Josefin Skogsberg

Evidence That Peroxisome Proliferator–Activated Receptor Delta Influences Cholesterol Metabolism in Men

Objective—The objective of this work was to explore the role of peroxisome proliferator–activated receptor delta (PPARD) in lipid metabolism in humans. Methods and Results—PPARD is a nuclear receptor involved in lipid metabolism in primates and mice. We screened the 5′-region of the human gene for polymorphisms to be used as tools in association studies. Four polymorphisms were detected: −409C/T in the promoter region, +73C/T in exon 1, +255A/G in exon 3, and +294T/C in exon 4. The frequencies of the rare alleles were 4.2%, 4.2%, 1.2% and 15.6%, respectively, in a population-based group of 543 healthy men. Only the +294T/C polymorphism showed significant association with a metabolic trait. Homozygotes for the rare C allele had a higher plasma LDL–cholesterol concentration than homozygotes for the common T allele, which was verified in an independent cohort consisting of 282 healthy men. Transfection studies showed that the rare C allele had higher transcriptional activity than the common T allele. Electrophoretic mobility shift assays demonstrated that the +294T/C polymorphism influenced binding of Sp-1. An interaction with the PPAR alpha L162V polymorphism was also detected for several lipid parameters. Conclusions—These findings suggest that PPARD plays a role in cholesterol metabolism in humans.

Multi-organ expression profiling uncovers a gene module in coronary artery disease involving transendothelial migration of leukocytes and LIM domain binding 2: the Stockholm Atherosclerosis Gene Expression (STAGE) study.

Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD). The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE) study was to determine whether there are functionally associated genes (rather than individual genes) important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n = 66/tissue) and atherosclerotic and unaffected arterial wall (n = 40/tissue) isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n = 15,042/12,621 RefSeqs/genes) in each tissue, resulted in a total of 60 tissue clusters (n = 3958 genes). In the second step (performed within tissue clusters), one atherosclerotic lesion (n = 49/48) and one visceral fat (n = 59) cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P = 0.008 and P = 0.00015). The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n = 55/54) relating to carotid stenosis (P = 0.04), 27 genes in the two clusters relating to coronary stenosis were confirmed (n = 16/17, P<10−27and−30). Genes in the transendothelial migration of leukocytes (TEML) pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module). In a second validation step, using three independent cohorts, the A-module was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004). The transcription co-factor LIM domain binding 2 (LDB2) was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2, and by the expression of 13 TEML genes in Ldb2–deficient arterial wall. Thus, the A-module appears to be important for atherosclerosis development and, together with LDB2, merits further attention in CAD research.

Peroxisome proliferator activated receptor delta genotype in relation to cardiovascular risk factors and risk of coronary heart disease in hypercholesterolaemic men

Objectives.  Peroxisome proliferator activated receptor delta (PPARD) is a transcription factor implicated in the regulation of genes involved in cholesterol metabolism. We recently discovered a common polymorphism in the 5′‐untranslated region (5′‐UTR) of the human PPARD, +294T/C, that is associated with an increased plasma low‐density lipoprotein cholesterol (LDL‐C) concentration in healthy subjects. Whether the +294C allele is associated with LDL‐C elevation independently of the background lipoprotein phenotype and whether it confers increased risk of coronary heart disease (CHD) is unknown. Against this background, we investigated the relationships between the PPARD polymorphism and plasma lipoprotein concentrations and the risk for contracting CHD in the West of Scotland Coronary Prevention Study (WOSCOPS).

Cardiometabolic risk loci share downstream cis- and trans-gene regulation across tissues and diseases

Genetic variation and coronary artery disease Most genetic variants lie outside protein-coding genes, but their effects, especially in human health, are not well understood. Franzén et al. examined gene expression in tissues affected by coronary artery disease (CAD). They found that individuals with loci that have been associated with CAD in genome-wide analyses had different patterns of tissue-specific gene expression than individuals without these genetic variants. Similarly, tissues not associated with CAD did not have CAD-like expression patterns. Thus, tissue-specific data can be used to dissect the genetic effects that predispose individuals to CAD. Science, this issue p. 827 A gene expression survey in patients with coronary artery disease reveals how genetic variation affects the risk of heart failure. Genome-wide association studies (GWAS) have identified hundreds of cardiometabolic disease (CMD) risk loci. However, they contribute little to genetic variance, and most downstream gene-regulatory mechanisms are unknown. We genotyped and RNA-sequenced vascular and metabolic tissues from 600 coronary artery disease patients in the Stockholm-Tartu Atherosclerosis Reverse Networks Engineering Task study (STARNET). Gene expression traits associated with CMD risk single-nucleotide polymorphism (SNPs) identified by GWAS were more extensively found in STARNET than in tissue- and disease-unspecific gene-tissue expression studies, indicating sharing of downstream cis-/trans-gene regulation across tissues and CMDs. In contrast, the regulatory effects of other GWAS risk SNPs were tissue-specific; abdominal fat emerged as an important gene-regulatory site for blood lipids, such as for the low-density lipoprotein cholesterol and coronary artery disease risk gene PCSK9. STARNET provides insights into gene-regulatory mechanisms for CMD risk loci, facilitating their translation into opportunities for diagnosis, therapy, and prevention.

Transcriptional profiling uncovers a network of cholesterol-responsive atherosclerosis target genes.

Despite the well-documented effects of plasma lipid lowering regimes halting atherosclerosis lesion development and reducing morbidity and mortality of coronary artery disease and stroke, the transcriptional response in the atherosclerotic lesion mediating these beneficial effects has not yet been carefully investigated. We performed transcriptional profiling at 10-week intervals in atherosclerosis-prone mice with human-like hypercholesterolemia and a genetic switch to lower plasma lipoproteins (Ldlr −/− Apo 100/100 Mttp flox/flox Mx1-Cre). Atherosclerotic lesions progressed slowly at first, then expanded rapidly, and plateaued after advanced lesions formed. Analysis of lesion expression profiles indicated that accumulation of lipid-poor macrophages reached a point that led to the rapid expansion phase with accelerated foam-cell formation and inflammation, an interpretation supported by lesion histology. Genetic lowering of plasma cholesterol (e.g., lipoproteins) at this point all together prevented the formation of advanced plaques and parallel transcriptional profiling of the atherosclerotic arterial wall identified 37 cholesterol-responsive genes mediating this effect. Validation by siRNA-inhibition in macrophages incubated with acetylated-LDL revealed a network of eight cholesterol-responsive atherosclerosis genes regulating cholesterol-ester accumulation. Taken together, we have identified a network of atherosclerosis genes that in response to plasma cholesterol-lowering prevents the formation of advanced plaques. This network should be of interest for the development of novel atherosclerosis therapies.

The Q/E27 polymorphism in the β2-adrenoceptor gene is associated with increased body weight and dyslipoproteinaemia involving triglyceride-rich lipoproteins

Abstract. Ehrenborg E, Skogsberg J, Ruotolo G, Large V, Eriksson P, Arner P, Hamsten A (Karolinska Hospital, Stockholm, and Huddinge University Hospital, Huddinge, Sweden; and Istuto Scientifico H San Raffaele, Milano, Italy). The Q/E27 polymorphism in the β2‐adrenoceptor gene is associated with increased body weight and dyslipoproteinaemia involving triglyceride‐rich lipoproteins. J Intern Med 2000; 247: 651–656.

Improved treatment satisfaction but no difference in metabolic control when using continuous subcutaneous insulin infusion vs. multiple daily injections in children at onset of type 1 diabetes mellitus

Objective:  The aim of this study was to compare safety, metabolic control, and treatment satisfaction in children/adolescents at onset of type 1 diabetes mellitus who were treated with either continuous subcutaneous insulin infusion (CSII) or multiple daily injections (MDI).

MMP-2 and MMP-9 are prominent matrix metalloproteinases during atherosclerosis development in the Ldlr-/-Apob100/100 mouse

Matrix-degrading proteases capable of degrading components of the extracellular matrix may play an important role in development and progression of atherosclerotic lesions. In the present study, we used the Ldlr(-/-)Apob(100/100) mouse model, which has a plasma lipoprotein profile similar to that of humans with atherosclerosis, to study the expression of matrix metalloproteinases (MMPs) during early stages of atherosclerosis development. We analyzed the expression of 11 proteases and three protease inhibitors in 5- to 40-week-old Ldlr(-/-)Apob(100/100) mice. Expression and activity of MMP-2 and MMP-9 was increased in advanced atherosclerotic lesions followed by macrophage infiltration as shown by real-time PCR, gel-based and in situ zymography and immunohistochemistry. Expression of other investigated MMPs did not increase during disease progression. However, the mRNA expression of MMP-8 and MMP-13 was down-regulated, which could explain the relatively high amount of collagen observed in the vessels in this model. In conclusion, low proteolytic expression at early stages of atherogenesis and a limited repertoire of proteolytic enzymes were associated with the progression of atherosclerosis in Ldlr(-/-)Apob(100/100) mice. The study suggests that MMP-2 and MMP-9 are the main proteases involved in atherogenesis in this mouse model.

Cross-Tissue Regulatory Gene Networks in Coronary Artery Disease

SUMMARY Inferring molecular networks can reveal how genetic perturbations interact with environmental factors to cause common complex diseases. We analyzed genetic and gene expression data from seven tissues relevant to coronary artery disease (CAD) and identified regulatory gene networks (RGNs) and their key drivers. By integrating data from genome-wide association studies, we identified 30 CAD-causal RGNs interconnected in vascular and metabolic tissues, and we validated them with corresponding data from the Hybrid Mouse Diversity Panel. As proof of concept, by targeting the key drivers AIP, DRAP1, POLR2I, and PQBP1 in a cross-species-validated, arterial-wall RGN involving RNA-processing genes, we re-identified this RGN in THP-1 foam cells and independent data from CAD macrophages and carotid lesions. This characterization of the molecular landscape in CAD will help better define the regulation of CAD candidate genes identified by genome-wide association studies and is a first step toward achieving the goals of precision medicine.

Plasma cholesterol-induced lesion networks activated before regression of early, mature, and advanced atherosclerosis.

Plasma cholesterol lowering (PCL) slows and sometimes prevents progression of atherosclerosis and may even lead to regression. Little is known about how molecular processes in the atherosclerotic arterial wall respond to PCL and modify responses to atherosclerosis regression. We studied atherosclerosis regression and global gene expression responses to PCL (≥80%) and to atherosclerosis regression itself in early, mature, and advanced lesions. In atherosclerotic aortic wall from Ldlr−/−Apob 100/100 Mttp flox/floxMx1-Cre mice, atherosclerosis regressed after PCL regardless of lesion stage. However, near-complete regression was observed only in mice with early lesions; mice with mature and advanced lesions were left with regression-resistant, relatively unstable plaque remnants. Atherosclerosis genes responding to PCL before regression, unlike those responding to the regression itself, were enriched in inherited risk for coronary artery disease and myocardial infarction, indicating causality. Inference of transcription factor (TF) regulatory networks of these PCL-responsive gene sets revealed largely different networks in early, mature, and advanced lesions. In early lesions, PPARG was identified as a specific master regulator of the PCL-responsive atherosclerosis TF-regulatory network, whereas in mature and advanced lesions, the specific master regulators were MLL5 and SRSF10/XRN2, respectively. In a THP-1 foam cell model of atherosclerosis regression, siRNA targeting of these master regulators activated the time-point-specific TF-regulatory networks and altered the accumulation of cholesterol esters. We conclude that PCL leads to complete atherosclerosis regression only in mice with early lesions. Identified master regulators and related PCL-responsive TF-regulatory networks will be interesting targets to enhance PCL-mediated regression of mature and advanced atherosclerotic lesions.