Denise Lau

Myeloperoxidase acts as a profibrotic mediator of atrial fibrillation

Observational clinical and ex vivo studies have established a strong association between atrial fibrillation and inflammation. However, whether inflammation is the cause or the consequence of atrial fibrillation and which specific inflammatory mediators may increase the atrias susceptibility to fibrillation remain elusive. Here we provide experimental and clinical evidence for the mechanistic involvement of myeloperoxidase (MPO), a heme enzyme abundantly expressed by neutrophils, in the pathophysiology of atrial fibrillation. MPO-deficient mice pretreated with angiotensin II (AngII) to provoke leukocyte activation showed lower atrial tissue abundance of the MPO product 3-chlorotyrosine, reduced activity of matrix metalloproteinases and blunted atrial fibrosis as compared to wild-type mice. Upon right atrial electrophysiological stimulation, MPO-deficient mice were protected from atrial fibrillation, which was reversed when MPO was restored. Humans with atrial fibrillation had higher plasma concentrations of MPO and a larger MPO burden in right atrial tissue as compared to individuals devoid of atrial fibrillation. In the atria, MPO colocalized with markedly increased formation of 3-chlorotyrosine. Our data demonstrate that MPO is a crucial prerequisite for structural remodeling of the myocardium, leading to an increased vulnerability to atrial fibrillation.

Heparins Increase Endothelial Nitric Oxide Bioavailability by Liberating Vessel-Immobilized Myeloperoxidase

Background— Neutrophils and monocytes are centrally linked to vascular inflammatory disease, and leukocyte-derived myeloperoxidase (MPO) has emerged as an important mechanistic participant in impaired vasomotor function. MPO binds to and transcytoses endothelial cells in a glycosaminoglycan-dependent manner, and MPO binding to the vessel wall is a prerequisite for MPO-dependent oxidation of endothelium-derived nitric oxide (NO) and impairment of endothelial function in animal models. In the present study, we investigated whether heparin mobilizes MPO from vascular compartments in humans and defined whether this translates into increased vascular NO bioavailability and function. Methods and Results— Plasma MPO levels before and after heparin administration were assessed by ELISA in 109 patients undergoing coronary angiography. Whereas baseline plasma MPO levels did not differ between patients with or without angiographically detectable coronary artery disease (CAD), the increase in MPO plasma content on bolus heparin administration was higher in patients with CAD (P=0.01). Heparin treatment also improved endothelial NO bioavailability, as evidenced by flow-mediated dilation (P<0.01) and by acetylcholine-induced changes in forearm blood flow (P<0.01). The extent of heparin-induced MPO release was correlated with improvement in endothelial function (r=0.69, P<0.01). Moreover, and consistent with this tenet, ex vivo heparin treatment of extracellular matrix proteins, cultured endothelial cells, and saphenous vein graft specimens from CAD patients decreased MPO burden. Conclusions— Mobilization of vessel-associated MPO may represent an important mechanism by which heparins exert antiinflammatory effects and increase vascular NO bioavailability. These data add to the growing body of evidence for a causal role of MPO in compromised vascular NO signaling in humans.

Myeloperoxidase attracts neutrophils by physical forces

Recruitment of polymorphonuclear neutrophils (PMNs) remains a paramount prerequisite in innate immune defense and a critical cofounder in inflammatory vascular disease. Neutrophil recruitment comprises a cascade of concerted events allowing for capture, adhesion and extravasation of the leukocyte. Whereas PMN rolling, binding, and diapedesis are well characterized, receptor-mediated processes, mechanisms attenuating the electrostatic repulsion between the negatively charged glycocalyx of leukocyte and endothelium remain poorly understood. We provide evidence for myeloperoxidase (MPO), an abundant PMN-derived heme protein, facilitating PMN recruitment by its positive surface charge. In vitro, MPO evoked highly directed PMN motility, which was solely dependent on electrostatic interactions with the leukocytes surface. In vivo, PMN recruitment was shown to be MPO-dependent in a model of hepatic ischemia and reperfusion, upon intraportal delivery of MPO and in the cremaster muscle exposed to local inflammation or to intraarterial MPO application. Given MPOs affinity to both the endothelial and the leukocytes surface, MPO evolves as a mediator of PMN recruitment because of its positive surface charge. This electrostatic MPO effect not only displays a so far unrecognized, catalysis-independent function of the enzyme, but also highlights a principal mechanism of PMN attraction driven by physical forces.

Myeloperoxidase deficiency preserves vasomotor function in humans

Aims Observational studies have suggested a mechanistic link between the leucocyte-derived enzyme myeloperoxidase (MPO) and vasomotor function. Here, we tested whether MPO is systemically affecting vascular tone in humans. Methods and results A total of 12 135 patients were screened for leucocyte peroxidase activity. We identified 15 individuals with low MPO expression and activity (MPOlow), who were matched with 30 participants exhibiting normal MPO protein content and activity (control). Nicotine-dependent activation of leucocytes caused attenuation of endothelial nitric oxide (NO) bioavailability in the control group (P < 0.01), but not in MPOlow individuals (P = 0.12); here the MPO burden of leucocytes correlated with the degree of vasomotor dysfunction (P = 0.008). To directly test the vasoactive properties of free circulating MPO, the enzyme was injected into the left atrium of anaesthetized, open-chest pigs. Myeloperoxidase plasma levels peaked within minutes and rapidly declined thereafter, reflecting vascular binding of MPO. Blood flow in the left anterior descending artery and the internal mammary artery (IMA) as well as myocardial perfusion decreased following MPO injection when compared with albumin-treated animals (P < 0.001). Isolated IMA-rings from animals subjected to MPO revealed markedly diminished relaxation in response to acetylcholine (P < 0.01) and nitroglycerine as opposed to controls (P < 0.001). Conclusion Myeloperoxidase elicits profound effects on vascular tone of conductance and resistance vessels in vivo. These findings not only call for revisiting the biological functions of leucocytes as systemic and mobile effectors of vascular tone, but also identify MPO as a critical systemic regulator of vasomotion in humans and thus a potential therapeutic target.

Pathogenic Cycle Between the Endogenous Nitric Oxide Synthase Inhibitor Asymmetrical Dimethylarginine and the Leukocyte-Derived Hemoprotein Myeloperoxidase

Background— The nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA) and the leukocyte-derived hemoprotein myeloperoxidase (MPO) are associated with cardiovascular diseases. Activation of monocytes and polymorphonuclear neutrophils (PMNs) with concomitant release of MPO is regulated in a nitric oxide–dependent fashion. The aim of the study was to investigate a potential 2-way interaction between ADMA and MPO. Methods and Results— Ex vivo, ADMA uptake by isolated human PMNs, the principal source of MPO in humans, significantly impaired nitric oxide synthase activity determined by gas chromatography–mass spectrometry. In humans, short-term ADMA infusion (0.0125 mg · kg−1 · min−1) significantly increased MPO plasma concentrations. Functionally, PMN exposure to ADMA enhanced leukocyte adhesion to endothelial cells, augmented NADPH oxidase activity, and stimulated PMN degranulation, resulting in release of MPO. In vivo, a 28-day ADMA infusion (250 &mgr;mol · kg−1 · d−1) in C57Bl/6 mice significantly increased plasma MPO concentrations, whereas this ADMA effect on MPO was attenuated by human dimethylarginine dimethylaminohydrolase1 (hDDAH1) overexpression. Moreover, the MPO-derived reactive molecule hypochlorous acid impaired recombinant hDDAH1 activity in vitro. In MPO−/− mice, the lipopolysaccharide-induced increase in systemic ADMA concentrations was abrogated. Conclusions— ADMA profoundly impairs nitric oxide synthesis of PMNs, resulting in increased PMN adhesion to endothelial cells, superoxide generation, and release of MPO. In addition, MPO impairs DDAH1 activity. Our data reveal an ADMA-induced cycle of PMN activation, enhanced MPO release, and subsequent impairment of DDAH1 activity. These findings not only highlight so far unrecognized cytokine-like properties of ADMA but also identify MPO as a regulatory switch for ADMA bioavailability under inflammatory conditions.

Uric acid modulates vascular endothelial function through the down regulation of nitric oxide production.

Abstract Endothelial dysfunction characterized by decreased nitric oxide (NO) bioavailability is the first stage of coronary artery disease. It is known that one of the factors associated with an increased risk of coronary artery disease is a high plasma level of uric acid. However, causative associations between hyperuricaemia and cardiovascular risk have not been definitely proved. In this work, we tested the effect of uric acid on endothelial NO bioavailability. Electrochemical measurement of NO production in acetylcholine-stimulated human umbilical endothelial cells (HUVECs) revealed that uric acid markedly decreases NO release. This finding was confirmed by organ bath experiments on mouse aortic segments. Uric acid dose-dependently reduced endothelium-dependent vasorelaxation. To reveal the mechanism of decreasing NO bioavailability we tested the effect of uric acid on reactive oxygen species production by HUVECs, on arginase activity, and on acetylcholine-induced endothelial NO synthase phosphorylation. It was found that uric acid increases arginase activity and reduces endothelial NO synthase phosphorylation. Interestingly, uric acid significantly increased intracellular superoxide formation. In conclusion, uric acid decreases NO bioavailability by means of multiple mechanisms. This finding supports the idea of a causal association between hyperuricaemia and cardiovascular risk.

Liberation of vessel adherent myeloperoxidase by enoxaparin improves endothelial function

BACKGROUND Myeloperoxidase (MPO), a leukocyte-derived heme enzyme binds to the endothelium and depletes vascular nitric oxide (NO) bioavailability in animal models. Unfractionated heparins release vessel-bound MPO and increase endothelial NO bioavailability. Whether low molecular weight heparins also affect circulating MPO levels and NO dependent vasoreactivity however remains elusive. METHODS AND RESULTS In a randomized, double-blind, placebo-controlled trial patients with stable coronary artery disease received either 1 mg/kg enoxaparin or an equivalent volume of sodium chloride (NaCl) subcutaneously. Enoxaparin led to a significant improvement of FMD (5.51+/-0.53% vs. 6.55+/-0.58%, p=0.01) accompanied by a significant increase in plasma MPO levels (2.51 [IR: 2.04-3.62] ng/ml vs. 3.70 [IR: 2.80-5.50] ng/ml; p<0.001) whereas NaCl revealed neither a change in FMD (5.56+/-0.67% vs. 5.34+/-0.61%, p=ns) nor in plasma MPO levels (3.04 [IR: 2.22-4.67] ng/ml vs. 2.90 [IR: 1.95-4.32] ng/ml; p=ns). The extent of enoxaparin-induced MPO release and the improvement in endothelial function showed a good correlation (r=0.67, p<0.001). DISCUSSION This study confirms the concept that heparins improve endothelial function, an established read-out of vascular NO bioavailability, by mobilizing vessel bound MPO. These data not only support the notion of extracoagulant, anti-inflammatory properties of heparins but reinforce the concept of MPO-dependent NO oxidation as a central mechanism for regulation of vascular tone in inflammatory vascular disease. (Eudra-CT number: 2005-006113-40).

Diagnostic value of MPO plasma levels in patients admitted for suspected myocardial infarction

BACKGROUND Besides its well-established role in atherosclerosis, myeloperoxidase (MPO) has gained attention as a prognostic indicator in cardiovascular disease. Previous studies assessed MPO retrospectively and at a single time point. The current study aimed to evaluate the prognostic information of MPO prospectively and in consecutive measurements in patients presenting with chest pain. METHODS MPO plasma levels were determined in 274 consecutive chest pain patients admitted to the emergency room. RESULTS A total of 100 patients (36.5%) were finally diagnosed for acute myocardial infarction (AMI). Patients with AMI had significantly higher MPO levels than patients without AMI. Importantly, MPO levels were elevated in patients finally diagnosed for AMI even when troponin I (TNI) was negative (cutoff: 0.032 ng/ml). Overall, MPO yielded a negative predictive value (NPV) of 85.5% (95% confidence interval (CI): 82.6-88.4) and a sensitivity for diagnosing AMI of 80.0% (95% CI: 75.8-84.2) compared to a NPV of 91.7% (95% CI: 89.5-94.0) and a sensitivity of 85.9% (95% CI: 82.3-89.5) for TNI. For patients with a symptom onset of ≤ 2 h the sensitivity of MPO increased to 95.8% (95% CI: 93.7-97.9) whereas the sensitivity of TNI dropped to 50.0% (95% CI: 44.8-55.2). The negative predictive value of MPO for this group of patients was 95.6% (95% CI: 94.0-97.3) compared to 73.3% (95% CI: 69.8-76.9) for TNI. DISCUSSION The current data underscore the role of MPO as diagnostic marker in acute coronary disease; however the additive information derived from MPO is restricted to patients presenting in the early phase of symptom onset.

New Role for L-Arginine in Regulation of Inducible Nitric-Oxide-Synthase-Derived Superoxide Anion Production in Raw 264.7 Macrophages

Dietary supplementation with L-arginine was shown to improve immune responses in various inflammatory models. However, the molecular mechanisms underlying L-arginine effects on immune cells remain unrecognized. Herein, we tested the hypothesis that a limitation of L-arginine could lead to the uncoupled state of murine macrophage inducible nitric oxide synthase and, therefore, increase inducible nitric-oxide-synthase-derived superoxide anion formation. Importantly, we demonstrated that L-arginine dose- and time dependently potentiated superoxide anion production in bacterial endotoxin-stimulated macrophages, although it did not influence NADPH oxidase expression and activity. Detailed analysis of macrophage activation showed the time dependence between LPS-induced iNOS expression and increased O2∙− formation. Moreover, downregulation of macrophage iNOS expression, as well as the inhibition of iNOS activity by NOS inhibitors, unveiled an important role of this enzyme in controlling O2∙− and peroxynitrite formation during macrophage stimulation. In conclusion, our data demonstrated that simultaneous induction of NADPH oxidase, together with the iNOS enzyme, can result in the uncoupled state of iNOS resulting in the production of functionally important levels of O2∙− soon after macrophage activation with LPS. Moreover, we demonstrated, for the first time that increased concentrations of L-arginine further potentiate iNOS-dependent O2∙− formation in inflammatory macrophages.

The potentiation of myeloperoxidase activity by the glycosaminoglycan-dependent binding of myeloperoxidase to proteins of the extracellular matrix.

BACKGROUND Myeloperoxidase (MPO) is an abundant hemoprotein expressed by neutrophil granulocytes that is recognized to play an important role in the development of vascular diseases. Upon degranulation from circulating neutrophil granulocytes, MPO binds to the surface of endothelial cells in an electrostatic-dependent manner and undergoes transcytotic migration to the underlying extracellular matrix (ECM). However, the mechanisms governing the binding of MPO to subendothelial ECM proteins, and whether this binding modulates its enzymatic functions are not well understood. METHODS We investigated MPO binding to ECM derived from aortic endothelial cells, aortic smooth muscle cells, and fibroblasts, and to purified ECM proteins, and the modulation of these associations by glycosaminoglycans. The oxidizing and chlorinating potential of MPO upon binding to ECM proteins was tested. RESULTS MPO binds to the ECM proteins collagen IV and fibronectin, and this association is enhanced by the pre-incubation of these proteins with glycosaminoglycans. Correspondingly, an excess of glycosaminoglycans in solution during incubation inhibits the binding of MPO to collagen IV and fibronectin. These observations were confirmed with cell-derived ECM. The oxidizing and chlorinating potential of MPO was preserved upon binding to collagen IV and fibronectin; even the potentiation of MPO activity in the presence of collagen IV and fibronectin was observed. CONCLUSIONS Collectively, the data reveal that MPO binds to ECM proteins on the basis of electrostatic interactions, and MPO chlorinating and oxidizing activity is potentiated upon association with these proteins. GENERAL SIGNIFICANCE Our findings provide new insights into the molecular mechanisms underlying the interaction of MPO with ECM proteins.