Ida Gregersen

Microbiota‐dependent metabolite trimethylamine‐N‐oxide is associated with disease severity and survival of patients with chronic heart failure

Recent metabolomic, experimental and clinical studies have demonstrated that trimethylamine‐N‐oxide (TMAO), a microbiota‐dependent metabolite from dietary phosphatidylcholine and carnitine, is a strong predictor of coronary artery disease (CAD). This finding suggests a link between the gut microbiota and atherosclerosis. The potential impact of TMAO in chronic heart failure (HF) is unknown. We hypothesized that TMAO levels would provide prognostic information about adverse outcomes in chronic HF.

Interleukin 23 Levels Are Increased in Carotid Atherosclerosis Possible Role for the Interleukin 23/Interleukin 17 Axis

Background and Purpose— Interleukin (IL)-23 is a cytokine in the IL-12 family, mainly produced by antigen-presenting cells with a central role in inflammation. We hypothesize that IL-23 is also important in atherogenesis and investigate this in a population with carotid atherosclerosis. Methods— Plasma levels of IL-23 were measured in patients with carotid artery stenosis and in healthy controls. The mRNA levels of IL-23 and its receptor, IL-23R, were measured in atherosclerotic plaques, nonatherosclerotic vessels, peripheral blood mononuclear cells, and plasmacytoid dendritic cells. Results— Our findings were as follows: (1) patients with carotid atherosclerosis (n=177) had significantly raised plasma levels of IL-23 when compared with healthy controls (n=24) with particularly high levels in those with the most recent symptoms. (2) mRNA levels of IL-23 and IL-23R were markedly increased in carotid plaques (n=68) when compared with nonatherosclerotic vessels (n=8–10). Immunostaining showed colocalization to plaque macrophages. (3) Patients with carotid atherosclerosis had increased mRNA levels of both IL-23 and IL-23R in plasmacytoid dendritic cells, but not in peripheral blood mononuclear cells. (4) IL-23 increased IL-17 release in monocytes and particularly in peripheral blood mononuclear cells from patients with carotid atherosclerosis, but not in cells from healthy controls. (5) IL-23 gave a prominent tumor necrosis factor release in monocytes from patients with carotid atherosclerosis but not in cells from healthy controls. (6) High plasma levels of IL-23 were associated with increased mortality during follow-up. Conclusions— We have shown an association between IL-23 and disease progression in patients with carotid atherosclerosis, potentially involving IL-17-related mechanisms.

The Carnitine-butyrobetaine-trimethylamine-N-oxide pathway and its association with cardiovascular mortality in patients with carotid atherosclerosis

BACKGROUND AND PURPOSE γ-butyrobetaine (γBB) is a metabolite from dietary Carnitine, involved in the gut microbiota-dependent conversion from Carnitine to the pro-atherogenic metabolite trimethylamine-N-oxide (TMAO). Orally ingested γBB has a pro-atherogenic effect in experimental studies, but γBB has not been studied in relation to atherosclerosis in humans. The aim of this study was to evaluate associations between serum levels of γBB, TMAO and their common precursors Carnitine and trimethyllysine (TML) and carotid atherosclerosis and adverse outcome. METHODS Serum γBB, Carnitine, TML and TMAO were quantified by high performance liquid chromatography in patients with carotid artery atherosclerosis (n = 264) and healthy controls (n = 62). RESULTS Serum γBB (p = 0.024) and Carnitine (p = 0.001), but not TMAO or TML, were increased in patients with carotid atherosclerosis. Higher levels of γBB and TML, but not TMAO or Carnitine were independently associated with cardiovascular death also after adjustment for age and eGFR (adjusted HR [95%] 3.3 [1.9-9.1], p = 0.047 and 6.0 [1.8-20.34], p = 0.026, respectively). CONCLUSIONS Patients with carotid atherosclerosis had increased serum levels of γBB, and elevated levels of γBB and its precursor TML were associated with cardiovascular mortality. Long-term clinical studies of γBB, as a cardiovascular risk marker, and safety studies regarding dietary supplementation of γBB, are warranted.

Increased Systemic and Local Interleukin 9 Levels in Patients with Carotid and Coronary Atherosclerosis

Objective Atherosclerosis is a chronic inflammatory disorder that involves a range of inflammatory mediators. Although interleukin (IL)-9 has been related to inflammation, there are at present no data on its role in atherosclerosis. Here we have examined IL-9 and IL-9 receptor (IL-9R) systemically and locally in patients with coronary and carotid atherosclerosis. Methods Plasma IL-9 was quantified by enzyme immunoassay and multiplex technology. IL-9 and IL-9R mRNA were quantified by real-time RT-PCR, and their localization within the lesion was assessed by immunohistochemistry. Results The main findings were: (i) Patients with carotid atherosclerosis had significantly raised IL-9 plasma levels compared with healthy controls (n = 28), with no differences between asymptomatic (n = 56) and symptomatic (n = 88) patients. (ii) On admission, patients with acute ST-elevation myocardial infarction (STEMI) (n = 42) had markedly raised IL-9 plasma levels which gradually declined during the first week post-MI. (iii) T cells and monocytes from patients with unstable angina (n = 17) had increased mRNA levels of IL-9 as compared with controls (n = 11). (iv) Carotid plaques (n = 68) showed increased mRNA levels of IL-9 and IL-9R compared to non-atherosclerotic vessels (n = 10). Co-localization to T cells (IL-9 and IL-9R) and macrophages (IL-9) were shown by immunohistochemistry. (v) IL-9 increased IL-17 release in peripheral blood mononuclear cells from patients with unstable angina (n = 5) and healthy controls (n = 5) with a particularly enhancing effect in cells from the patient group. Conclusion Our findings show increased IL-9 levels in different atherosclerotic disorders both systemically and within the lesion, suggesting a role for the IL-9/IL-9R axis in the atherosclerotic process, potentially involving IL-17 mediated mechanisms. However, the functional consequences of these findings should be further investigated.

LIGHT/TNFSF14 is increased in patients with type 2 diabetes mellitus and promotes islet cell dysfunction and endothelial cell inflammation in vitro.

Aims/hypothesisActivation of inflammatory pathways is involved in the pathogenesis of type 2 diabetes mellitus. On the basis of its role in vascular inflammation and in metabolic disorders, we hypothesised that the TNF superfamily (TNFSF) member 14 (LIGHT/TNFSF14) could be involved in the pathogenesis of type 2 diabetes mellitus.MethodsPlasma levels of LIGHT were measured in two cohorts of type 2 diabetes mellitus patients (191 Italian and 40 Norwegian). Human pancreatic islet cells and arterial endothelial cells were used to explore regulation and relevant effects of LIGHT in vitro.ResultsOur major findings were: (1) in both diabetic cohorts, plasma levels of LIGHT were significantly raised compared with sex- and age-matched healthy controls (n = 32); (2) enhanced release from activated platelets seems to be an important contributor to the raised LIGHT levels in type 2 diabetes mellitus; (3) in human pancreatic islet cells, inflammatory cytokines increased the release of LIGHT and upregulated mRNA and protein levels of the LIGHT receptors lymphotoxin β receptor (LTβR) and TNF receptor superfamily member 14 (HVEM/TNFRSF14); (4) in these cells, LIGHT attenuated the insulin release in response to high glucose at least partly via pro-apoptotic effects; and (5) in human arterial endothelial cells, glucose boosted inflammatory response to LIGHT, accompanied by an upregulation of mRNA levels of HVEM (also known as TNFRSF14) and LTβR (also known as LTBR).Conclusions/interpretationOur findings show that patients with type 2 diabetes mellitus are characterised by increased plasma LIGHT levels. Our in vitro findings suggest that LIGHT may contribute to the progression of type 2 diabetes mellitus by attenuating insulin secretion in pancreatic islet cells and by contributing to vascular inflammation.

A focus on inflammation as a major risk factor for atherosclerotic cardiovascular diseases.

ABSTRACT Atherosclerosis is a dynamic, pathogenic process in the artery wall, with potential adverse outcome for the host. Acute events such as myocardial infarction and ischemic stroke often result from rupture of unstable atherosclerotic lesions. Understanding the underlying pathology of such lesions and why and when they rupture, is therefore of great interest for the development of new diagnostics and treatment. Inflammation is one of the key drivers of atherosclerotic plaque development and the interplay between inflammation and lipids constitutes the hallmark of atherosclerotic disease. This review summarizes the role of inflammation in atherosclerosis and presents some of the latest discoveries as well as unmet needs regarding the role of inflammation as major risk factor in atherosclerotic disease.

Neil3-dependent base excision repair regulates lipid metabolism and prevents atherosclerosis in Apoe-deficient mice

Increasing evidence suggests that oxidative DNA damage accumulates in atherosclerosis. Recently, we showed that a genetic variant in the human DNA repair enzyme NEIL3 was associated with increased risk of myocardial infarction. Here, we explored the role of Neil3/NEIL3 in atherogenesis by both clinical and experimental approaches. Human carotid plaques revealed increased NEIL3 mRNA expression which significantly correlated with mRNA levels of the macrophage marker CD68. Apoe−/−Neil3−/− mice on high-fat diet showed accelerated plaque formation as compared to Apoe−/− mice, reflecting an atherogenic lipid profile, increased hepatic triglyceride levels and attenuated macrophage cholesterol efflux capacity. Apoe−/−Neil3−/− mice showed marked alterations in several pathways affecting hepatic lipid metabolism, but no genotypic alterations in genome integrity or genome-wide accumulation of oxidative DNA damage. These results suggest a novel role for the DNA glycosylase Neil3 in atherogenesis in balancing lipid metabolism and macrophage function, potentially independently of genome-wide canonical base excision repair of oxidative DNA damage.

Interleukin 27 is increased in carotid atherosclerosis and promotes NLRP3 inflammasome activation.

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.

Enhanced base excision repair capacity in carotid atherosclerosis may protect nuclear DNA but not mitochondrial DNA

BACKGROUND Lesional and systemic oxidative stress has been implicated in the pathogenesis of atherosclerosis, potentially leading to accumulation of DNA base lesions within atherosclerotic plaques. Although base excision repair (BER) is a major pathway counteracting oxidative DNA damage, our knowledge on BER and accumulation of DNA base lesions in clinical atherosclerosis is scarce. Here, we evaluated the transcriptional profile of a wide spectrum of BER components as well as DNA damage accumulation in atherosclerotic and non-atherosclerotic arteries. METHODS BER gene expression levels were analyzed in 162 carotid plaques, 8 disease-free carotid specimens from patients with carotid plaques and 10 non-atherosclerotic control arteries. Genomic integrity, mitochondrial (mt) DNA copy number, oxidative DNA damage and BER proteins were evaluated in a subgroup of plaques and controls. RESULTS Our major findings were: (i) The BER pathway showed a global increased transcriptional response in plaques as compared to control arteries, accompanied by increased expression of several BER proteins. (ii) Whereas nuclear DNA stability was maintained within carotid plaques, mtDNA integrity and copy number were decreased. (iii) Within carotid plaques, mRNA levels of several BER genes correlated with macrophage markers. (iv) In vitro, some of the BER genes were highly expressed in the anti-inflammatory and pro-resolving M2 macrophages, showing increased expression upon exposure to modified lipids. CONCLUSIONS The increased transcriptional response of BER genes in atherosclerosis may contribute to lesional nuclear DNA stability but appears insufficient to maintain mtDNA integrity, potentially influencing mitochondrial function in cells within the atherosclerotic lesion.

Increased Levels of Lectin‐Like Oxidized Low‐Density Lipoprotein Receptor‐1 in Ischemic Stroke and Transient Ischemic Attack

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, OLR1) 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 OLR1 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 OLR1 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 OLR1 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.