Gene expression of fibrinolytic markers in coronary thrombi.

BACKGROUND: The fibrinolytic system plays an important role in coronary artery atherothrombosis, and especially circulating plasminogen-activator inhibitor (PAI) type 1 (PAI-1) associates with increased mortality, infarct size and heart failure in patients with myocardial infarction (MI). In a cross-sectional study, we aimed to study whether genes encoding tissue plasminogen activator (tPA), urinary-type plasminogen activator (uPA), PAI-1 and PAI-2 are expressed in coronary thrombi from acute ST-elevation MI (STEMI) patients. Any relations to myocardial injury measured by peak troponin T, time from symptom onset to Percutaneous Coronary Intervention (PCI), and to different cell types present in the thrombi were also explored. METHODS: Intracoronary thrombi were aspirated from 33 STEMI patients treated with primary PCI. The thrombi were snap-frozen for gene expression analyses, relatively quantified by RT PCR. Peripheral blood samples were drawn. Correlations were performed by Spearmans rho. RESULTS: The genes were present in 74-94% of the thrombi. Median peak troponin T was 3434 µ/L and median ischemic time 152 min. There were no significant correlations between the measured genes and troponin T, or ischemic time. Genes encoding tPA, u-PA, PAI-1 and PAI-2 all correlated significantly to the presence of monocytes/macrophages (CD68) in the thrombi (p = 0.028, p < 0.001, p = 0.003, p < 0.001). PAI-1 and PAI-2 also correlated to endothelial cells (CD31) (p = 0.002, p = 0.016). uPA associated with neutrophil granulocytes (CD 66b) (p = 0.019). CONCLUSION: Genes encoding tPA, uPA, PAI-1 and PAI-2 were highly expressed in human coronary thrombi from STEMI patients, indicating fibrinolytic regulators playing active roles in the thrombi, although not related to myocardial injury. All markers related to the presence of monocytes/macrophages, indicating connection to local inflammatory cells. TRIAL REGISTRATION: The study is registered at clinicaltrials.gov with identification number NCT02746822 .

The Inflammasome Signaling Pathway Is Actively Regulated and Related to Myocardial Damage in Coronary Thrombi from Patients with STEMI.

BACKGROUND: The Nod-Like-Receptor-Protein-3 (NLRP3) inflammasome and the Interleukin-6 (IL-6) pathways are central mechanisms of the inflammatory response in myocardial reperfusion injury. Expanding our knowledge about the inflammasome signaling axis is important to improve treatment options. In a cross-sectional study, we aimed to study presence, localization, and genetic expression of inflammasome- and IL-6- signaling-related proteins in coronary thrombi and circulating leukocytes from ST-elevation myocardial infarction (STEMI) patients, with relation to myocardial injury and time from symptoms to PCI. METHODS: Intracoronary thrombi were aspirated from 33 STEMI patients. Blood samples were drawn. mRNA of Toll-Like-Receptor-4 (TLR4), NLRP3, caspase 1, Interleukin-1ß (IL1-ß), Interleukin-18 (IL-18), IL-6, IL-6-receptor (IL-6R), and glycoprotein 130 (gp130) were isolated from thrombi and circulating leukocytes and relatively quantified by RT-PCR. A part of each thrombus was embedded in paraffin for histology and immunohistochemistry analyses. RESULTS: Genes encoding the 8 markers were present in 76-100% of thrombi. Expression of TLR4 in thrombi significantly correlated to troponin T (r = 0.455, p = 0.013), as did NLRP3 (r = 0.468, p = 0.024). Troponin T correlated with expression in circulating leukocytes of TLR4 (r = 0.438, p = 0.011), NLRP3 (r = 0.420, p = 0.0149), and IL-1ß (r = 0.394, p = 0.023). IL-6R expression in thrombi correlated significantly to troponin T (r = 0.434, p = 0.019), whereas gp130 was inversely correlated (r = -0.398, p = 0.050). IL-6 in circulating leukocytes correlated inversely to troponin T (r = -0.421, p = 0.015). There were no significant correlations between genes expressed in thrombi and time from symptom to PCI. CONCLUSIONS: The inflammasome signaling pathway was actively regulated in coronary thrombi and in circulating leukocytes from patients with STEMI, in association with myocardial damage measured by troponin T. This supports the strategy of medically targeting this pathway in treating myocardial infarction and contributes to sort out optimal timing and targets for anti-inflammatory treatment. The study is registered at clinicaltrials.gov with identification number NCT02746822.

Complement Activation in Association with Markers of Neutrophil Extracellular Traps and Acute Myocardial Infarction in Stable Coronary Artery Disease.

Complement activation and neutrophil extracellular traps (NETs) have both been suggested to drive atherosclerotic plaque progression. Although experimental studies suggest interplay between these two innate immunity components, the relevance in patients with coronary artery disease (CAD) is unclear. The aim of this study was to assess associations between complement activation and NETs in patients with stable CAD and examine the role of complement activation on clinical outcome. Blood samples from a cohort of patients with angiographically verified stable CAD (n = 1001) were analyzed by ELISA for the terminal complement complex (TCC) and by relative quantification for gene expression of the C5a receptor 1 (C5aR1) as markers of complement activation. As markers of NETs, dsDNA was analyzed by fluorescent nucleic acid stain and myeloperoxidase-DNA (MPO-DNA) by ELISA. Clinical outcome was defined as unstable angina, nonhemorrhagic stroke, acute myocardial infarction (MI), or death (n = 106, whereof 36 MI). Levels of TCC and C5aR1 were not significantly correlated to dsDNA (TCC: r = -0.045, p = 0.153; C5aR1: r = -0.060, p = 0.434) or MPO-DNA (TCC: r = 0.026, p = 0.414; C5aR1: r = 0.123, p = 0.107). When dividing TCC and C5aR1 levels into quartiles (Q), levels of MPO-DNA differed significantly across quartiles (TCC: p = 0.008, C5aR1: 0.049), while dsDNA did not (TCC: p = 0.181, C5aR1: p = 0.771). Patients with TCC levels in Q4 had significantly higher levels of MPO-DNA than Q1-3 (p = 0.019), and C5aR1 levels in Q3-4 had significantly higher levels of MPO-DNA than Q1-2 (p = 0.046). TCC levels did not differ between patients experiencing a clinical endpoint or not, but high levels were associated with increased risk of acute MI (OR. 1.97, 95% CI: 0.99-3.90, p = 0.053) during two-year follow up, also when adjusted for relevant covariates. In conclusion, TCC and C5aR1 were moderately associated with the NET marker MPO-DNA, and TCC levels were related to the risk of future MI in this cohort of patients with stable CAD.

Glucose associated NETosis in patients with ST-elevation myocardial infarction: an observational study.

BACKGROUND: Neutrophil extracellular traps (NETs) have recently been identified as mediators in atherothrombosis. Although NETosis in general has been suggested to be glucose dependent, the transferability to patients with acute ST-elevation myocardial infarction (STEMI) is unclear. We assessed whether the NETs markers double-stranded deoxyribonucleid acid (dsDNA) and myeloperoxidase-DNA (MPO-DNA) associated with plasma glucose and the glucometabolic status in the acute phase and 3 months after a STEMI. We also explored whether an acute glucose load resulted in upregulated NETosis by assessment of peptidylarginine deiminase 4 (PAD4) gene expression. METHODS: In total, 224 STEMI patients were prospectively enrolled and underwent blood sampling acutely (median 16.5 h after PCI) and after 3 months. Glucometabolic status was defined based on the results of an oral glucose tolerance test (OGTT) as normal glucose regulation (NGR), impaired fasting glucose (IFG), impaired glucose tolerance (IGT) or type 2 diabetes (T2DM). dsDNA and MPO-DNA were measured in serum, while PAD4 mRNA was measured in circulating leukocytes by RT-PCR. RESULTS: dsDNA levels were significantly correlated to plasma glucose both acutely and after 3 months (r = 0.12 and r = 0.17, both p < 0.02), whereas MPO-DNA was not. No associations with the glucometabolic status were encountered for dsDNA and MPO-DNA acutely, but after 3 months dsDNA levels were elevated in patients with IFG and T2DM vs. NGR (428 vs. 371 ng/ml and 408 vs. 371 ng/ml, both p < 0.045). During the acute glucose load after 3 months, dsDNA and MPO-DNA remained unchanged while PAD4 mRNA increased significantly (RQ 0.836 vs. 0.920, p = 0.02). CONCLUSIONS: In this cohort of STEMI patients, levels of dsDNA associated with plasma glucose both in the acute and stable condition. The glucometabolic status was not substantially related to the selected NETs markers, however, an acute glucose load by OGTT performed after 3 months resulted in increased PAD4 expression, suggestive of enhanced NETosis in the aftermath of STEMI. TRIAL REGISTRATION: www.clinicaltrials.gov, NCT00926133 . Registered June 23, 2009.

Neutrophil Extracellular Trap Components Associate with Infarct Size, Ventricular Function, and Clinical Outcome in STEMI.

BACKGROUND: The relevance of neutrophil extracellular traps (NETs) in acute ST-elevation myocardial infarction (STEMI) is unclear. We explored the temporal profile of circulating NET markers and their associations to myocardial injury and function and to adverse clinical events in STEMI patients. METHODS AND RESULTS: In 259 patients, blood samples were drawn before and after PCI, on day 1, and after 4 months. Double-stranded deoxyribonucleic acid (dsDNA) and myeloperoxidase-DNA (MPO-DNA) were measured in serum by a nucleic acid stain and ELISA. Cardiac magnetic resonance imaging assessed microvascular obstruction (MVO), area at risk, infarct size, myocardial salvage index, left ventricular ejection fraction (LVEF), and change in indexed left ventricular end-diastolic volume (LVEDVi). Clinical events were registered after 12 months. dsDNA and MPO-DNA levels were highest before PCI, with reduced levels thereafter (all p ≤ 0.02). Patients with high vs. low day 1 dsDNA levels (>median; 366 ng/ml) more frequently had MVO, larger area at risk, larger infarct size acutely and after 4 months, and lower myocardial salvage index (all p < 0.03). Moreover, they had lower LVEF acutely and after 4 months, and larger change in LVEDVi (all p ≤ 0.014). High day 1 dsDNA levels also associated with risk of having a large infarct size (>75th percentile) and low LVEF (≤49%) after 4 months when adjusted for gender, time from symptoms to PCI, and infarct localization (OR 2.3 and 3.0, both p < 0.021), and patients with high day 1 dsDNA levels were more likely to experience an adverse clinical event, also when adjusting for peak troponin T (hazard ratio 5.1, p = 0.012). No such observations were encountered for MPO-DNA. CONCLUSIONS: High day 1 dsDNA levels after STEMI were associated with myocardial infarct size, adverse left ventricular remodeling, and clinical outcome. Although the origin of dsDNA could be discussed, these observations indicate a potential role for dsDNA in acute myocardial ischemia. This trial is registered with S-08421d, 2008/10614 (Regional Committee for Medical Research Ethics in South-East Norway (2008)).

Managing patients with prediabetes and type 2 diabetes after coronary events: individual tailoring needed - a cross-sectional study.

BACKGROUND: Understanding the determinants associated with prediabetes and type 2 diabetes in coronary patients may help to individualize treatment and modelling interventions. We sought to identify sociodemographic, medical and psychosocial factors associated with normal blood glucose (HbA1c < 5.7%), prediabetes (HbA1c 5.7-6.4%), and type 2 diabetes. METHODS: A cross-sectional explorative study applied regression analyses to investigate the factors associated with glycaemic status and control (HbA1c level) in 1083 patients with myocardial infarction and/or a coronary revascularization procedure. Data were collected from hospital records at the index event and from a self-report questionnaire and clinical examination with blood samples at 2-36 months follow-up. RESULTS: In all, 23% had type 2 diabetes, 44% had prediabetes, and 33% had normal blood glucose at follow-up. In adjusted analyses, type 2 diabetes was associated with larger waist circumference (Odds Ratio 1.03 per 1.0 cm, p = 0.001), hypertension (Odds Ratio 2.7, p < 0.001), lower high-density lipoprotein cholesterol (Odds Ratio 0.3 per1.0 mmol/L, p = 0.002) and insomnia (Odds Ratio 2.0, p = 0.002). In adjusted analyses, prediabetes was associated with smoking (Odds Ratio 3.3, p = 0.001), hypertension (Odds Ratio 1.5, p = 0.03), and non-participation in cardiac rehabilitation (Odds Ratio 1.7, p = 0.003). In patients with type 2 diabetes, a higher HbA1c level was associated with ethnic minority background (standardized beta [ß] 0.19, p = 0.005) and low drug adherence (ß 0.17, p = 0.01). In patients with prediabetes or normal blood glucose, a higher HbA1c was associated with larger waist circumference (ß 0.13, p < 0.001), smoking (ß 0.18, p < 0.001), hypertension (ß 0.08, p = 0.04), older age (ß 0.16, p < 0.001), and non-participation in cardiac rehabilitation (ß 0.11, p = 0.005). CONCLUSIONS: Along with obesity and hypertension, insomnia and low drug adherence were the major modifiable factors associated with type 2 diabetes, whereas smoking and non-participation in cardiac rehabilitation were the factors associated with prediabetes. Further research on the effect of individual tailoring, addressing the reported significant predictors of failure, is needed to improve glycaemic control. TRIAL REGISTRATION: Retrospectively registered at ClinicalTrials.gov: NCT02309255 , December 5th 2014.

The Time Course of Markers of Neutrophil Extracellular Traps in Patients Undergoing Revascularisation for Acute Myocardial Infarction or Stable Angina Pectoris.

Background and Aims. Neutrophil extracellular traps (NETs) have been identified in acute myocardial infarction. We assessed the time profile and association with infarct size for NETs markers in ST-elevation myocardial infarction (STEMI) and stable angina pectoris (AP). Methods. In 20 patients with STEMI and 10 with AP undergoing percutaneous coronary intervention (PCI), blood samples were collected before PCI (only AP group) and after 3 and 12 hours, days 1, 3, 5, 7, and 14 for measurement of NETs markers. Results. Double-stranded deoxyribonucleic acid (dsDNA) and nucleosome levels were higher in STEMI than AP until day 3 and 12 hours (p < 0.03, all). DsDNA declined after day 5 in both groups (p < 0.04, all), while nucleosomes declined until day 3 only in the AP group (p < 0.05, all). DsDNA correlated with peak troponin T and creatine kinase MB (CKMB) at day 5 (r = 0.48, p = 0.03, both) and with MRI-measured infarct size at days 5 and 7 (r = 0.61, p = 0.01 and r = 0.52, p = 0.04, resp.), while nucleosomes correlated with infarct size at day 5 (r = 0.58, p = 0.02). Conclusions. High levels of NETs markers were observed in STEMI shortly after revascularisation and were partly associated with infarct size. The decline thereafter in both groups indicates a role for NETs in both acute and chronic atherothrombosis.

Gestational diabetes mellitus among Nordic Caucasian women: prevalence and risk factors according to WHO and simplified IADPSG criteria.

BACKGROUND: Gestational diabetes mellitus (GDM) is associated with both maternal and offspring adverse effects. The World Health Organization (WHO) has recently adopted novel GDM criteria. The aim of this study was to evaluate the former WHO and a simplified version of the new International Association for Diabetes in Pregnancy Study Group (IADPSG) criteria as to prevalence of and risk factors for GDM in a Nordic Caucasian population. METHODS: A 75 g oral glucose tolerance test was performed in 687 women at 18-22 and 32-36 pregnancy weeks. GDM was defined according to the WHO criteria as fasting plasma glucose ≥ 7.0 mmol/L and/or 2-hour plasma glucose ≥ 7.8 mmol/L and by a simplified version of the IADPSG criteria as either fasting glucose ≥ 5.1 mmol/L and/or 2-h plasma glucose ≥ 8.5 mmol/L. One-hour glucose values were not available and were thus not included in the diagnosis of GDM by IADPSG. Prevalence of GDM during pregnancy and risk factors for GDM at 18-22 weeks were studied in retrospect according to each of the two criteria. RESULTS: The total prevalence of GDM during pregnancy was 6.1% (42/687) for the WHO criteria and 7.4% (51/687) for the simplified IADPSG criteria. High maternal age and short stature were independently associated with WHO GDM. Maternal age, fasting insulin and no regular exercise at 18-22 pregnancy weeks associated with simplified IADPSG GDM. CONCLUSIONS: Simplified IADPSG criteria moderately increase GDM prevalence compared with the WHO criteria. Risk factors for GDM differ with the diagnostic criteria used.

The time profile of Pentraxin 3 in patients with acute ST-elevation myocardial infarction and stable angina pectoris undergoing percutaneous coronary intervention.

BACKGROUND: High levels of Pentraxin 3 (PTX3) are reported in acute myocardial infarction (AMI). AIM: To investigate circulating levels and gene expression of PTX3 in patients with AMI and stable angina pectoris (AP) undergoing PCI. METHODS: Ten patients with AP and 20 patients with AMI were included. Blood samples were drawn before PCI in the AP group and after 3 and 12 hours and days 1, 3, 5, 7, and 14 in both groups. RESULTS: Circulating PTX3 levels were higher in AMI compared to AP at 3 and 12 hours (P < 0.001 and P = 0.003). Within the AMI group, reduction from 3 hours to all later time points was observed (all P ≤ 0.001). Within the AP group, increase from baseline to 3 hours (P = 0.022), followed by reductions thereafter (all P < 0.05), was observed. PTX3 mRNA increased in the AMI group from 3 hours to days 7 and 14 in a relative manner of 62% and 73%, while a relative reduction from baseline to 3 and 12 hours of 29% and 37% was seen in the AP group. CONCLUSION: High circulating PTX3 levels shortly after PCI in AMI indicate that AMI itself influences PTX3 levels. PTX3 mRNA might be in response to fluctuations in circulating levels.

Links Between Adipose Tissue Gene Expression of Gut Leakage Markers, Circulating Levels, Anthropometrics, and Diet in Patients with Coronary Artery Disease.

Background: Recent studies suggest gut-derived lipopolysaccharide (LPS)-translocation to play a role in both systemic inflammation and in inflammatory adipose tissue. We aimed to investigate whether circulating LPS-related inflammatory markers and corresponding genetic expression in adipose tissue were associated with obesity, cardiometabolic risk factors, and dietary habits in patients with coronary artery disease. Methods: Patients (n=382) suffering a myocardial infarction 2-8 weeks prior to inclusion were enrolled in this cross-sectional study. Subcutaneous adipose tissue (SAT), taken from the gluteal region, and fasting blood samples were collected at inclusion for determination of genetic expression of LPS-binding protein (LBP), CD14, toll-like receptor 2 (TLR2), and TLR4 in SAT, and LPS, LBP, and soluble cluster of differentiation 14 (sCD14) in the circulation. All patients filled out a dietary registration form. Results: Patients (median age 74 years, 25% women), had a median body mass index (BMI) of 25.9 kg/m2. Circulating levels of LBP correlated to BMI (p=0.02), were significantly higher in overweight or obese (BMI≥25 kg/m2) compared to normal- or underweight patients (BMI<25 kg/m2), and were significantly elevated in patients with T2DM, hypertension, and MetS, compared to patients without (p≤0.04, all). In SAT, gene expression of CD14 and LBP correlated significantly to BMI (p≤0.001, both), and CD14 and TLR2 expressions were significantly higher in patients with T2DM and MetS compared to patients without (p≤0.001, both). Circulating and genetically expressed CD14 associated with use of n-3 PUFAs (p=0.008 and p=0.003, respectively). No other significant associations were found between the measured markers and dietary habits. Conclusion: In patients with established CAD, circulating levels of LBP and gene expression of CD14 and TLR2 in SAT were related to obesity, MetS, T2DM, and hypertension. This suggests that the LPS-LBP-CD14 inflammatory axis is activated in the chronic low-grade inflammation associated with cardiometabolic abnormalities, whereas no significant associations with dietary habits were observed.

Gut Leakage and Cardiac Biomarkers after Prolonged Strenuous Exercise.

PURPOSE: Transient increase in the cardiac biomarkers troponin T (cTnT) and NT-proBNP are observed during strenuous exercise, even in healthy athletes. Gut leakage, the translocation of bacterial lipopolysaccharide (LPS) into the circulation, is associated with atherosclerosis and cardiovascular disease but has also been reported after prolonged endurance exercise. We aimed to explore the link between exercise-induced gut leakage and cardiac biomarker release. METHODS: Participants in Norseman Xtreme Triathlon (Norseman) were included ( n = 44, age 43 ± 9 yr, 9 [21%] women). Blood samples were taken before and immediately after the race for the determination of biomarkers. cTnT and NT-proBNP were measured by conventional methods. Gut leakage marker LPS was measured by the kinetic, chromogenic limulus amebocyte lysate assay method, whereas LPS-binding protein (LBP), soluble cluster of differentiation 14 (sCD14), and intestinal injury marker intestinal fatty acid-binding protein (I-FABP) were measured by enzyme-linked immunosorbent assay. RESULTS: Median (25, 75 percentiles) finish time was 14 h 33 min (13 h 42 min, 15 h 29 min). TnT and NT-proBNP increased significantly to 38 ng·L -1 (27, 56) and 495 ng·L -1 (310, 828) after the race ( P < 0.001, both). LBP and sCD14 also increased significantly ( P < 0.001, both), as did I-FABP ( P = 0.003), whereas LPS remained unchanged ( P = 0.13). No significant correlations between changes in gut leakage markers and changes in cardiac biomarkers were observed after adjusting for multiple testing. CONCLUSIONS: Cardiac and gut leakage biomarkers increased after Norseman Xtreme triathlon. However, changes in these biomarkers were not intercorrelated, suggesting that the exercise-induced increase in cardiac and gut leakage biomarkers occurs independently of each other.

Coagulation factors XI and XII as possible targets for anticoagulant therapy.

In this review, we give an overview over observational and experimental studies supporting factors XI and XII as targets for anticoagulant therapy. The majority of observational studies on FXI report low concentrations of FXI to be protective against ischemic stroke and venous thrombosis. There is also extensive evidence from experimental and animal studies supporting FXI inhibition as a target for anticoagulant therapy, alone or in combination with other antithrombotic treatments. Four Phase 2 clinical trials on patients undergoing total knee arthroplasty showed non-inferiority or superiority of FXI inhibition compared to enoxaparin for the primary outcome, which was incidence of venous thromboembolism. One Phase 2 trial reported that FXI inhibition is associated with fewer bleeding events than apixaban. The results from observational studies on FXII are more conflicting. Some show that low FXII concentrations confer protection against thrombosis, while others have found it to be deleterious. Results from experimental studies are inconclusive, but suggest that FXII inhibition might be useful in preventing thrombosis caused by foreign objects like catheters or mechanical heart valves. One Phase 2 study not conducted on thrombosis has reported FXII inhibition as safe. In conclusion, FXI seems to be a promising target for antithrombotic therapy, both alone and in combination with existing therapies, while the potential of targeting FXII is still unclear.

TIMP-1 expression in coronary thrombi associate with myocardial injury in ST-elevation myocardial infarction patients.

BACKGROUND: Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are considered important both in atherosclerosis and remodeling after acute myocardial infarction (AMI). We aimed to study genetic expression and presence of MMP-2, MMP-9, TIMP-1, TIMP-2 and the extracellular MMP-inducer (EMMPRIN) in coronary thrombi. Circulating levels and genetic expression in circulating leukocytes were also assessed, and relations to degree of myocardial injury measured by troponin T and time from symptom to PCI were explored. Expression of cell markers were also analyzed, indicating relations to cell types. METHODS: Intracoronary thrombi were aspirated from 33 patients with ST-elevation myocardial infarction (STEMI). Blood samples with Pax-gene tubes were drawn at end of PCI and the next day. RNA was isolated from thrombi and leukocytes, and genes were relatively quantified by RT-PCR. Each thrombus was preserved for histology and immunohistochemistry analyzes. RESULTS: Genes coding for the five markers were present in 84-100% of thrombi and immunohistochemically stained in 96-100%. Expression of TIMP-1 in thrombi and in leukocytes correlated significantly to peak troponin T ( r = 0.393 P = 0.026, r = 0.469 P = 0.006, respectively). No significant correlations between genes expressed in thrombi and time from symptom to PCI were observed. TIMP-1 was connected mainly to monocytes/macrophages in the thrombi. CONCLUSION: MMP-2, MMP-9, TIMP-1, TIMP-2 and EMMPRIN were highly expressed in human coronary thrombi. The correlation between troponin T and the expression of TIMP-1 both in thrombi and in leukocytes at time of PCI indicates that TIMP-1 plays a role in myocardial damage early post-MI.

The NLRP3 Genetic Variant (rs10754555) Reduces the Risk of Adverse Outcome in Middle-Aged Patients with Chronic Coronary Syndrome.

Background: Inflammation is central in development of cardiovascular disease (CVD). Aberrant function of the Nod-Like Receptor Protein 3 (NLRP3) inflammasome, a central mediator in the proinflammatory response, has been associated with atherosclerosis. The influence of genetic determinants on this inflammatory pathway and its downstream effects is less known. We aimed to investigate the frequency of a single NLRP3 gene variant according to clinical outcome in CVD and its influence on NLRP3-related markers. Methods: In this observational study, we included 1001 patients with chronic coronary syndrome. Blood samples were drawn at inclusion, including whole-blood and PAXgene tubes for DNA and RNA isolation, respectively. Allelic discrimination of the NLRP3 single nucleotide polymorphism rs10754555 was performed; and gene expression of NLRP3, Toll-Like Receptor 4, Interleukin- (IL-) 1ß, and IL-18 was relatively quantified, both methods by RT-PCR. Circulating IL-6, high-sensitivity (hs) C-reactive protein, IL-18, and IL-12 were measured by enzyme-like immunosorbent assays. Clinical endpoints during 2 years (n = 106) were a composite of unstable angina pectoris, myocardial infarction, nonhemorrhagic stroke, and death. Results: Minor allele frequency of the NLRP3 variant was 0.36. In all, no association of the NLRP3 variant with clinical subgroups or outcome was found, neither any significant influence on the genes' mRNA expression or circulating protein. However, in subjects < 56 years (25 percentile), the variant G-allele is associated with significant lower risk of suffering a composite event (OR = 0.43 (95% CI 0.19, 0.97), p = 0.043, adjusted). In the same age group, the NLRP3 gene was accordingly downregulated in G-allele carriers vs. noncarriers, and circulating IL12 was significantly reduced (p < 0.05, both). In subjects > 56 years, no significant effect of the variant was observed. Conclusion: The age-related reduced risk of composite endpoint in rs10754555 G-allele carriers accompanied by diminished NLRP3 mRNA expression is hypothesis generating and needs to be further explored. The study is registered at http://www.clinicaltrials.gov, with identification number NCT00222261.

Circulating Microvesicles in Association with the NLRP3 Inflammasome in Coronary Thrombi from STEMI Patients.

Microvesicles (MVs) are actively secreted by cells. The NLRP3-inflammasome and the interleukin 6 (IL-6)-pathways are central in cardiovascular disease. Knowledge of how the inflammasome influences the MVs is limited. In a cross-sectional study, we assessed whether MVs in plasma associate with genes encoding inflammasome signalling in coronary thrombi. Moreover, any relationships between inflammasome activation and phosphatidylserine (PS) externalization, determined through Annexin V (AV+) labelling, and myocardial injury, assessed by cardiac troponin T (cTnT), were analysed. Intracoronary thrombi and blood samples from STEMI patients (n = 33) were investigated. mRNA of NLRP3, caspase-1, interleukin-1ß (IL-1ß), interleukin-18 (IL-18), IL-6, soluble IL-6-receptor (sIL-6R), and glycoprotein-130 (gp130) were isolated from the thrombi and relatively quantified by RT-PCR. MVs were analysed by flow cytometry. Total AV+ MVs, mainly reflecting hypercoagulability, correlated positively to NLRP3 gene expression (r = 0.545, p = 0.009). A similar pattern was seen for platelet, endothelial and leukocyte derived MVs, separately. The majority of the MVs were AV− (96%). Total and AV− MVs correlated inversely with IL-1ß (r = −0.399 and −0.438, respectively, p < 0.05, both) and gp130 (r = −0.457 and −0.502, respectively, p < 0.05, both). No correlations between MVs and cTnT were observed. Our findings indicate an association between NLRP3-inflammasome in coronary thrombi and procoagulant AV+ MVs in STEMI patients. The inverse relationships between AV− MVs and the gene expression of inflammasome activation may indicate an immuno-dampening role of this subpopulation.

Complement activation in association with clinical outcomes in ST-elevation myocardial infarction.

Introduction: The complement system and neutrophil extracellular traps (NETs) might contribute to ischemia-reperfusion injury in ST-elevation myocardial infarction (STEMI). We aimed to estimate associations between complement activation and NETs in STEMI, and their prognostic value on clinical endpoints. Methods: In this cohort study, 864 patients admitted for PCI during STEMI were included. Complement activation was analyzed by the terminal complement complex (TCC), while NETs were analyzed by myeloperoxidase-DNA, citrullinated histone 3 (CitH3) and dsDNA. The composite endpoint was reinfarction, unscheduled revascularization, stroke, hospitalization due to heart failure, or death, and the secondary endpoint was total mortality. The association between TCC and clinical endpoints was assessed by Cox regression and ROC curve analysis. Results: TCC was weakly correlated to dsDNA (r = 0.127, p < 0.001) and CitH3 (r = 0.102, p = 0.003). After a median follow-up time of 4.6 years, 184 (21.3 %) patients had reached a clinical endpoint. TCC was not associated with the composite endpoint, but with total mortality (HR: 1.673, 95 % CI: [1.014, 2.761], p = 0.044). The significant association was lost when adjusting for CRP, NT-proBNP, LVEF and time from symptoms to PCI. In ROC curve analysis of total mortality, the AUC for TCC alone was 0.549 (95 % CI: [0.472, 0.625]), AUC for dsDNA alone was 0.653 (95 % CI: [0.579, 0.720]), while AUC for TCC and dsDNA combined was 0.660 (95 % CI: [0.590, 0.730]). Conclusions: In this STEMI cohort, TCC was not associated with the composite endpoint, but somewhat with total mortality. Combining TCC and dsDNA did not increase the prognostic value compared to dsDNA alone.

Gut related inflammation and cardiorespiratory fitness in patients with CAD and type 2 diabetes: a sub-study of a randomized controlled trial on exercise training.

AIM: Gut leakage has been shown to associate with low-grade inflammation and lower cardiorespiratory fitness in diabetic subjects. We aimed to investigate whether gut leakage markers related to cardiorespiratory fitness in patients with both coronary artery disease and type 2 diabetes, and whether these were affected by long-term exercise training. METHODS: Patients with angiographically verified coronary artery disease and type 2 diabetes mellitus (n = 137) were randomized to either 12 months exercise intervention or conventional follow-up. A cardiopulmonary exercise test and fasting blood samples were obtained before and after intervention to assess VO2peak and the biomarkers soluble CD14, lipopolysaccharide-binding protein and intestinal fatty-acid binding protein as markers of gut leakage. RESULTS: 114 patients completed the intervention satisfactory. VO2peak correlated inversely to sCD14 (r = - 0.248, p = 0.004) at baseline. Dividing sCD14 into quartiles (Q), VO2peak was significantly higher in Q1 vs. Q2-4 (p = 0.001), and patients in Q2-4 (sCD14 > 1300 ng/mL) had an OR of 2.9 (95% CI 1.2-7.0) of having VO2peak below median (< 23.8 ml/kg/min) at baseline. There were no statistically significant differences in changes in gut leakage markers between the two randomized groups (all p > 0.05) after 12 months. CONCLUSIONS: Cardiorespiratory fitness related inversely to sCD14, suggesting physical capacity to be associated with gut leakage in patients with CAD and T2DM. Long-term exercise training did not affect circulating gut leakage markers in our population. Trial registration NCT01232608, Registered 02 November 2010-Retrospectively registered at https://clinicaltrials.gov/ct2/show/NCT01232608?term=NCT01232608&draw=2&rank=1.

Gut Leakage Markers in Response to Strenuous Exercise in Patients with Suspected Coronary Artery Disease.

Elevated levels of gut leakage markers have been shown after strenuous exercise in healthy individuals. Any association between a temporary increase in these markers and the presence of coronary artery disease (CAD) is unknown. We therefore aimed to explore circulating gut leakage markers in response to a bout of strenuous exercise in patients with symptoms of CAD. Patients referred to exercise stress testing due to symptoms of CAD were included (n = 287). A maximal exercise ECG stress test was performed and venous blood samples were drawn at rest and within five minutes after, for analysis of soluble cluster of differentiation 14 (sCD14), lipopolysaccharide-binding protein (LBP), intestinal fatty-acid binding protein (I-FABP), lipopolysaccharide (LPS) and gene expression of toll-like receptor 4 (TLR4) in circulating leukocytes. Patients then underwent coronary angiography. LPS, LBP and sCD14 increased significantly after strenuous exercise in patients with symptoms of CAD, suggesting that even short bouts of vigorous exercise are associated with gut leakage. The gene expression of TLR4 decreased significantly after exercise, possibly as a negative feedback to the increase in LPS. There were no differences in exercise-induced changes between the groups of CAD, suggesting gut leakage to be independent of the presence of CAD.

NETosis in Long-Term Type 1 Diabetes Mellitus and Its Link to Coronary Artery Disease.

Background: Neutrophil extracellular traps NETs have been linked to glucose and the pathogenesis of type 1 diabetes mellitus (T1DM). NETs also play a role in vascular inflammation and the development of coronary artery disease (CAD). The role of NETs in CAD progression in patients with long-term T1DM is unclear. We aimed to 1) investigate whether levels of circulating NETs markers were elevated in long-term T1DM subjects compared to controls, and 2) explore whether levels of NETs were related to the presence of CAD. Material and Methods: 102 patients with > 45 years of T1DM and 75 age-matched controls were enrolled in a cross-sectional study. Median age was 62 years. Computed tomography coronary angiography (CTCA) was performed in 148 subjects without established coronary heart disease. For the current study, CAD was defined as a coronary artery stenosis >50%. Double-stranded deoxyribonucleic acid (dsDNA) was measured by a nucleic acid stain, myeloperoxidase-DNA (MPO-DNA), citrullinated histone 3 (H3Cit) and peptidylarginine deiminase 4 (PAD4) by ELISAs, while gene expression of PAD4 was measured in leukocytes from PAXgene tubes. Results: Circulating MPO-DNA levels were significantly lower in patients with T1DM than in controls (0.17 vs 0.29 OD, p<0.001), while dsDNA, H3Cit, PAD4 and gene expression of PAD4 did not differ with respect to the presence of T1DM. There were no significant associations between NETs markers and HbA1c in the T1DM group. None of the NETs markers differed according to the presence of CAD in patients with T1DM. While all circulating NETs markers correlated significantly with circulating neutrophils in the control group (r=0.292-393, p<0.014), only H3Cit and PAD4 correlated with neutrophils in the T1DM group (r= 0.330-0.449, p ≤ 0.001). Conclusions: In this cross-sectional study of patients with long-term T1DM and age-matched controls, circulating NETs levels were not consistently associated with the presence of T1DM or glycemic status, and did not differ according to the presence of CAD in patients with T1DM. Our results entail the possibility of altered neutrophil function and reduced NETosis in T1DM. This warrants further investigation.