Concomitant Coronary Atheroma Regression and Stabilization in Response to Lipid-Lowering Therapy.

BACKGROUND: The frequency, characteristics, and outcomes of patients treated with high-intensity lipid-lowering therapy and showing concomitant atheroma volume reduction, lipid content reduction, and increase in fibrous cap thickness (ie, triple regression) are unknown. OBJECTIVES: This study was designed to investigate rates, determinants, and prognostic implications of triple regression in patients presenting with acute myocardial infarction and treated with high-intensity lipid-lowering therapy. METHODS: The PACMAN-AMI (Effects of the PCSK9 Antibody Alirocumab on Coronary Atherosclerosis in Patients with Acute Myocardial Infarction) trial used serial intravascular ultrasound, near-infrared spectroscopy, and optical coherence tomography to compare the effects of alirocumab vs placebo in patients receiving high-intensity statin therapy. Triple regression was defined by the combined presence of percentage of atheroma volume reduction, maximum lipid core burden index within 4 mm reduction, and minimal fibrous cap thickness increase. Clinical outcomes at 1-year follow-up were assessed. RESULTS: Overall, 84 patients (31.7%) showed triple regression (40.8% in the alirocumab group vs 23.0% in the placebo group; P = 0.002). On-treatment low-density lipoprotein cholesterol levels were lower in patients with vs without triple regression (between-group difference: -27.1 mg/dL; 95% CI: -37.7 to -16.6 mg/dL; P < 0.001). Triple regression was independently predicted by alirocumab treatment (OR: 2.83; 95% CI: 1.57-5.16; P = 0.001) and a higher baseline maximum lipid core burden index within 4 mm (OR: 1.03; 95% CI: 1.01-1.06; P = 0.013). The composite clinical endpoint of death, myocardial infarction, and ischemia-driven revascularization occurred less frequently in patients with vs without triple regression (8.3% vs 18.2%; P = 0.04). CONCLUSIONS: Triple regression occurred in one-third of patients with acute myocardial infarction who were receiving high-intensity lipid-lowering therapy and was associated with alirocumab treatment, higher baseline lipid content, and reduced cardiovascular events. (Vascular Effects of Alirocumab in Acute MI-Patients [PACMAN-AMI]; NCT03067844).

Neutrophils in lung cancer patients: Activation potential and neutrophil extracellular trap formation.

Background: Patients with cancer have an increased risk of developing venous thromboembolism. Neutrophils and neutrophil extracellular traps (NETs) reportedly influence the risk of cancer-associated thrombosis. Subpopulations of high and low-density neutrophils (HDN/LDN) are of specific interest, as they might have different functions in cancer patients. Objectives: We aimed to investigate differences between HDNs and LDNs of patients with lung cancer and healthy controls, and their ability of activation and NET formation. Methods: Within the framework of the Vienna Cancer and Thrombosis Study, a prospective observational cohort study, HDNs and LDNs from 20 patients with lung cancer and 20 controls were isolated by density gradient centrifugation. The ability of neutrophil subpopulations for activation and NET formation was investigated by flow cytometry. Results: Compared to controls, patients with cancer had higher numbers of total leukocytes, HDNs, and LDNs. LDNs of patients were more frequently in an activated state (CD62L↓/CD16↑) at baseline (median [IQR] 5.9% [3.4-8.8] vs 2.5% [1.6-6.7]). HDNs and LDNs from patients showed a significantly increased response to stimulation with ionomycin (CD11b HDN: 98.5 [95.4-99.4] vs 41.7 [13.4-91.6]; LDN: 82.9 [63-94] vs 39.6 [17.3-72.1]). In addition, HDNs from patients showed a higher capability of NET formation after ionomycin stimulation compared to HDNs from healthy controls (18509.5 [12242.5-29470.3] vs 10001 [6618.8-18384.3]). Conclusion: Protumorigenic LDNs were elevated, and neutrophil subpopulations showed an increased activation profile and ability for NET formation in patients with cancer. These mechanisms might be involved in tumor promotion and contribute to the prothrombotic phenotype of neutrophils in cancer.

Left Ventricular Filling Pressure in Chronic Thromboembolic Pulmonary Hypertension.

BACKGROUND: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by obstruction of major pulmonary arteries with organized thrombi. Clinical risk factors for pulmonary hypertension due to left heart disease including metabolic syndrome, left-sided valvular heart disease, and ischemic heart disease are common in CTEPH patients. OBJECTIVES: The authors sought to investigate prevalence and prognostic implications of elevated left ventricular filling pressures (LVFP) in CTEPH. METHODS: A total of 593 consecutive CTEPH patients undergoing a first diagnostic right and left heart catheterization were included in this study. Mean pulmonary arterial wedge pressure (mPAWP) and left ventricular end-diastolic pressure (LVEDP) were utilized for assessment of LVFP. Two cutoffs were applied to identify patients with elevated LVFP: 1) for the primary analysis mPAWP and/or LVEDP >15 mm Hg, as recommended by the current pulmonary hypertension guidelines; and 2) for the secondary analysis mPAWP and/or LVEDP >11 mm Hg, representing the upper limit of normal. Clinical and echocardiographic features, and long-term mortality were assessed. RESULTS: LVFP was >15 mm Hg in 63 (10.6%) and >11 mm Hg in 222 patients (37.4%). Univariable logistic regression analysis identified age, systemic hypertension, diabetes, atrial fibrillation, calcific aortic valve stenosis, mitral regurgitation, and left atrial volume as significant predictors of elevated LVFP. Atrial fibrillation, calcific aortic valve stenosis, mitral regurgitation, and left atrial volume remained independent determinants of LVFP in adjusted analysis. At follow-up, higher LVFPs were measured in patients who had meanwhile undergone pulmonary endarterectomy (P = 0.002). LVFP >15 mm Hg (P = 0.021) and >11 mm Hg (P = 0.006) were both associated with worse long-term survival. CONCLUSIONS: Elevated LVFP is common, appears to be due to comorbid left heart disease, and predicts prognosis in CTEPH.

Metabolomics implicate eicosanoids in severe functional mitral regurgitation.

AIMS: Secondary, or functional, mitral regurgitation (FMR) was recently recognized as a separate clinical entity, complicating heart failure with reduced ejection fraction (HFrEF) and entailing particularly poor outcome. Currently, there is a lack of targeted therapies for FMR due to the fact that pathomechanisms leading to FMR progression are incompletely understood. In this study, we sought to perform metabolomic profiling of HFrEF patients with severe FMR, comparing results to patients with no or mild FMR. METHODS AND RESULTS: Targeted plasma metabolomics and untargeted eicosanoid analyses were performed in samples drawn from HFrEF patients (n = 80) on optimal guideline-directed medical therapy. Specifically, 17 eicosanoids and 188 metabolites were analysed. Forty-seven patients (58.8%) had severe FMR, and 33 patients (41.2%) had no or non-severe FMR. Comparison of eicosanoid levels between groups, accounting for age, body mass index, and sex, revealed significant up-regulation of six eicosanoids (11,12-EET, 13(R)-HODE, 12(S)-HETE, 8,9-DiHETrE, metPGJ2, and 20-HDoHE) in severe FMR patients. Metabolites did not differ significantly. In patients with severe FMR, but not in those without severe FMR, levels of 8,9-DiHETrE above a cut-off specified by receiver-operating characteristic analysis independently predicted all-cause mortality after a median follow-up of 43 [interquartile range 38, 48] months [hazard ratio 12.488 (95% confidence interval 3.835-40.666), P < 0.0001]. CONCLUSIONS: We report the up-regulation of various eicosanoids in patients with severe FMR, with 8,9-DiHETrE appearing to predict mortality. Our observations may serve as a nucleus for further investigations into the causes and consequences of metabolic derangements in this important valvular abnormality.

Cell-Free Double-Stranded DNA to DNase Ratio Predicts Outcome after Primary Survived Cardiac Arrest.

(1) Double-stranded DNA (dsDNA) and deoxyribonuclease (DNase) as surrogate parameters for accumulating inflammatory hazards are insufficiently studied in resuscitation research. (2) Blood samples of 76 individuals after CA were analyzed 24 and 96 h after ICU admission. Plasma levels of dsDNA, interleukin-8, and monocyte chemoattractant protein-1 and activity of DNase were assessed along with baseline characteristics, intensive care measures, and outcome data. DsDNA/DNase ratio was used as main prognostication parameter. After calculating an optimal empirical cut-off for outcome prediction (death or Cerebral Performance Category ≥3 at 6 months), multivariable logistic regression was applied. (3) Using receiver operating characteristic (ROC) analysis, an area under the curve (AUC) of 0.65 (95% CI 0.50-0.79) was found for dsDNA/DNase after 24 h versus 0.83 (95% CI 0.73-0.92) after 96 h (p = 0.03). The empirical cut-off for dsDNA/DNase ratio after 96 h was 149.97 (Youden). DsDNA/DNase ratio was associated with unfavorable outcome at six months (aOR 1.006, 95% CI 1.0017-1.0094, p = 0.005). In multivariable analysis, the association of dsDNA/DNase ratio independently predicted outcome as a continuous variable (aOR 1.004, 95% CI 1.0004-1.0079, p = 0.029) after adjusting for potential confounders. (4) DsDNA/DNase ratio at 96 h demonstrates good predictive performance for estimating outcome after CA.

The age-specific prognostic impact of the platelet-to-lymphocyte ratio on long-term outcome after acute coronary syndrome.

Aims: Personalized risk stratification within the ageing society after acute coronary syndrome (ACS) remains scarce but in urgent need. Increased platelet activity together with inflammatory activation play a key role during ACS. We aimed to evaluate the age-specific prognostic potential of the platelet to lymphocyte ratio (PLR) on long-term cardiovascular mortality after ACS. Methods and results: Patients presenting with ACS admitted to the Vienna General Hospital between December 1996 and January 2010 were enrolled within a clinical registry including assessment of peripheral blood samples. The impact of the PLR on survival was assessed by Cox-regression hazard analysis. We included a total of 681 patients with a median age of 64 years (interquartile range: 45-84). Two hundred (29.4%) individuals died during the median follow-up time of 8.5 years. A strong and independent association of the PLR with cardiovascular mortality was found in the total study population [adjusted (adj.) hazard ratio (HR) per 1 standard deviation (1 SD) of 1.07 (95% confidence interval, CI: 1.03-1.10); P < 0.001]. After stratification in individuals <65 years (n = 339) and ≥65 years (n = 342), a prognostic effect of the PLR on cardiovascular mortality was solely observed in elderly patients ≥65 years [adj. HR per 1 SD of 1.04 (95% CI: 1.00-1.08); P = 0.039], but not in their younger counterparts <65 years [adj. HR per 1 SD of 0.97 (95% CI: 0.83-1.14); P = 0.901]. Conclusion: The present investigation highlights a strong and independent age-specific association of the PLR with cardiovascular mortality in patients with ACS. The PLR only allows to identify patients ≥65 years at high risk for fatal events after ACS-even from a long-term perspective.

The Effect of Paracrine Factors Released by Irradiated Peripheral Blood Mononuclear Cells on Neutrophil Extracellular Trap Formation.

Neutrophil extracellular trap (NET)-formation represents an important defence mechanism for the rapid clearance of infections. However, exaggerated NET formation has been shown to negatively affect tissue-regeneration after injury. As our previous studies revealed the strong tissue-protective and regenerative properties of the secretome of stressed peripheral blood mononuclear cells (PBMCsec), we here investigated the influence of PBMCsec on the formation of NETs. The effect of PBMCsec on NET formation was assessed ex vivo in ionomycin stimulated neutrophils derived from healthy donors using flow cytometry, image stream analysis, and quantification of released extracellular DNA. The effect of PBMCsec on molecular mechanisms involved in NET formation, including Ca-flux, protein kinase C activity, reactive oxygen species production, and protein arginine deiminase 4 activity, were analysed. Our results showed that PBMCsec significantly inhibited NET formation. Investigation of the different biological substance classes found in PBMCsec revealed only a partial reduction in NET formation, suggesting a synergistic effect. Mechanistically, PBMCsec treatment did not interfere with calcium signalling and PKC-activation, but exerted anti-oxidant activity, as evidenced by reduced levels of reactive oxygen species and upregulation of heme oxygenase 1 and hypoxia inducible-factor 1 in PBMCsec-treated neutrophils. In addition, PBMCsec strongly inhibited the activation of protein arginine deiminase 4 (PAD4), ultimately leading to the inhibition of NET formation. As therapeutics antagonizing excessive NET formation are not currently available, our study provides a promising novel treatment option for a variety of conditions resulting from exaggerated NET formation.

Culprit site extracellular DNA and microvascular obstruction in ST-elevation myocardial infarction.

AIMS: Extracellular chromatin and deoxyribonuclease (DNase) have been identified as important players of thrombosis, inflammation, and homeostasis in a murine model. We previously demonstrated that activated neutrophils release neutrophil extracellular traps (NETs) at the culprit site in ST-elevation myocardial infarction (STEMI), which significantly contribute to extracellular chromatin burden, and are associated with larger infarcts. To understand the correlation between neutrophil activation, extracellular chromatin, and infarct size (IS), we investigated these parameters in a porcine myocardial infarction model, and at different time points and sites in a prospective STEMI trial with cardiac magnetic resonance (CMR) endpoints. METHODS AND RESULTS: In a prospective STEMI trial (NCT01777750), 101 STEMI patients were included and blood samples were obtained from first medical contact until 6 months after primary percutaneous coronary intervention (pPCI) including direct sampling from the culprit site. CMR was performed 4 ± 2 days and 6 months after pPCI. Neutrophil counts, markers of extracellular chromatin, and inflammation were measured. Double-stranded deoxyribonucleic acid (dsDNA), citrullinated histone 3, nucleosomes, myeloperoxidase, neutrophil elastase, and interleukin (IL)-6 were significantly increased, while DNase activity was significantly decreased at the culprit site in STEMI patients. High neutrophil counts and dsDNA levels at the culprit site correlated with high microvascular obstruction (MVO) and low ejection fraction (EF). High DNase activity at the culprit site correlated with low MVO and high EF. In correspondence, dsDNA correlated with IS in the porcine myocardial infarction model. In porcine infarcts, neutrophils and extracellular chromatin were detected in congested small arteries corresponding with MVO. Markers of neutrophil activation, extracellular chromatin, DNase activity and CMR measurements correlated with markers of systemic inflammation C-reactive protein and IL-6 in patients. CONCLUSIONS: NETs and extracellular chromatin are important determinants of MVO in STEMI. Rapid degradation of extracellular chromatin by DNases appears to be crucial for microvascular patency and outcome.

Neutrophil Extracellular Traps in Atherosclerosis and Thrombosis.

Despite effective therapeutic and preventive strategies, atherosclerosis and its complications still represent a substantial health burden. Leukocytes and inflammatory mechanisms are increasingly recognized as drivers of atherosclerosis. Neutrophil granulocytes within the circulation were recently shown to undergo neutrophil extracellular trap (NET) formation, linking innate immunity with acute complications of atherosclerosis. In this chapter, we summarize mechanisms of NET formation, evidence for their involvement in atherosclerosis and thrombosis, and potential therapeutic regimens specifically targeting NET components.

Comparison of SARS-CoV-2 Antibody Response 4 Weeks After Homologous vs Heterologous Third Vaccine Dose in Kidney Transplant Recipients: A Randomized Clinical Trial.

Importance: Fewer than 50% of kidney transplant recipients (KTRs) develop antibodies against the SARS-CoV-2 spike protein after 2 doses of an mRNA vaccine. Preliminary data suggest that a heterologous vaccination, combining mRNA and viral vector vaccines, may increase immunogenicity. Objective: To assess the effectiveness of a third dose of an mRNA vs a vector vaccine in KTRs who did not have antibodies against the SARS-CoV-2 spike protein after 2 doses of an mRNA vaccine. Design, Setting, and Participants: This was a single center, single-blinded, 1:1 randomized clinical trial of a third dose of vaccine against SARS-CoV-2, conducted from June 15 to August 16, 2021, in 201 KTRs who had not developed SARS-CoV-2 spike protein antibodies after 2 doses of an mRNA vaccine. Data analyses were performed from August 17 to August 31, 2021. Interventions: mRNA (BNT162b2 or mRNA-1273) or vector (Ad26COVS1) as a third dose of a SARS-CoV-2 vaccine. Main Outcomes and Measures: The primary study end point was seroconversion after 4 weeks (29-42 days) following the third vaccine dose. Secondary end points included neutralizing antibodies and T-cell response assessed by interferon-γ release assays (IGRA). In addition, the association of patient characteristics and vaccine response was assessed using logistic regression, and the reactogenicity of the vaccines was compared. Results: Among the study population of 197 kidney transplant recipients (mean [SD] age, 61.2 [12.4] years; 82 [42%] women), 39% developed SARS-CoV-2 antibodies after the third vaccine. There was no statistically significant difference between groups, with an antibody response rate of 35% and 42% for the mRNA and vector vaccines, respectively. Only 22% of seroconverted patients had neutralizing antibodies. Similarly, T-cell response assessed by IGRA was low with only 17 patients showing a positive response after the third vaccination. Receiving nontriple immunosuppression (odds ratio [OR], 3.59; 95% CI, 1.33-10.75), longer time after kidney transplant (OR, 1.44; 95% CI, 1.15-1.83, per doubling of years), and torque teno virus plasma levels (OR, 0.92; 95% CI, 0.88-0.96, per doubling of levels) were associated with vaccine response. The third dose of an mRNA vaccine was associated with a higher frequency of local pain at the injection site compared with the vector vaccine, while systemic symptoms were comparable between groups. Conclusions and Relevance: This randomized clinical trial found that 39% of KTRs without an immune response against SARS-CoV-2 after 2 doses of an mRNA vaccine developed antibodies against the SARS-CoV-2 spike protein 4 weeks after a third dose of an mRNA or a vector vaccine. The heterologous vaccination strategy with a vector-based vaccine was well tolerated and safe but not significantly better than the homologous mRNA-based strategy. Trial Registration: EudraCT Identifier: 2021-002927-39.

Clinical Impact of Pre-Procedural Percutaneous Coronary Intervention in Low- and Intermediate-Risk Transcatheter Aortic Valve Replacement Recipients.

The clinical relevance of as well as the optimal treatment strategy for coronary artery disease (CAD) in patients undergoing transcatheter aortic valve replacement (TAVR) for severe aortic stenosis (AS) are unclear. Current data are conflicting, and mainly derived from high-risk patients. We aimed to investigate the feasibility and safety of complete revascularization prior to TAVR for severe AS in low- and intermediate-risk patients. We enrolled 449 patients at low (STS score < 4%) and intermediate risk (STS score 4-8%) undergoing TAVR for severe AS and investigated the influence of recent (<3 months) and prior (>3 months) complete revascularization on clinical outcome. Primary study endpoint was all-cause mortality. Overall, 58% of patients had no or non-significant CAD; 18% had a history of complete revascularization prior to TAVR and 24% had complete revascularization shortly before TAVR. Two-year all-cause mortality was not different between patients with recent revascularization prior to TAVR and patients with no or non-significant CAD (13.7% vs. 14.2%, p = 0.905). Cox regression did not reveal an effect on all-cause mortality for recent revascularization. The present analysis reassures that percutaneous complete revascularization prior to TAVR procedures is neutral in terms of all-cause mortality in patients at low and intermediate surgical risk.

Mild Therapeutic Hypothermia Alters Hemostasis in ST Elevation Myocardial Infarction Patients.

Background and Rationale: Mild therapeutic hypothermia (MTH) is a concept to reduce infarct size and improve outcome after ST-segment elevation myocardial infarction (STEMI). In the STATIM trial, we investigated MTH as an additional therapy for STEMI patients. In the intention-to-treat set, 101 patients were included. No difference in primary and secondary endpoints measured by cardiac magnetic resonance imaging was found. Platelet activation and plasmatic coagulation are key in the pathophysiology of STEMI. In the present study, we investigated the effect of MTH on primary and secondary hemostasis in STEMI patients. Methods and Results: Platelet function and morphology were assessed by routine blood count, aggregometry testing, and flow cytometry. Soluble platelet markers were determined by enzyme-linked immunosorbent assay (ELISA) testing. Plasmatic coagulation was measured throughout the study. Platelet count remained unchanged, irrespective of treatment, whereas platelet size decreased in both patient groups. Platelet aggregometry indicated increased platelet reactivity in the MTH group. Furthermore, higher adenosine diphosphate (ADP) plasma levels were found in MTH patients. Expression of glycoprotein IIb/IIIa was increased on platelets of STEMI patients treated with MTH. Lower patient temperatures correlated with longer clotting times and resulted in reduced pH. Lower pH values were positively correlated with longer clotting times. Conclusion: Present data indicate longer clotting times and higher platelet reactivity in STEMI patients treated with MTH. These changes did not correspond to clinical bleeding events or larger infarct size.

Deoxyribonuclease is prognostic in patients undergoing transcatheter aortic valve replacement.

Degenerative aortic valve stenosis is an inflammatory process that resembles atherosclerosis. Neutrophils release their DNA upon activation and form neutrophil extracellular traps (NETs), which are present on degenerated aortic valves. NETs correlate with pressure gradients in severe aortic stenosis. Transcatheter aortic valve replacement (TAVR) is an established treatment option for aortic valve stenosis. Bioprosthetic valve deterioration promoted by inflammatory, fibrotic and thrombotic processes limits outcome. Deoxyribonuclease is a natural counter mechanism to degrade DNA in circulation. In the present observational study, we investigated plasma levels of double-stranded DNA, deoxyribonuclease activity and outcome after TAVR. 345 consecutive patients undergoing TAVR and 100 healthy reference controls were studied. Double-stranded DNA was measured by fluorescence assays in plasma obtained at baseline and after TAVR. Deoxyribonuclease activity was measured at baseline using single radial enzyme diffusion assays. Follow-up was performed at 12 months, and mean aortic pressure gradient and survival were evaluated. Receiver operating characteristic, Kaplan-Meier curves and Cox regression models were calculated. Baseline double-stranded DNA in plasma was significantly higher compared to healthy controls, was increased at 3 and 7 days after TAVR, and declined thereafter. Baseline deoxyribonuclease activity was decreased compared to healthy controls. Interestingly, low deoxyribonuclease activity correlated with higher C-reactive protein and higher mean transaortic gradient after 12 months. Finally, deoxyribonuclease activity was a strong independent predictor of outcome 12 months after TAVR. Deoxyribonuclease activity is a potential biomarker for risk stratification after TAVR. Pathomechanisms of bioprosthetic valve deterioration involving extracellular DNA and deoxyribonuclease merit investigation.

ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo.

Over the past years, neutrophil extracellular traps (NETs) were shown to contribute to states of acute and chronic inflammatory disease. They are composed of expelled chromatin and decorated by neutrophil-derived proteins. Therefore, the analysis of DNA complexes with myeloperoxidase (MPO) by ELISA has become an attractive tool to measure NET formation in in vitro and in vivo samples. When we used a published MPO-DNA ELISA protocol and included an isotype control for the anti-MPO coating antibody, we observed high assay specificity for in vitro prepared NET samples, whereas the specificity for in vivo plasma samples was low. In addition, the assay failed to detect in vitro generated MPO-DNA complexes when spiked into plasma. Therefore, we set out to improve the specificity of the MPO-DNA ELISA for plasma samples. We found that the use of Fab fragments or immunoglobulins from different species or reversal of the antibody pair led to either a high background or a low dynamic range of detection that did not improve the specificity for plasma samples. Also, the use of higher plasma dilutions or pre-clearing of plasma immunoglobulins were ineffective. Finally, we found that a commercial reagent designed to block human anti-mouse antibodies and multivalent substances increased the detection window between the MPO antibody and isotype control for highly diluted plasma. We applied this modified ELISA protocol to analyze MPO-DNA complexes in human blood samples of acute and chronic inflammatory conditions. While markers of neutrophil activation and NET formation such as MPO, elastase and citrullinated histone H3 correlated significantly, we observed no correlation with the levels of MPO-DNA complexes. Therefore, we conclude that ELISA measurements of MPO-DNA complexes in human plasma are highly questionable regarding specificity of NET detection. In general, plasma analyses by ELISA should more frequently include isotype controls for antibodies to demonstrate target specificity.

Deoxyribonuclease 1 Q222R single nucleotide polymorphism and long-term mortality after acute myocardial infarction.

Upon activation, neutrophils release neutrophil extracellular traps (NETs), which contribute to circulating DNA burden and thrombosis, including ST-segment elevation myocardial infarction (STEMI). Deoxyribonuclease (DNase) 1 degrades circulating DNA and NETs. Lower DNase activity correlates with NET burden and infarct size. The DNase 1 Q222R single nucleotide polymorphism (SNP), impairing DNase 1 function, is linked with myocardial infarction. We assessed whether the Q222R SNP is connected to increased NET burden in STEMI and influences long-term outcomes. We enrolled 711 STEMI patients undergoing primary percutaneous coronary intervention (pPCI), and 1422 controls. Genotyping was performed for DNase 1 Q222R SNP. DNase activity, double-stranded (ds)DNA and citrullinated histone H3 were determined in culprit site and peripheral plasma during pPCI. The association of the Q222R variant on cardiovascular and all-cause mortality was assessed by multivariable Cox regression adjusted for cardiovascular risk factors. Homozygous Q222R DNase 1 variant was present in 64 (9.0%) STEMI patients, at the same frequency as in controls. Patients homozygous for Q222R displayed less DNase activity and increased circulating DNA burden. In overall patients, median survival was 60 months. Homozygous Q222R variant was independently associated with cardiovascular and all-cause mortality after STEMI. dsDNA/DNase ratio independently predicted cardiovascular and all-cause mortality. These findings highlight that the Q222R DNase 1 SNP is associated with increased NET burden and decreased compensatory DNase activity, and may serve as an independent risk factor for poor outcome after STEMI.

Neutrophil extracellular traps promote fibrous vascular occlusions in chronic thrombosis.

Acute pulmonary embolism generally resolves within 6 months. However, if the thrombus is infected, venous thrombi transform into fibrotic vascular obstructions leading to chronic deep vein thrombosis and/or chronic thromboembolic pulmonary hypertension (CTEPH), but precise mechanisms remain unclear. Neutrophils are crucial in sequestering pathogens; therefore, we investigated the role of neutrophil extracellular traps (NETs) in chronic thrombosis. Because chronic pulmonary thrombotic obstructions are biologically identical to chronic deep venous thrombi, the murine inferior vena cava ligation model was used to study the transformation of acute to chronic thrombus. Mice with staphylococcal infection presented with larger thrombi containing more neutrophils and NETs but less resolution. Targeting NETs with DNase1 diminished fibrosis and promoted thrombus resolution. For translational studies in humans, we focused on patients with CTEPH, a severe type of deep venous and pulmonary artery fibrotic obstruction after thrombosis. Neutrophils, markers of neutrophil activation, and NET formation were increased in CTEPH patients. NETs promoted the differentiation of monocytes to activated fibroblasts with the same cellular phenotype as fibroblasts from CTEPH vascular occlusions. RNA sequencing of fibroblasts isolated from thrombo-endarterectomy specimens and pulmonary artery biopsies revealed transforming growth factor-ß (TGF-ß) as the central regulator, a phenotype which was replicated in mice with fibroblast-specific TGF-ß overactivity. Our findings uncover a role of neutrophil-mediated inflammation to enhance TGF-ß signaling, which leads to fibrotic thrombus remodeling. Targeting thrombus NETs with DNases may serve as a new therapeutic concept to treat thrombosis and prevent its sequelae.

Neutrophil subpopulations and their activation potential in patients with antiphospholipid syndrome and healthy individuals.

OBJECTIVES: Patients with APS are at increased risk of thromboembolism. Neutrophils have been shown to play a role in inducing thrombosis. We aimed to investigate differences in neutrophil subpopulations, their potential of activation and neutrophil extracellular trap (NET) formation comparing high and low-density neutrophils (HDNs/LDNs) as well as subpopulations in patients with APS and controls to gain deeper insight into their potential role in thrombotic manifestations in patients with APS. METHODS: HDNs and LDNs of 20 patients with APS and 20 healthy donors were isolated by density gradient centrifugation and stimulated. Neutrophil subpopulations, their activation and NET release were assessed by flow cytometry. RESULTS: LDNs of both groups showed higher baseline activation, lower response to stimulation (regulation of activation markers CD11b/CD66b), but higher NET formation compared with HDNs. In patients with APS, the absolute number of LDNs was higher compared with controls. HDNs of APS patients showed higher spontaneous activation [%CD11b high: median (interquartile range): 2.78% (0.58-10.24) vs 0.56% (0.19-1.37)] and response to stimulation with ionomycin compared with HDNs of healthy donors [%CD11b high: 98.20 (61.08-99.13) vs 35.50% (13.50-93.85)], whereas no difference was found in LDNs. NET formation was increased in patients' HDNs upon stimulation. CONCLUSION: HDNs and LDNs act differently, unstimulated and upon various stimulations in both healthy controls and APS patients. Differences in HDNs and LDNs between patients with APS and healthy controls indicate that neutrophils may enhance the risk of thrombosis in these patients and could thus be a target for prevention of thrombosis in APS.

Neutrophil Extracellular Traps Induce MCP-1 at the Culprit Site in ST-Segment Elevation Myocardial Infarction.

BACKGROUND: Leukocyte-mediated inflammation is crucial in ST-segment elevation myocardial infarction (STEMI). We recently observed that neutrophil extracellular traps (NETs) are increased at the culprit site, promoting activation and differentiation of fibrocytes, cells with mesenchymal and leukocytic properties. Fibrocyte migration is mediated by monocyte chemoattractant protein (MCP)-1 and C-C chemokine receptor type 2 (CCR2). We investigated the interplay between NETs, fibrocyte function, and MCP-1 in STEMI. METHODS: Culprit site and peripheral blood samples of STEMI patients were drawn during primary percutaneous coronary intervention. MCP-1 and the NET marker citrullinated histone H3 (citH3) were measured by ELISA while double-stranded DNA was stained with a fluorescent dye. The influence of MCP-1 on NET formation in vitro was assessed using isolated healthy donor neutrophils. Human coronary artery endothelial cells (hCAECs) were stimulated with isolated NETs, and MCP-1 gene expression was measured by ELISA and qPCR. CCR2 expression of culprit site and peripheral blood fibrocytes was characterized by flow cytometry. Healthy donor fibrocyte receptor expression and chemotaxis were investigated in response to stimulation with MCP-1 and NETs in vitro. RESULTS: NETs and concentrations of MCP-1 were increased at the culprit site of 50 consecutive STEMI patients. NET stimulation of hCAECs induced transcription of ICAM-1, IL-6, and MCP-1, and secretion of MCP-1. MCP-1 promoted NET formation of healthy donor neutrophils in vitro. An increasing MCP-1 gradient correlated with fibrocyte accumulation at the culprit site. Locally increased MCP-1 levels were negatively correlated with CCR2 expression on fibrocytes. MCP-1 and NETs induced CCR2 downregulation on fibrocytes in vitro. NETs did not function as a chemotactic stimulus for fibrocytes or monocytes and could block migration in response to MCP-1 for both cell populations. CONCLUSION: NETs function as signaling scaffolds at the culprit site of STEMI. NETs assist MCP-1 and ICAM-1 release from culprit site coronary artery endothelial cells. MCP-1 facilitates further NETosis. Monocytes enter the culprit site along an MCP-1 gradient, to transdifferentiate into fibrocytes in the presence of NETs.

Neutrophil extracellular traps and monocyte subsets at the culprit lesion site of myocardial infarction patients.

Neutrophils release their chromatin into the extracellular space upon activation. These web-like structures are called neutrophil extracellular traps (NETs) and have potent prothrombotic and proinflammatory properties. In ST-elevation myocardial infarction (STEMI), NETs correlate with increased infarct size. The interplay of neutrophils and monocytes impacts cardiac remodeling. Monocyte subsets are classified as classical, intermediate and non-classical monocytes. In the present study, in vitro stimulation with NETs led to an increase of intermediate monocytes and reduced expression of CX3CR1 in all subsets. Intermediate monocytes have been associated with poor outcome, while non-classical CX3CR1-positive monocytes could have reparative function after STEMI. We characterized monocyte subsets and NET markers at the culprit lesion site of STEMI patients (n = 91). NET surrogate markers were increased and correlated with larger infarct size and with fewer non-classical monocytes. Intermediate and especially non-classical monocytes were increased at the culprit site compared to the femoral site. Low CX3CR1 expression of monocytes correlated with high NET markers and increased infarct size. In this translational system, causality cannot be proven. However, our data suggest that NETs interfere with monocytic differentiation and receptor expression, presumably promoting a subset shift at the culprit lesion site. Reduced monocyte CX3CR1 expression may compromise myocardial salvage.

Neutrophil extracellular traps and fibrocytes in ST-segment elevation myocardial infarction.

Leukocyte-mediated inflammation is central in atherothrombosis and ST-segment elevation myocardial infarction (STEMI). Neutrophil extracellular traps (NETs) have been shown to enhance atherothrombosis and stimulate fibroblast function. We analyzed the effects of NETs on cardiac remodeling after STEMI. We measured double-stranded (ds)DNA and citrullinated histone H3 (citH3) as NET surrogate markers in human culprit site and femoral blood collected during primary percutaneous coronary intervention (n = 50). Fibrocytes were characterized in whole blood by flow cytometry, and in culprit site thrombi and myocardium by immunofluorescence. To investigate mechanisms of fibrocyte activation, isolated NETs were used to induce fibrocyte responses in vitro. Enzymatic infarct size was assessed using creatine-phosphokinase isoform MB area under the curve. Left ventricular function was measured by transthoracic echocardiography. NET surrogate markers were increased at the culprit site compared to the femoral site and were positively correlated with infarct size and left ventricular dysfunction at follow-up. In vitro, NETs promoted fibrocyte differentiation from monocytes and induced fibrocyte activation. Highly activated fibrocytes accumulated at the culprit site and in the infarct transition zone. Our data suggest that NETs might be important mediators of fibrotic remodeling after STEMI, possibly by stimulating fibrocytes.