Menno P. J. de Winther

Accumulation of Myeloperoxidase-Positive Neutrophils in Atherosclerotic Lesions in LDLR−/− Mice

Objective—Atherosclerosis is a chronic inflammatory disease in which the immune system plays an important role. Neutrophils have not been thoroughly studied in the context of atherogenesis. Here, we investigated neutrophils in the development of murine atherosclerotic lesions. Methods and Results—LDLR−/− mice were given a high-fat diet for different time periods and subsequently atherosclerotic lesions were studied by immunohistochemistry. Staining with anti–Ly-6G monoclonal antibody, a specific marker for neutrophils, revealed a marked accumulation of neutrophils during atherosclerosis development. Neutrophils were observed in the lesion, attached to the cap, and in the arterial adventitia. In addition, at some sites, neutrophil accumulation colocalized with endothelial E-selectin expression. Immunofluorescence double staining with anti-myeloperoxidase and anti–Ly-6G antibodies demonstrated the presence of myeloperoxidase in atherosclerotic lesions and its colocalization with neutrophils. After introducing the high-fat diet, levels of circulating myeloperoxidase in plasma strongly increased, with a peak at 6 weeks and a subsequent decrease to almost normal levels after 16 weeks of diet. Conclusions—We here demonstrate for the first time the presence of neutrophils and myeloperoxidase in murine atherosclerotic lesions. As a major cell type in inflammatory responses the neutrophil may also be an important mediator in the development of atherosclerosis.

Myeloid Type I Interferon Signaling Promotes Atherosclerosis by Stimulating Macrophage Recruitment to Lesions

Inflammatory cytokines are well-recognized mediators of atherosclerosis. Depending on the pathological context, type I interferons (IFNs; IFNalpha and IFNbeta) exert either pro- or anti-inflammatory immune functions, but their exact role in atherogenesis has not been clarified. Here, we demonstrate that IFNbeta enhances macrophage-endothelial cell adhesion and promotes leukocyte attraction to atherosclerosis-prone sites in mice in a chemokine-dependent manner. Moreover, IFNbeta treatment accelerates lesion formation in two different mouse models of atherosclerosis and increases macrophage accumulation in the plaques. Concomitantly, absence of endogenous type I IFN signaling in myeloid cells inhibits lesion development, protects against lesional accumulation of macrophages, and prevents necrotic core formation. Finally, we show that type I IFN signaling is upregulated in ruptured human atherosclerotic plaques. Hereby, we identify type I IFNs as proatherosclerotic cytokines that may serve as additional targets for prevention or treatment.

Macrophage MicroRNA-155 Promotes Cardiac Hypertrophy and Failure

Background— Cardiac hypertrophy and subsequent heart failure triggered by chronic hypertension represent major challenges for cardiovascular research. Beyond neurohormonal and myocyte signaling pathways, growing evidence suggests inflammatory signaling pathways as therapeutically targetable contributors to this process. We recently reported that microRNA-155 is a key mediator of cardiac inflammation and injury in infectious myocarditis. Here, we investigated the impact of microRNA-155 manipulation in hypertensive heart disease. Methods and Results— Genetic loss or pharmacological inhibition of the leukocyte-expressed microRNA-155 in mice markedly reduced cardiac inflammation, hypertrophy, and dysfunction on pressure overload. These alterations were macrophage dependent because in vivo cardiomyocyte-specific microRNA-155 manipulation did not affect cardiac hypertrophy or dysfunction, whereas bone marrow transplantation from wild-type mice into microRNA-155 knockout animals rescued the hypertrophic response of the cardiomyocytes and vice versa. In vitro, media from microRNA-155 knockout macrophages blocked the hypertrophic growth of stimulated cardiomyocytes, confirming that macrophages influence myocyte growth in a microRNA-155-dependent paracrine manner. These effects were at least partly mediated by the direct microRNA-155 target suppressor of cytokine signaling 1 (Socs1) because Socs1 knockdown in microRNA-155 knockout macrophages largely restored their hypertrophy-stimulating potency. Conclusions— Our findings reveal that microRNA-155 expression in macrophages promotes cardiac inflammation, hypertrophy, and failure in response to pressure overload. These data support the causative significance of inflammatory signaling in hypertrophic heart disease and demonstrate the feasibility of therapeutic microRNA targeting of inflammation in heart failure.

Rupture of the Atherosclerotic Plaque. Does a Good Animal Model Exist

Abstract—By its very nature, rupture of the atherosclerotic plaque is difficult to study directly in humans. A good animal model would help us not only to understand how rupture occurs but also to design and test treatments to prevent it from happening. However, several difficulties surround existing models of plaque rupture, including the need for radical interventions to produce the rupture, lack of direct evidence of rupture per se, and absence of convincing evidence of platelet- and fibrin-rich thrombus at the rupture site. At the present time, attention should therefore focus on the processes of plaque breakdown and thrombus formation in humans, whereas the use of animal models should probably be reserved for studying the function of particular genes and for investigating isolated features of plaques, such as the relationship between cap thickness and plaque stability.

Hematopoietic miR155 Deficiency Enhances Atherosclerosis and Decreases Plaque Stability in Hyperlipidemic Mice

microRNA-155 (miR155) is a central regulator of immune responses that is induced by inflammatory mediators. Although miR155 is considered to be a pro-inflammatory microRNA, in vitro reports show anti-inflammatory effects in lipid-loaded cells. In this study we examined the role of miR155 in atherosclerosis in vivo using bone marrow transplantation from miR155 deficient or wildtype mice to hyperlipidemic mice. Hematopoietic deficiency of miR155 enhanced atherosclerotic plaque development and decreased plaque stability, as evidenced by increased myeloid inflammatory cell recruitment to the plaque. The increased inflammatory state was mirrored by a decrease in circulating CD4+CD25+FoxP3+ regulatory T cells, and an increase in granulocytes (CD11b+Ly6G+) in blood of miR155−/− transplanted mice. Moreover, we show for the first time a crucial role of miR155 in monocyte subset differentiation, since hematopoietic deficiency of miR155 increases the ‘inflammatory’ monocyte subset (CD11b+Ly6G−Ly6Chi) and reduces ‘resident’ monocytes (CD11b+Ly6G−Ly6Clow) in the circulation. Furthermore, cytokine production by resident peritoneal macrophages of miR155−/− transplanted hyperlipidemic mice was skewed towards a more pro-inflammatory state since anti-inflammatory IL-10 production was reduced. In conclusion, in this hyperlipidemic mouse model miR155 acts as an anti-inflammatory, atheroprotective microRNA. Additionally, besides a known role in lymphoid cell development, we show a crucial role of miR155 in myeloid lineage differentiation.

MicroRNA Profiling Identifies MicroRNA-155 as an Adverse Mediator of Cardiac Injury and Dysfunction During Acute Viral Myocarditis

Rationale: Viral myocarditis results from an adverse immune response to cardiotropic viruses, which causes irreversible myocyte destruction and heart failure in previously healthy people. The involvement of microRNAs and their usefulness as therapeutic targets in this process are unknown. Objective: To identify microRNAs involved in viral myocarditis pathogenesis and susceptibility. Methods and Results: Cardiac microRNAs were profiled in both human myocarditis and in Coxsackievirus B3-injected mice, comparing myocarditis-susceptible with nonsusceptible mouse strains longitudinally. MicroRNA responses diverged depending on the susceptibility to myocarditis after viral infection in mice. MicroRNA-155, -146b, and -21 were consistently and strongly upregulated during acute myocarditis in both humans and susceptible mice. We found that microRNA-155 expression during myocarditis was localized primarily in infiltrating macrophages and T lymphocytes. Inhibition of microRNA-155 by a systemically delivered LNA-anti-miR attenuated cardiac infiltration by monocyte-macrophages, decreased T lymphocyte activation, and reduced myocardial damage during acute myocarditis in mice. These changes were accompanied by the derepression of the direct microRNA-155 target PU.1 in cardiac inflammatory cells. Beyond the acute phase, microRNA-155 inhibition reduced mortality and improved cardiac function during 7 weeks of follow-up. Conclusions: Our data show that cardiac microRNA dysregulation is a characteristic of both human and mouse viral myocarditis. The inflammatory microRNA-155 is upregulated during acute myocarditis, contributes to the adverse inflammatory response to viral infection of the heart, and is a potential therapeutic target for viral myocarditis.

Role of Scavenger Receptor A and CD36 in Diet-Induced Nonalcoholic Steatohepatitis in Hyperlipidemic Mice

BACKGROUND & AIMSnNonalcoholic steatohepatitis (NASH) is a disorder that consists of steatosis and hepatic inflammation. It is not known why only some people with steatosis develop NASH. Recently, we identified dietary cholesterol as a factor that directly leads to hepatic inflammation and hepatic foam cell formation. We propose a mechanism by which Kupffer cells (KCs) take up modified cholesterol-rich lipoproteins via scavenger receptors (SRs). KCs thereby accumulate cholesterol, become activated, and may then trigger an inflammatory reaction. Scavenging of modified lipoproteins mainly depends on CD36 and macrophage scavenger receptor 1.nnnMETHODSnTo evaluate the involvement of SR-mediated uptake of modified lipoproteins by KCs in the development of diet-induced NASH, female low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice were lethally irradiated and transplanted with bone marrow from Msr1(+/+)/Cd36(+/+)or Msr1(-/-)/Cd36(-/-) mice and fed a Western diet.nnnRESULTSnMacrophage and neutrophil infiltration revealed that hepatic inflammation was substantially reduced by approximately 30% in Msr1(-/-)/Cd36(-/-)-transplanted mice compared with control mice. Consistent with this, the expression levels of well-known inflammatory mediators were reduced. Apoptotis and fibrosis were less pronounced in Msr1(-/-)/Cd36(-/-)-transplanted mice, in addition to the protective phenotype of natural antibodies against oxidized low-density lipoprotein in the plasma. Surprisingly, the effect on hepatic inflammation was independent of foam cell formation.nnnCONCLUSIONSnTargeted inactivation of SR pathways reduces the hepatic inflammation and tissue destruction associated with NASH, independent of hepatic foam cell formation.

Macrophage secretory phospholipase A2 group X enhances anti-inflammatory responses, promotes lipid accumulation, and contributes to aberrant lung pathology

Secreted phospholipase A2 group X (sPLA2-X) is one of the most potent enzymes of the phospholipase A2 lipolytic enzyme superfamily. Its high catalytic activity toward phosphatidylcholine (PC), the major phospholipid of cell membranes and low-density lipoproteins (LDL), has implicated sPLA2-X in chronic inflammatory conditions such as atherogenesis. We studied the role of sPLA2-X enzyme activity in vitro and in vivo, by generating sPLA2-X-overexpressing macrophages and transgenic macrophage-specific sPLA2-X mice. Our results show that sPLA2-X expression inhibits macrophage activation and inflammatory responses upon stimulation, characterized by reduced cell adhesion and nitric oxide production, a decrease in tumor necrosis factor (TNF), and an increase in interleukin (IL)-10. These effects were mediated by an increase in IL-6, and enhanced production of prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2). Moreover, we found that overexpression of active sPLA2-X in macrophages strongly increases foam cell formation upon incubation with native LDL but also oxidized LDL (oxLDL), which is mediated by enhanced expression of scavenger receptor CD36. Transgenic sPLA2-X mice died neonatally because of severe lung pathology characterized by interstitial pneumonia with massive granulocyte and surfactant-laden macrophage infiltration. We conclude that overexpression of the active sPLA2-X enzyme results in enhanced foam cell formation but reduced activation and inflammatory responses in macrophages in vitro. Interestingly, enhanced sPLA2-X activity in macrophages in vivo leads to fatal pulmonary defects, suggesting a crucial role for sPLA2-X in inflammatory lung disease.

NF-κB Activation Is Required for the Transition of Pulmonary Inflammation to Muscle Atrophy

Disease exacerbations and muscle wasting comprise negative prognostic factors of chronic obstructive pulmonary disease (COPD). Transient systemic inflammation and malnutrition have been implicated in skeletal muscle wasting after acute exacerbations of COPD. However, the interactions between systemic inflammation and malnutrition in their contributions to muscle atrophy, as well as the molecular basis underlying the transition of systemic inflammation to muscle atrophy, remain unresolved. Pulmonary inflammation was induced in mice by an intratracheal instillation of LPS to model acute disease exacerbation. Systemic inflammation, nutritional intake, and body and muscle weights were determined. Muscle inflammatory signaling and atrophy signaling were examined, and the effect of the muscle-specific inactivation of NF-κB on muscle atrophy was assessed in genetically modified mice. The intratracheal LPS instillation was followed by markedly elevated circulating cytokine concentrations and NF-κB activation in extrapulmonary tissues, including skeletal muscle. The administration of intratracheal LPS increased the expression of muscle E3 ubiquitin ligases, which govern muscle proteolysis, in particular MuRF1, and caused a rapid loss of muscle mass. Reduced food intake only partly accounted for the observed muscle atrophy, and did not activate NF-κB in muscle. Rather, plasma transfer experiments revealed the presence of NF-κB-signaling and atrophy-signaling properties in the circulation of intratracheal LPS-treated mice. The genetic inhibition of muscle NF-κB activity suppressed intratracheal LPS-induced MuRF1 expression and resulted in a significant sparing of muscle tissue. Systemic inflammation and malnutrition contribute to the muscle wasting induced by acute pulmonary inflammation via distinct mechanisms, and muscle NF-κB activation is required for the transition from inflammatory to muscle atrophy signaling.

A disintegrin and metalloproteases: Molecular scissors in angiogenesis, inflammation and atherosclerosis

A disintegrin and metalloproteases (ADAMs) are enzymes that cleave (shed) the extracellular domains of various cell surface molecules, e.g. adhesion molecules, cytokine/chemokine and growth factor receptors, thereby releasing soluble molecules that can exert agonistic or antagonistic functions or serve as biomarkers. By functioning as such molecular scissors, ADAM proteases have been implicated in various diseases, e.g. cancer, and their role in cardiovascular diseases is now emerging. This review will focus on the role of ADAM proteases in molecular mechanisms of angiogenesis and inflammation in relation to atherosclerosis. Besides a concise overview of the current state and recent advances of this research area, we will discuss key questions about redundancy, specificity and regulation of ADAM proteases and emphasize the importance of confirmation of in vitro findings in in vivo models.