Martin Mollenhauer

Neutrophil-derived myeloperoxidase promotes atherogenesis and neointima formation in mice

BACKGROUND Myeloperoxidase (MPO), expressed mainly in neutrophils, is an enzyme linked to inflammation and oxidative stress. MPO is an independent prognostic marker in healthy individuals as well as in patients with coronary artery disease. In this present study we analyze the role of MPO in experimental atherogenesis and neointima formation after vascular injury in mice. METHODS AND RESULTS 6-8 weeks old apolipoprotein E-deficient (ApoE(-/-)) mice were fed a high-cholesterol diet for 8 weeks with concomitant treatment with two different doses (10 μg/mg bw vs. 20 μg/mg bw) of 4-ABAH (MPO inhibitor). Application at lower dosage did not affect oxidative stress, endothelial function and atherosclerotic plaque development. 4-ABAH in higher dosage decreased inflammatory markers and vascular oxidative stress, consecutively improved endothelial function and reduced significantly atherosclerotic plaque development. To assess the role of circulating intracellular MPO, irradiated ApoE(-/-) mice were repopulated with bone marrow-derived cells from MPO(-/-) mice and were fed a high-cholesterol diet for 8 weeks. This MPO deficiency resulted in alleviated inflammation, reduced oxidative stress and improved endothelial function with a significant impact on plaque formation. To understand the possible role of MPO in vascular remodeling, we tested its effects on neointima formation following vascular injury in mice. MPO inhibition by 4-ABAH reduced significantly neointima formation. It was significantly reduced in MPO deficient mice, whereas transfer of spleen-derived neutrophils from WT mice enhanced it. CONCLUSION Our data suggests a central role of MPO in the pathogenesis of atherogenesis and prefers pharmacological MPO inhibition as a therapeutic strategy for prevention and therapy of atherosclerosis and restenosis.

Induction of Atrial Fibrillation by Neutrophils Critically Depends on CD11b/CD18 Integrins

Background Recent observational clinical and ex-vivo studies suggest that inflammation and in particular leukocyte activation predisposes to atrial fibrillation (AF). However, whether local binding and extravasation of leukocytes into atrial myocardium is an essential prerequisite for the initiation and propagation of AF remains elusive. Here we investigated the role of atrial CD11b/CD18 mediated infiltration of polymorphonuclear neutrophils (PMN) for the susceptibility to AF. Methods and Results C57bl/6J wildtype (WT) and CD11b/CD18 knock-out (CD11b−/−) mice were treated for 14 days with subcutaneous infusion of angiotensin II (Ang II), a known stimulus for PMN activation. Atria of Ang II-treated WT mice were characterized by increased PMN infiltration assessed in immunohistochemically stained sections. In contrast, atrial sections of CD11b−/− mice lacked a significant increase in PMN infiltration upon Ang II infusion. PMN infiltration was accompanied by profoundly enhanced atrial fibrosis in Ang II treated WT as compared to CD11b−/− mice. Upon in-vivo electrophysiological investigation, Ang II treatment significantly elevated the susceptibility for AF in WT mice if compared to vehicle treated animals given an increased number and increased duration of AF episodes. In contrast, animals deficient of CD11b/CD18 were entirely protected from AF induction. Likewise, epicardial activation mapping revealed decreased electrical conduction velocity in atria of Ang II treated WT mice, which was preserved in CD11b−/− mice. In addition, atrial PMN infiltration was enhanced in atrial appendage sections of patients with persistent AF as compared to patients without AF. Conclusions The current data critically link CD11b-integrin mediated atrial PMN infiltration to the formation of fibrosis, which promotes the initiation and propagation of AF. These findings not only reveal a mechanistic role of leukocytes in AF but also point towards a potential novel avenue of treatment in AF.

Nitrated fatty acids suppress angiotensin II-mediated fibrotic remodelling and atrial fibrillation

AIM Atrial fibrosis, one of the most striking features in the pathology of atrial fibrillation (AF), is promoted by local and systemic inflammation. Electrophilic fatty acid nitroalkenes, endogenously generated by both metabolic and inflammatory reactions, are anti-inflammatory mediators that in synthetic form may be useful as drug candidates. Herein we investigate whether an exemplary nitro-fatty acid can limit atrial fibrosis and AF. METHODS AND RESULTS Wild-type C57BL6/J mice were treated for 2 weeks with angiotensin II (AngII) and vehicle or nitro-oleic acid (10-nitro-octadec-9-enoic acid, OA-NO2, 6 mg/kg body weight) via subcutaneous osmotic minipumps. OA-NO2 significantly inhibited atrial fibrosis and depressed vulnerability for AF during right atrial electrophysiological stimulation to levels observed for AngII-naive animals. Left atrial epicardial mapping studies demonstrated preservation of conduction homogeneity by OA-NO2. The protection from fibrotic remodelling was mediated by suppression of Smad2-dependent myofibroblast transdifferentiation and inhibition of Nox2-dependent atrial superoxide formation. CONCLUSION OA-NO2 potently inhibits atrial fibrosis and subsequent AF. Nitro-fatty acids and possibly other lipid electrophiles thus emerge as potential therapeutic agents for AF, either by increasing endogenous levels through dietary modulation or by administration as synthetic drugs.

Redox-sensitive mechanisms underlying vascular dysfunction in heart failure

Abstract Regardless of the progress in therapeutic drugs and devices to treat heart failure (HF) during the last few years, the clinical outcome of this disease remains deleterious. Impaired left ventricular function leads to neurohumoral activation, altered local shear forces, and hypoxia, which might give rise to inflammatory processes within the vasculature. Among those, the imbalance of the redox equilibrium toward increased concentrations of reactive oxygen species (ROS) is particularly important, as it affects the integrity of vascular function. Apart from injured or dysfunctional cardiomyocytes, vascular dysfunction has been demonstrated to play a crucial role in the development and progression of HF, which makes it an interesting target for new HF therapies. The mechanisms that initiate vascular dysfunction in HF pathogenesis and the processes leading to oxidative stress are not yet fully elucidated. However, oxidative stress promotes a variety of redox-sensitive mechanisms contributing to vascular dysfunction in HF. Here, we will summarize the sources of ROS in the vasculature, elucidate the impact of oxidative stress on functional and structural vascular remodeling, and consider the link to vascular dysfunction. Furthermore, we will point out the importance of vascular dysfunction in HF and discuss therapeutic options.

Myeloperoxidase Mediates Postischemic Arrhythmogenic Ventricular Remodeling

Rationale: Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase, a heme enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling. Objective: To reveal the role of myeloperoxidase for the development of ventricular arrhythmias. Methods and Results: In different murine models of myocardial ischemia, myeloperoxidase deficiency profoundly decreased vulnerability for ventricular tachycardia on programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry after isoproterenol injection. Experiments using CD11b/CD18 integrin–deficient (CD11b−/−) mice and intravenous myeloperoxidase infusion revealed that neutrophil infiltration is a prerequisite for myocardial myeloperoxidase accumulation. Ventricles from myeloperoxidase-deficient (Mpo−/−) mice showed less pronounced slowing and decreased heterogeneity of electric conduction in the peri-infarct zone than wild-type mice. Expression of the redox-sensitive gap junctional protein Cx43 (Connexin 43) was reduced in the peri-infarct area of wild-type compared with Mpo−/− mice. In isolated wild-type cardiomyocytes, Cx43 protein content decreased on myeloperoxidase/H2O2 incubation. Mapping of induced pluripotent stem cell–derived cardiomyocyte networks and in vivo investigations linked Cx43 breakdown to myeloperoxidase-dependent activation of matrix metalloproteinase 7. Moreover, Mpo−/− mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, myeloperoxidase was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases resulting in upregulated collagen generation. In support of our experimental findings, baseline myeloperoxidase plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter–defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction >35% undergoing elective diagnostic cardiac evaluation. Conclusions: Myeloperoxidase emerges as a crucial mediator of postischemic myocardial remodeling and may evolve as a novel pharmacological target for secondary disease prevention after myocardial ischemia.

Myeloperoxidase aggravates pulmonary arterial hypertension by activation of vascular Rho-kinase

Pulmonary arterial hypertension (PAH) remains a disease with limited therapeutic options and dismal prognosis. Despite its etiologic heterogeneity, the underlying unifying pathophysiology is characterized by increased vascular tone and adverse remodeling of the pulmonary circulation. Myeloperoxidase (MPO), an enzyme abundantly expressed in neutrophils, has potent vasoconstrictive and profibrotic properties, thus qualifying as a potential contributor to this disease. Here, we sought to investigate whether MPO is causally linked to the pathophysiology of PAH. Investigation of 2 independent clinical cohorts revealed that MPO plasma levels were elevated in subjects with PAH and predicted adverse outcome. Experimental analyses showed that, upon hypoxia, right ventricular pressure was less increased in Mpo-/- than in WT mice. The hypoxia-induced activation of the Rho-kinase pathway, a critical subcellular signaling pathway yielding vasoconstriction and structural vascular remodeling, was blunted in Mpo-/- mice. Mice subjected to i.v. infusion of MPO revealed activation of Rho-kinase and increased right ventricular pressure, which was prevented by coinfusion of the Rho-kinase inhibitor Y-27632. In the Sugen5416/hypoxia rat model, PAH was attenuated by the MPO inhibitor AZM198. The current data demonstrate a tight mechanistic link between MPO, the activation of Rho-kinase, and adverse pulmonary vascular function, thus pointing toward a potentially novel avenue of treatment.

Prasugrel as opposed to clopidogrel improves endothelial nitric oxide bioavailability and reduces platelet-leukocyte interaction in patients with unstable angina pectoris: A randomized controlled trial

BACKGROUND Platelet inhibition has been linked to improved endothelial function, a prognostic factor in coronary artery disease. Whether prasugrel, a potent platelet inhibitor, affects endothelial function remains unknown. METHODS This was a double-blind, randomized, active-controlled, parallel trial. Patients with unstable angina pectoris undergoing percutaneous coronary intervention (PCI) received either a daily dose of clopidogrel 75mg (n=23) or prasugrel 10mg (n=22). Flow-mediated dilation (FMD), circulating nitrate and nitrite, inflammatory markers and platelet-leukocyte aggregates (PLAs) were assessed the day after PCI and after 3months. RESULTS Baseline patient demographics were well matched between treatment groups. Prasugrel led to a significant improvement of FMD after 3months (9.01±3.64% vs. 6.65±3.24%, p=0.001). In contrast, no significant change was observed in the clopidogrel group (7.21±2.84% vs. 6.30±2.97%, p=0.187). Adjusted for baseline FMD, hyperlipidemia and statin use, the treatment effect on change in FMD favoured prasugrel by an absolute 1.97% (95% CI 0.29% to 3.66%, p=0.023). A significant reduction of plasma hsCRP, myeloperoxidase and neutrophil elastase and an increase of nitrate levels were noted in both treatment arms. Interestingly, only prasugrel significantly reduced sCD40 ligand and RANTES and increased nitrite levels. Prasugrel reduced the ADP-stimulated increase in PLAs by 40% (IR: 82 to 13), whereas clopidogrel revealed no such effect (1% increase (IR: 13 to 50) (p=0.01). CONCLUSION Prasugrel exhibits beneficial mid-term effects on endothelial nitric oxide bioavailability and inflammatory markers. (EudraCT number: 2009-015406-19).

Nitrated fatty acids in cardiovascular diseases

Cardiovascular disease (CVD) is the leading cause of death and accounts for one third of disease-related mortality worldwide. Dysregulated redox mechanisms, in particular the formation of reactive oxygen species (ROS) play a pivotal pathogenetic role in CVD. Nitro-fatty acids (NO2-FAs) are electrophilic molecules which have a NO2-group bound to one of their olefinic carbons. They are endogenously formed by the reaction of reactive nitrogen species with unsaturated fatty acids. Basal levels of NO2-FAs are in the low nanomolar range and higher concentrations can be encountered under acidic (stomach) and inflammatory (e.g. ischemia/reperfusion) conditions. Dietary intake of polyunsaturated fatty acids in combination with nitrites raises circulating NO2-FAs to a clinically relevant level in mice. NO2-FAs undergo reversible covalent binding to cysteine residues and by virtue of these posttranslational protein modifications act as potent anti-inflammatory signaling mediators via modulation of various critical pathways like nuclear factor E2-related factor 2 (Nrf2)- and peroxisome proliferator-activated receptor γ (PPARγ) activation, nuclear factor-kappa B (NF-κB) inhibition and hem oxygenase-1 (HO-1)- and heat shock protein (HSP) induction. In this review article, we summarize recent findings about the effects and underlying molecular mechanisms of NO2-FAs from a variety of pre-clinical cardiovascular disease models. The described findings suggest the potential of NO2-FAs to emerge as therapeutic agents with a broad range of potential clinical applications for CVD.

MPO (Myeloperoxidase) Reduces Endothelial Glycocalyx Thickness Dependent on Its Cationic Charge

Objective— The leukocyte heme-enzyme MPO (myeloperoxidase) exerts proinflammatory effects on the vascular system primarily linked to its catalytic properties. Recent studies have shown that MPO, depending on its cationic charge, mediates neutrophil recruitment and activation. Here, we further investigated MPO’s extracatalytic properties and its effect on endothelial glycocalyx (EG) integrity. Approach and Results— In vivo staining of murine cremaster muscle vessels with Alcian Blue 8GX provided evidence of an MPO-dependent decrease in anionic charge of the EG. MPO binding to the glycocalyx was further characterized using Chinese hamster ovary cells and its glycosaminoglycan mutants—pgsA-745 (mutant Chinese hamster ovary cells lacking heparan sulfate and chondroitin sulfate glycosaminoglycan) and pgsD-677 (mutant Chinese hamster ovary cells lacking heparan sulfate glycosaminoglycan), which revealed heparan sulfate as the main mediator of MPO binding. Further, EG integrity was assessed in terms of thickness using intravital microscopy of murine cremaster muscle. A significant reduction in EG thickness was observed on infusion of catalytically active MPO, as well as mutant inactive MPO and cationic polymer polylysine. Similar effects were also observed in wild-type mice after a local inflammatory stimulus but not in MPO-knockout mice. The reduction in EG thickness was reversed after removal of vessel-bound MPO, suggesting a possible physical collapse of the EG. Last, experiments with in vivo neutrophil depletion revealed that MPO also induced neutrophil-mediated shedding of the EG core protein, Sdc1 (syndecan-1). Conclusions— These findings provide evidence that MPO, via ionic interaction with heparan sulfate side chains, can cause neutrophil-dependent Sdc1 shedding and collapse of the EG structure.