Gene Expression Profiling of Human Atherosclerosis

Atherosclerosis is a progressive inflammatory disease that causes lipid accumulation in the arterial wall, leading to the formation of plaques. The clinical manifestations of plaque rupture—stroke and myocardial infarction—are increasing worldwide and pose an enormous economic burden for society. Atherosclerosis development reflects a complex interaction between environmental exposures and genetic predisposition. To understand this complexity, we hypothesized that a top-down approach—one in which all molecular activities that drive atherosclerosis are examined simultaneously—is necessary to highlight those that are clinically relevant. To this end, we performed whole-genome expression profiling in multiple tissues isolated from patients with coronary artery disease (CAD). In the Stockholm Atherosclerosis Gene Expression (STAGE) study, biopsies of five tissues (arterial wall with and without atherosclerotic lesions, liver, skeletal muscle and visceral fat) were isolated from 124 CAD patients undergoing coronary artery bypass grafting surgery (CABG) at the Karolinska University Hospital, Solna and carotid lesions from 39 patients undergoing carotid artery surgery at Stockholm Söder Hospital. Detailed clinical characteristics of these patients were assembled together with a total of 303 global gene expression profiles obtained with the Affymetrix GeneChip platform. In paper 1, a two-way clustering analysis of the data identified 60 tissue clusters of functionally related genes. One cluster, partly present in both visceral fat and atherosclerotic lesions, related to atherosclerosis severity as judged by coronary angiograms. Many of the genes in that cluster were also present in a carotid lesion cluster relating to intima-media thickness (IMT) in the carotid patients. The union of all three clusters relating to extent of atherosclerosis—referred to as the “A-module”—was overrepresented with genes belonging to the transendothelial migration of leukocyte (TEML) pathway. The transcription co-factor, Lim domain binding 2 (LDB2), was identified as putative regulator of the A-module and TEML pathway in validation studies including Ldb2 mice. In paper 2, we investigated the increased incidence of postoperative complications in CABG patients with diabetes. Using the STAGE compendium, we identified an anti-inflammatory marker, dual-specificity phosphatase 1 (DUSP1), as a novel preoperative blood marker of risk for a prolonged hospital stay after CABG. In paper 3, plaque age was determined with C-dating in the carotid patients. Interestingly, the strongest correlation with plaque age was not the age of the patients or IMT. Rather, the strongest correlations were with plasma insulin levels and inflammatory gene expression. Taken together, the findings in this thesis show that a top-down approach using multi-tissue gene expression profiling in CAD and C-dating of plaques can contribute to a better understanding of the molecular processes underlying atherosclerosis development and to the identification of clinically useful biomarkers. POPULÄRVETENSKAPLIG SAMMANFATTNING Allt fler människor i världen utvecklar idag hjärt-kärlsjukdom och drabbas av dess följder som hjärtinfarkt och stroke. Den bakomliggande orsaken är främst förträngningar i kärlen som ger upphov till minskad blodtillförsel i organ och vävnader. Förträngningarna uppstår när kolesteroler i blodet tar sig in i kärlväggen och lagras i fettdepåer. Denna process involverar även immunförsvaret som genom sitt angrepp mot kolesterolet startar en kronisk inflammation i kärlväggen. Genom en förbättrad livsstil, dvs. genom att äta hälsosam mat, öka sin fysiska aktivitet och genom att inte röka kan man minska det sjukliga förloppet av fettinlagringar i kärlen. Tyvärr finns det också ärftliga anlag som påverkar benägenheten att få förträngningar och genom att studera dessa kan vi bättre förstå nya mekanismer bakom sjukdomen. Vi har studerat arvsmassan, dvs. generna, hos kranskärlssjuka patienter för att hitta nya gener som är viktiga för sjukdomsförloppet. Vi har mätt alla geners uttryck och kopplat detta till graden av förträngning hos varje patient. På så sätt har vi lyckats identifiera en grupp av gener som är kopplade till hur celler i vårt immunförsvar tar sig in i kärlväggen och reagerar på fettinlagringarna. Vi har också hittat en gen vars proteinnivåer i blod tyder på en inflammation hos vissa patienter. Denna inflammation förefaller leda till ökad risk för komplikationer och ökat behov av vård efter genomgången kranskärlsoperation. Slutligen så har vi, med hjälp av Cdateringsmetoden, för första gången lyckats mäta hur många år sedan det var som fettinlagringen startade hos en grupp patienter. På så vis har vi kunnat konstatera att en snabbare utveckling av kärlförträngningar hos vissa individer är kopplad till en högre grad av inflammation i kärlet. Genom att dela med oss av dessa nya kunskaper hoppas vi kunna öka förståelsen för sjukdomsmekanismen bakom hjärt-kärlsjukdomar. En förståelse som i förlängningen kan leda till förbättrade behandlingar och nya mediciner. PREFACE Many people have contributed to the studies in this thesis, which is collaboration between Linköping University, Karolinska Institutet (KI) and Clinical Gene Networks AB. The main co-workers, all with different skills, are presented here. Biopsies were assembled by surgeons at the Karolinska University Hospital Solna (Torbjörn Ivert, Anders Franco-Cereceda, Jan Liska, Ulf Lockowandt) and at the Stockholm Söder Hospital (Peter Konrad, Rabbe Takolander). Blood sampling of the Tartu cohort in Estonia was done by another surgeon (Arno Ruusalepp). Patient characteristics was collected by a research nurse at the Thoracic research clinic at the Karolinska University Hospital (Merja Heinonen). I gathered additional clinical characterizations and performed angiographic measurements of patients. Intimamedia thickness measurements were done by Stefan Rosfors at the Stockholm Söder Hospital. Plaque age estimation by C-dating was done by Mehran Salehpour at Uppsala University. RNA isolation was performed in our own laboratory at KI mainly by Peri Noori (PN) and Josefin Skogsberg (JS). Assessment of RNA quality and quantity was performed by me, PN and JS. The expression profiling was also done in our laboratory by PN, JS, myself and Roland Nilsson (RN). Jesper Lundström (JL), RN, myself and Björn Brinne did data pre-processing. As for the analyses, JL and myself worked in collaboration where JL did the two-way clustering and network analysis. I did most of the other calculations such as the gene function annotation, genetic enrichment (together with Judy Zhong from the Schadt group), Spearman rank correlation and statistical analysis. The experimental validation was carried out by JS, PN, Shohreh Maleki and Ming-Mei Shang. I wrote the papers together with Johan Björkegren (JB) and JS. JB designed and had the overall responsibility for the clinical studies, JS for the validation experiments and Jesper Tegnér (JT) for data analysis. JB and JT have joint overall responsibility for the laboratory. PUBLICATIONS INCLUDED IN THE THESIS I. Hägg S*, Skogsberg J*, Lundström J*, Noori P, Nilsson R, Zhong H, Maleki S, Shang M-M, Brinne B, Bradshaw M, Bajic V, Samnegård A, Silveira A, Kaplan LM, Gigante B, Leander K, de Faire U, Rosfors S, Lockowandt U, Liska J, Konrad P, Takolander R, Franco-Cereceda A, Schadt EE, Ivert T, Hamsten A, Tegnér J, Björkegren J. 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. PLoS Genetics. In press. * Shared 1 authors. II. Hägg S, Alserius T, Noori P, Skogsberg J, Ruusalepp A, Ivert T, Tegnér J, Björkegren J. Dual-Specificity Phosphatase-1—An Anti-Inflammatory Marker in Blood Independently Predicting Prolonged Postoperative Stay after Coronary Artery Bypass Grafting. Submitted to JACC III. Hägg S, Salehpour M, Noori P, Lundström J, Skogsberg J, Konrad P, Rosfors S, Tegnér J, Björkegren J. Carbon-14 Dating to Determine Carotid Plaque Age. Manuscript