Biomarkers predictive of atrial fibrillation in patients with cryptogenic stroke. Insights from the Nordic Atrial Fibrillation and Stroke (NOR-FIB) study.

BACKGROUND AND PURPOSE: There are currently no biomarkers to select cryptogenic stroke (CS) patients for monitoring with insertable cardiac monitors (ICMs), the most effective tool for diagnosing atrial fibrillation (AF) in CS. The purpose of this study was to assess clinically available biomarkers as predictors of AF. METHODS: Eligible CS and cryptogenic transient ischaemic attack patients underwent 12-month monitoring with ICMs, clinical follow-up and biomarker sampling. Levels of cardiac and thromboembolic biomarkers, taken within 14 days from symptom onset, were compared between patients diagnosed with AF (n = 74) during monitoring and those without AF (n = 185). Receiver operating characteristic curves were created. Biomarkers reaching area under the receiver operating characteristic curve ≥ 0.7 were dichotomized by finding optimal cut-off values and were used in logistic regression establishing their predictive value for increased risk of AF in unadjusted and adjusted models. RESULTS: B-type natriuretic peptide (BNP), N-terminal pro-brain natriuretic peptide (NT-proBNP), creatine kinase, D-dimer and high-sensitivity cardiac troponin I and T were significantly higher in the AF than non-AF group. BNP and NT-proBNP reached the predefined area under the curve level, 0.755 and 0.725 respectively. Optimal cut-off values were 33.5 ng/l for BNP and 87 ng/l for NT-proBNP. Regression analysis showed that NT-proBNP was a predictor of AF in both unadjusted (odds ratio 7.72, 95% confidence interval 3.16-18.87) and age- and sex-adjusted models (odds ratio 4.82, 95% confidence interval 1.79-12.96). CONCLUSION: Several clinically established biomarkers were associated with AF. NT-proBNP performed best as AF predictor and could be used for selecting patients for long-term monitoring with ICMs.

Increased plasma levels of non-sugar sweeteners in patients with symptomatic carotid atherosclerosis.

Purpose. The non-sugar sweeteners acesulfame K and saccharin are considered safe, but there is conflicting evidence on their effects on cardiovascular health. Materials and methods. In this explorative pilot study, we measured plasma levels of acesulfame K and saccharin in 15 patients with symptomatic carotid atherosclerosis, 18 asymptomatic patients and 15 control subjects. Fecal microbiota and short-chain fatty acids were analyzed. Dietary and medical history was assessed. Results. Symptomatic patients had higher levels of acesulfame K and saccharin compared to controls. Acesulfame K was associated with increased leukocyte count. Saccharin was associated with more severe carotid stenosis, as well as lower fecal butyric acid.

Tissue factor pathway inhibitor attenuates ER stress-induced inflammation in human M2-polarized macrophages.

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.

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

AIMS/HYPOTHESIS: Activation 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. METHODS: Plasma 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. RESULTS: Our 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 ß receptor (LTßR) 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 LTßR (also known as LTBR). CONCLUSIONS/INTERPRETATION: Our 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.

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.

Relationship between fibroblast growth factor in plasma and carotid plaque neovascularization: a pilot study.

Background: Unstable atherosclerotic carotid plaques with intraplaque neovascularization (IPN) carry a substantial risk for ischemic stroke. Conventional ultrasound methods fall short in detecting IPN, where superb microvascular imaging (SMI) has emerged as a promising tool for both visualizing and quantification. High levels of fibroblast growth factor 23 (FGF-23) have, in observational studies, been suggested as related to cardiovascular morbidity and mortality. The association of FGF-23 to atherosclerotic carotid plaque instability remains relatively unexplored. Methods: A cohort of twenty-nine patients with ≥50% atherosclerotic carotid stenosis underwent conventional carotid ultrasound, SMI, and blood tests, including measurement of FGF-23 in plasma. Nineteen patients were characterized as symptomatic and ten as asymptomatic. Results: Our major findings were: i) Higher FGF-23 levels were strongly correlated with increased SMI-assessed IPN. ii) Neo-vessel count recorded by quantitative SMI was positively correlated to increased FGF-23 levels, but not with basic FGF levels. (iii) In contrast, traditional risk factors for plaque instability exhibited no noteworthy associations with SMI-assessed IPN or with FGF-23 levels. Conclusion: This pilot study suggest the potential of FGF-23 as a valuable marker for neovascularization and atherosclerotic carotid plaque instability as a risk factor for ischemic stroke. Further research involving larger cohorts and prospective data is necessary to understand FGF-23's role in this context comprehensively.

Extracellular matrix remodelling pathway in peripheral blood mononuclear cells from severe COVID-19 patients: an explorative study.

There is a reciprocal relationship between extracellular matrix (ECM) remodelling and inflammation that could be operating in the progression of severe COVID-19. To explore the immune-driven ECM remodelling in COVID-19, we in this explorative study analysed these interactions in hospitalised COVID-19 patients. RNA sequencing and flow analysis were performed on peripheral blood mononuclear cells. Inflammatory mediators in plasma were measured by ELISA and MSD, and clinical information from hospitalised COVID-19 patients (N=15) at admission was included in the analysis. Further, we reanalysed two publicly available datasets: (1) lung tissue RNA-sequencing dataset (N=5) and (2) proteomics dataset from PBCM. ECM remodelling pathways were enriched in PBMC from COVID-19 patients compared to healthy controls. Patients treated at the intensive care unit (ICU) expressed distinct ECM remodelling gene profiles compared to patients in the hospital ward. Several markers were strongly correlated to immune cell subsets, and the dysregulation in the ICU patients was positively associated with plasma levels of inflammatory cytokines and negatively associated with B-cell activating factors. Finally, our analysis of publicly accessible datasets revealed (i) an augmented ECM remodelling signature in inflamed lung tissue compared to non-inflamed tissue and (ii) proteomics analysis of PBMC from severe COVID-19 patients demonstrated an up-regulation in an ECM remodelling pathway. Our results may suggest the presence of an interaction between ECM remodelling, inflammation, and immune cells, potentially initiating or perpetuating pulmonary pathology in severe COVID-19.

High levels of S100A12 are associated with recent plaque symptomatology in patients with carotid atherosclerosis.

BACKGROUND AND PURPOSE: Atherosclerosis is a progressive chronic disease, in which inflammation plays a key role. The calcium-binding proteins calgranulins including S100A8, S100A9, and S100A12 are involved in many cellular activities and pathological processes including inflammation. We therefore hypothesized that calgranulins may be markers of plaque instability in patients with carotid atherosclerosis. METHODS: Plasma levels of S100A8/A9 and S100A10 were measured in 159 consecutive patients with high-grade carotid stenosis and in 22 healthy control subjects. The mRNA levels of calgranulins were also measured within the atherosclerotic carotid plaques, and their regulation was analyzed in vitro in monocytes. RESULTS: Our main findings were: (1) plasma levels of S100A12 were significantly higher in patients with carotid atherosclerosis compared with healthy control subjects with the highest levels in patients with the most recent symptoms (ie, within 2 months); (2) plasma levels of S100A8/S100A9 showed a modest increase in patients with symptoms in the previous 2 to 6 months but not in the other patients; (3) mRNA levels of S100A8, S100A9, and S100A12 showed increased expression in atherosclerotic carotid plaques from patients with the most recent symptoms compared with the remaining patients; (4) in THP-1 monocytes, activation of Toll-like receptors 2 and 4 increased mRNA levels of S100A8, S100A9, and S10012 and interleukin-1ß, interferon γ, and releasate from thrombin-activated platelets significantly enhanced the expression of S100A12. CONCLUSIONS: Our findings support a link between calgranulins and atherogenesis and suggest that these mediators, and in particular S100A12, may be related to plaque instability.

Fecal level of butyric acid, a microbiome-derived metabolite, is increased in patients with severe carotid atherosclerosis.

The short-chain fatty acid (SCFA) butyric acid maintains a healthy gut barrier and vascular endothelium. We aimed to investigate the association between fecal butyric acid, carotid atherosclerosis and risk factors for ischemic stroke. Patients with severe carotid atherosclerosis (i.e. ≥ 50% stenosis) (n = 43) were compared with healthy controls (n = 38). We analyzed fecal SCFAs by gas chromatography, microbiota composition by 16S rRNA sequencing, markers of gut barrier damage and inflammasome activation by immunoassay, and plasma SCFAs by ultra-high performance liquid chromatography-tandem mass spectroscopy. Patients had higher fecal butyric acid level (p = 0.024), along with increased functional potential of microbial butyric acid production (p = 0.031), compared with controls. Dietary fiber intake was comparable. Patients had higher levels of gut barrier damage markers CCL25 and IFABP, and the inflammasome activation marker IL-18, whereas plasma level of butyric was similar. Increased fecal butyric acid was associated with higher BMI, waist-hip ratio, HbA1c, CRP and leukocyte count. Contrary to our hypothesis, patients with severe carotid atherosclerosis had higher fecal butyric acid level, and increased microbial production, compared with controls. Gut barrier damage in patients might indicate decreased absorption of butyric acid and hence contribute to the higher fecal level.

Interleukin-6 inhibition in ST-elevation myocardial infarction: Immune cell profile in the randomised ASSAIL-MI trial.

BACKGROUND: We recently showed that interleukin (IL)-6 inhibition by tocilizumab improves myocardial salvage in ST-elevation myocardial infarction (STEMI). However, the mechanisms for this effect are not clear. METHODS: In this exploratory sub-study of the ASSAIL-MI trial, we examined leukocyte differential counts and their relation to myocardial salvage and peak troponin T (TnT) in STEMI patients randomised to tocilizumab (n = 101) or placebo (n = 98). We performed RNA-sequencing on whole blood (n = 40) and T cells (n = 20). B and T cell subpopulations were examined by flow cytometry (n = 69). FINDINGS: (i) STEMI patients had higher neutrophil counts at hospitalisation compared with stable angina patients. (ii) After percutaneous coronary intervention there was a gradual decline in neutrophils, which was significantly more pronounced in the tocilizumab group. (iii) The decrease in neutrophils in the tocilizumab group was associated with improved myocardial salvage and lower peak TnT. (iv) RNA-sequencing suggested that neutrophil function was also attenuated by tocilizumab. (v) B and T cell sub-populations changed only minimally after STEMI with minor effects of tocilizumab, supported as well by RNA-sequencing analyses of T cells. (vi) However, a low CD8+ count was associated with improved myocardial salvage in patients admitted to the hospital > 3 h after symptom onset. INTERPRETATION: Tocilizumab induced a rapid reduction in neutrophils and seemed to attenuate neutrophil function in STEMI patients potentially related to the beneficial effects of tocilizumab on myocardial salvage. FUNDING: South-Eastern Norway Regional Health Authority (Nos. 2019067, 2017084), the Central Norway Regional Health Authority and Norwegian Research Council (No. 283867).

DNA glycosylase Neil3 regulates vascular smooth muscle cell biology during atherosclerosis development.

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.

Biomarkers Associated with Atrial Fibrillation in Patients with Ischemic Stroke: A Pilot Study from the NOR-FIB Study.

BACKGROUND AND PURPOSE: Cardioembolic stroke due to paroxysmal atrial fibrillation (AF) may account for 1 out of 4 cryptogenic strokes (CS) and transient ischemic attacks (TIAs). The purpose of this pilot study was to search for biomarkers potentially predicting incident AF in patients with ischemic stroke or TIA. METHODS: Plasma samples were collected from patients aged 18 years and older with ischemic stroke or TIA due to AF (n = 9) and large artery atherosclerosis (LAA) with ipsilateral carotid stenosis (n = 8) and age- and sex-matched controls (n = 10). Analyses were performed with the Olink technology simultaneously measuring 184 biomarkers of cardiovascular disease. For bioinformatics, acquired data were analyzed using gene set enrichment analysis (GSEA). Selected proteins were validated using ELISA. Individual receiver operating characteristic (ROC) curves and odds ratios from logistic regression were calculated. A randomForest (RF) model with out-of-bag estimate was applied for predictive modeling. RESULTS: GSEA indicated enrichment of proteins related to inflammatory response in the AF group. Interleukin (IL)-6, growth differentiation factor (GDF)-15, and pentraxin-related protein PTX3 were the top biomarkers on the ranked list for the AF group compared to the LAA group and the control group. ELISA validated increased expression of all tested proteins (GDF-15, PTX3, and urokinase plasminogen activator surface receptor [U-PAR]), except for IL-6. 19 proteins had the area under the ROC curve (AUC) over 0.85 including all of the proteins with significant evolution in the logistic regression. AUCs were very discriminant in distinguishing patients with and without AF (LAA and control group together). GDF-15 alone reached AUC of 0.95. Based on RF model, all selected participants in the tested group were classified correctly, and the most important protein in the model was GDF-15. CONCLUSIONS: Our results demonstrate an association between inflammation and AF and that multiple proteins alone and in combination may potentially be used as indicators of AF in CS and TIA patients. However, further studies including larger samples sizes are needed to support these findings. In the ongoing NOR-FIB study, we plan further biomarker assessments in patients with CS and TIA undergoing long-term cardiac rhythm monitoring with insertable cardiac monitors.

Chemokines and common variable immunodeficiency; possible contribution of the fractalkine system (CX3CL1/CX3CR1) to chronic inflammation.

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by defective immunoglobulin production and high frequency of bacterial infections, autoimmunity and manifestations of chronic inflammation. The chemokine Fractalkine (CX3CL1) and its receptor CX3CR1 is suggested to play an important role in the pathogenesis of several inflammatory disorders. We hypothesized that enhanced CX3CL1/CX3CR1 interaction could be involved in the chronic inflammation characterising subgroups of CVID. CVID patients were characterized by raised plasma levels of CX3CLl and enhanced expression of its corresponding receptor CX3CR1 on CD4(+) and CD8(+) T cells, including both CD45RA(+) and CD45RA(-) subsets. CX3CR1 expression was particularly enhanced in patients characterized by chronic inflammation in vivo. The high expression of the receptor in CVID patients was accompanied by enhanced chemotactic, adhesive, and other inflammatory cell responses to stimulation with CX3CL1. Our findings suggest that increased CX3CL1/CX3CR1 interaction could contribute to the inflammatory phenotype seen in subgroups of CVID patients.

Atrial fibrillation in cryptogenic stroke and transient ischaemic attack - The Nordic Atrial Fibrillation and Stroke (NOR-FIB) Study: Rationale and design.

PURPOSE: Paroxysmal atrial fibrillation is often suspected as a probable cause of cryptogenic stroke. Continuous long-term ECG monitoring using insertable cardiac monitors is a clinically effective technique to screen for atrial fibrillation and superior to conventional follow-up in cryptogenic stroke. However, more studies are needed to identify factors which can help selecting patients with the highest possibility of detecting atrial fibrillation with prolonged rhythm monitoring. The clinical relevance of short-term atrial fibrillation, the need for medical intervention and the evaluation as to whether intervention results in improved clinical outcomes should be assessed. METHOD: The Nordic Atrial Fibrillation and Stroke Study is an international, multicentre, prospective, observational trial evaluating the occurrence of occult atrial fibrillation in cryptogenic stroke and transient ischaemic attack. Patients with cryptogenic stroke or transient ischaemic attack from the Nordic countries are included and will have the Reveal LINQ® Insertable cardiac monitor system implanted for 12 months for atrial fibrillation detection. Biomarkers which can be used as predictors for atrial fibrillation and may identify patients, who could derive the most clinical benefit from the detection of atrial fibrillation by prolonged monitoring, are being studied. CONCLUSION: The primary endpoint is atrial fibrillation burden within 12 months of continuous rhythm monitoring. Secondary endpoints are atrial fibrillation burden within six months, levels of biomarkers predicting atrial fibrillation, CHA2DS2-VASc score, incidence of recurrent stroke or transient ischaemic attack, use of anticoagulation and antiarrhythmic drugs, and quality of life measurements. The clinical follow-up period is 12 months. The study started in 2017 and the completion is expected at the end of 2020.

The antiatherogenic function of HDL is impaired in hyperhomocysteinemic subjects.

High plasma homocysteine concentrations have been associated with increased risk of cardiovascular disease, whereas plasma HDL concentration is inversely correlated to such disorders. We hypothesized that hyperhomocysteinemic subjects may have dysfunctional HDL. We therefore investigated the ability of serum from hyperhomocysteinemic male and female subjects (n = 10) and control subjects (n = 10) to induce cholesterol efflux and to inhibit release of inflammatory mediators from human umbilical vein endothelial cell. We found that serum from hyperhomocysteinemic subjects had impaired ability to induce cholesterol efflux from lipid-loaded macrophages compared with healthy controls. HDL from those with markedly raised homocysteine concentrations had a reduced antiinflammatory effect in tumor necrosis factor-alpha-activated endothelial cells with an attenuated suppressive effect on interleukin-6 growth-related oncogene-alpha release. Also, the activity of paraoxonase in serum, a multifunctional enzyme with antioxidative effects in relation to the function of HDL, was significantly reduced in hyperhomocysteinemic subjects, in particular those with markedly raised homocysteine concentration. Our findings suggest that hyperhomocysteinemic individuals have dysfunctional HDL particles with attenuated antiatherogenic activity and may represent a novel explanation for the increased risk of cardiovascular events in these individuals.

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.

Increased expression of TFPI in human carotid stenosis.

INTRODUCTION: Tissue factor (TF) pathway inhibitor (TFPI) is the physiological inhibitor of TF induced blood coagulation and two isoforms exists, TFPIα and TFPIß. In atherosclerotic plaques, TFPI may inhibit TF activity and thrombus formation, which is the main cause of ischemic stroke in carotid artery disease. We aimed to identify the isoforms of TFPI present in human carotid plaques and potential sources of TFPI. MATERIALS AND METHODS: Human atherosclerotic plaques from carotid endarterectomies were used for mRNA and immunohistochemistry analyses. hPBMCs isolated from buffy coats and THP-1 cells were differentiated and polarized into M1 or M2 macrophages, and subsequently cultured with or without cholesterol crystals (CC). mRNA and protein expression were measured with qRT-PCR and ELISA, respectively, and procoagulant activity was assessed using a two-stage chromogenic assay. RESULTS: TFPIα and TFPIß mRNA levels were significantly increased in carotid plaques, whereas TF levels were unchanged as compared to healthy arteries. Antibodies against total TFPI showed elevated levels compared to antibodies against free TFPIα, both by immunohistochemical and ELISA detection in plaques. The antibody against total TFPI also co-localized with CD68 and the M1 and M2 markers CD80 and CD163, respectively. The TFPI mRNA expression was elevated and the procoagulant activity was decreased in M2 compared to M1 polarized human macrophages. TFPI was present in early foam cell formation and CC treatment increased the TFPI mRNA expression even further in M2 macrophages. CONCLUSIONS: Our data indicate that both isoforms of TFPI are present in advanced plaques and that anti-inflammatory M2 macrophages may be a potential source of TFPI.

Impaired inhibitory effect of interleukin-10 on the balance between matrix metalloproteinase-9 and its inhibitor in mononuclear cells from hyperhomocysteinemic subjects.

BACKGROUND AND PURPOSE: Homocysteine has been linked to increased risk of ischemic stroke and other cardiovascular events, but the mechanism by which elevated plasma levels of homocysteine promotes atherogenesis remains unclear. Matrix degradation, partly regulated by the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), plays an important role in atherogenesis and plaque destabilization, and we hypothesized an imbalance between MMPs and TIMPs in hyperhomocysteinemia. METHODS: Serum MMP-9 and TIMP-1 was measured in 12 hyperhomocysteinemic and 12 control subjects. The release of MMP-9 and TIMP-1, with and without interleukin-10 (IL-10), and the effect of IL-10 on signal transducer and activator of transcription 3 (STAT3) phosphorylation were measured in peripheral blood mononuclear cells (PBMCs) from hyperhomocysteinemic and control subjects. RESULTS: Our main findings were: (1) hyperhomocysteinemic subjects had raised serum levels of MMP-9 and MMP-9/TIMP-1 ratio comparing healthy controls; (2) although IL-10 markedly suppressed MMP-9 release from PBMCs in controls, no or only minor effect was seen in hyperhomocysteinemic subjects; (3) although IL-10 enhanced TIMP-1 levels in PBMCs from both hyperhomocysteinemic and control subjects, the increase was more prominent in controls, resulting in a marked difference in IL-10-induced changes in MMP-9/TIMP-1 ratio between these 2 groups; and (4) comparing PBMCs from controls, cells from hyperhomocysteinemic individuals had impaired IL-10-induced STAT3 phosphorylation. CONCLUSIONS: Our findings suggest an attenuated inhibitory response to IL-10 on MMP-9 activity in hyperhomocysteinemic subjects, potentially promoting atherogenesis and plaque instability, representing a novel explanation for increased risk for atherosclerotic disease in these individuals.

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.