Anders Gabrielsen

Expression of Neutrophil Gelatinase–Associated Lipocalin in Atherosclerosis and Myocardial Infarction

Objective—Neutrophil gelatinase-associated lipocalin (NGAL) modulates the activity of matrix metalloproteinase (MMP) 9, an important mediator of vascular remodeling and plaque instability in atherosclerosis. This study aimed to analyze the expression of NGAL in atherosclerotic plaques and myocardial infarction (MI). Methods and Results—Atherosclerotic apolipoprotein E (apoE)−/− × low-density lipoprotein receptor (LDLR)−/− and C57BL/6J control mice were exposed to brief hypoxic stress (10 minutes of 10% oxygen). Expression of the mouse NGAL homolog (24p3) and MMP-9 was analyzed 48 hours later by quantitative RT-PCR, immunohistochemistry, and zymography. Hypoxic stress increased NGAL/24p3 mRNA in the cardiac vasculature. NGAL/24p3 was also increased in atherosclerotic plaques of apolipoprotein E−/− × LDLR−/− mice compared with C57BL/6J mice. Mice developing MI exhibited the highest plaque mRNA expression of NGAL/24p3 and MMP-9. Zymography revealed strong proteolytic activity in areas rich in 24p3 and MMP-9 protein. Immunohistochemistry performed on human carotid endarterectomy specimens and control tissue from the internal mammary artery showed colocalization of MMP-9 and NGAL with macrophages in the atherosclerotic plaques. Conclusions—NGAL/24p3 is increased in atherosclerotic plaques and MI. Colocalization with MMP-9 in areas with high-proteolytic activity suggests a role for NGAL/24p3 in modulating the MMP-9-mediated remodeling of plaques and infarcted hearts.

Relationship between CAD Risk Genotype in the Chromosome 9p21 Locus and Gene Expression. Identification of Eight New ANRIL Splice Variants

Background Several genome-wide association studies have recently linked a group of single nucleotide polymorphisms in the 9p21 region with cardiovascular disease. The molecular mechanisms of this link are not fully understood. We investigated five different expression microarray datasets in order to determine if the genotype had effect on expression of any gene transcript in aorta, mammary artery, carotid plaque and lymphoblastoid cells. Methodology/Principal Findings After multiple testing correction, no genes were found to have relation to the rs2891168 risk genotype, either on a genome-wide scale or on a regional (8 MB) scale. The neighbouring ANRIL gene was found to have eight novel transcript variants not previously known from literature and these varied by tissue type. We therefore performed a detailed probe-level analysis and found small stretches of significant relation to genotype but no consistent associations. In all investigated tissues we found an inverse correlation between ANRIL and the MTAP gene and a positive correlation between ANRIL and CDKN2A and CDKN2B. Conclusions/Significance Investigation of relation of the risk genotype to gene expression is complicated by the transcript complexity of the locus. With our investigation of a range of relevant tissue we wish to underscore the need for careful attention to the complexity of the alternative splicing issues in the region and its implications to the design of future gene expression studies.

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.

Dickkopf-1 Enhances Inflammatory Interaction Between Platelets and Endothelial Cells and Shows Increased Expression in Atherosclerosis

Objective—Based on the emerging importance of the wingless (Wnt) pathways in inflammation and vascular biology, we hypothesized a role for Dickkopf-1 (DKK-1), a major modulator of Wnt signaling, in atherogenesis and plaque destabilization. Methods and Results—We report increased levels of DKK-1 in experimental (ApoE−/− mice) and clinical (patients with coronary artery disease [n=80] and patients with carotid plaque [n=47]) atherosclerosis, both systemically (serum) and within the lesion, with particularly high levels in advanced and unstable disease. We identified platelets as an important cellular source of DKK-1 as shown by in vitro experiments and by immunostaining of thrombus material obtained at the site of plaque rupture in patients with acute ST-elevation myocardial infarction, with strong immunoreactivity in platelet aggregates. Our in vitro experiments identified a role for platelet- and endothelial-derived DKK-1 in platelet-dependent endothelial activation, promoting enhanced release of inflammatory cytokines. These inflammatory effects of DKK-1 involved inhibition of the Wnt/&bgr;-catenin pathway and activation of nuclear factor &kgr;B. Conclusion—Our findings identify DKK-1 as a novel mediator in platelet-mediated endothelial cell activation. The demonstration of enhanced DKK-1 expression within advanced carotid plaques may suggest that this DKK-1-driven inflammatory loop could be operating within the atherosclerotic lesion.

YKL-40 a new biomarker in patients with acute coronary syndrome or stable coronary artery disease.

Background. YKL-40 is involved in remodelling and angiogenesis in non-cardiac inflammatory diseases. Aim was to quantitate plasma YKL-40 in patients with ST-elevation myocardial infarction (STEMI) or stable chronic coronary artery disease (CAD), and YKL-40 gene activation in human myocardium. Methods and results. We included 73 patients: I) 20 patients with STEMI; II) 28 patients with stable CAD; III) 15 CAD patients referred for coronary by-pass surgery. YKL-40 mRNA expression was measured in myocardium subtended by stenotic or occluded arteries and areas with no apparent disease; and IV) 10 age-matched healthy controls. Plasma YKL-40 was significantly increased in patients with STEMI (88 µg/l, median) and CAD (66 µg/l) compared to controls (16 µg/l, p<0.01 for both). Plasma YKL-40 correlated with CRP at baseline in STEMI (r=0.53, p=0.02) and CAD patients (r=0.41, p=0.031).YKL-40 gene expression was similar in ischemic and non-ischemic myocardium. Conclusions. Plasma YKL-40 was significantly increased in patients with STEMI and stable CAD. Further studies will define the role of YKL-40 as a clinically useful marker for myocardial ischemia, remodelling and maybe prognosis.

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.

Vasorelaxation in space

During everyday life, gravity constantly stresses the cardiovascular system in upright humans by diminishing venous return. This decreases cardiac output and induces systemic vasoconstriction to prevent blood pressure from falling. We therefore tested the hypothesis that entering weightlessness leads to a prompt increase in cardiac output and to systemic vasodilatation and that these effects persist for at least a week of weightlessness in space. Cardiac output and mean arterial pressure were measured in 8 healthy humans during acute 20-s periods of weightlessness in parabolic airplane flights and on the seventh and eighth day of weightlessness in 4 astronauts in space. The seated 1-G position acted as reference. Entering weightlessness promptly increased cardiac output by 29±7%, from 6.6±0.7 to 8.4±0.9 L min−1 (mean±SEM; P=0.003), whereas mean arterial pressure and heart rate were unaffected. Thus, systemic vascular resistance decreased by 24±4% (P=0.017). After a week of weightlessness in space, cardiac output was increased by 22±8% from 5.1±0.3 to 6.1±0.1 L min−1 (P=0.021), with mean arterial pressure and heart rate being unchanged so that systemic vascular resistance was decreased by 14±9% (P=0.047). In conclusion, entering weightlessness promptly increases cardiac output and dilates the systemic circulation. This vasorelaxation persists for at least a week into spaceflight. Thus, it is probably healthy for the human cardiovascular system to fly in space.

Unraveling divergent gene expression profiles in bicuspid and tricuspid aortic valve patients with thoracic aortic dilatation: the ASAP study.

Thoracic aortic aneurysm (TAA) is a common complication in patients with a bicuspid aortic valve (BAV), the most frequent congenital heart disorder. For unknown reasons TAA occurs at a younger age, with a higher frequency in BAV patients than in patients with a tricuspid aortic valve (TAV), resulting in an increased risk for aortic dissection and rupture. To investigate the increased TAA incidence in BAV patients, we obtained tissue biopsy samples from nondilated and dilated aortas of 131 BAV and TAV patients. Global gene expression profiles were analyzed from controls and from aortic intima-media and adventitia of patients (in total 345 samples). Of the genes found to be differentially expressed with dilation, only a few (<4%) were differentially expressed in both BAV and TAV patients. With the use of gene set enrichment analysis, the cell adhesion and extracellular region gene ontology sets were identified as common features of TAA in both BAV and TAV patients. Immune response genes were observed to be particularly overexpressed in the aortic media of dilated TAV samples. The divergent gene expression profiles indicate that there are fundamental differences in TAA etiology in BAV and TAV patients. Immune response activation solely in the aortic media of TAV patients suggests that inflammation is involved in TAA formation in TAV but not in BAV patients. Conversely, genes were identified that were only differentially expressed with dilation in BAV patients. The result has bearing on future clinical studies in which separate analysis of BAV and TAV patients is recommended.

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.

12- and 15-lipoxygenases in human carotid atherosclerotic lesions: Associations with cerebrovascular symptoms

Lipoxygenase (ALOX) enzymes are implicated in both pro- and anti-atherogenic processes. The aim of this study was to investigate mRNA expression of 12- and 15-lipoxygenases (ALOX12, ALOX12B, ALOX15, ALOX15B) and the atypical ALOXE3 in human carotid atherosclerotic lesions, in relation to cerebrovascular symptoms and risk factors. The Biobank of Karolinska Endarterectomies (BiKE) collection of human carotid plaque tissue and associated clinical data was utilized (n=132). Lesion mRNA levels were analyzed by TaqMan qPCR (n=132) and microarray hybridization (n=77). Of the investigated mRNAs, only ALOX15B (15-LOX-2; epidermis-type 15-LOX) was readily detected in all plaque samples by qPCR, and thus suitable for quantitative statistical evaluation. ALOX12, ALOX12B, ALOX15 and ALOXE3 were detected with lower frequency and at lower levels, or virtually undetected. Microarray analysis confirmed ALOX15B as the most abundant 12- or 15-lipoxygenase mRNA in carotid lesions. Comparing plaques with or without attributable cerebrovascular symptoms (amaurosis fugax, transient ischemic attack, or stroke), ALOX15B mRNA levels were higher in symptomatic than asymptomatic plaques (1.31 [1.11-1.56], n=102; and 0.79 [0.55-1.15], n=30, respectively; p=0.008; mean [95% CI], arbitrary units). Multiple regression analysis confirmed symptomatic/asymptomatic status as a significant determinant of ALOX15B mRNA levels, independently of potentially confounding factors. Immunohistochemical analyses showed abundant ALOX15B expression in macrophage-rich areas of carotid lesions, and lipidomic analyses demonstrated the presence of typical ALOX15B products in plaque tissue. In summary, we observed associations between high ALOX15B expression in carotid lesions and a history of cerebrovascular symptoms. These findings suggest a link between ALOX15B and atherothrombotic events that merits further investigation.