken together, our data suggest that PMVs are potential biomarkers of antiplatelet responses to clopidogrel. If PMVs also have prognostic value after, ACS should be tested in future studies.

CD40L expression in plasma of volunteers following LPS administration: A comparison between assay of CD40L on platelet microvesicles and soluble CD40L.

CD40 ligand (CD40L) is a transmembrane protein that is mainly expressed on activated T cells and platelets. This protein, however, may also be shed from cells and circulate in the blood in a soluble form. "Soluble CD40L" has attracted interest as a biomarker as it can interact with CD40 and elicit cellular responses involved in the pathophysiology of various thrombotic and inflammatory conditions. As platelets can release microvesicles following activation, we investigated the expression of CD40L on circulating microvesicles as well as CD40L in plasma, in an experimental model of inflammation in healthy volunteers (i.e., intravenous lipopolysaccharide administration). We studied CD40L quantified as CD40L-positive platelet microvesicles by flow cytometry, and as CD40L in plasma ("soluble CD40L") by an ELISA. Results of these studies showed that levels of CD40L exposed on platelet microvesicles were significantly increased after lipopolysaccharide administration. ELISA measurements of CD40L in plasma ("soluble CD40L") did not show any significant increase in plasma levels over time. Separation of soluble and vesicle-bound CD40L by high-speed centrifugation indicated that the ELISA can also detect CD40L on microvesicles, as a trend toward increased concentrations were observed in the pellet of high-speed centrifuged samples (i.e., in samples in which microvesicles are enriched). Together, these findings suggest that platelet microvesicles are a source of CD40L in the circulation and that CD40L exposure on platelet microvesicles increases following experimentally induced inflammation. Our data also suggest that determining levels of CD40L on microvesicles in plasma samples may provide a more sensitive detection of changes in CD40L expression than measurement of "soluble CD40L" in plasma with an ELISA. In addition, information regarding the cellular source of CD40L can be obtained with a flow cytometry-based microvesicle assay in a way not possible with an ordinary ELISA.

Lipid-lowering treatment and inflammatory mediators in diabetes and chronic kidney disease.

BACKGROUND: Inflammation may contribute to the high cardiovascular risk in diabetes mellitus (DM) and chronic kidney disease (CKD). Monocyte chemoattractant protein-1 (MCP-1) facilitates the recruitment of monocytes into atherosclerotic lesions and is involved in diabetic nephropathy. Interferon gamma (IFNγ) is important in atherosclerosis and increases the synthesis of chemokines including MCP-1. Lipid-lowering treatment (LLT) with statins may have anti-inflammatory effects, and ezetimibe cotreatment provides additional cholesterol lowering. METHODS: After a placebo run-in period, the effects of simvastatin alone (S) or simvastatin + ezetimibe (S+E) were compared in a randomized, double-blind, cross-over study on inflammatory parameters. Eighteen DM patients with estimated glomerular filtration rate (eGFR) 15-59 mL/min × 1·73 m(2) (CKD stages 3-4) (DM-CKD) and 21 DM patients with eGFR > 75 mL/min (DM only) were included. RESULTS: At baseline, monocyte chemoattractant protein 1 (MCP-1) (P = 0·03), IFNγ (P = 0·02), tumour necrosis factor-α (TNFα) (P < 0·01) and soluble vascular adhesion molecule (sVCAM) (P = 0·001) levels were elevated in DM-CKD compared with DM-only patients. LLT with S and S+E reduced MCP-1 levels (P < 0·01 by anova) and IFNγ levels (P < 0·01) in DM-CKD patients but not in DM-only patients. Reductions were most pronounced with the combination treatment. CONCLUSIONS: DM patients with CKD stages 3-4 had increased inflammatory activity compared with DM patients with normal GFR. Lipid-lowering treatment decreased the levels of MCP-1 and IFNγ in DM patients with concomitant CKD, which may be beneficial with regard to the progression of both atherosclerosis and diabetic nephropathy.

Blood brain barrier permeability and astrocyte-derived extracellular vesicles in children with juvenile idiopathic arthritis: a cross-sectional study.

BACKGROUND: Juvenile idiopathic arthritis (JIA) is the most prevalent rheumatic disease in children, and the inflammatory process is widely studied, primarily characterized by its impact on joint health. Emerging evidence suggests that JIA may also affect the central nervous system (CNS). This study investigates the potential CNS involvement in JIA by analyzing the presence of astrocyte-derived extracellular vesicles (EVs) and the S100B protein in plasma, both of which are indicative of astrocyte activity and blood-brain barrier (BBB) integrity. METHODS: EDTA plasma from 90 children diagnosed with JIA and 10 healthy controls, matched by age and gender, was analyzed for extracellular vesicles by flow cytometric measurement. Astrocyte-derived EVs were identified using flow cytometry with markers for aquaporin 4 (AQP-4) and glial fibrillary acidic protein (GFAP). Levels of the S100B protein were measured using a commercial ELISA. Disease activity was assessed using the Juvenile Arthritis Disease Activity Score (JADAS27, 0-57), and pain levels were measured using a visual analogue scale (VAS, 0-10 cm). RESULTS: Our analyses revealed a significantly higher concentration of astrocyte-derived EVs in the plasma of children with JIA compared with healthy controls. Furthermore, children with JADAS27 scores of 1 or higher exhibited notably higher levels of these EVs. The S100B protein was detectable exclusively in the JIA group. CONCLUSION: The elevated levels of astrocyte-derived EVs and the presence of S100B in children with JIA provide evidence of BBB disruption and CNS involvement, particularly in those with higher disease activity. These findings underscore the importance of considering CNS health in the comprehensive management of JIA. Further research is required to elucidate the mechanisms behind CNS engagement in JIA and to develop treatments that address both joint and CNS manifestations of the disease.

Electronic Cigarette Vaping with Nicotine Causes Increased Thrombogenicity and Impaired Microvascular Function in Healthy Volunteers: A Randomised Clinical Trial.

Electronic cigarette (EC) vaping is increasingly popular, despite growing evidence of adverse health effects. To further evaluate the impact of EC use on vascular health, we investigated the effects of brief EC inhalation on flow-dependent thrombus formation and microcirculation in healthy volunteers. The study was performed with a randomised double-blind crossover design. Twenty-two healthy subjects aged between 18 and 45 years with occasional tobacco use were recruited. Subjects inhaled 30 puffs of EC aerosol with and without nicotine on two occasions separated by a wash-out period of at least 1 week. Blood samples were collected at baseline and at 15 and 60 min following exposure and analysed with the Total-Thrombus-formation analysis system evaluating fibrin-rich thrombus formation and platelet thrombus formation in whole blood under flow. Microvascular function was assessed at baseline and 30 min after exposure by laser speckle contrast imaging and iontophoresis of acetylcholine and sodium nitroprusside (SNP) to evaluate the endothelium-dependent and independent pathways of vasodilation. Compared with nicotine free EC aerosol, exposure to EC aerosol with nicotine significantly increased platelet thrombus formation and fibrin-rich thrombus formation at 15 min (p = 0.017 and p = 0.037, respectively) with normalisation after 60 min. Peak SNP-mediated microvascular perfusion, i.e. endothelium-independent vasodilation, was reduced following EC vaping with nicotine compared with baseline (p = 0.006). Thirty puffs of EC aerosol with nicotine increased platelet and fibrin-dependent thrombus formation and reduced microvascular dilatation capacity. No compelling effects of EC vaping without nicotine were observed, indicating nicotine as the main effector. Trial registration: ClinicalTrials.gov Identifier: NCT04175457 URL: https://clinicaltrials.gov/ct2/show/NCT04175457.

Augmented thrombin formation is related to circulating levels of extracellular vesicles exposing tissue factor and citrullinated histone-3 in anti-neutrophil cytoplasmic antibody-associated vasculitides.

Objectives: To study circulating myeloperoxidase (MPO)-positive extracellular vesicles (MPO+EVs) exposing citrullinated histone-3 (H3Cit), tissue factor (TF), and plasminogen (Plg) in association to thrombin generation in patients with anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV). Methods: We have involved well-characterized patients with AAV together with population-based controls. Flow cytometry was used to assess the levels of MPO+EVs in citrated plasma. MPO+EVs were phenotyped by anti-MPO-antibodies together with anti-CD142 (anti-TF), anti-H3Cit, and anti-Plg antibodies. A modified Calibrated Automated Thrombogram (CAT) assay was utilized to measure thrombin generation in plasma initiated by EVs-enriched pellets. The activity of AAV was evaluated with the Birmingham Vasculitis Activity Score (BVAS). Results: This study comprised 46 AAV patients, 23 in the active stage of the disease and 23 in remission, as well as 23 age- and sex matched population-based controls. Augmented levels of all investigated MPO+ EVs were found in active AAV patients in comparison to the subgroup of patients in remission and controls. Thrombin generation, measured by endogenous thrombin potential (ETP) and peak of thrombin formation, was higher in plasma when triggered by EVs-enriched pellet from AAV patients. ETP and peak were associated with the levels of MPO+TF+ and MPO+H3Cit+ EVs. Additionally, MPO+TF+ EVs correlated with the disease activity evaluated with BVAS. Conclusion: Augmented thrombin generation is found in AAV patients regardless of disease activity and is associated with higher exposure of TF and H3Cit on MPO+EVs. This may contribute to the increased risk of thrombosis seen in AAV patients.

Plasma levels of S100B and neurofilament light chain protein in stress-related mental disorders.

The pathophysiological changes underlying stress-related mental disorders remain unclear. However, research suggests that alterations in astrocytes and neurons may be involved. This study examined potential peripheral markers of such alterations, including S100B and neurofilament light chain (NF-L). We compared plasma levels of S100B and NF-L in patients with chronic stress-induced exhaustion disorder (SED), patients with major depressive disorder (MDD), and healthy controls. We also investigated whether levels of S100B and NF-L correlated with levels of astrocyte-derived extracellular vesicles (EVs that indicate astrocyte activation or apoptosis) and with symptom severity. Only women had measurable levels of S100B. Women with SED had higher plasma levels of S100B than women with MDD (P < 0.001) and healthy controls (P < 0.001). Self-rated symptoms of cognitive failures were positively correlated with levels of S100B (rs = 0.434, P = 0.005) as were depressive symptoms (rs = 0.319, P < 0.001). Plasma levels of astrocyte-derived EVs were correlated with levels of S100B (rs = 0.464, P < 0.001). Plasma levels of NF-L did not differ between the groups and were not correlated with symptom severity or EV levels. Thus, long-term stress without sufficient recovery and SED may be associated with raised plasma levels of S100B, which may be evidence of pathophysiological changes in astrocytes. The findings also support the hypothesis that plasma levels of S100B are associated with cognitive dysfunction.

Extracellular vesicles in heart failure - A study in patients with heart failure with preserved ejection fraction or heart failure with reduced ejection fraction characteristics undergoing elective coronary artery bypass grafting.

Aims: Extracellular vesicles (EVs) were investigated as potential biomarkers associated with heart failure (HF) pathophysiology in patients undergoing elective coronary artery bypass surgery characterized by HF phenotype. Materials and methods: Patients with preoperative proxy-diagnoses of HF types i.e., preserved (HFpEF; n = 19) or reduced ejection fraction (HFrEF; n = 20) were studied and compared to patients with normal left ventricular function (n = 42). EVs in plasma samples collected from the coronary sinus, an arterial line, and from the right atrium were analyzed by flow cytometry. We studied EVs of presumed cardiomyocyte origin [EVs exposing Connexin-43 + Caveolin-3 (Con43 + Cav3) and Connexin-43 + Troponin T (Con43 + TnT)], of endothelial origin [EVs exposing VE-Cadherin (VE-Cad)] and EVs exposing inflammatory markers [myeloperoxidase (MPO) or pentraxin3 (PTX3)]. Results: Median concentrations of EVs exposing Con43 + TnT and Con43 + Cav3 were approximately five to six times higher in coronary sinus compared to radial artery indicative of cardiac release. Patients with HFrEF had high trans-coronary gradients of both Con43 + TnT and Con43 + Cav3 EVs, whereas HFpEF had elevated gradients of Con43 + Cav3 EVs but lower gradients of Con43 + TnT. Coronary sinus concentrations of both Con43 + TnT and Con43 + Cav3 correlated significantly with echocardiographic and laboratory measures of HF. MPO-EV concentrations were around two times higher in the right atrium compared to the coronary sinus, and slightly higher in HFpEF than in HFrEF. EV concentrations of endothelial origin (VE-Cad) were similar in all three patient groups. Conclusion: Con43 + TnT and Con43 + Cav3 EVs are released over the heart indicating cardiomyocyte origin. In HFrEF the EV release profile is indicative of myocardial injury and myocardial stress with elevated trans-coronary gradients of both Con43 + TnT and Con43 + Cav3 EVs, whereas in HFpEF the profile indicates myocardial stress with less myocardial injury.

Comparison of venous and arterial blood sampling for the assessment of platelet aggregation with whole blood impedance aggregometry.

Platelet monitoring is presently under evaluation in the clinic as a tool to improve antiplatelet treatment in patients with coronary artery disease (CAD). Measuring platelet function has, however, many inherent problems. It is important not only to evaluate the method used, but also to evaluate and standardize sampling and sample handling. As platelet monitoring is often performed in connection to coronary angiography and percutaneous coronary interventions, arterial sampling may be more convenient. However, in the outpatient follow-up setting venous sampling is, for obvious reasons, more practical and convenient. In the present study we compared platelet aggregation in blood collected from the arterial sheath to blood collected from the antecubital vein using multiple electrode aggregometry in whole blood in 28 patients with CAD. We found that sampling from artery and vein give similar data and that an identical number of patients with insufficient antiplatelet responses ('low responders' to aspirin and clopidogrel, respectively, according to predefined criteria) were detected with respect to adenosine diphosphate induced and arachidonic-acid induced aggregation. Thus both arterial and venous blood samples can be used in the monitoring of platelet function when multiple electrode aggregometry is applied to detect 'low responders'.