Sirtuin1, not NAMPT, possesses anti-inflammatory effects in epicardial, pericardial and subcutaneous adipose tissue in patients with CHD.

BACKGROUND: Inflammation in cardiac adipose tissue (AT) is associated with atherosclerosis. We investigated whether the epicardial-, pericardial and pre-sternal subcutaneous AT (EAT, PAT and SAT) expression of Sirtuin1 (SIRT1) and nicotinamide phosphoribosyl transferase (NAMPT) are involved in the inflammatory process in coronary heart disease (CHD), and potentially associated to nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-related markers, macrophage polarization markers, cell markers and the cardiometabolic profile. METHODS: In this cohort study performed between 2016 and 2018, EAT, PAT and SAT biopsies were retrieved from 52 CHD patients (77% men, median age 67) undergoing open-chest coronary artery bypass grafting (CABG), and 22 patients (50% men, median age 69) undergoing aortic valve replacement serving as controls. AT samples were snap-frozen at - 80 °C until RNA extraction and AT expression of actual markers, relatively quantified by PCR. Circulating SIRT1 and NAMPT were measured with Enzyme-linked immunosorbent assays (ELISAs). Non-parametric statistical tests were mainly used, including Friedman's test coupled to Wilcoxon signed-rank test and Spearman Correlation. RESULTS: SIRT1 and NAMPT levels were similar in CHD and controls. In CHD, SIRT1 and NAMPT were inter-correlated in all AT compartments (r = 0.37-0.56, p < 0.01, all), and differently expressed between compartments, with the highest expression in SAT, significantly different from EAT (p < 0.01, both). Circulating SIRT1 and NAMPT levels were inversely associated (r = - 0.32, p = 0.024). In EAT and SAT, SIRT1 expression was inversely associated with IL-18 (r = - 0.43 and r = - 0.38, p < 0.01, both), whereas NAMPT expression was positively associated with the NLRP3 inflammasome-related markers in all compartments (r = 0.37-0.55, p < 0.01, all). While SIRT1 and NAMPT correlated to nitric oxide synthase 2 (NOS2), especially in SAT (r = 0.50-0.52, p ≤ 0.01, both), SIRT1 expression was related to endothelial cells, and NAMPT to macrophages. SIRT1 levels were correlated to weight and waist (r = 0.32 and r = 0.38, p < 0.03, both) and inversely to triglycerides and glycated haemoglobin (HbA1c) (r = - 0.33-- 0.37, p < 0.03, all), the latter positively correlated to NAMPT concentration (r = 0.39, p = 0.010). CONCLUSION: The study indicates that targeting SIRT1, with its anti-inflammatory properties, may be a novel anti-inflammatory strategy in preventing atherosclerosis and CHD progression. NAMPT may be an early player in AT inflammation, mediating/reflecting a pro-inflammatory state. TRIAL REGISTRATION: Registration: Clinicaltrials.gov ID: NCT02760914, registered the 5th of February 2016, http://clinicaltrials.gov/NCT02760914.

Omega-3 fatty acid supplements and risk of atrial fibrillation and 'micro-atrial fibrillation': A secondary analysis from the OMEMI trial.

BACKGROUND & AIMS: Recent randomized clinical trials have raised concerns regarding potential off target adverse effects from supplementation of n-3 polyunsaturated fatty acids (PUFA) on atrial fibrillation (AF) risk. We aimed to assess risk and potential mediators of AF and 'micro-AF' from n-3 PUFA in post-myocardial infarction (MI) patients. METHODS: In the OMEMI trial, 70-82 y. o. patients with a recent MI were randomized to 1.8 g/day of eicosapentaenoic-/docosahexaenoic acid (EPA/DHA) or placebo (corn oil) for two years. New-onset AF and 'micro-AF' was recorded by clinical detection and by screening with Zenicor thumb-ECG (adjudicated by blinded investigators). Serum EPA and DHA were measured at baseline and study end. RESULTS: At baseline, 759 of 1014 (75%) patients had no AF history. These patients were aged 75 ± 4 years and 71% were male. During follow-up, 43 patients developed new-onset AF (39 clinically-detected and 4 by thumb-ECG screening). In addition, 27 patients had episodes of micro-AF, yielding a total of 70 patients with new-onset AF or 'micro-AF'. In the n-3 PUFA group 46 (11.9%) had AF/'micro-AF' (28 AF, 18 'micro-AF') and in the placebo group 24 (6.5%) had AF/micro-AF (15 AF, 9 micro-AF); HR 1.90 (95%CI 1.16-3.11), P = 0.011. Changes in serum EPA (but not DHA) mediated the effect from n-3 PUFA on AF risk, explaining 65% of the association. CONCLUSION: Supplementation of n-3 PUFA post MI increases the risk of 'micro-AF' and AF, and increases in EPA seems to be an important mediator of the treatment effect from n-3 PUFA on the risk of AF. STUDY REGISTRATION: OMEMI Study; ClinicalTrails.gov identifier: NCT0184194.

Total Bilirubin Yields Prognostic Information Following a Myocardial Infarction in the Elderly.

Total bilirubin consists of an unconjugated form, solubilized by its binding to albumin, and a conjugated form representing a minor part of the circulating bilirubin. As total bilirubin in physiological concentrations is a powerful antioxidant, its concentration gradient may reflect the health status of an individual, and serve as a prognostic indicator of outcome in primary and secondary cardiovascular disease prevention. The aim of this study was to assess the association between total bilirubin and incident cardiovascular events following a myocardial infarction. Total bilirubin in serum was measured at baseline 2-8 weeks after hospitalization for an MI in 881 patients, aged 70 to 82 years, included in the OMEMI (Omega-3 Fatty acids in Elderly with Myocardial Infarction) study, where patients were followed-up for up to 2 years. The first major adverse clinical event (MACE) was the primary endpoint and consisted of nonfatal MI, unscheduled coronary revascularization, stroke, hospitalization for heart failure or all-cause death. As total bilirubin was non-normally distributed, log-transformed values and quartiles of bilirubin were analyzed using Cox regression models. The median (Q1, and Q3) baseline concentration of bilirubin was 11 (9, and 14) µmol/L, and higher log-transformed concentrations were associated with male sex, lower New York Heart Association (NYHA) class and non-smoking. MACE occurred in 177 (20.1%) patients during the follow-up. Higher concentrations of bilirubin were associated with a lower risk of MACE: HR 0.67 (95%CI 0.47-0.97) per log-unit increase, p = 0.032. Patients in the lowest quartile of bilirubin (<9 µmol/L) had the highest risk with HR 1.61 (95%CI 1.19-2.18), p = 0.002, compared to quartiles 2-4. This association remained significant even after adjusting for age, sex, body mass index (BMI), smoking status, NYHA class and treatment allocation: HR 1.52 (1.21-2.09), p = 0.009. Low concentrations of bilirubin (<9 µmol/L) are associated with increased nonfatal cardiovascular events or death in elderly patients with a recent myocardial infarction.

von Willebrand factor, ADAMTS-13, and thrombospondin 1 in relation to clinical outcomes in elderly patients with a recent myocardial infarction.

Background: von Willebrand factor (VWF) multimers are cleaved by A disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS-13) into less active fragments. Thrombospondin 1 (TSP-1) competes with VWF's cleavage site, protecting it from degradation. Low ADAMTS-13 and high VWF have been associated with cardiovascular disease and atrial fibrillation (AF). Objectives: We aimed to investigate whether VWF, ADAMTS-13, and TSP-1 are associated with clinical outcome. Methods: Elderly patients with a recent myocardial infarction (MI) (n = 1027) were followed for 2 years. Blood was collected 2 to 8 weeks after the MI for ADAMTS-13, VWF, and TSP-1 measures. The primary endpoints (major adverse cardiovascular events; n = 210) included the first event of MI, stroke, heart failure hospitalization, coronary revascularization, and all-cause death. Total mortality was also registered (n = 56). The secondary endpoint was new-onset AF (n = 43). Results: Concentrations of VWF, ADAMTS-13, and TSP-1 did not intercorrelate. The risk of major adverse cardiovascular events was altered in patients with VWF ≥ median (hazard ratio [HR], 1.4; 95% CI, 1.0-1.8; P = .03) and ADAMTS-13 ≥ median (HR, 0.7; 95% CI, 0.5-0.9; P = .02); however, it was not significant in adjusted models. VWF and ADAMTS-13 were significantly associated with total mortality, with a HR of 2.7 (95% CI, 1.6-4.6; P < .001) for VWF (Q4 vs. Q1-Q3) and HR of 0.3 (95% CI, 0.2-0.5; P < .001) for ADAMTS-13 (Q2-4 vs. Q1). The associations persisted in multivariable analysis, but the significance disappeared for VWF after correcting for high-sensitivity C-reactive protein. The risk of new-onset AF was lower in patients with VWF ≥ median (HR, 0.5; 95% CI, 0.3-1.0; P = .04]), and this was still significant after adjustments. Conclusion: Although low ADAMTS-13 predicted death, the cardiovascular risk associated with VWF and ADAMTS-13 was weaker than previously reported. Low VWF is associated with new-onset AF and needs further research.

Macrophage polarization markers in subcutaneous, pericardial, and epicardial adipose tissue are altered in patients with coronary heart disease.

Background: Epicardial and pericardial adipose tissue (EAT and PAT) surround and protect the heart, with EAT directly sharing the microcirculation with the myocardium, possibly presenting a distinct macrophage phenotype that might affect the inflammatory environment in coronary heart disease (CHD). This study aims to investigate the expression of genes in different AT compartments driving the polarization of AT macrophages toward an anti-inflammatory (L-Galectin 9; CD206) or pro-inflammatory (NOS2) phenotype. Methods: EAT, PAT, and subcutaneous (SAT) biopsies were collected from 52 CHD patients undergoing coronary artery bypass grafting, and from 22 CTRLs undergoing aortic valve replacement. L-Galectin9 (L-Gal9), CD206, and NOS2 AT gene expression and circulating levels were analyzed through RT-PCR and ELISA, respectively. Results: L-Gal9, CD206, and NOS2 gene expression was similar in all AT compartments in CHD and CTRLs, as were also L-Gal9 and CD206 circulating levels, while NOS2 serum levels were higher in CHD (p = 0.012 vs. CTRLs). In CTRLs, NOS2 expression was lower in EAT vs. SAT (p = 0.007), while in CHD patients CD206 expression was lower in both SAT and EAT as compared to PAT (p = 0.003, p = 0.006, respectively), suggestive of a possible macrophage reprogramming toward a pro-inflammatory phenotype in EAT. In CHD patients, NOS2 expression in SAT correlated to that in PAT and EAT (p = 0.007, both), CD206 expression correlated positively to L-Gal9 (p < 0.001) only in EAT, and CD206 expression associated with that of macrophage identifying markers in all AT compartments (p < 0.001, all). In CHD patients, subjects with LDL-C above 1.8 mmol/L showed significantly higher NOS2 expression in PAT and EAT as compared to subjects with LDL-C levels below (p < 0.05), possibly reflecting increased cardiac AT pro-inflammatory activation. In SAT and PAT, CD206 expression associated with BMI in both CHD and CTRLs (p < 0.05, all), and with L-Gal9 in EAT, however only in CTRLs (p = 0.002). Conclusion: CHD seems to be accompanied by an altered cardiac, and especially epicardial AT macrophage polarization. This may represent an important pathophysiological mechanism and a promising field of therapy targeting the excessive AT inflammation, in need of further investigation.

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.

Screen-detected atrial fibrillation and 'micro-atrial fibrillation' and risk of cardiovascular events after myocardial infarction in elderly patients.

BACKGROUND: Incident atrial fibrillation (AF) occurs in 5-10% of patients after acute myocardial infarction (AMI) and is associated with adverse outcomes. Guidelines now recommend screening for AF in all elderly patients. However, the relevance of screen-detected AF and short episodes of irregular supraventricular ectopic beats ('micro-AF') after AMI is unknown. OBJECTIVES: To investigate the value of two-week intermittent ECG screening to detect incident AF and 'micro-AF' in elderly patients 12 months after an AMI, and its association with risk of cardiovascular events. METHODS: This was an investigator-initiated, multicenter substudy of the OMega-3 fatty acids in Elderly patients with Myocardial Infarction (OMEMI) trial, in Norway. Women and men aged 70-82 years, with a recent AMI, were recruited during 2012-2018. All participants had a 12-lead ECG performed at 3, 12 and 24 months. Patients without AF one year after the index AMI underwent 2 weeks of intermittent 30-second 'thumb ECG' screening. Incident AF and 'micro-AF' (episodes of ≥3 consecutive irregular supraventricular ectopic beats) were registered, and the association with risk of major cardiovascular events (MACE; non-fatal AMI, stroke, coronary revascularization, hospitalization for heart failure, or all-cause death) was analyzed with logistic regression. RESULTS: Among 1014 patients (198 (28.7%) women), 255 (25.1%) had known AF or AF identified at baseline. New-onset AF was detected clinically or at study visits in 39 (3.8%) patients. By screening participants without AF (n=567), unknown AF was identified in 4 (0.7%) and 'micro-AF' in 27 (4.8%) patients. Among 43 patients with incident AF, 21 (48.8%) experienced a MACE, which was significantly higher than those without AF (n=114, 15.9%; p<0.001), driven by a higher risk of AMI or revascularization. Nine (33.3%) patients with 'micro-AF' and 75 (13.9%) without 'micro-AF' experienced a MACE (p=0.002), explained mostly by a higher risk of heart failure hospitalization (p<0.001). Using patients without AF and 'micro-AF' as reference, 'micro-AF' was associated with an intermediate risk of MACE (OR 2.8; 95% CI 1.2-6.4) and new-onset AF with a high risk of MACE (OR 5.3; 95% CI 2.8-10.0). CONCLUSIONS: Two-week intermittent ECG screening identified few cases of new-onset AF, but a substantial number of patients with 'micro-AF'. 'Micro-AF' was associated with an increased risk of major cardiovascular events, albeit with an intermediate risk compared to those with new-onset AF.

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.

TERT and TET2 Genetic Variants Affect Leukocyte Telomere Length and Clinical Outcome in Coronary Artery Disease Patients-A Possible Link to Clonal Hematopoiesis.

Inherited and acquired mutations in hematopoietic stem cells can cause clonal expansion with increased risk of cardiovascular disease (CVD), a condition known for the clonal hematopoiesis of indeterminate potential (CHIP). Inherited genetic variants in two CHIP-associated genome loci, the telomerase gene telomerase enzyme reverse transcriptase (TERT) (rs7705526) and the epigenetic regulator ten−eleven translocation 2 (TET2) (rs2454206), were investigated in 1001 patients with stable coronary artery disease (CAD) (mean age 62 years, 22% women), with regards to cardiovascular outcome, comorbidities, and leukocyte telomere length. Over 2 years, mutated TERT increased the risk two-fold for major clinical events (MACEs) in all patients (p = 0.004), acute myocardial infarction (AMI) in male patients (p = 0.011), and stroke in female patients (p < 0.001). Mutated TET2 correlated with type 2 diabetes (p < 0.001), the metabolic syndrome (p = 0.002), as well as fasting glucose, HbA1c, and shorter telomeres (p = 0.032, p = 0.003, and p = 0.016, respectively). In conclusion, our results from stable CAD patients highlight TERTs' role in CVD, and underline TET2s' role in the epigenetic regulation of lifestyle-related diseases.

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 .

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.

Changes in eicosapentaenoic acid and docosahexaenoic acid and risk of cardiovascular events and atrial fibrillation: A secondary analysis of the OMEMI trial.

BACKGROUND: The cardiovascular benefit from n-3 polyunsaturated fatty acids (PUFAs) after acute myocardial infarction (AMI) is controversial, and the importance of serum eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrations for clinical events is unclear. OBJECTIVES: To assess changes in EPA and DHA serum concentrations during n-3 PUFA supplementation and their association with incident cardiovascular events. METHODS: In the OMEMI trial, elderly patients with a recent AMI were randomized to 1.8 g/day of EPA/DHA or control (corn oil) for 2 years. The primary outcome was a composite of AMI, coronary revascularization, stroke, heart failure hospitalization, or all-cause death (major adverse cardiovascular event [MACE]) and the secondary outcome was new-onset atrial fibrillation (AF). RESULTS: EPA and DHA measurements were available in 881 (92% of survivors) participants at randomization and study completion. EPA and DHA increased in the active treatment arm (n = 438) by a median of 87% and 16%, respectively. Greater on-treatment increases in EPA and DHA were associated with decreasing triglycerides, increasing high-density lipoprotein cholesterol, and lower baseline EPA and DHA concentrations. Greater on-treatment increases in EPA were associated with lower risk of MACE (adjusted hazard ratio 0.86 [95% confidence interval, CI, 0.75-0.99], p = 0.034), and higher risk of AF (adjusted hazard ratio (HR) 1.36 [95% CI 1.07-1.72], p = 0.011). Although there were similar tendencies for DHA changes and outcomes, these associations were not statistically significant (HR 0.84 [0.66-1.06] for MACE and 1.39 [0.90-2.13] for AF). CONCLUSION: Greater on-treatment increases in EPA were associated with lower risk of MACE and higher risk of new-onset AF. These data suggest that the cardiovascular effects of increasing n-3 PUFA levels through supplements are complex, involving both potential benefits and harm.

The NLRP3 inflammasome activation in subcutaneous, epicardial and pericardial adipose tissue in patients with coronary heart disease undergoing coronary by-pass surgery.

Background and aims: Epicardial and pericardial adipose tissue (EAT and PAT) associate with atherosclerosis, however, discussed to have different inflammatory properties. We examined the NLRP3 inflammasome related pathway, playing a pivotal role in atherosclerosis, in EAT, PAT and subcutaneous AT (SAT), their relationship to cell types and anthropometric measures in patients undergoing coronary artery bypass grafting. Methods: Biopsies from EAT, PAT and SAT were collected from 52 patients with coronary heart disease (CHD) (median body weight 85.0 kg) and 22 controls. RNA was extracted and expression of interleukin (IL)-1ß, IL-18, NLRP3, Caspase-1, toll-like receptor 4 (TLR4), IL-6, IL-6 receptor and gp130 were analyzed by RT-PCR. Results: Limited differences in any genes between CHD patients and controls. IL-18 and IL-6 were 4-fold higher expressed in EAT versus PAT (p < 0.01, both) and SAT (p < 0.001, both), whereas caspase-1, IL-6R and gp130 were higher expressed in SAT compared to the other compartments (all p = 0.06-<0.001). Significant correlations between SAT and PAT gene expressions (r = 0.358-0.579, all p ≤ 0.01). Especially NLRP3 and TLR4 associated with the expression of macrophages in all compartments (all p < 0.001). In EAT IL-18 correlated inversely with the expression of macrophages and T-cells. In SAT and PAT most of the mediators associated with body weight. Conclusions: Higher expression of IL-18 and IL-6 was observed in EAT in our non-obese CHD patients, not related to inflammatory cells. The NLRP3 inflammasome activation in SAT that mirrored PAT, both related to anthropometrics, suggest that SAT samples, being easily available, to a certain degree, represent adipose tissue inflammation in general.

Adiponectin in relation to exercise and physical performance in patients with type 2 diabetes and coronary artery disease.

INTRODUCTION: Adipokines, expressed by adipose tissue (AT), have been associated with metabolic disturbances and coronary artery disease (CAD). The impact of exercise training on the AT in patients suffering from both diabetes and CAD is unknown. To gain knowledge on changes in ATs' inflammatory profile in such a population, we investigated the effects of long-term exercise on selected adipokines and their associations with physical performance and glucometabolic variables. Adiponectin was selected based on its anti-atherogenic and anti-diabetic properties and visfatin and tumour necrosis factor (TNF) for their association with atherosclerosis and metabolic disorders. Not many studies have focused on the effects of long-term exercise training on adipokines in patients with concomitant T2DM and CAD. METHODS: Patients with type 2 diabetes and CAD (n = 137), 41-81 years, 17.2% females, were randomized in a 1:1 manner to an exercise group, who underwent 1 year of 150 min weekly combined strength and endurance exercise, or a control group. AT from the gluteal region and blood samples were obtained at baseline and after 12 months, along with a physical performance test, assessed by the VO2 peak. Circulating protein levels were measured by ELISA. RNA was extracted from AT and expression levels were relatively quantified by PCR. RESULTS: After 1 year, no significant difference in the change in the investigated markers between the intervention group and the control group was observed. Changes in circulating adiponectin and VO2 peak correlated in the total population (r = 0.256, p = 0.008). At baseline, circulating adiponectin and TNF correlated inversely with insulin and with C-peptide and VO2peak, respectively (p < 0.001, all). CONCLUSION: In this population with concomitant diabetes and CAD, ATs' inflammatory profile remained unchanged apparently after 1 year of exercise intervention. Changes in the VO2peak were nevertheless, related to changes in circulating adiponectin levels. TRIAL REGISTRATION: http://www.clinicaltrials.gov NCT01232608.

One year of omega 3 polyunsaturated fatty acid supplementation does not reduce circulating prothrombotic microvesicles in elderly subjects after suffering a myocardial infarction.

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

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.

Rifaximin or Saccharomyces boulardii in heart failure with reduced ejection fraction: Results from the randomized GutHeart trial.

BACKGROUND: The gut microbiota represents a potential treatment target in heart failure (HF) through microbial metabolites such as trimethylamine N-oxide (TMAO) and systemic inflammation. Treatment with the probiotic yeast Saccharomyces boulardii have been suggested to improve left ventricular ejection fraction (LVEF). METHODS: In a multicentre, prospective randomized open label, blinded end-point trial, we randomized patients with LVEF <40% and New York Heart Association functional class II or III, despite optimal medical therapy, to treatment (1:1:1) with the probiotic yeast Saccharomyces boulardii, the antibiotic rifaximin, or standard of care (SoC) only. The primary endpoint, the baseline-adjusted LVEF at three months, was assessed in an intention-to-treat analysis. FINDINGS: We enrolled a total of 151 patients. After three months' treatment, the LVEF did not differ significantly between the SoC arm and the rifaximin arm (mean difference was -1•2 percentage points; 95% CI -3•2 - 0•7; p=0•22) or between the SoC arm and the Saccharomyces boulardii arm (mean difference -0•2 percentage points; 95% CI -2•2 - 1•9; p=0•87). We observed no significant between-group differences in changes in microbiota diversity, TMAO, or C-reactive protein. INTERPRETATION: Three months' treatment with Saccharomyces boulardii or rifaximin on top of SoC had no significant effect on LVEF, microbiota diversity, or the measured biomarkers in our population with HF. FUNDING: The trial was funded by the Norwegian Association for Public Health, the Blix foundation, Stein Erik Hagen's Foundation for Clinical Heart Research, Ada og Hagbart Waages humanitære og veldedige stiftelse, Alfasigma, and Biocodex.

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.

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.

Biomarkers of ageing and cardiac remodeling are associated with atrial fibrillation.

Objectives. Ageing is one of the strongest risk factors for atrial fibrillation (AF), and additional risk factors are also closely related to ageing. Remodeling is part of the pathophysiology of AF, and a possible common denominator of ageing and other AF risk factors. The aim of this study was to investigate any association between the presence of AF and the ageing biomarkers, leukocyte telomere length (LTL) and sirtuin-1 (SIRT-1), and the cardiac remodeling biomarkers Galectin-3 and sST2 in elderly myocardial infarction (MI) patients. Design. Patients were included after admission for MI. Diagnosis of AF was retrieved from medical records and classified as either history of AF before MI or new onset from admission to study inclusion. SIRT-1, sST2 and Galectin-3 were analyzed by ELISAs and LTL by qPCR. Results. In total, 299 patients were included, median age 75 years, 70.2% male. A history of AF was recorded in 38 patients and 30 patients experienced new onset AF. Higher levels of SIRT-1 were associated with lower risk of having a history of AF (OR = 0.46 (95% CI 0.26, 0.81), p = 0.007), whereas higher sST2 levels were associated with higher risk of AF (OR = 4.13 (95% CI 1.69, 10.13), p = 0.002). Results remained significant after adjustment for other AF risk factors. No significant associations with AF were found for Galectin-3 or LTL. None of the biomarkers associated with new onset AF. Conclusion. In elderly patients with MI, higher ST2 and lower SIRT-2 levels were associated with higher prevalence of AF, possibly reflecting both ageing and the remodeling phenomena in AF. Clinical trials registration: ClinicalTrials.gov (NCT01841944).