Abstract Aims We aimed to study the long-term effect of neutrophils on cardiac health during the process of natural aging. We hypothesized that neutrophil PAD4, via its role in neutrophil extracellular trap (NET) formation, is involved in myocardial remodeling and cardiac fibrosis development, resulting in turn in impaired cardiac function. Methods and results We generated mice with deletion of Padi4 , a NET-essential gene, under the neutrophil-specific promoter S100A8 (PAD4 fl/fl MRP8Cre + ). These mice and their littermate controls were aged for two years (coinciding with approximately 70 years of age in humans; the age at which HF is the number one cause of hospitalization), after which cardiac function and remodeling were evaluated. We performed a comprehensive echocardiography analysis including both structural and functional parameter measurements. Deletion of PAD4 in neutrophils resulted in a protection against both systolic, and diastolic dysfunction. Interestingly, these mice showed protection against age induced fibrosis, detected as through the absence of cardiac collagen deposition. To explore this further, cardiac gene expression and plasma cytokine levels were evaluated. Here we saw a clear impact of PAD4-deficiency on cardiac neutrophil recruitment, with both cardiac genes as well as plasma cytokines involved in neutrophil recruitment being downregulated in aged PAD4 fl/fl MRP8Cre + animals in comparison to littermate PAD4 fl/fl controls, including decreased plasma levels of C-X-C ligand 1 (CXCL1). Conclusion Our data confirms neutrophil PAD4 involvement in heart failure progression by promoting cardiac remodeling, leading to cardiac dysfunction with old age. We saw that the deletion of PAD4 specifically in neutrophils had an influence on the CXCL1-CXCR2 axis, which is known to be involved in HF development. Translational perspective In the developed world, an estimated 2% of the population lives with heart failure (HF). HF can be viewed as an upcoming pandemic, which is only expected to increase due to the aging of the global population. Therefore, research in HF development and progression are needed to establish new avenues for treatment and improved therapies. In our study, we were able to show the contribution of neutrophil PAD4 to HF pathogenesis, providing new supporting evidence for the involvement of NETs in detrimental cardiac remodeling.
Background Platelets are key components in atherogenesis and determine the course of its clinical sequelae acute coronary syndrome (ACS). Components of the innate immune system—the superfamily of TLR receptors–are present in platelets and represent a link between atherothrombosis and inflammation. We hypothesize that alteration in platelet TLR mRNA expression is a result of inflammation driving coronary atherosclerosis and may represent an alternative platelet activation pathway in ACS. TLR2-, TLR4- and TLR9- mRNA-expression was determined in ACS patients and compared to patients with invasive exclusion of atherosclerotic lesions of coronary arteries. Methods A total of fifty-four patients were enrolled in this clinical retrospective cohort single centre study. Total RNA from sepharose-filtered highly purified platelets was isolated using acid guanidinium thiocyanate-phenol-chloroform extraction and transcribed to cDNA using a first strand cDNA synthesis kit. To determine absolute copy numbers of TLR2, TLR4 and TLR9 we used plasmid based quantitative PCR with normalisation to an internal control. Results We found that mRNA expression levels of TLR2 but not TLR 4 and 9 are up-regulated in platelets of patients with ACS when compared to patients without coronary atherosclerosis. Conclusion Our results suggest elevated TLR2 mRNA expression in platelets as a biomarker reflecting the underlying inflammation in ACS and possibly severity of coronary atherosclerosis. Platelet TLR2 may represent a link between inflammation and atherothrombosis in ACS.
Objective Activated platelets release serotonin at sites of inflammation where it acts as inflammatory mediator and enhances recruitment of neutrophils. Chronic treatment with selective serotonin reuptake inhibitors (SSRI) depletes the serotonin storage pool in platelets, leading to reduced leukocyte recruitment in murine experiments. Here, we examined the direct and acute effects of SSRI on leukocyte recruitment in murine peritonitis. Methods C57Bl/6 and Tph1−/− (Tryptophan hydroxylase1) mice underwent acute treatment with the SSRI fluoxetine or vehicle. Serotonin concentrations were measured by ELISA. Leukocyte rolling and adhesion on endothelium was analyzed by intravital microscopy in mesentery venules with and without lipopolysaccharide challenge. Leukocyte extravasation in sterile peritonitis was measured by flow cytometry of abdominal lavage fluid. Results Plasma serotonin levels were elevated 2 hours after fluoxetine treatment (0.70±0.1 µg/ml versus 0.27±0.1, p = 0.03, n = 14), while serum serotonin did not change. Without further stimulation, acute fluoxetine treatment increased the number of rolling leukocytes (63±8 versus 165±17/0.04 mm2min−1) and decreased their velocity (61±6 versus 28±1 µm/s, both p<0.0001, n = 10). In Tph1−/− mice leukocyte rolling was not significantly influenced by acute fluoxetine treatment. Stimulation with lipopolysaccharide decreased rolling velocity and induced leukocyte adhesion, which was enhanced after fluoxetine pretreatment (27±3 versus 36±2/0.04 mm2, p = 0.008, n = 10). Leukocyte extravasation in sterile peritonitis, however, was not affected by acute fluoxetine treatment. Conclusions Acute fluoxetine treatment increased plasma serotonin concentrations and promoted leukocyte-endothelial interactions in-vivo, suggesting that serotonin is a promoter of acute inflammation. E-selectin was upregulated on endothelial cells in the presence of serotonin, possibly explaining the observed increase in leukocyte-endothelial interactions. However transmigration of neutrophils in sterile peritonitis was not affected by higher serotonin concentrations, indicating that the effect of fluoxetine was restricted to early steps in the leukocyte recruitment. Whether SSRI use in humans alters leukocyte recruitment remains to be investigated.
The following funder is missing from the Funding section: Deutsche Herzstiftung. The correct funding information is as follows: This work was funded by the Deutsche Forschungsgemeinschaft (DFG) and the Deutsche Herzstiftung. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Ein neuer Name für eine alte Krankheit: Die stabile koronare Herzerkrankung wird in den aktualisierten Richtlinien der Europäischen Gesellschaft für Kardiologie nun als chronisches Koronarsyndrom (CCS) bezeichnet. Damit soll der fortschreitende Verlauf der Erkrankung hervorgehoben werden. Auch in den aktualisierten Therapieempfehlungen wird die neue Sichtweise auf die Erkrankungen deutlich, die wir in dieser SOP berücksichtigt haben.
Abstract Background Emerging data suggest an association between left atrial (LA) enlargement, thrombus formation, and ischemic stroke. However, it is unknown what may mediate such clot formation in LA dysfunction. Neutrophils promote large vessel occlusion and microthrombosis via neutrophil extracellular trap (NET) release, thus lying at the interface of inflammation, thrombosis, and fibrosis. Approach We conducted a prospective all-comers cohort study in patients undergoing catheterization procedures with atrial transseptal access (MitraClip, MC; left atrial appendage closure, LAAC; pulmonary vein ablation, PVA; patent foramen ovale closure, PFO). We measured NETs, cytokines, thrombotic factors, and cardiac injury markers in paired blood samples collected from peripheral blood and within the left atrium. We correlated these biomarkers with echocardiographic measures of LA structure and function (including left atrial volume index, LAVI). Data were analyzed by procedure type, and stratified by LAVI or atrial fibrillation (AF) status. Results We enrolled 70 patients (mean age 64 years, 53% women). NETs, but not other markers, were elevated in LA compared to peripheral blood samples. Most thrombotic, inflammatory, and cardiac damage markers were elevated in LAs from MC or LAAC compared to PFO patients. Overall, NET biomarkers positively correlated with VWF, LAVI, and markers of cardiac injury and negatively with ADAMTS13 activity. LA enlargement and the presence of AF similarly stratified patients based on thromboinflammation measurements, but this was not limited to AF at the time of sample collection. Conclusion Elevated NETs and VWF in patients with enlarged LA or AF suggest enhanced thromboinflammation within the LA.
Timely diagnosis of heart failure (HF) in patients with a systemic right ventricle (sRV) is difficult but important since clinical deterioration is fast once HF develops. We aimed to compare echocardiography and biomarker profile between sRV patients with and without HF and patients with a systemic left ventricle diagnosed with HF (sLV-HF). Eighty-seven sRV patients and 30 sLV-HF patients underwent echocardiographic evaluation and blood sampling. Compared to sRV patients without HF, sRV-HF patients had more remodeling of the subpulmonary LV (spLV) (internal diameter 3.9 cm [3.3–5.7] vs 3.4 cm [2.9–3.9], P = 0.03, posterior wall 0.93 cm [0.76–1.20] vs 0.71 cm [0.59–0.91], P = 0.006) and lower spLV systolic function: ejection fraction (59 % ± 14 vs 70 % ± 10, P = 0.011), mitral annular plane systolic excursion (1.7 cm ± 0.5 vs 2.1 cm ± 0.4, P = 0.003), fractional area change (47 % [38–58] vs 59 % [51–70], P = 0.002) and lateral strain rate (−1.2/s ± 0.46 vs −1.5/s ± 0.39, P = 0.016). Inflammatory biomarkers were higher in sRV-HF patients compared to those without HF: red cell distribution width (13.3 fL [12.8–14.1] vs 12.6 fL [12.3–13.1], P < 0.001), neutrophil lymphocyte ratio (NLR, 3.7 [2.2–4.9] vs 2.4 [1.9–3.0], P = 0.015), C-reactive protein (CRP, 2.5 mg/dL [1.0–4.2] vs 1.2 mg/dL [0.0–2.0], P = 0.005) and compared to sLV-HF patients (NLR (3.7 [2.2–4.9] vs 2.5 [1.7–3.3], P = 0.044) and CRP (2.5 mg/dL [1.0–4.2] vs 0.85 mg/dL [0.6–2.0], P = 0.006). Biventricular echocardiographic evaluation with a focus on the subpulmonary LV together with assessing inflammatory status in sRV patients could help in an earlier detection of HF.
Atrial cardiopathy results in disturbed blood flow, providing an ideal thrombogenic environment. Neutrophils promote large vessel occlusions and microthrombosis via release of neutrophil extracellular traps (NETs) and thus lie at the interface of (sterile) inflammation, thrombosis, and fibrosis. We profiled NETs, cytokines, thrombotic factors, and cardiac damage markers in left atrial (LA) cardiopathies together with LA structural analysis. A total of 71 patients (median age 66, 46.5% male) undergoing catheterization procedures with atrial transseptal access at the Freiburg Heart Center were enrolled in this prospective study. Paired samples were collected from peripheral blood draws or from within the left atrium (LA). NET biomarkers, sP-selectin, PAD4, proinflammatory cytokines, thrombotic factors (D-dimers, VWF, ADAMTS13), and cardiac damage markers (TropT, proBNP, ICTP) were measured in plasma or serum as appropriate. Echocardiography was performed to assess LA structure and left ventricular function. Data were analyzed by the type of procedure (MitraClip (MC), left atrial appendage closure (LAAC), pulmonary vein ablation (PVA), or patent foramen ovale closure (PFOC), and by atrial fibrillation (Afib) status at time of procedure. NETs, but not other markers, were elevated in local LA samples as compared to peripheral blood. Conversely, most thrombotic, inflammatory, and cardiac damage markers, but not NETs, were elevated in MC or LAAC as compared to PFOC or PVA patients. Across all patients, NET biomarkers positively correlated with IL-6/TNF-α, VWF, and cardiac damage biomarkers. PAD4/NETs negatively correlated with ADAMTS13 activity, indicating that citrullination may contribute to the prothrombotic phenotype of these patients. Afib was not key to these observations. NETs are locally elevated within the left atrium, with elevation of thrombosis markers including VWF in patients with a diseased left atrium. These elevations partly correlate with structural changes of the left atrium and, therefore, may point toward a disturbed local blood flow. This may provide insight on mechanisms that drive detrimental left atrial remodeling as well as clues regarding thromboembolic risks that are rooted in LA changes rather than Afib.
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