Relationship between iron deficiency and expression of genes involved in iron metabolism in human myocardium and skeletal muscle.

BACKGROUND: Iron deficiency (ID) is associated with adverse prognosis in patients with heart failure. This study aims to investigate the relationship between ID and expression of genes involved in iron metabolism in human myocardium and skeletal muscle, focusing on Transferrin 1 receptor (TfR1), the main pathway of cellular iron uptake. METHODS: Patients undergoing elective CABG were assessed prior to surgery with echocardiography and serum iron parameters. Core needle biopsies were collected from the left and right ventricle (LV, RV), the right atrium and intercostal skeletal muscle (SM). Gene expression analyses were done by mRNA sequencing. RESULTS: Of 69 patients (median age 69 years, 91% men), 28% had ID. 26% had HFrEF, 25% had HFpEF physiology according to echocardiographic findings and NT-proBNP levels, and 49% had normal LV function. The expression of TfR1 was increased in patients with ID compared to patients without ID in ventricular tissue (p = 0.04) and in intercostal SM (p = 0.01). The increase in TfR1 expression in LV and RV was more pronounced when analysing patients with absolute ID (S-Ferritin<100 µg/L). Analysing the correlation between various iron parameters, S-Ferritin levels showed the strongest correlation with TfR1 expression. There was no correlation with NT-proBNP levels and no difference in TfR1 expression between different HF phenotypes. CONCLUSIONS: In patients undergoing elective CABG we found an association between ID and increased TfR1 expression in myocardium regardless of LV function, indicating physiologically upregulated TfR1 expression in the presence of ID to restore intracellular iron needs. CLINICAL TRIAL REGISTRATION: Clinicaltrials.govNCT03671122.

Neutrophil extracellular traps promote cancer-associated inflammation and myocardial stress.

Cancer is associated with systemic pathologies that contribute to mortality, such as thrombosis and distant organ failure. The aim of this study was to investigate the potential role of neutrophil extracellular traps (NETs) in myocardial inflammation and tissue damage in treatment-naïve individuals with cancer. Mice with mammary carcinoma (MMTV-PyMT) had increased plasma levels of NETs measured as H3Cit-DNA complexes, paralleled with elevated coagulation, compared to healthy littermates. MMTV-PyMT mice displayed upregulation of pro-inflammatory markers in the heart, myocardial hypertrophy and elevated cardiac disease biomarkers in the blood, but not echocardiographic heart failure. Moreover, increased endothelial proliferation was observed in hearts from tumor-bearing mice. Removal of NETs by DNase I treatment suppressed the myocardial inflammation, expression of cardiac disease biomarkers and endothelial proliferation. Compared to a healthy control group, treatment-naïve cancer patients with different malignant disorders had increased NET formation, which correlated to plasma levels of the inflammatory marker CRP and the cardiac disease biomarkers NT-proBNP and sTNFR1, in agreement with the mouse data. Altogether, our data indicate that NETs contribute to inflammation and myocardial stress during malignancy. These findings suggest NETs as potential therapeutic targets to prevent cardiac inflammation and dysfunction in cancer patients.

The Total Thrombus Formation (T-TAS) platelet (PL) assay, a novel test that evaluates whole blood platelet thrombus formation under physiological conditions.

Dual antiplatelet therapy (DAPT, aspirin, and a P2Y12 inhibitor) reduces thrombotic events in patients with coronary artery disease (CAD). The T-TAS PL assay uses arterial shear flow over collagen surface, better mimicking in vivo conditions compared to conventional agonist-based platelet function assays, to evaluate platelet function. Here, the platelet function in patients taking DAPT is evaluated with the T-TAS PL assay. In 57 patients with CAD, taking DAPT ≥7 days (n = 22 for clopidogrel, n = 15 for prasugrel, n = 20 for ticagrelor), T-TAS PL assessments were performed in duplicate, and expressed as area under the flow pressure curve within a 10-minute period (AUC10). The duplicate measurements were strongly correlated (r = 0.90, p < .001), with an intra-assay coefficient of variation (CV) of 11,5%. For clopidogrel, the median AUC10 was 11.5 (IQR5.9-41.8), for prasugrel 28.8 (IQR10.3-37.6), and for ticagrelor 8.9 (IQR 6.4-10.9). All measurements were below the AUC10 cutoff of 260 measured in healthy volunteers, suggesting excellent discrimination of DAPT-treated and untreated persons. The new T-TAS PL assay demonstrated complete discrimination of platelet function in patients on DAPT based on an established cutoff. Ticagrelor showed lower levels of platelet function and a more uniform response compared to prasugrel and clopidogrel.

Use of proteomics to identify biomarkers associated with chronic kidney disease and long-term outcomes in patients with myocardial infarction.

BACKGROUND: Patients with chronic kidney disease (CKD) have poor outcomes following myocardial infarction (MI). We performed an untargeted examination of 175 biomarkers to identify those with the strongest association with CKD and to examine the association of those biomarkers with long-term outcomes. METHODS: A total of 175 different biomarkers from MI patients enrolled in the Swedish Web-System for Enhancement and Development of Evidence-Based Care in Heart Disease Evaluated According to Recommended Therapies (SWEDEHEART) registry were analysed either by a multiple reaction monitoring mass spectrometry assay or by a multiplex assay (proximity extension assay). Random forests statistical models were used to assess the predictor importance of biomarkers, CKD and outcomes. RESULTS: A total of 1098 MI patients with a median estimated glomerular filtration rate of 85 mL min-1 /1.73 m2 were followed for a median of 3.2 years. The random forests analyses, without and with adjustment for differences in demography, comorbidities and severity of disease, identified six biomarkers (adrenomedullin, TNF receptor-1, adipocyte fatty acid-binding protein-4, TNF-related apoptosis-inducing ligand receptor 2, growth differentiation factor-15 and TNF receptor-2) to be strongly associated with CKD. All six biomarkers were also amongst the 15 strongest predictors for death, and four of them were amongst the strongest predictors of subsequent MI and heart failure hospitalization. CONCLUSION: In patients with MI, a proteomic approach could identify six biomarkers that best predicted CKD. These biomarkers were also amongst the most important predictors of long-term outcomes. Thus, these biomarkers indicate underlying mechanisms that may contribute to the poor prognosis seen in patients with MI and CKD.

Rapid determination of anticoagulating effects of dabigatran in whole blood with rotational thromboelastometry and a thrombin-based trigger.

Essentials A rapid test to detect thrombin inhibition by dabigatran would be valuable in acute situations. A thrombin-based trigger was applied in whole blood using rotation thromboelastometry. Effects of dabigatran were assessed in vitro and in samples from patients on dabigatran. The test produced data rapidly and was sensitive to dabigatran concentrations from 20 to 500 ng mL-1 . SUMMARY: Background Rapid determination of the anticoagulant effect of dabigatran is essential in emergency situations. Objective To study a viscoelastic test (rotational thromboelastometry [ROTEM]) for rapid determination of dabigatran effects in whole blood samples. Method ROTEM measurements were performed with comparison of two triggers (thrombin-based versus the commercial tissue factor-based trigger Ex-tem) in samples from 10 healthy donors spiked with dabigatran (20-500 ng mL-1 ) and in samples from 35 patients receiving dabigatran treatment; 10 healthy subjects served as controls. Clotting time (CT) and the difference in CT without versus with addition of the dabigatran antidote idarucizumab (CTdiff ) were measured. Addition of idarucizumab reveals the contribution of dabigatran to ROTEM measurements and its potential reversibility. Results In vitro studies showed that thrombin CT and thrombin CTdiff were more sensitive than Ex-tem CT and Ex-tem CTdiff in detecting dabigatran in whole blood samples. In patient samples, when thrombin CT and thrombin CTdiff were used, it was possible to detect dabigatran with a cut-off of dabigatran at 20 ng mL-1 , whereas, when Ex-tem CT and Ex-tem CTdiff were used, the method was less sensitive. Data from patient samples were obtained within 15 min of blood sampling. Conclusions ROTEM CT with a thrombin-based trigger is more sensitive to dabigatran effects than Ex-tem CT, and detects anticoagulant effects of drug concentrations in the low-very low therapeutic range. Analysis with idarucizumab (CTdiff ) reveals dabigatran-specific effects. As data are rapidly obtained, this method could, with further development and validation of its performance, be suitable for detecting clinically significant dabigatran effects in emergency situations.

An increased tendency in fibrinogen activity and its association with a hypo-fibrinolytic state in early stages after injury in patients without acute traumatic coagulopathy (ATC).

Acute traumatic coagulopathy (ATC) diagnosed by prolongation of APTT and/or PT/INR involves alterations in platelet activity, coagulation and fibrinolysis. However, data showing the haemostatic situation in injured patients without ATC are scarce. To assess whether haemostatic impairment is also present in injured patients without ATC, ten injured patients without ATC and ten normal individuals were examined. The patients were sampled on arrival at the emergency department 0, 2, 12 h after surgical or other intervention. Thrombin generation, fibrin formation and fibrin proteolysis were determined via several laboratory methods, using tissue factor as the coagulation trigger. Thrombograms demonstrated that trauma accelerated both thrombin generation and decay. In the presence of unaffected peak thrombin levels, these two contradictory effects cancelled each other out, leading to the global endogenous thrombin potential (ETP) remaining normal. Under the mediation of normal ETP, fibrin network permeability (Ks) kept the reference levels in the two groups of subjects. Fibrinogen (FBG) activity (Clauss) rose with time from 0 to 2 h and 12 h, which significantly slowed down Clot Lysis Potential as determined by an in vitro method with exogenous t-PA. SUMMARY: the main haemostatic impairment in the present patients concerned an increased tendency in FBG activity. Since an increase in FBG is a biomarker of acute inflammation and also predicts greater fibrin production which down-regulates fibrinolysis, we suggest that during early stages after injury, patients without ATC may suffer from worsening inflammation and confront enhancement of thrombosis risk due to dysfunction of fibrinolysis.

Brain-Derived Microparticles in Patients with Severe Isolated TBI.

PRIMARY OBJECTIVE: to investigate the presence of circulating microparticles (MPs) of brain tissue origin in the systemic and cerebrovenous blood of patients with severe traumatic brain injury (TBI). RESEARCH DESIGN: Prospective observational study in 15 consecutive patients with severe isolated TBI. METHODS AND PROCEDURES: We repeatedly measured concentrations of MPs expressing glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE) and aquaporin-4 (AQP4), in arterial and cerebrovenous blood at admittance to hospital and up to 72 hours after the injury. MAIN OUTCOMES AND RESULTS: Concentrations of MPs expressing GFAP and AQP4 were significantly higher in the TBI group compared with healthy controls: GFAP 2.0 [1.1-7.9] vs. 1.3 [1-2.1] × 106/mL, p < 0.001; AQP4 0.1 [0.07-0.22] vs. 0.08 [0.06-0.11] × 106/mL, p < 0.001 (median, range). No transcranial gradients were found. Levels of NSE-expressing MPs were also higher in the TBI group compared with healthy controls: 0.4 [0.25-2.1] vs. 0.26 [0.13-0.98] × 106/mL, p < 0.05; however, regarding NSE-positive non-platelet MPs, there were no differences between patients and controls. CONCLUSIONS: Patients with TBI have higher numbers of brain-derived MPs. Further studies are needed, however, to identify specific and sensitive MP markers of brain injury.

The expression of HMGB1 on microparticles from Jurkat and HL-60 cells undergoing apoptosis in vitro.

HMGB1 is a highly conserved nuclear protein that displays important biological activities inside as well as outside the cell and serves as a prototypic alarmin to activate innate immunity. The translocation of HMGB1 from inside to outside the cell occurs with cell activation as well as cell death, including apoptosis. Apoptosis is also a setting for the release of cellular microparticles (MPs), which are small membrane-bound vesicles that represent an important source of extracellular nuclear molecules. To investigate whether HMGB1 released from cells during apoptosis is also present on MPs, we determined the presence of HMGB1 on particles released from Jurkat and HL-60 cells induced to undergo apoptosis in vitro by treatment with either etoposide or staurosporine; MPs released from cells undergoing necrosis by freeze-thaw were also characterized. As shown by both Western blot analysis and flow cytometry, MPs from apoptotic cells contain HMGB1, with binding by antibodies indicating an accessible location in the particle structure. These results indicate that HMGB1, like other nuclear molecules, can translocate into MPs during apoptosis and demonstrate another biochemical form of this molecule that may be immunologically active.

Fibrinogen depletion after plasma-dilution: impairment of proteolytic resistance and reversal via clotting factor concentrates.

In trauma patients, resuscitation treatment of intravascular volume may cause haemodilution including blood cell- and plasma-dilution. After plasma-dilution, fibrinogen is the first factor that decreases to critically low concentrations. Fibrin formed in lowered levels is susceptible to fibrinolysis, a natural forerunner for bleeding. To assess whether a fibrinogen concentrate or a factor XIII (FXIII) concentrate can reverse the impairment of fibrin properties after plasma dilution, different laboratory methods were used to determine thrombin generation and fibrin quantity/quality in a normal plasma sample diluted in vitro. Coagulation and clot lysis by plasmin were triggered with tissue factor and rt-PA, respectively.We found that while the endogenous thrombin potential (ETP) was unaffected after plasma-dilution due to postponement of thrombin decay, levels of fibrinogen and hence fibrin were decreased in dilution degree-dependency. The imbalance between influence of the dilution on thrombin activity and fibrin formation brought unexpected outcomes of fibrin properties: the formed clots favoured the degradation by plasminbut the fibrin networks remained tighter/less permeable. This proteolytic tendency was partly overturned by the fibrinogen concentrate added (total fibrinogen ≥ 2 g/l), and much more affected if used in combination with tranexamic acid (a fibrinolysis inhibitor) at small doses. No reversal effect resulted from the FXIII concentrate added. We conclude that plasma-dilution did reduce the proteolytic resistance of formed clots. The fibrinogen concentrate, better together with small doses of tranexamic acid, may reverse the impairment of fibrin property.The FXIII concentrate is not effective in this regard in our in vitro model using platelet-poor plasma.

Effects of ex vivo platelet supplementation on platelet aggregability in blood samples from patients treated with acetylsalicylic acid, clopidogrel, or ticagrelor.

BACKGROUND: Transfusion of platelet concentrate is often used to treat bleeding in patients on platelet inhibitors, but little is known about its efficacy between different inhibitors. We assessed the effect of ex vivo platelet supplementation on platelet aggregability in blood samples from patients treated with acetylsalicylic acid (ASA), clopidogrel, or ticagrelor. METHODS: Platelet aggregability was investigated with multiple electrode aggregometry with adenosine diphosphate (ADP), arachidonic acid (to assess ASA-dependent aggregability), and thrombin receptor activating peptide-6 (TRAP) as activators in whole-blood samples from patients treated with ASA (n=10), ASA+clopidogrel (n=15), or ASA+ticagrelor (n=15), and from healthy controls (n=10). Aggregability was measured before and after supplementation of AB0-compatible fresh apheresis platelets (+46, +92, and +138×10(9) litre(-1)). RESULTS: Both ASA-dependent and ADP-dependent aggregability improved in a dose-dependent fashion after platelet supplementation. ASA-dependent aggregability was completely restored in all patient groups, but there was only a small improvement in ADP-dependent aggregability in patients on dual antiplatelet therapy. There was less effect of platelet supplementation on ADP- and ASA-dependent aggregability in ticagrelor-treated patients than in clopidogrel-treated patients [3.9 (95% confidence interval 1.6-6.3) vs 9.0 (5.2-12.8) AU×min (P=0.021) and 48 (36-59) vs 69 (60-78) AU×min (P=0.004), respectively, at the highest platelet dose]. CONCLUSIONS: Platelet supplementation improved platelet aggregability independently of antiplatelet therapy. The effect on ADP-dependent platelet inhibition was limited however. Reduced effect of platelet transfusion is more likely within 2 h of drug intake in patients treated with ASA+ticagrelor compared with ASA+clopidogrel.

Effect of lipopolysaccharide administration on the number, phenotype and content of nuclear molecules in blood microparticles of normal human subjects.

Microparticles (MPs) are small membrane-bound vesicles that arise from activated and dying cells and promote inflammation and thrombosis. To characterize the in vivo release of MPs, we used flow cytometry to measure MPs in the blood of 15 healthy volunteers administered bacterial endotoxin (lipopolysaccharide or LPS) in the presence of a low dose of hydrocortisone with or without inhaled nitric oxide. MPs, defined as particles less than 1.0 µm in size, were assessed following labelling for CD42a, CD14 and CD62E or CD144 antibodies to identify MPs from platelets (PMPs), monocytes (MMPs) and endothelial cells (EMPs). In addition, PMPs and MMPs were labelled with anti-HMGB1 and stained with SYTO13 to assess nuclear acid content. Administration of LPS led to an increase in the numbers of PMPs, MMPs and EMPs as defined by CD62E, as well as the number of MMPs and PMPs staining with anti-HMGB1 and SYTO13. Inhalation of NO did not influence these findings. Together, these studies show that LPS can increase levels of blood MPs and influence phenotype, including nuclear content. As such, particles may be a source of HMGB1 and other nuclear molecules in the blood during inflammation.

Studies of microparticles in patients with the antiphospholipid syndrome (APS).

OBJECTIVES: To study circulating platelet, monocyte and endothelial microparticles (PMPs, MMPs and EMPs) in patients with antiphospholipid syndrome (APS) in comparison with healthy controls. MATERIAL AND METHOD: Fifty-two patients with APS and 52 healthy controls were investigated. MPs were measured on a flow cytometer (Beckman Gallios) and defined as particles sized < 1.0 µm, negative to phalloidin, positive to lactadherin and positive to either CD42a (PMPs), CD144 (EMPs) or CD14 (MMPs). Exposure of CD142 (TF) was measured on CD144 positive MPs. RESULTS: Total number of MPs (i.e. lactadherin positive particles) was higher in APS patients versus controls (p < 0.001). An increased number of EMPs (p < 0.001), increased TF-positive EMPs (p < 0.001) and increased MMPs (p < 0.001) were also observed. PMP numbers did not differ between the groups. None of the MP types differed in numbers between obstetric and thrombotic APS patients. CONCLUSION: We observed a high number of EMPs expressing TF in APS patients. The numbers of MMPs and total EMPs were also higher as compared with healthy controls but in contrast to previous reports, the number of PMPs did not differ between groups.

Concentration-dependent stimulation of intestinal phase III of migrating motor complex by circulating serotonin in humans.

1. The influence of circulating 5-hydroxytryptamine (serotonin) on small intestinal motility was investigated in healthy volunteers. 2. Small intestinal motility was studied by means of a constantly perfused multi-channel manometry tube, connected to a computer system. 3. Intravenous infusions of either 5-hydroxytryptamine at increasing doses or saline were given over a period of 4 h. 4. 5-Hydroxytryptamine infusion dose-dependently increased plasma 5-hydroxytryptamine from approximately 2 to 10 and 25 nmol/l respectively, as well as urinary excretions of 5-hydroxytryptamine and 5-hydroxyindole acetic acid, a major 5-hydroxytryptamine metabolite. 5. The number of phase III of the migrating motor complex originating in the small intestine was dose-dependently increased by 5-hydroxytryptamine, and found to correlate to the plasma concentration of 5-hydroxytryptamine. The fraction of phase III also increased at the expense of phase II activity. In addition, 5-hydroxytryptamine increased the motility index, propagation velocity of phase III activity and the amplitude of contractions during phase III. 6. Whereas the low dose of 5-hydroxytryptamine (15 nmol.min-1.kg-1) had no haemodynamic effects, an increase in heart rate by approximately 20 beats/min, without change in blood pressure, was observed at the higher dose (60 nmol.min-1.kg-1). Respiratory parameters did not change during infusion of 5-hydroxytrytamine at either dose. 7. In conclusion, elevation of circulating 5-hydroxytryptamine by intravenous infusion results in more frequent and faster propagating migrating motor complexes in the human small intestine during the inter-digestive period.

E1B 19,000-molecular-weight protein interacts with and inhibits CED-4-dependent, FLICE-mediated apoptosis.

Genetic studies of the nematode Caenorhabditis elegans (C. elegans) have identified several important components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homologue CED-9 (or the mammalian Bcl-2 family member Bcl-xL) and the caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a potent apoptosis inhibitor and the adenovirus homologue of Bcl-2-related apoptosis inhibitors. Since E1B 19K and Bcl-xL have functional similarity, we determined if E1B 19K interacts with CED-4 and regulates CED-4-dependent caspase activation. Binding analysis indicated that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 was dramatically changed by E1B 19K, supporting the theory of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could not induce cell death, coexpression of CED-4 and FLICE augmented cell death induction by FLICE, which was blocked by expression of E1B 19K. Even though E1B 19K did not prevent FLICE-induced apoptosis, it did inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggested that CED-4 was required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting with CED-4, and presumably a mammalian homologue of CED-4, to inhibit caspase activation and apoptosis.

Interaction and regulation of the Caenorhabditis elegans death protease CED-3 by CED-4 and CED-9.

In the nematode Caenorhabditis elegans, three genes, ced-3, ced-4, and ced-9, play critical roles in the induction and execution of the death pathway. Genetic studies have suggested that ced-9 controls programmed cell death by regulating ced-4 and ced-3. However, the mechanism by which CED-9 controls the activities of CED-4 and the cysteine protease CED-3, the effector arm of the cell-death pathway, remains poorly understood. Immunoprecipitation analysis demonstrates that CED-9 forms a multimeric protein complex with CED-4 and CED-3 in vivo. Expression of wild-type CED-4 promotes the ability of CED-3 to induce apoptosis in mammalian cells, which is inhibited by CED-9. The pro-apoptotic activity of CED-4 requires the expression of a functional CED-3 protease. Significantly, loss-of-function CED-4 mutants are impaired in their ability to promote CED-3-mediated apoptosis. Expression of CED-4 enhances the proteolytic activation of CED-3. We also show that CED-9 inhibits the formation of p13 and p15, two cleavage products of CED-3 associated with its proteolytic activation in vivo. Moreover, CED-9 inhibits the enzymatic activity of CED-3 promoted by CED-4. Thus, these results provide evidence that CED-4 and CED-9 regulate the activity of CED-3 through physical interactions, which may provide a molecular basis for the control of programmed cell death in C. elegans.

Interaction and regulation of subcellular localization of CED-4 by CED-9.

The Caenorhabditis elegans survival gene ced-9 regulates ced-4 activity and inhibits cell death, but the mechanism by which this occurs is unknown. Through a genetic screen for CED-4-binding proteins, CED-9 was identified as an interacting partner of CED-4. CED-9, but not loss-of-function mutants, associated specifically with CED-4 in yeast or mammalian cells. The CED-9 protein localized primarily to intracellular membranes and the perinuclear region, whereas CED-4 was distributed in the cytosol. Expression of CED-9, but not a mutant lacking the carboxy-terminal hydrophobic domain, targeted CED-4 from the cytosol to intracellular membranes in mammalian cells. Thus, the actions of CED-4 and CED-9 are directly linked, which could provide the basis for the regulation of programmed cell death in C. elegans.

Primary prevention in patients with coronary heart disease: the APSIS study.

Symptomatic treatment of patients with angina pectoris is well established and consists of nitrates, calcium antagonists, or beta-blockers. All these drugs improve symptomatology and reduce signs of ischemia on exercise test or long-term electrocardiogram recordings. It is not known, however, whether these drugs also improve prognosis. The only drug shown to improve prognosis is aspirin. In order to study the prognostic effect of calcium antagonists and beta-blockers, these two drugs were compared in the APSIS study.