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Background and aims: Atherogenesis involves a complex interaction between immune cells and lipids, processes greatly influenced by the vascular smooth muscle cell (VSMC) phenotype. The DNA glycosylase NEIL3 has previously been shown to have a role in atherogenesis, though whether this is due to its ability to repair DNA damage or to other non-canonical functions is not yet clear. Hereby, we investigate the role of NEIL3 in atherogenesis, specifically in VSMC phenotypic modulation, which is critical in plaque formation and stability.Methods: Chow diet-fed atherosclerosis-prone Apoe(-/-) mice deficient in Neil3, and NEIL3-abrogated human primary aortic VSMCs were characterized by qPCR, and immunohistochemical and enzymatic-based assays; moreover, single-cell RNA sequencing, mRNA sequencing, and proteomics were used to map the molecular effects of Neil3/NEIL3 deficiency in the aortic VSMC phenotype. Furthermore, BrdU-based proliferation assays and Western blot were performed to elucidate the involvement of the Akt signaling pathway in the transdifferentiation of aortic VSMCs lacking Neil3/NEIL3.Results: We show that Neil3 deficiency increases atherosclerotic plaque development without affecting systemic lipids. This observation was associated with a shift in VSMC phenotype towards a proliferating, lipid-accumulating and secretory macrophage-like cell phenotype, without changes in DNA damage. VSMC transdifferentiation in Neil3-deficient mice encompassed increased activity of the Akt signaling pathway, supported by cell experiments showing Akt-dependent proliferation in NEIL3-abrogated human primary aortic VSMCs.Conclusions: Our findings show that Neil3 deficiency promotes atherosclerosis development through non-canonical mechanisms affecting VSMC phenotype involving activation of the Akt signaling pathway.

Background In cardiovascular diseases, atherosclerotic disorder are the most frequent and important with respect to morbidity and mortality. Inflammation mediated by immune cells is central in all parts of the atherosclerotic progress, and further understanding of the underlying mechanisms is needed. Growing evidence suggests that deamination of adenosine‐to‐inosine in RNA is crucial for a correct immune response; nevertheless, the role of adenosine‐to‐inosine RNA editing in atherogenesis has barely been studied. Several proteins have affinity for inosines in RNA, one being ENDOV (endonuclease V), which binds and cleaves RNA at inosines. Data on ENDOV in atherosclerosis are lacking. Methods and Results Quantitative polymerase chain reaction on ENDOV mRNA showed an increased level in human carotid atherosclerotic plaques compared with control veins. Inosine‐ribonuclease activity as measured by an enzyme activity assay is detected in immune cells relevant for the atherosclerotic process. Abolishing EndoV in atherogenic apolipoprotein E‐deficient ( ApoE −/− ) mice reduces the atherosclerotic plaque burden, both in size and lipid content. In addition, in a brain stroke model, mice without ENDOV suffer less damage than control mice. Finally, lack of EndoV reduces the recruitment of monocytes to atherosclerotic lesions in atherogenic ApoE −/− mice. Conclusions ENDOV is upregulated in human atherosclerotic lesions, and data from mice suggest that ENDOV promotes atherogenesis by enhancing the monocyte recruitment into the atherosclerotic lesion, potentially by increasing the effect of CCL2 activation on these cells.

Background The immune response during falciparum malaria mediates both harmful and protective effects on the host; however the participating molecules have not been fully defined. Interleukin (IL)-27 is a pleiotropic cytokine exerting both inflammatory and anti-inflammatory effects, but data on IL-27 in malaria patients are scarce. Methods Clinical data and blood samples were collected from adults in Mozambique with P. falciparum infection, with (n = 70) and without (n = 61) HIV-1 co-infection, from HIV-infected patients with similar symptoms without malaria (n = 58) and from healthy controls (n = 52). In vitro studies were performed in endothelial cells and PBMC using hemozoin crystals. Samples were analyzed using enzyme immunoassays and quantitative PCR. Results (i) IL-27 was markedly up-regulated in malaria patients compared with controls and HIV-infected patients without malaria, showing no relation to HIV co-infection. (ii) IL-27 was correlated with P. falciparum parasitemia and von Willebrand factor as a marker of endothelial activation, but not with disease severity. (iii) In vitro, IL-27 modulated the hemozoin-mediated cytokine response in endothelial cells and PBMC with enhancing effects on IL-6 and attenuating effects on IL-8. Conclusion Our findings show that IL-27 is regulated during falciparum malaria, mediating both inflammatory and anti-inflammatory effects, potentially playing an immune-regulatory role during falciparum malaria.

Endoplasmic reticulum (ER) stress has been shown to play a key role during the initiation and clinical progression of the cardiovascular diseases, such as atherosclerosis. We have recently shown that expression of tissue factor pathway inhibitor (TFPI) in human monocyte-derived macrophages (MDMs) was induced by cholesterol crystals (CC). In the present study we aimed to determine the role of TFPI under ER stress conditions using human MDMs. qRT-PCR and immunohistochemistry analysis were performed to determine the presence of the ER stress marker CCAAT/enhancer binding protein homologous protein (CHOP) and TFPI in human carotid plaque material and also in human MDMs polarized into pro-inflammatory M1 or anti-inflammatory M2 populations. CHOP mRNA levels were upregulated in the plaques compared to healthy vessels, and CHOP protein was localized in the same area as TFPI in the plaques. Both CHOP and TFPI mRNA levels were upregulated after CC treatment, especially in the M2 phenotype, and the ER stress inhibitor 4-phenylbutyric acid (PBA) reversed this effect. Furthermore, CC treatment increased the levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-8, which for TNF-α and IL-8 was inhibited by PBA, and reduced the levels of the anti-inflammatory cytokine IL-10 in M2-polarized macrophages. Knockdown of TFPI prior to CC treatment exacerbated TNF-α and IL-6 levels, but reduced IL-8 and IL-10 levels. Our results show that CC induce TFPI and cytokine expression in M2-polarized macrophages through activation of the ER stress pathway and that TFPI has a protective effect against TNF-α and IL-6 mediated inflammation. These mechanisms may have implications for the pathogenesis of atherosclerosis.

People with human immunodeficiency virus (HIV) infection (PWH) have an increased risk of cardiovascular disease (CVD), compared with the general population. In a nested case-control study of 55 PWH with first-time myocardial infarction (MI; cases) and 182 PWH with no CVD (controls), we measured soluble markers of interleukin 1 (IL-1) activation at 4 different time points before the case's MI. Cases had higher levels of IL-1 receptor antagonist (IL-1Ra) at all time points leading up to first-time MI, and higher levels of IL-1Ra were associated with an approximately 1.5-fold increased risk of MI, supporting the rationale to target IL-1 activation to reduce cardiovascular risk in PWH.

Background Termination of acute inflammation is an active process orchestrated by lipid mediators (LM) derived from polyunsaturated fatty acids, referred to as specialized pro-resolving mediators (SPM). These mediators also provide novel therapeutic opportunities for treating inflammatory disease. However, the regulation of these molecules following acute myocardial infarction (MI) remains of interest. Methods In this prospective observational study we aimed to profile plasma levels of SPMs in ST-elevation MI (STEMI) patients during the first week following MI. Plasma LM concentrations were measured in patients with STEMI (n = 15) at three time points and compared with stable coronary artery disease (CAD; n = 10) and healthy controls (n = 10). Findings Our main findings were: (i) Immediately after onset of MI and before peak troponin T levels, STEMI patients had markedly increased levels of SPMs as compared with healthy controls and stable CAD patients, with levels of these mediators declining during follow-up. (ii) The increase in SPMs primarily reflected an increase in docosapentaenoic acid- and docosahexaenoic acid-derived protectins. (iii) Several individual protectins were correlated with the rapid increase in neutrophil counts, but not with CRP. (iv) A shift in 5-LOX activity from the leukotriene B4 pathway to the pro-resolving RvTs was observed. Interpretation The temporal regulation of SPMs indicates that resolution mechanisms are activated early during STEMI as part of an endogenous mechanism to initiate repair. Thus strategies to boost the activity and/or efficacy of these endogenous mechanisms may represent novel therapeutic opportunities for treatment of patients with MI. Fund This work was supported by grants from the South-Eastern Norwegian regional health authority, the European Research Council under the European Union's Horizon 2020 research and innovation program, a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society, and the Barts Charity.

Background: Following birth asphyxia there is a robust inflammatory response. NLRP3 is a receptor of the innate immune system. Upon activation, NLRP3 forms an inflammasome together with ASC and procaspase-1 to mediate release of IL-1β and IL-18. NLRP3 has previously been shown to be upregulated following neonatal hypoxic-ischemic (HI) brain injury in mice, but with no early effect on brain injury. Objective: We aimed to evaluate if deficiency of NLRP3 or ASC protects against neonatal HI brain damage 7 days after hypoxia-ischemia. Methods: C57BL/6J, NLRP3–/–, and ASC–/– mice were subjected to unilateral common carotid artery ligation followed by hypoxia at P9. Brain infarction, apoptosis, and microglial response were evaluated, as well as total RNA sequencing and examination of plasma levels of systemic proinflammatory cytokines. Results: NLRP3–/– mice showed significantly increased brain infarction volumes compared to wild-type (Wt) mice, while ASC–/– mice showed reduced brain infarction volumes after neonatal hypoxia-ischemia. The amount of activated microglia was increased in NLRP3–/– mice, while decreased in ASC–/– mice compared to Wt mice. Total RNA sequencing showed an impaired inflammatory transcriptional response in the hippocampus of NLRP3–/– mice. Plasma levels of IL-1β and IL-18 were not affected, but TNF was lower in NLRP3–/– and ASC–/– mice compared to Wt mice. Conclusion: ASC deficiency is neuroprotective in neonatal HI brain damage in mice, while NLRP3 deficiency increases brain damage.

Background Soluble lectin‐like oxidized low‐density lipoprotein receptor‐1 ( sLOX ‐1) has been shown to be increased in patients with acute ischemic stroke. Here, we evaluated plasma sLOX ‐1 levels and vascular carotid plaque LOX ‐1 (ie, OLR 1 ) gene expression in patients with ischemic stroke and transient ischemic attack ( TIA ) with particular focus on their relation to time since symptom onset. Methods and Results Plasma sLOX ‐1 (n=232) and carotid plaque OLR 1 gene expression (n=146) were evaluated in patients who were referred to evaluation for carotid endarterectomy, as well as in healthy control plasma (n=81). Patients were categorized according to presence of acute ischemic stroke or transient ischemic attack (n=35) ≤7 days, >7 days ≤3 months (n=90), >3 months (n=40), or no reported symptoms before study inclusion (n=67). Our major findings were the following: (1) Patients with carotid atherosclerosis had increased plasma sLOX ‐1 levels as compared with controls. (2) Plaque OLR 1 mRNA levels were increased in carotid plaques (n=146) compared with nonatherosclerotic vessels (ie, common iliac arteries of organ donors, n=10). (3) There were no differences in sLOX plasma levels or OLR 1 gene expression when analyzed according to the time since relevant cerebral ischemic symptoms. (4) Also patients with severe carotid atherosclerosis without any previous ischemic events had raised sLOX ‐1 levels. (5) Immunostaining showed colocalization between LOX ‐1 and macrophages within the carotid plaques. (6) Also patients with acute stroke (within 7 days) caused by atrial fibrillation (n=22) had comparable raised sLOX ‐1 levels. Conclusions sLOX ‐1 levels are elevated in patients with ischemic stroke and transient ischemic attack independent of cause and time since the ischemic event.

Aim The aim of the present study were to elucidate the role of NAMPT in atherosclerosis, by examine NAMPT expression in peripheral blood mononuclear cells (PBMC) in patients with coronary artery disease (CAD) and healthy controls and by examining the regulation and effect of NAMPT on macrophage polarization, hypothesizing that it could influence the polarization to inflammatory and resolving macrophages. Method and Results We analyzed RNA levels of NAMPT in PBMC from CAD and healthy controls and found NAMPT to be increased in PBMC from patients with acute coronary syndrome (n = 39) compared to healthy controls (n = 20) and patients with stable CAD (n = 22). Within the PBMC NAMPT was correlated to several inflammatory cytokines and the antioxidant enzyme superoxide dismutase 2. In vitro cell experiments revealed that NAMPT is increased both intracellular and extracellular in inflammatory M1 macrophages compared to in anti-inflammatory M2 macrophages. In addition, inhibiting NAMPT enzymatic activity inhibited M1 polarization in macrophages. Conclusion Based on our in vivo and in vitro findings we suggest that NAMPT could contribute to systemic and plaque inflammation in atherosclerotic disorders at least partly through effect on macrophages.

Aim Interleukin-27 (IL-27) is involved in different inflammatory diseases; however, its role in atherosclerosis is unclear. In this study we investigated the expression of IL-27 and its receptor in patients with carotid atherosclerosis and if IL-27 could modulate the inflammatory effects of the NLRP3 inflammasome in vitro. Methods Plasma IL-27 was measured by enzyme immunoassay in patients with carotid stenosis (n = 140) and in healthy controls (n = 19). Expression of IL-27 and IL-27R was analyzed by quantitative PCR and immunohistochemistry in plaques from patients and in non-atherosclerotic vessels. THP-1 monocytes, primary monocytes and peripheral blood mononuclear cells (PBMCs) were used to study effects of IL-27 in vitro. Results Our main findings were: (i) Plasma levels of IL-27 were significantly elevated in patients with carotid atherosclerotic disease compared to healthy controls. (ii) Gene expression of IL-27 and IL-27R was significantly elevated in plaques compared to control vessels, and co-localized to macrophages. (iii) In vitro, IL-27 increased NLRP3 inflammasome activation in monocytes with enhanced release of IL-1 β. Conclusions We demonstrate increased levels of IL-27 and IL-27R in patients with carotid atherosclerosis. Our in vitro findings suggest an inflammatory role for IL-27, which can possibly be linked to atherosclerotic disease development. © 2017 Gregersen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Interleukin (IL)-10 is a prototypical anti-inflammatory cytokine that has been shown to attenuate atherosclerosis development. In addition to its anti-inflammatory properties, the anti-atherogenic effect of IL-10 has recently also been suggested to reflect a complex effect of IL-10 on lipid metabolism in macrophages. In the present study we examined the effects of IL-10 on cholesterol efflux mechanism in lipid-loaded THP-1 macrophages. Our main findings were: (i) IL-10 significantly enhanced cholesterol efflux induced by fetal-calf serum, high-density lipoprotein (HDL)2 and apolipoprotein A-1. (ii) The IL-10-mediated effects on cholesterol efflux were accompanied by an increased IL-10-mediated expression of the ATP-binding cassette transporters ABCA1 and ABCG1, that was further enhanced when the cells were co-activated with the liver X receptor (LXR)α agonist (22R)-hydroxycholesterol. (iii) The effect of LXRα activation on the IL-10-mediated effects on the ATP-binding cassette transporters seems to include enhancing effects on the IL-10 receptor 1 (IL10R1) expression and interaction with STAT-3 signaling. (iv) These enhancing effects on ABCA1 and ABCG1 was not seen when the cells were stimulated with the IL-10 family members IL-22 and IL-24. This study suggests that the anti-atherogenic properties of IL-10 may include enhancing effects on cholesterol efflux mechanism that involves cross-talk with LXRα activation.

Objectives Based on the newly recognized role of the homeostatic chemokines in inflammation, we hypothesized that CXCL13 could modulate atherogenesis and plaque destabilization. Methods The study included in vivo analyses in patients with carotid atherosclerosis and in vitro experiments in cells involved in atherogenesis (ie, monocytes/macrophages, vascular smooth muscle cells [SMC], and platelets). Results Our main findings were: (i) Patients with carotid atherosclerosis ( n = 130) had increased plasma levels of CXCL13 with particularly high levels in symptomatic disease. (ii) CXCL13 showed increased expression within atherosclerotic carotid plaques as compared with non-atherosclerotic vessels. (iii) Within the atherosclerotic lesions, CXCR5 and CXCL13 were expressed by macrophages and SMC in all stages of plaque progression. (iv) Releasate from activated platelets and toll-like receptor activation enhanced the expression of CXCL13 in THP-1 monocytes and primary monocytes. (v) In vitro, CXCL13 exerted anti-apoptotic effects in primary monocytes, THP-1 macrophages, and vascular SMC. (vi) CXCL13 increased arginase-1, transforming growth factor-β, and interleukin-10 expression in THP-1 cells and in samples from isolated carotid plaques. Conclusion Levels of CXCL13 are increased in carotid atherosclerosis both systemically and within the atherosclerotic lesion. Based on our in vitro findings, we hypothesize a potential plaque stabilizing effects of CXCL13-CXCR5 interaction.

Background We have previously shown that the homeostatic chemokine CXCL13 is up-regulated in monocytes in atherosclerosis, mediating anti-apoptotic and anti-inflammatory effects. Objective To investigate the regulation of CXCL13s receptor, CXCR5. Methods/patients In vitro studies in THP-1 and primary monocytes and studies of CXCR5 expression in thrombus material obtained at the site of plaque rupture during myocardial infarction (MI). Results Our major findings were: (i) toll-like receptor agonists and particularly β-adrenergic receptor activation and releasate from thrombin-activated platelets increased CXCR5 mRNA levels in monocytes. (ii) The platelet-mediated induction of CXCR5 involved prostaglandin E2/cAMP/protein kinase A-dependent as well as RANTES-dependent pathways with NFκB activation as a potential common down-stream mediator. (iii) Releasate from thrombin-activated platelets augmented the anti-inflammatory effects of CXCL13 in monocytes at least partly by enhancing the effects of CXCL13 on CXCR5 expression. (iv) We found strong immunostaining of CXCR5 in thrombus material obtained at the site of plaque rupture in patients with ST elevation MI (STEMI) and in unstable carotid lesions, co-localized with platelets. Conclusion Our findings suggest that platelet-mediated signaling through CXCR5 may be active in vivo during plaque destabilization, potentially representing a counteracting mechanism to inflammation.

Background The purpose of this study was to investigate mediators of inflammation and haemostasis in patients with chronic necrotizing pulmonary aspergillosis (CNPA), a locally, destructive process of the lung due to invasion by Aspergillus species. Methods Measurements of selected biomarkers in 10 patients with CNPA and 19 healthy, matched controls were performed with enzyme-linked immunosorbent assay (ELISA) and multiplex methodology. The gene expressions of relevant biomarkers were analyzed with real-time quantitative RT-PCR. Results Increased concentrations of circulating mediators of inflammation interleukin (IL)-6, IL-8, RANTES, TNF-α, ICAM-1 and mediators involved in endothelial activation and thrombosis (vWF, TF and PAI-1) were observed in patients with CNPA. The concentration of the anti-inflammatory cytokine IL-10 was increased both in plasma and in PBMC in the patient population. The gene expression of CD40L was decreased in PBMC from the patient group, accompanied by decreased concentrations of soluble (s) CD40L in the circulation. Conclusions The proinflammatory response against Aspergillus may be counteracted by reduced CD40L and sCD40L, as well as increased IL-10, which may compromise the immune response against Aspergillus in patients with CNPA.

Background and Purpose— The risk for cardiovascular events is related to the composition and stability of an atherosclerotic plaque driven by inflammation and deposition of lipids. Scavenger receptors are a family of cell surface receptors involved in lipid uptake and inflammation. Recently, we found that soluble CD36 is increased in plasma from patients with diabetes strongly correlated with insulin resistance. Methods— We tested whether soluble CD36 is a marker of plaque stability in patients with high-grade internal carotid stenoses (n=62). The patients were classified according to plaque symptomatology and plaque echogenicity on ultrasound examination. Results— When patients were divided into 3 groups according to the latest clinical symptoms from plaques (ie, symptoms within the last 2 months [n=16], symptoms within the last 2 to 6 months [n=15], or asymptomatic [n=31]), the former group had significantly raised plasma levels of soluble CD36 as compared with the other 2 groups. In contrast, we found no differences in plasma levels of C-reactive protein, β-thromboglobulin, lipid parameters, or HbA1C between these groups. The patients with echolucent carotid plaques (n=20) tended to have higher soluble CD36 levels in plasma compared with those with echogenic/heterogenic plaque (n=39; P =0.087). By immunohistochemistry, CD36 was localized to macrophages-rich area of intima within the atherosclerotic lesion. Conclusion— We propose that sCD36 may be a marker of plaque instability and symptomatic carotid atherosclerosis, possibly at least partly as a result of CD36 release to the circulation from the foam cells within the atherosclerotic lesion.

Cytokine systems are activated in heart failure, and it is believed that interaction between such systems may be important during progression of this disorder. We have previously shown that failing hearts have increased levels of the interleukin-6 related cytokine leukemia inhibitory factor (LIF) and activin A, a member of the transforming growth factor-beta family. The aim of this study was to examine the effects of activin A on cardiomyocytes and a potential interaction with LIF-mediated changes in cell signaling and growth. Cardiomyocytes were isolated from 1- to 3-day-old Wistar rats, and the cells were treated with LIF, activin A or a combination thereof. Our main findings were: (i) activin A treatment reduced the LIF-mediated increase in cardiomyocyte length, perimeter and sarcomeric organization and was accompanied by a substantially decreased alpha-skeletal actin gene expression. (ii) The activin A-mediated phosphorylation of Smad2 was markedly enhanced by LIF. (iii) Activin A markedly induced SOCS3 gene expression, while LIF potently increased the expression of Smad7 mRNA, representing inhibitors of LIF and activin A signaling pathways, respectively. (iv) Inhibiting activation of the Smad2/3 pathway abolished the effects of activin A on LIF-induced changes in cell length, perimeter and sarcomeric organization. In conclusion, activin A markedly attenuates LIF-induced changes in cardiomyocytes, reflecting a potentially important role for both activin A and the Smad2/3 pathway in regulation of myocardial remodeling.

Background Atherosclerosis is a major cause of cerebrovascular disease. Matrix metalloproteinases (MMPs) play an important role in matrix degradation within the atherosclerotic lesion leading to plaque destabilization and ischemic stroke. We hypothesized that MMP-7 could be involved in this process. Methods Plasma levels of MMP-7 were measured in 182 consecutive patients with moderate (50–69%) or severe (≥70%) internal carotid artery stenosis, and in 23 healthy controls. The mRNA levels of MMP-7 were measured in atherosclerotic carotid plaques with different symptomatology, and based on its localization to macrophages, the in vitro regulation of MMP-7 in primary monocytes was examined. Results Our major findings were (i) Patients with carotid atherosclerosis had markedly increased plasma levels of MMP-7 compared to healthy controls, with particularly high levels in patients with recent symptoms (i.e., within the last 2 months). (ii) A similar pattern was found within carotid plaques with markedly higher mRNA levels of MMP-7 than in non-atherosclerotic vessels. Particularly high protein levels of MMP-7 levels were found in those with the most recent symptoms. (iii) Immunhistochemistry showed that MMP-7 was localized to macrophages, and in vitro studies in primary monocytes showed that the inflammatory cytokine tumor necrosis factor-α in combination with hypoxia and oxidized LDL markedly increased MMP-7 expression. (iv) During the follow-up of patients with carotid atherosclerosis, high plasma levels of MMP-7 were independently associated with total mortality. Conclusion Our findings suggest that MMP-7 could contribute to plaque instability in carotid atherosclerosis, potentially involving macrophage-related mechanisms.