Ekaterina Chernogubova

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.

miR-24 limits aortic vascular inflammation and murine abdominal aneurysm development

Identification and treatment of abdominal aortic aneurysm (AAA) remain among the most prominent challenges in vascular medicine. MicroRNAs (miRNAs) are crucial regulators of cardiovascular pathology and represent intriguing targets to limit AAA expansion. Here we show, by using two established murine models of AAA disease along with human aortic tissue and plasma analysis, that miR-24 is a key regulator of vascular inflammation and AAA pathology. In vivo and in vitro studies reveal chitinase 3-like 1 (Chi3l1) to be a major target and effector under the control of miR-24, regulating cytokine synthesis in macrophages as well as their survival, promoting aortic smooth muscle cell migration and cytokine production, and stimulating adhesion molecule expression in vascular endothelial cells. We further show that modulation of miR-24 alters AAA progression in animal models, and that miR-24 and CHI3L1 represent novel plasma biomarkers of AAA disease progression in humans.

Local MicroRNA Modulation Using a Novel Anti-miR-21–Eluting Stent Effectively Prevents Experimental In-Stent Restenosis

Objective—Despite advances in stent technology for vascular interventions, in-stent restenosis (ISR) because of myointimal hyperplasia remains a major complication. Approach and Results—We investigated the regulatory role of microRNAs in myointimal hyperplasia/ISR, using a humanized animal model in which balloon-injured human internal mammary arteries with or without stenting were transplanted into Rowett nude rats, followed by microRNA profiling. miR-21 was the only significantly upregulated candidate. In addition, miR-21 expression was increased in human tissue samples from patients with ISR compared with coronary artery disease specimen. We systemically repressed miR-21 via intravenous fluorescein-tagged-locked nucleic acid-anti-miR-21 (anti-21) in our humanized myointimal hyperplasia model. As expected, suppression of vascular miR-21 correlated dose dependently with reduced luminal obliteration. Furthermore, anti-21 did not impede reendothelialization. However, systemic anti-miR-21 had substantial off-target effects, lowering miR-21 expression in liver, heart, lung, and kidney with concomitant increase in serum creatinine levels. We therefore assessed the feasibility of local miR-21 suppression using anti-21–coated stents. Compared with bare-metal stents, anti-21–coated stents effectively reduced ISR, whereas no significant off-target effects could be observed. Conclusion—This study demonstrates the efficacy of an anti-miR–coated stent for the reduction of ISR.

Common and Low-Frequency Genetic Variants in the PCSK9 Locus Influence Circulating PCSK9 Levels

Objective —Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein that influences plasma low-density lipoprotein concentration and susceptibility to coronary heart disease. Circulating PCSK9 levels show considerable interindividual differences, but the factors responsible for this variation are largely unknown. Methods and Results—We analyzed circulating PCSK9 levels in 4 cohorts of healthy, middle-aged Swedes (n=5722) and found that PCSK9 levels varied over ≈50-fold range, showed a positive relationship with plasma low-density lipoprotein–cholesterol concentration, and were associated with plasma triglyceride, fibrinogen, insulin, and glucose concentrations. A genome-wide association study conducted in 2 cohorts (n=1215) failed to uncover common genetic variants robustly associated with variation in circulating PCSK9 level. As expected, the minor allele of the PCSK9 R46L variant was in all cohorts associated with reduced PCSK9 levels and decreased plasma low-density lipoprotein–cholesterol concentrations, but no relationship was observed with the plasma triglyceride concentration. Further mapping of the PCSK9 locus revealed a common polymorphism (rs2479415, minor allele frequency 43.9%), located ≈6 kb upstream from PCSK9, which is independently associated with increased circulating PCSK9 levels. Conclusion—Common and low-frequency genetic variants in the PCSK9 locus influence the pronounced interindividual variation in circulating PCSK9 levels in healthy, middle-aged white (predominantly Swedish) subjects.

Insulin-Induced Gene 2 Involvement in Human Adipocyte Metabolism and Body Weight Regulation

BACKGROUND Insulin-induced genes (INSIGs) encode proteins that block proteolytic activation of sterol regulatory element-binding proteins, transcription factors that regulate lipogenic enzymes, and adipocyte differentiation. OBJECTIVE Here, we analyzed the relative significance of INSIG1 and INSIG2 in human liver and adipocyte metabolism, and defined a novel, functional polymorphism in the promoter of INSIG2 associated with body mass index. RESEARCH METHODS Variations in gene expression of different human tissues, of hepatoma cells exposed to INSIG1 and INSIG2 gene silencing probes, and of differentiating 3T3-L1 adipocytes were determined by real-time quantitative PCR. The functional significance of a novel polymorphism in the promoter of INSIG2 was analyzed using in vitro methods and gene expression analysis of human adipose tissue, whereas the phenotype associated with this polymorphism was studied in two cohorts of middle-aged men. RESULTS Gene expression analysis of 17 human tissues demonstrated that INSIG1 is highly expressed in the liver, whereas INSIG2 is ubiquitously expressed. Gene silencing experiments confirmed that INSIG1, but not INSIG2, regulates the expression of sterol regulatory element-binding proteins target genes in human hepatoma cells. In contrast, adipocyte differentiation of 3T3-L1 cells was associated with a 13-fold increase in expression of INSIG2. Significant relationships between the INSIG2-102G/A polymorphism and body mass index were observed in two cohorts of middle-aged men (ANOVA P = 0.017 and 0.044, respectively). In vitro studies and analysis of allele-specific expression in human adipose tissue substantiated the functional significance of the INSIG2-102G/A polymorphism. CONCLUSION INSIG2 is involved in adipocyte metabolism and body weight regulation.

Variants in the coagulation factor 2 receptor (F2R) gene influence the risk of myocardial infarction in men through an interaction with interleukin 6 serum levels.

Thrombin-activated factor 2 receptor (F2R) links thrombosis to inflammation modulating interleukin (IL)6 synthesis. We have investigated the role of F2R genetic variants and their interaction with IL6 serum levels in the occurrence of myocardial infarction (MI) in the Stockholm Heart Epidemiology Program (SHEEP). Seven SNPs -1738 G/A, -506-/GGCCGCGGGAAGC (D/I), 2860 G/A, 2930 T/C, 9113 C/A, 9333 C/T and 120813 T/C within F2R locus were genotyped in the SHEEP (n=2,774). The C allele at position 2930 was associated with a slight reduction in MI risk in men. IL6 serum levels were higher in male cases carrying genotypes AA at the -1738 (p= 0.01) and GG at the 2860 loci (p= 0.03) and both alleles were found to differentially modulate IL6 serum levels in the context of selective haplotypes. High IL6 serum levels (>75(th) percentile), were independently associated with an increased risk of MI in men with an odds ratio (OR) (95% confidence interval [CI]) of 2.44 (1.72-3.46), (p=0.0016), but not in women ( OR 0.83 [95%CI 0.50-1.36], p=0.64). In the presence of high IL6 serum levels, the -1738A allele increased and the 2860A allele reduced the risk of MI (all p < or = 0.02). Consistently, the AG diplotype increased MI risk (OR 1.71 [95%CI 1.17-2.51], p=0.005). The -1738 and 2860 loci association with IL6 serum levels was replicated in men in the Stockholm Coronary Artery Risk Factor (SCARF) study (both p < or = 0.04). In the pooled data from the two populations, the A and G allele modulated the risk of MI in men with high IL6 serum levels (p < or = 0.03). Our results demonstrate that in men F2R genetic variants influence the risk of MI mainly through an interaction with IL6 serum levels.

Transcription Factor Runx2 Promotes Aortic Fibrosis and Stiffness in Type 2 Diabetes Mellitus

RATIONALE Accelerated arterial stiffening is a major complication of diabetes mellitus with no specific therapy available to date. OBJECTIVE The present study investigates the role of the osteogenic transcription factor runt-related transcription factor 2 (Runx2) as a potential mediator and therapeutic target of aortic fibrosis and aortic stiffening in diabetes mellitus. METHODS AND RESULTS Using a murine model of type 2 diabetes mellitus (db/db mice), we identify progressive structural aortic stiffening that precedes the onset of arterial hypertension. At the same time, Runx2 is aberrantly upregulated in the medial layer of db/db aortae, as well as in thoracic aortic samples from patients with type 2 diabetes mellitus. Vascular smooth muscle cell-specific overexpression of Runx2 in transgenic mice increases expression of its target genes, Col1a1 and Col1a2, leading to medial fibrosis and aortic stiffening. Interestingly, increased Runx2 expression per se is not sufficient to induce aortic calcification. Using in vivo and in vitro approaches, we further demonstrate that expression of Runx2 in diabetes mellitus is regulated via a redox-sensitive pathway that involves a direct interaction of NF-κB with the Runx2 promoter. CONCLUSIONS In conclusion, this study highlights Runx2 as a previously unrecognized inducer of vascular fibrosis in the setting of diabetes mellitus, promoting arterial stiffness irrespective of calcification.

Human evidence for the involvement of insulin-induced gene 1 in the regulation of plasma glucose concentration.

Aims/hypothesisInsulin-induced gene 1 (INSIG1) is a protein that blocks proteolytic activation of sterol regulatory element-binding proteins (SREBPs), transcription factors that activate genes regulating cholesterol and fatty acid metabolism and possibly genes involved in glucose homeostasis. In search of genetic regulation of these processes we examined human INSIG1 for common polymorphisms and analysed their associations with biochemical parameters related to lipid and glucose metabolism.MethodsAssociations between common polymorphisms in INSIG1 and several biochemical parameters were analysed in a group of 618 healthy, 50-year-old men. A replication analysis was performed in a cohort of 472 healthy, middle-aged men. The impact of one promoter polymorphism on oral glucose tolerance was analysed in a subset of 181 subjects. Small interfering RNA (siRNA) inhibition was used to test the significance of INSIG1 for gene expression in human Huh7 hepatoma cells.ResultsA potentially functional polymorphism, a C to T substitution at position −169, was discovered in a highly conserved section of the promoter. Significant relationships between the −169C>T polymorphism and plasma glucose concentration were found in two cohorts of healthy, middle-aged men (p < 0.01 and p < 0.02, respectively). The −169T allele was associated with significantly lower post-load plasma glucose concentrations. A significant (p = 0.02) reduction in expression of phosphoenolpyruvate carboxykinase (PCK2) was observed following siRNA inhibition of INSIG1 in human Huh7 hepatoma cells.Conclusions/interpretationPopulation studies demonstrate that INSIG1 plays a role in glucose homeostasis. Experiments with siRNA suggest that this action of INSIG1 is related to SREBP-mediated regulation of PCK2.

MicroRNA-210 Enhances Fibrous Cap Stability in Advanced Atherosclerotic Lesions

Rationale: In the search for markers and modulators of vascular disease, microRNAs (miRNAs) have emerged as potent therapeutic targets. Objective: To investigate miRNAs of clinical interest in patients with unstable carotid stenosis at risk of stroke. Methods and Results: Using patient material from the BiKE (Biobank of Karolinska Endarterectomies), we profiled miRNA expression in patients with stable versus unstable carotid plaque. A polymerase chain reaction–based miRNA array of plasma, sampled at the carotid lesion site, identified 8 deregulated miRNAs (miR-15b, miR-29c, miR-30c/d, miR-150, miR-191, miR-210, and miR-500). miR-210 was the most significantly downregulated miRNA in local plasma material. Laser capture microdissection and in situ hybridization revealed a distinct localization of miR-210 in fibrous caps. We confirmed that miR-210 directly targets the tumor suppressor gene APC (adenomatous polyposis coli), thereby affecting Wnt (Wingless-related integration site) signaling and regulating smooth muscle cell survival, as well as differentiation in advanced atherosclerotic lesions. Substantial changes in arterial miR-210 were detectable in 2 rodent models of vascular remodeling and plaque rupture. Modulating miR-210 in vitro and in vivo improved fibrous cap stability with implications for vascular disease. Conclusions: An unstable carotid plaque at risk of stroke is characterized by low expression of miR-210. miR-210 contributes to stabilizing carotid plaques through inhibition of APC, ensuring smooth muscle cell survival. We present local delivery of miR-210 as a therapeutic approach for prevention of atherothrombotic vascular events.

Making Sense in Antisense: Therapeutic Potential of Noncoding RNAs in Diabetes-Induced Vascular Dysfunction

The rapid rise of type II diabetes mellitus and its accompanying vascular complications call for novel approaches in unravelling its pathophysiological mechanisms and designing new treatment modalities. Noncoding RNAs represent a class of previously unknown molecular modulators of this disease. The most important features of diabetes-induced vascular disease, which include metabolic deregulation, increased oxidative stress, release of inflammatory mediators like adipokines, and pathologic changes in vascular cells, all are depicted and governed by a certain set of noncoding RNAs. While these mechanisms are being unravelled, new diagnostic and therapeutic opportunities to treat diabetes-induced vascular disease emerge.