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Characterisation of the P19CL6 Cardiovascular Cell-Line at the Gene Expression Level; Effects of Long-Chain Fatty Acids

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Characterisation of the P19CL6 Cardiovascular Cell-Line at the Gene Expression Level; Effects of Long-Chain Fatty Acids Characterisation of the P19CL6 cardiovascular cell-line at the gene expression level; effects of long-chain fatty acids A thesis submitted in accordance with the requirements of the University of Surrey for the degree of Doctor of Philosophy by Iraj Khodadadi Kohlan September 2005 Centre of Nutrition and Food Safety School of Biomedical and Molecular Sciences University of Surrey Guildford Surrey GU2 7XH ProQuest Number: 27610157 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 27610157 Published by ProQuest LLO (2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 48106- 1346 Dedication This thesis is dedicated to my parents and my wife for their enormous support, and to Alisa and Parita 11 Acknowledgment I would like to thank my two supervisors: Dr Bruce Griffin and Dr Alfred Thumser for helping me to conduct this research in such an interesting and rapidly expanding field of science. I have been fortunate to use an excellent selection of techniques in this study. I am honoured for having worked with Dr Bruce Griffin, who has been an encouraging supervisor and great support during my study. I am also extremely grateful to Dr Alfred Thumser, for his invaluable guidance and help throughout my research at the University of Surrey. He was a great support at the times when I needed him most, and I would consider him a good fiiend as well as an excellent supervisor. I am also very grateful to Dr Nick Plant, who frequently helped during this project, to Dr George Kass for his kind help in fluorescent microscopy, and to Dr David Lovell for his advice in statistical analysis. Much gratitude is also due to Dr Vassilios Mersinias, whose help and advice were invaluable throughout the microarray experiments. Many thanks go to Prof. Colin Smith and Dr Giselda Bucca for their help in conducting the microarray experiments, and to Mr Ben Routley for his help in performing microarray data analysis. I am also thankful to Miss Victoria Leggett for DNA sequencing, and to Mr Peter Kentish for mouse tissue dissection. Finally, a big thank you to my sponsors: the Iranian Ministry of Health and Medical Sciences and the Hamadan University of Medical Sciences. Ill Abstract Dietary fats have been implicated in the progression of coronary heart disease and clear differences have been shown for the effects of saturated, monounsaturated and polyunsaturated fatty acids on the different stages of disease. In part, these effects can be explained by the regulation of gene transcription. In this study, a novel cardiac cell-line was targeted for the investigation of fatty acid effects on gene transcription. The aims of this study were to (i) characterise the P19CL6 cell-line at the transcriptional level, (ii) investigate the effects of long-chain fatty acids and clofibrate on mRNA levels of specific lipid metabolism-related genes, such as heart-type fatty acid-binding protein and peroxisome proliferator-activated receptor-a, -|3, and -y in the P19CL6 cell-line, and (iii) determine the effects of long chain fatty acids and clofibrate on global transcriptome levels in P19CL6 cells, using cDNA microarray analysis. P19CL6 cells displayed characteristics indicative of a cardiomyocyte phenotype including: the expression of cardiac-specific markers (a-MHC, P-MHC and CaCh-h), an increase in pulse rate with increasing adrenaline concentration and the display of mononuclear cardiomyocyte morphology. However, spontaneous contraction was inconsistent between cultures of P19CL6 cells and pulse rate decreased significantly with increasing passage number until no “beating” was eventually recorded. These observations led us to conclude that P19CL6 cells may display a heterogeneous phenotype, which was investigated further. Analysis of microarray data indicated that global transcriptome profiles observed in the P19CL6 cell-line are not indicative of a pure adult cardiac or skeletal muscle phenotype, and more closely related to mouse embryonic heart tissue. However, the H9C2(2-1) cell-line was more closely related to adult and embryonic heart tissue than the P19CL6 cell-line. Therefore it was concluded that the H9C2(2-1) cell-line represents a better cardiomyocyte model system than the P19CL6 cell-line. The results of this study also showed that in P19CL6 cells long-chain fatty acids (but not clofibrate) significantly increased the abundance of peroxisome proliferator-activated receptor-a and -y, whereas no changes were observed in the expression levels of heart-type fatty acid-binding protein and peroxisome proliferator-activated receptor-P, under the conditions used. The non-response of the latter genes can probably be explained by the relatively short time of exposure of cells in culture. Microarray analysis IV showed that linoleic and a-linolenic acids and clofibrate had similar effects, and that these differed fi’om those of palmitic and oleic acids. These results are in agreement with other studies showing that cellular responses to polyunsaturated fatty acids differ fi*om those observed with saturated and monounsaturated fatty acids. V Abbreviations: AA arachidonic acid ABCAl ATP-binding cassette transporter 1 ACBP acyl-Coenzyme A binding protein ACS acyl-Coenzyme A synthetase Ad-heart adult heart ADRP adipocyte differentiation related protein AF-1,2 activation factor-1, 2 A-I, II apolipoprotein A-I, II ALA a-Iinolenic acid A-LBP adipocyte lipid-binding protein ANF atrial natriuretic factor B-FABP brain fatty acid-binding protein BSA bovine serum albumin CaCh-h calcium channel (heart isoform) CaCh-m calcium channel (skeletal muscle isoforr CETP cholesterol ester transfer protein CHD coronary heart disease C-II, III apolipoprotein C-II, III Clo clofibrate CM chylomicron COX cyclooxygenase CPT carnitine palmitoyltransferase CRABP cellular retinoic acid binding-protein CRBP cellular retinol binding-protein DART diet and re-infarction trial DBD DNA binding domain DHA docosahexaenoic acid DNA deoxyribonucleic acid EFA essential fatty acid E-FABP epidermal fatty acid binding-protein VI Em-heart embryonic heart EPA eicosapentaenoic acid FABP fatty acid-binding protein FABPpm plasmalemmal fatty acid-binding protein FAR fatty acid receptor FAT fatty acid translocase FATP fatty acid transport protein HAT histone acetyltransferase HDL high-density lipoprotein HDLC HDL-Cholesterol H-FABP heart fatty acid-binding protein HGMP human genome mapping project HNF-4a hepatic nuclear factor-4a IDL intermediate-density lipoprotein I-FABP intestinal fatty acid-binding protein IL interleukin iLBP intracellular lipid-binding protein Kd dissociation constant LA linoleic acid LBD ligand binding domain LCAT lecithin-cholesterol acyltransferase LCFA long-chain fatty acid LDL low-density lipoprotein LDL-C LDL-Cholesterol 1-FABP ileal fatty acid binding-protein L-FABP liver fatty acid-binding protein LLAMA live linear analysis of mocroarray LOX lipooxygenase LPL lipoprotein lipase LT leukotriene LXR liver X receptor MHC myosin heavy chain M-LBP myelin-lipid binding protein; M-FABP vu MLC2 myosin light chain-2 MUFA monounsaturated fatty acid NFkB nuclear factor k B OA oleic acid PA palmitic acid PCA principal components analysis PG prostaglandins PLTP phospholipid transfer protein PPAR peroxisome proliferator-activated receptor PPRE peroxisome proliferator regulatory (responsive) element PUFA polyunsaturated fatty acid RXR retinoic X receptor SDS sodium dodecyl sulphate SFA saturated fatty acid SR-Bl scavenger receptor SREBP sterol regulatory element binding protein SSC sodium chloride and sodium citrate TAG triacylglycerol TFA trans fatty acid T-FABP testis fatty acid binding-protein TNFa tumour necrosis factor-a TX thromboxane TZD thiazollidinedione VCAM-1 vascular cell adhesion molecule-1 VLDL very low-density lipoprotein WHO world health organization V lll Content Chapter 1 Introduction page 1.1 Introduction 2 1.2 Coronary Heart Disease 3 1.2.1 Definition 3 1.2.2 Risk Factors 4 1.2.3 Atherosclerosis 5 1.3 Properties of fatty acids 5 1.3.1 Fatty acids function as an energy source in the cardiomyocytes 7 1.3.2 p-oxidation of fatty acids 7 1.3.3 Lipoprotein metabolism 8 1.3.4 Effect of fatty acids on PUFA metabolism 10 1.3.5 The link between dietary lipids and CHD 12 1.3.6 Cardioprotective effect of PUFA 13 1.3.7 Delivery of fatty acids to the cardiomyocytes 15 1.4 Membrane-associated fatty acid transporters 15 1.5 Intracellular fatty acid-binding proteins (FABPs) 19 1.5.1 Classification of Intracellular FABPs 19 1.5.2 Ligand-binding characteristics of FABPs 21 1.6 Nuclear Receptors 25 1.6.1 Classification of nuclear receptors 25 1.6.2 Structure of nuclear receptors 27 1.7 Peroxisome proliferator-activated receptors (PPARs) 28 1.7.1 Distribution and PPAR isoforms 28 1.7.2 Transcriptional activity of PPARs 29 1.7.3 PPARs ligands 30 1.7.4 Synthetic ligands of PPARs 33 1.7.5 Complementary roles of PPARs in lipid metabolism 34 1.7.6 PPARs and atherosclerosis 36 1.8 Hypothesis, aim and objectives 38 IX Chapter 2 Materials and Methods page 2.1 Materials 41 2.2 Methods 42 2.2.1 Cell culture 42 2.2.1.1
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