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Background: Inflammation is a key process during atherosclerotic lesion development and propagation. Recent evidence showed clearly that especially the inhibition of interleukin (IL)-1β reduced atherosclerotic adverse events in human patients. Fatty acid oxidation (FAO) was previously demonstrated to interact with the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) pathway which is required for mature IL-1β secretion. To understand possible anti-inflammatory properties of FAO inhibition, we tested the effect of pharmacological FAO inhibition using the inhibitor for long-chain 3-ketoacyl coenzyme A thiolase trimetazidine on atherosclerotic plaque development and inflammation., Experimental Approach: The effect of FAO inhibition was determined in LDL-R -/- male mice on a C57/BL6 background. In vitro effects of trimetazidine treatment were analyzed in human umbilical vein endothelial cells and human monocyte derived macrophages., Key Results: We were able to demonstrate that inhibition of FAO reduced atherosclerotic plaque growth. We did not find direct anti-inflammatory properties of trimetazidine in endothelial cells or macrophages in vitro. However, we found that the activation of the NLRP3 system and the secretion of IL-1β were significantly reduced in macrophages after FAO inhibition. These results were confirmed in atherosclerotic lesions of mice treated with trimetazidine as they showed a significant reduction of IL-1β and cleaved caspase-1 in the atherosclerotic lesion as well as of IL-1β and IL-18 in the circulation., Conclusion: Overall, we therefore suggest that the main mechanism of reducing inflammation of trimetazidine and FAO inhibition is the reduction of the NLRP-3 activation leading to reduced levels of the proinflammatory cytokine IL-1β., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: Macrophages are immune cells, capable to remodel the extracellular matrix, which can harbor extracellular DNA incorporated into neutrophil extracellular traps (NETs). To study the breakdown of NETs we studied the capability of macrophage subsets to degrade these structures in vitro and in vivo in a murine thrombosis model. Furthermore, we analyzed human abdominal aortic aneurysm samples in support of our in vitro and in vivo results. Approach and Results: Macrophages were seeded onto blood clots or isolated NETs and polarized. All macrophages were capable to degrade NETs. For initial breakdown, macrophages relied on extracellular deoxyribonucleases. Proinflammatory polarization enhanced NET degradation. The boost in degradation was because of increased macropinocytosis, as inhibition by imipramine diminished their NET breakdown. Inhibition of macropinocytosis in a murine thrombosis model led to increased NET burden and reduced thrombus resolution in vivo. When analyzing abdominal aortic aneurysm samples, macrophage density furthermore corresponded negatively with the amount of local NETs in the intraluminal thrombi as well as in the vessel wall, as increased macrophage density was associated with a reduction in NET burden., Conclusions: We provide evidence that macrophages degrade NETs by extracellular predigestion and subsequent uptake. Furthermore, we show that proinflammatory macrophages increase NET degradation through enhanced macropinocytosis, priming them for NET engulfment. Based on our findings, that inhibition of macropinocytosis in mice corresponded to increased NET amounts in thrombi and that local macrophage density in human abdominal aortic aneurysm is negatively associated with surrounding NETs, we hypothesize, that macrophages are able to degrade NETs in vivo.

Membrane-bound plasmin is used by immune cells to degrade extracellular matrices, which facilitates migration. The plasminogen receptor Plg-R KT is expressed by immune cells, including monocytes and macrophages. Among monocytes and macrophages, distinct subsets can be distinguished based on cell surface markers and pathophysiological function. We investigated expression of Plg-R KT by monocyte and macrophage subsets and whether potential differential expression might have functional consequences for cell migration. Proinflammatory CD14 ++ CD16 + human monocytes and Ly6C high mouse monocytes expressed the highest levels of Plg-R KT and bound significantly more plasminogen compared with the other respective subsets. Proinflammatory human macrophages, generated by polarization with lipopolysaccharide and interferon-γ, showed significantly higher expression of Plg-R KT compared with alternatively activated macrophages, polarized with interleukin-4 and interleukin-13. Directional migration of proinflammatory monocytes was plasmin dependent and was abolished by anti-Plg-R KT monoclonal antibody, ε-amino-caproic acid, aprotinin, and the aminoterminal fragment of urokinase-type plasminogen activator. In an in vivo peritonitis model, significantly less Ly6C high monocyte recruitment was observed in Plg-R KT -/- mice. Immunohistochemical analysis of human carotid plaques and adipose tissue showed that proinflammatory macrophages also exhibited high levels of Plg-R KT +/+ mice. Immunohistochemical analysis of human carotid plaques and adipose tissue showed that proinflammatory macrophages also exhibited high levels of Plg-R KT in vivo. Our data demonstrate higher expression of Plg-R KT on proinflammatory monocyte and macrophage subsets that impacts their migratory capacity., (© 2019 by The American Society of Hematology.)

Following the implantation of biodegradable vascular grafts, macrophages and fibroblasts are the major two cell types recruited to the host-biomaterial interface. In-vitro biocompatibility assessment usually involves one cell type, predominantly macrophages. In this study, macrophage and fibroblast mono- and co-cultures, in paracrine and juxtacrine settings, were used to evaluate a new biodegradable thermoplastic polyurethane (TPU) vascular graft. Expanded-polytetrafluoroethylene (ePTFE) grafts served as controls. Pro/anti-inflammatory gene expression of macrophages and cytokines was assessed in vitro and compared to those of an in vivo rat model. Host cell infiltration and the type of proliferated cells was further studied in vivo. TPU grafts revealed superior support in cell attachment, infiltration and proliferation compared with ePTFE grafts. Expression of pro-inflammatory TNF-α/IL-1α cytokines was significantly higher in ePTFE, whereas the level of IL-10 was higher in TPU. Initial high expression of pro-inflammatory CCR7 macrophages was noted in TPU, however there was a clear transition from CCR7 to anti-inflammatory CD163 expression in vitro and in vivo only in TPU, confirming superior cell-biomaterial response. The co-culture models, especially the paracrine model, revealed higher fidelity to the immunomodulatory/biocompatibility behavior of degradable TPU grafts in vivo. This study established an exciting approach developing a co-culture model as a tool for biocompatibility evaluation of degradable biomaterials.

The complement component C5a is formed during activation of the complement cascade and exerts chemotactic and proinflammatory effects. Macrophages, which are localized in the rupture-prone shoulder regions of coronary plaques, are thought to play a major role in plaque destabilization and rupture through the production of matrix metalloproteinases (MMPs). When human monocyte-derived macrophages were stimulated in vitro with C5a, MMP-1 and MMP-9 mRNA levels were significantly increased. Furthermore, C5a up-regulated MMP-1 and MMP-9 antigens and activity, as determined by ELISA and specific activity assays. These effects were blocked by antibodies against the receptor C5aR/CD88. In addition, blocking experiments revealed that MMP-1 expression was mediated by activation of the transcription factor AP-1, and MMP-9 expression was induced by activation of NF-κB and AP-1. Immunohistochemical analysis of human coronary plaques demonstrated the colocalization of C5a, MMP-1, and MMP-9 in vivo. Together, these observations indicate that activation of the complement cascade and formation of C5a may play a role in the onset of acute coronary events by induction of MMPs in atherosclerotic lesions.

Macrophages as inflammatory cells are involved in the pathogenesis of atherosclerosis that today is recognized as an inflammatory disease. Activation of coagulation leads to the late complication of atherosclerosis, namely atherothrombosis with its clinical manifestations stroke, unstable angina, myocardial infarction, and sudden cardiac death. Thus inflammation and coagulation play fundamental roles in the pathogenesis of atherosclerosis. We show that the coagulation enzyme thrombin up-regulates oncostatin M (OSM), a pleiotropic cytokine implicated in the pathophysiology of vascular disease, in human monocyte-derived macrophages (MDMs) up to 16.8-fold. A similar effect was seen in human peripheral blood monocytes and human plaque macrophages. In MDMs, the effect of thrombin on OSM was abolished by PPACK and mimicked by a PAR-1-specific peptide. Thrombin induced phosphorylation of ERK1/2 and p38 in MDMs. The ERK1/2 inhibitor PD98059 blocked the effect of thrombin on OSM production in MDMs, whereas the p38 inhibitor SB202190 had no effect. Thrombin induced translocation of c-fos and c-jun to the nucleus of MDMs. Using OSM promoter-luciferase reporter constructs transfected into MDMs, we show that a functional AP-1 site is required for promoter activation by thrombin. We present another link between coagulation and inflammation, which could impact on the pathogenesis of atherosclerosis.

Objective: Macrophages produce the cytokine oncostatin M (OSM), which beside other functions is also involved in inflammation. The complement component C5a mobilizes and activates these cells at inflammatory sites. We examined the effect of C5a on OSM production in human monocytes and in human monocyte-derived macrophages., Methods and Results: For macrophage transformation peripheral blood monocytes were cultivated for 8 to 10 days in the presence of human serum. C5a significantly increased in these cells OSM antigen as determined by specific ELISA and mRNA as quantitated by real-time polymerase chain reaction in these cells as well as in plaque macrophages. This effect was blocked by antibodies against the receptor C5aR/CD88 and by pertussis toxin. The C5a-induced phosphorylation of p38 and JNK and the C5a-induced increase in OSM production in macrophages was abolished by 2 p38 inhibitors and by a JNK inhibitor. Furthermore C5a increased the nuclear translocation of c-fos and c-jun. Using different OSM promoter deletion mutant constructs we show that the putative AP-1 element is responsible for activation of OSM promoter activity by C5a., Conclusions: Our data establish a link between the complement system and the gp130 receptor cytokine family with possible implications for the pathology of inflammatory diseases.

Background: Atherosclerosis is considered to be a chronic inflammatory disorder. Activation of the complement cascade is a major aspect of chronic inflammatory diseases. Complement components were identified in atherosclerotic plaques, and a correlation between adverse events and C5a plasma levels was found. These findings support the notion that complement activation contributes to development and progression of atherosclerotic lesions., Objectives: We investigated whether complement components C3a and C5a regulate plasminogen activator inhibitor (PAI-1) in human macrophages., Methods: Human monocyte-derived macrophages (MDM) and human plaque macrophages were cultured and incubated with the complement component C5a., Results: C5a increased PAI-1 up to 11-fold in human MDM and up to 2.7-fold in human plaque macrophages. These results were confirmed at the mRNA level using real time-polymerase chain reaction. Pertussis toxin or anti-C5aR/CD88 antibody completely abolished the effect of recombinant human C5a on PAI-1 production, suggesting a role of the C5a receptor. Experiments with antitumor necrosis factor (TNF)-alpha antibodies and tiron showed that the effect of C5a was not mediated by TNF-alpha or oxidative burst. Furthermore C5a induced NF-kappaB binding to the cis element in human macrophages and the C5a-induced increase in PAI-1 was completely abolished by an NF-kappaB inhibitor., Conclusions: We conclude that C5a upregulates PAI-1 in macrophages via NF-kappaB activation. We hypothesize that - if operative in vivo- this effect could favor thrombus development and thrombus stabilization in the lesion area. On the other hand one could speculate that C5a-induced upregulation of PAI-1 in plaque macrophages could act as a defense mechanism against plaque destabilization and rupture.

Plaque destabilization leading to myocardial infarction is observed after surgery even if the intervention is of noncardiovascular nature. Mediators of peri- or postoperative stress responsible for such events could include catecholamines and lipopolysaccharide (LPS). Monocytes may be involved in destabilization of atherosclerotic plaques by production of matrix metalloproteinases (MMP). We examined whether catecholamines could affect the expression of MMPs in human monocytes/macrophages and whether catecholamines could modulate LPS-stimulated expression of particular MMPs in these cells. Epinephrine and norepinephrine up-regulated MMP-1 and potentiated LPS-induced expression of MMP-1 in peripheral blood monocytes and monocyte-derived macrophages. We further characterized this effect employing the monocytic cell line U937 and showed that catecholamines potentiate LPS-induced effects on MMP-1 and MMP-9 antigen and activity. mRNA levels of the respective MMPs also increased. These effects did not result from higher mRNA stability but rather from increased transcription possibly induced by enhanced DNA binding of AP-1 and were mediated by either beta1- or beta 2-receptors. If this mechanism is also effective in vivo, our findings might, at least in part, help to explain the observation that cardiac events are important causes of morbidity and mortality after noncardiac surgery and support the findings that peri-operative beta-blockade has been shown to reduce postoperative mortality from cardiac events.