Hanna E. Agardh

Association of Genetic Risk Variants With Expression of Proximal Genes Identifies Novel Susceptibility Genes for Cardiovascular Disease

Background—Population-based genome-wide association studies have identified several single nucleotide polymorphisms (SNPs) associated with cardiovascular disease or its risk factors. Genes in close proximity to these risk-SNPs are often thought to be pathogenetically important based on their location alone. However, the actual connections between SNPs and disease mechanisms remain largely unknown. Methods and Results—To identify novel susceptibility genes, we investigated how 166 SNPs previously found to be associated with increased cardiovascular risk and/or predisposing metabolic traits relate to the expression of nearby genes. Gene expression in 577 samples of aorta, liver, mammary artery, and carotid atherosclerotic plaque was measured using expression arrays. For 47 SNPs, the expression levels of proximal genes (located within 200 kb) were affected (P<0.005). More than 20 of these genes had not previously been identified as candidate genes for cardiovascular or related metabolic traits. SNP-associated gene effects were tissue-specific and the tissue specificity was phenotype-dependent. Conclusions—This study demonstrates several instances of association between risk-SNPs and genes immediately adjacent to them. It also demonstrates instances in which the associated gene is not the immediately proximal and obvious candidate gene for disease. This shows the necessity of careful studies of genetic marker data as a first step toward application of genome-wide association studies findings in a clinical setting.

Sustained Inflammation Due to Nuclear Factor-Kappa B Activation in Irradiated Human Arteries

OBJECTIVES The aim of this study was to investigate gene expression networks related to cardiovascular disease in radiated human arteries. BACKGROUND Recent epidemiological studies have shown that radiotherapy is associated with cardiovascular disease years after treatment. However, the molecular mechanisms underlying late effects of radiation are poorly described. METHODS Arterial biopsies from radiated and nonradiated human conduit arteries, from the same patient, were simultaneously harvested during microvascular free tissue transfer for cancer-reconstruction in 13 patients, 4 to 500 weeks from radiation treatment. Radiated and nonradiated arteries were compared, with Affymetrix (Santa Clara, California) microarrays on a subset of the material to generate candidate genes. A Taqman (Applied Biosystems, Foster City, California) low-density array of 45 selected genes was designed for analysis of the whole material. RESULTS Thirteen genes were synchronously expressed in all patients (p = 0.0015), including CCL8, CCL3, CXCL2, DUSP5, FGFR2, HMOX1, HOXA9, IL-6, MMP-1, PTX3, RDH10, SOD2, and TNFAIP3. A majority of differentially regulated genes related to the nuclear factor-kappa B (NF-kappaB) signaling pathway and were dysregulated even years after radiation. The NF-kappaB activation was confirmed by immunohistochemistry and immunofluorescence. CONCLUSIONS In the present study, we found sustained inflammation due to NF-kappaB activation in human radiated arteries. The results are supported by previous in vitro findings suggesting that deoxyribonucleic acid injury, after radiation, activates NF-kappaB. We also suggest that HOXA9 might be involved in the regulation of NF-kappaB activation. The observed sustained inflammatory response can explain cardiovascular disease years after radiation.

Expression of fatty acid–binding protein 4/aP2 is correlated with plaque instability in carotid atherosclerosis

Abstract.  Agardh HE, Folkersen L, Ekstrand J, Marcus D, Swedenborg J, Hedin U, Gabrielsen A, Paulsson‐Berne G (Department of Medicine, Experimental Cardiovascular Research, Karolinska Institutet; Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden). Expression of fatty acid–binding protein 4/aP2 is correlated with plaque instability in carotid atherosclerosis. J Intern Med 2011; 269: 200–210.

Correlations between clinical variables and gene-expression profiles in carotid plaque instability.

OBJECTIVE Strokes, a major cause of disability, are often caused by embolism from unstable carotid plaques. The aim of this study was to validate a biobank of human carotid endarterectomies as a platform for further exploration of pathways for plaque instability. For this purpose, we investigated the relationship between clinical parameters of plaque instability and expression of genes previously shown to be associated with either plaque instability or healing processes in the vessel wall. METHODS A database of clinical information and gene-expression microarray data from 106 carotid endarterectomies were used. RESULTS Expression of matrix metalloproteinase (MMP)-9 and MMP-7 was 100-fold higher in plaques than in normal artery. In general, genes associated with inflammation (such as RANKL and CD68) were overexpressed in symptomatic compared with asymptomatic plaques. Plaques obtained from patients undergoing surgery within 2 weeks after an embolic event showed up-regulation of genes involved in healing reactions in the vessel wall (including elastin and collagen). Statin treatment, as well as echodense lesions, were associated with a more stable phenotype. CONCLUSION Here, we demonstrate that gene-expression profiles reflect clinical parameters. Our results suggest that microarray technology and clinical variables can be used for the future identification of central molecular pathways in plaque instability.

Thromboxane synthase expression and thromboxane A2 production in the atherosclerotic lesion.

Thromboxane A2 (TXA2) is a potent prothrombotic and immune modulating lipid mediator, which is implicated in cardiovascular diseases, in particular, atherosclerotic lesion development and thrombogenicity. Here, we tested the hypothesis that thromboxane synthase (TXAS), the obligate enzyme required to synthesize TXA2, is expressed within the human atherosclerotic lesion, thus potentially contributing to TXA2 synthesis and disease development. In an animal study, different atherosclerosis-prone mouse strains were investigated and compared with control mice. In a patient study (n = 134), endarterectomies of carotid atherosclerotic lesions were compared with non-atherosclerotic arteries (n = 11). Expression of TXAS was evaluated by real-time quantitative reverse transcription PCR and immunohistochemistry. TXAS mRNA expression was increased within the vascular wall in mouse models of atherosclerosis with advanced lesions. In humans, TXAS was expressed in the atherosclerotic lesion, associated with increased inflammatory cells, in particular M2 polarized macrophages, and increased in atherosclerotic lesions of patients with recent symptoms of thrombotic events. Production of TXA2 by plaque tissue, verified by gas chromatography–mass spectrometry, increased after addition of arachidonic acid or lipopolysaccharide, and was inhibited by the TXAS inhibitor furegrelate. The findings suggest that intraplaque TXA2 generation may contribute to the development of atherosclerosis and its thrombotic complications in humans.

Inhibition of indoleamine 2,3-dioxygenase promotes vascular inflammation and increases atherosclerosis in Apoe-/- mice.

AIMS Atherosclerosis is a chronic inflammatory disease that is initiated by the retention and accumulation of low-density lipoprotein in the artery, leading to maladaptive response of cells from the immune system and vessel wall. Strong evidence implicates indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of the kynurenine pathway of tryptophan (Trp) degradation, with immune regulation and anti-inflammatory mechanisms in different diseases. However, the role of IDO and the endogenous degradation of Trp have never been directly examined in atherosclerosis development. We used the IDO inhibitor 1-methyl-Trp (1-MT) to determine the role of IDO-mediated Trp metabolism in vascular inflammation and atherosclerosis. METHODS AND RESULTS Apoe(-/-) mice were treated with 1-MT in drinking water for 8 weeks. Systemic IDO inhibition led to a significant increase in atherosclerotic lesions that were ∼58 and 54% larger in the aortic arch and root, respectively. 1-MT treatment enhanced vascular inflammation, up-regulated VCAM-1 and CCL2, and increased CD68 macrophage accumulation into the plaque. Notably, the rise in VCAM-1 expression was not limited to the plaque but also found in smooth muscle cells (SMCs) of the tunica media. Furthermore, we found that IDO-dependent Trp metabolism by SMCs regulates VCAM-1 expression, and that 1-MT-induced acceleration of atherosclerosis and vascular inflammation can be reversed by exogenous administration of the Trp metabolite 3-hydroxyanthranilic acid (3-HAA). CONCLUSION IDO-mediated Trp metabolism regulates vascular inflammation and plaque formation in hypercholesterolaemic Apoe(-/-) mice. Our data establish that this pathway plays a major role in the pathological process of atherogenesis.

Lack of Invariant Natural Killer T Cells Affects Lipid Metabolism in Adipose Tissue of Diet-Induced Obese Mice

Objective—Obesity promotes a chronic inflammatory condition in adipose tissue (AT). Impairment of insulin sensitivity coincides with infiltration of T cells into AT in early stages of obesity, when macrophages are not yet present. Here, we examine the role of invariant natural killer T (iNKT) cells, a subtype of T cells activated by lipid antigens, on glucose and lipid metabolism in obesity. Approach and Results—J&agr;18−/− mice, specifically lacking iNKT cells, and wild-type mice consumed a chow or high-fat diet for 10 weeks. One third of all T lymphocytes in the liver of wild-type mice were iNKT cells, whereas few were detected in AT. Diet-induced obesity increased blood glucose in both genotypes of mice, whereas glucose tolerance test revealed similar kinetics of glucose clearance in J&agr;18−/− and wild-type mice. Under obese conditions, expression of inflammatory cytokines in AT did not differ between the groups, although the number of T cells and macrophages was lower in J&agr;18−/− mice. Nonetheless, AT homeostasis in J&agr;18−/− mice was altered evidenced by lower AT weight, smaller adipocytes, accelerated lipogenesis, increased expression of hormone-sensitive lipase, and accelerated basal lipolysis. Conclusions—iNKT cells do not affect glucose clearance but rather modulate lipid metabolism in both liver and AT. Only few iNKT cells are found in AT under lean and obese conditions, suggesting that their effects on lipid metabolism are mainly mediated in the liver, their primary host organ.

Sterile inflammation in the spleen during atherosclerosis provides oxidation-specific epitopes that induce a protective B-cell response

Significance In this study we investigate the origin of the protective B-cell response in the spleen in atherosclerosis. We find an ongoing B-cell activation with production of antibodies against oxidation-specific epitopes. In addition, this response can be accelerated using apoptotic cells alone that reduce lesion development and serum cholesterol in a B-cell–dependent manner. This study pinpoints the spleen as an important organ for atherosclerosis-associated immunity and provides novel pathways to use for treatment. The B-cell response in atherosclerosis is directed toward oxidation-specific epitopes such as phosphorylcholine (PC) that arise during disease-driven oxidation of self-antigens. PC-bearing antigens have been used to induce atheroprotective antibodies against modified low-density lipoproteins (oxLDL), leading to plaque reduction. Previous studies have found that B-cell transfer from aged atherosclerotic mice confers protection to young mice, but the mechanism is unknown. Here, we dissected the atheroprotective response in the spleen and found an ongoing germinal center reaction, accumulation of antibody-forming cells, and inflammasome activation in apolipoprotein E-deficient mice (Apoe−/−). Specific B-cell clone expansion involved the heavy chain variable region (Vh) 5 and Vh7 B-cell receptor families that harbor anti-PC reactivity. oxLDL also accumulated in the spleen. To investigate whether protection could be induced by self-antigens alone, we injected apoptotic cells that carry the same oxidation-specific epitopes as oxLDL. This treatment reduced serum cholesterol and inhibited the development of atherosclerosis in a B-cell–dependent manner. Thus, we conclude that the spleen harbors a protective B-cell response that is initiated in atherosclerosis through sterile inflammation. These data highlight the importance of the spleen in atherosclerosis-associated immunity.

Endogenous control genes in complex vascular tissue samples

BackgroundGene expression microarrays and real-time PCR are common methods used to measure mRNA levels. Each method has a fundamentally different approach of normalization between samples. Relative quantification of gene expression using real-time PCR is often done using the 2^(-ΔΔCt) method, in which the normalization is performed using one or more endogenous control genes. The choice of endogenous control gene is often arbitrary or bound by tradition. We here present an analysis of the differences in expression results obtained with microarray and real-time PCR, dependent on different choices of endogenous control genes.ResultsIn complex tissue, microarray data and real-time PCR data show the best correlation when endogenous control genes are omitted and the normalization is done relative to total RNA mass, as measured before reverse transcription.ConclusionWe have found that for real-time PCR in heterogeneous tissue samples, it may be a better choice to normalize real-time PCR Ct values to the carefully measured mass of total RNA than to use endogenous control genes. We base this conclusion on the fact that total RNA mass normalization of real-time PCR data shows better correlation to microarray data. Because microarray data use a different normalization approach based on a larger part of the transcriptome, we conclude that omitting endogenous control genes will give measurements more in accordance with actual concentrations.

Fatty acid binding protein 4 in circulating leucocytes reflects atherosclerotic lesion progression in Apoe / mice

Discovery of novel biomarkers for atherosclerosis is important to aid in early diagnosis of pre‐symptomatic patients at high risk of cardiovascular events. The aim of the present study was therefore to identify potential biomarkers in circulating cells reflecting atherosclerotic lesion progression in the vessel wall. We performed gene arrays on circulating leucocytes from atherosclerosis prone Apoe−/− mice with increasing ages, using C57BL/6 mice as healthy controls. We identified fatty acid binding protein 4 (FABP4) mRNA to be augmented in mice with established disease compared with young Apoe−/− or controls. Interestingly, the transcript FABP4 correlated significantly with lesion size, further supporting a disease associated increase. In addition, validation of our finding on protein level showed augmented FABP4 in circulating leucocytes whereas, importantly, no change could be observed in plasma. Immunofluorescence analysis demonstrated FABP4 to be present mainly in circulating neutrophils and to some extent in monocytes. Moreover, FABP4‐positive neutrophils and macrophages could be identified in the subintimal space in the plaque. Using human circulating leucocytes, we confirmed the presence of FABP4 protein in neutrophils and monocytes. In conclusion, we have showed that cellular levels of FABP4 in circulating leucocytes associate with lesion development in the experimental Apoe−/− model. The increased expression is primarily localized to neutrophils, but also in monocytes. We have identified FABP4 in leucocytes as a potential and easy accessible biomarker of atherosclerosis which could be of future clinical relevance.