Rationale Healing after myocardial infarction (MI) involves the biphasic accumulation of inflammatory Ly-6Chigh and reparative Ly-6Clow monocytes/macrophages (Mo/MΦ). According to one model, Mo/MΦ heterogeneity in the heart originates in the blood and involves the sequential recruitment of distinct monocyte subsets that differentiate to distinct macrophages. Alternatively, heterogeneity may arise in tissue from one circulating subset via local macrophage differentiation and polarization. The orphan nuclear hormone receptor, Nr4a1, is essential to Ly-6Clow monocyte production but dispensable to Ly-6Clow macrophage differentiation; dependence on Nr4a1 can thus discriminate between systemic and local origins of macrophage heterogeneity. Objective This study tested the role of Nr4a1 in MI in the context of the two Mo/MΦ accumulation scenarios. Methods and Results We show that Ly-6Chigh monocytes infiltrate the infarcted myocardium and, unlike Ly-6Clow monocytes, differentiate to cardiac macrophages. In the early, inflammatory phase of acute myocardial ischemic injury, Ly-6Chigh monocytes accrue in response to a brief Ccl2 burst. In the second, reparative phase, accumulated Ly-6Chigh monocytes give rise to reparative Ly-6Clow F4/80high macrophages that proliferate locally. In the absence of Nr4a1, Ly-6Chigh monocytes express heightened levels of Ccr2 on their surface, avidly infiltrate the myocardium, and differentiate to abnormally inflammatory macrophages, which results in defective healing and compromised heart function. Conclusions Ly-6Chigh monocytes orchestrate both inflammatory and reparative phases during MI and depend on Nr4a1 to limit their influx and inflammatory cytokine expression.

The perpetuation of inflammation is an important pathophysiological contributor to the global medical burden. Chronic inflammation is promoted by non-programmed cell death1,2; however, how inflammation is instigated, its cellular and molecular mediators, and its therapeutic value are poorly defined. Here we use mouse models of atherosclerosis—a major underlying cause of mortality worldwide—to demonstrate that extracellular histone H4-mediated membrane lysis of smooth muscle cells (SMCs) triggers arterial tissue damage and inflammation. We show that activated lesional SMCs attract neutrophils, triggering the ejection of neutrophil extracellular traps that contain nuclear proteins. Among them, histone H4 binds to and lyses SMCs, leading to the destabilization of plaques; conversely, the neutralization of histone H4 prevents cell death of SMCs and stabilizes atherosclerotic lesions. Our data identify a form of cell death found at the core of chronic vascular disease that is instigated by leukocytes and can be targeted therapeutically.

Rationale: Atheroprogression is a consequence of nonresolved inflammation, and currently a comprehensive overview of the mechanisms preventing resolution is missing. However, in acute inflammation, resolution is known to be orchestrated by a switch from inflammatory to resolving lipid mediators. Therefore, we hypothesized that lesional lipid mediator imbalance favors atheroprogression. Objective: To understand the lipid mediator balance during atheroprogression and to establish an interventional strategy based on the delivery of resolving lipid mediators. Methods and Results: Aortic lipid mediator profiling of aortas from Apoe −/− mice fed a high-fat diet for 4 weeks, 8 weeks, or 4 months revealed an expansion of inflammatory lipid mediators, Leukotriene B4 and Prostaglandin E2, and a concomitant decrease of resolving lipid mediators, Resolvin D2 (RvD2) and Maresin 1 (MaR1), during advanced atherosclerosis. Functionally, aortic Leukotriene B4 and Prostaglandin E2 levels correlated with traits of plaque instability, whereas RvD2 and MaR1 levels correlated with the signs of plaque stability. In a therapeutic context, repetitive RvD2 and MaR1 delivery prevented atheroprogression as characterized by halted expansion of the necrotic core and accumulation of macrophages along with increased fibrous cap thickness and smooth muscle cell numbers. Mechanistically, RvD2 and MaR1 induced a shift in macrophage profile toward a reparative phenotype, which secondarily stimulated collagen synthesis in smooth muscle cells. Conclusions: We present evidence for the imbalance between inflammatory and resolving lipid mediators during atheroprogression. Delivery of RvD2 and MaR1 successfully prevented atheroprogression, suggesting that resolving lipid mediators potentially represent an innovative strategy to resolve arterial inflammation.

Onset of cardiovascular complications as a consequence of atherosclerosis exhibits a circadian incidence with a peak in the morning hours. Although development of atherosclerosis extends for long periods of time through arterial leukocyte recruitment, we hypothesized that discrete diurnal invasion of the arterial wall could sustain atherogenic growth. Here, we show that myeloid cell recruitment to atherosclerotic lesions oscillates with a peak during the transition from the activity to the resting phase. This diurnal phenotype is regulated by rhythmic release of myeloid cell-derived CCL2, and blockade of its signaling abolished oscillatory leukocyte adhesion. In contrast, we show that myeloid cell adhesion to microvascular beds peaks during the early activity phase. Consequently, timed pharmacological CCR2 neutralization during the activity phase caused inhibition of atherosclerosis without disturbing microvascular recruitment. These findings demonstrate that chronic inflammation of large vessels feeds on rhythmic myeloid cell recruitment, and lay the foundation for chrono-pharmacology-based therapy.

Background: The majority of the human genome comprises noncoding sequences, which are in part transcribed as long noncoding RNAs (lncRNAs). lncRNAs exhibit multiple functions, including the epigenetic control of gene expression. In this study, the effect of the lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) on atherosclerosis was examined. Methods: The effect of MALAT1 on atherosclerosis was determined in apolipoprotein E–deficient (Apoe − /− ) MALAT1-deficient (Malat1 −/− ) mice that were fed with a high-fat diet and by studying the regulation of MALAT1 in human plaques. Results: Apoe −/− Malat1 −/− mice that were fed a high-fat diet showed increased plaque size and infiltration of inflammatory CD45 + cells compared with Apoe −/− Malat1 +/+ control mice. Bone marrow transplantation of Apoe −/− Malat1 −/− bone marrow cells in Apoe −/− Malat1 +/+ mice enhanced atherosclerotic lesion formation, which suggests that hematopoietic cells mediate the proatherosclerotic phenotype. Indeed, bone marrow cells isolated from Malat1 −/− mice showed increased adhesion to endothelial cells and elevated levels of proinflammatory mediators. Moreover, myeloid cells of Malat1 −/− mice displayed enhanced adhesion to atherosclerotic arteries in vivo. The anti-inflammatory effects of MALAT1 were attributed in part to reduction of the microRNA miR-503. MALAT1 expression was further significantly decreased in human plaques compared with normal arteries and was lower in symptomatic versus asymptomatic patients. Lower levels of MALAT1 in human plaques were associated with a worse prognosis. Conclusions: Reduced levels of MALAT1 augment atherosclerotic lesion formation in mice and are associated with human atherosclerotic disease. The proatherosclerotic effects observed in Malat1 −/− mice were mainly caused by enhanced accumulation of hematopoietic cells.

Background Atherosclerotic lesions grow via the accumulation of leukocytes and oxidized lipoproteins in the vessel wall. Leukocytes can attenuate or augment atherosclerosis through the release of cytokines, chemokines, and other mediators. Deciphering how leukocytes develop, oppose and complement each other’s function, and shape the course of disease, can illuminate understanding of atherosclerosis. Innate response activator (IRA) B cells are a recently described population of GM-CSF-secreting cells of hitherto unknown function in atherosclerosis. Methods and Results Here we show that IRA B cells arise during atherosclerosis in mice and humans. In response to high cholesterol diet, IRA B cell numbers increase preferentially in secondary lymphoid organs via Myd88-dependent signaling. Mixed chimeric mice lacking B cell-derived GM-CSF develop smaller lesions with fewer macrophages and effector T cells. Mechanistically, IRA B cells promote the expansion of classical dendritic cells, which then generate IFNγ-producing TH1 cells. This IRA B cell-dependent TH1 skewing manifests in an IgG1 to IgG2c isotype switch in the immunoglobulin response against oxidized lipoproteins. Conclusions GM-CSF-producing IRA B cells alter adaptive immune processes and shift the leukocyte response toward a TH1-associated mileu that aggravates atherosclerosis.

Background: Acute infection is a well-established risk factor of cardiovascular inflammation increasing the risk for a cardiovascular complication within the first weeks after infection. However, the nature of the processes underlying such aggravation remains unclear. Lipopolysaccharide (LPS) derived from Gram-negative bacteria is a potent activator of circulating immune cells including neutrophils, which foster inflammation through discharge of neutrophil extracellular traps (NETs). Here we utilize a model of endotoxinemia to link acute infection and subsequent neutrophil activation with acceleration of vascular inflammation. Methods: Acute infection was mimicked by injection of a single dose of LPS into hypercholesterolemic mice. Atherosclerosis burden was studied by histomorphometric analysis of the aortic root. Arterial myeloid cell adhesion was quantified by intravital microscopy. Results: LPS treatment rapidly enhanced atherosclerotic lesion size by expansion of the lesional myeloid cell accumulation. LPS treatment led to the deposition of NETs along the arterial lumen and inhibition of NET release annulled lesion expansion during endotoxinemia, thus suggesting that NETs regulate myeloid cell recruitment. To study the mechanism of monocyte adhesion to NETs, we employed in vitro adhesion assays and biophysical approaches. In these experiments, NET-resident histone H2a attracted monocytes in a receptor-independent, surface charge-dependent fashion. Therapeutic neutralization of histone H2a by antibodies or by in silico designed cyclical peptides enables us to reduce luminal monocyte adhesion and lesion expansion during endotoxinemia. Conclusions: Our study shows, that NET-associated histone H2a mediates charge-dependent monocyte adhesion to NETs and accelerates atherosclerosis during endotoxinemia.

Physiological cardiovascular functions show daily diurnal variations, which are synchronized by intrinsic molecular clocks and environment-driven cues. The clinical manifestation of cardiovascular disease also exhibits diurnal variation, with an increased incidence in the early morning. This coincides with circadian oscillations of circulating parameters, such as hormones and leukocyte counts. We are just at the beginning of understanding how circadian rhythms of immune functions are related to cardiovascular disease progression and outcome after an acute ischemic event. Here, we briefly summarize clinical data on oscillations of circulating inflammatory parameters, as well as experimental evidences for the role of circadian clocks in atherosclerosis, postmyocardial infarction inflammatory responses, and cardiac healing.