A functional study of ADAMTS7 gene variants Xiangyuan Pu A thesis submitted to the University of London for the degree of Doctor of Philosophy Centre for Clinical Pharmacology William Harvey Research Institute Barts and the London School of Medicine and Dentistry Queen Mary, University of London March, 2014 I dedicate this thesis to my parents and my fiancée. Without their endless love, support and encouragement, none of my achievements would be possible. 2 Abstract Background: Recent studies have revealed an association between genetic variants at the ADAMTS7 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, 7) locus and susceptibility to coronary artery disease (CAD). ADAMTS-7 has been reported to facilitate vascular smooth muscle cell (VSMC) migration and promote neointima formation. We sought to study the functional mechanisms underlying this relationship and to further investigate the role of ADAMTS-7 in atherosclerosis. Methods and Results: In vitro assays showed that the CAD-associated non-synonymous single nucleotide polymorphism rs3825807, which results in a serine to proline (Ser-to- Pro) substitution at residue 214 in the ADAMTS-7 pro-domain, affected ADAMTS-7 pro- domain cleavage. Immunohistochemical analyses showed that ADAMTS-7 localised to vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) in human coronary and carotid atherosclerotic plaques. Cell migration assays demonstrated that VSMCs and ECs from individuals who were homozygous for the adenine (A) allele (encoding the Ser214 isoform) had increased migratory ability compared with cells from individuals who were homozygous for the G allele (encoding the Pro214 isoform). Western blot analyses revealed that media conditioned by VSMCs of the A/A genotype contained more cleaved ADAMTS-7 pro-domain and more of the cleaved form of thrombospondin-5 (TSP-5, an ADAMTS-7 substrate that had been shown to be produced by VSMCs and inhibit VSMC migration). In in vitro angiogenesis assays, ECs of the A/A genotype exhibited increased capillary-like network formation. ADAMTS-7 over-expression in ECs by transfection of an ADAMTS7-214Ser expressing plasmid significantly accelerated 3 EC migration and in vitro angiogenesis, whereas ADAMTS-7 knockdown by shRNA had opposite effects. Preliminary proteomics analyses of conditioned media of ECs over- expressing ADAMTS-7 and ECs with ADAMTS-7 knockdown indicated that ADAMTS- 7 can cleave thrombospondin-1 (TSP-1), a well-recognised angiogenesis inhibitor. Conclusion: The results of this study indicate that rs3825807 has a functional effect on ADAMTS-7 maturation, TSP-5 cleavage, VSMC and EC migration, and angiogenesis. As VSMC migration and angiogenesis play important roles in atherosclerosis, these results provide a mechanistic explanation for the association between rs3825807 and CAD. 4 Table of Contents Abstract .............................................................................................................................. 3 Table of Contents .............................................................................................................. 5 Acknowledgements ......................................................................................................... 10 Index of figures ................................................................................................................ 12 Index of tables.................................................................................................................. 15 List of abbreviations ........................................................................................................ 16 Chapter 1 Introduction .................................................................................................... 21 1.1 Cardiovascular diseases (CVDs) – the number one global killer ........................... 21 1.2 Atherosclerosis – common cause of CVDs .............................................................. 23 1.2.1 LDL retention and EC dysfunction ................................................................... 24 1.2.2 Leukocyte activation and foam cell formation ................................................. 27 1.2.3 SMC migration and fibrous cap formation ....................................................... 29 1.2.4 Extracellular Matrix proteins and atherosclerosis ............................................ 30 1.2.5 Intraplaque angiogenesis and plaque progression ............................................ 34 1.2.6 Plaque rupture ..................................................................................................... 35 1.3 Genetic basis of CVDs .............................................................................................. 37 1.3.1 Familial clustering and heritable basis of CVDs .............................................. 37 1.3.2 Genetic risk factors ............................................................................................. 38 1.3.3 Monogenic and polygenic CVD ........................................................................ 38 1.4 Genetic approaches to study CVDs .......................................................................... 39 1.4.1 Linkage study ...................................................................................................... 39 1.4.2 Candidate gene association study ...................................................................... 41 1.4.3 Genome-Wide Association Study (GWAS) ..................................................... 42 1.4.4 GWAS and the ADAMTS7 gene locus .............................................................. 45 1.5 Extracellular matrix proteases and atherosclerosis .................................................. 49 1.5.1 Matrix metalloproteinase family (MMPs) ........................................................ 50 1.5.2 The “A disintegrin and metalloproteinase” (ADAM) family .......................... 51 1.5.3 The “ADAM with thrombospondin motifs” (ADAMTS) family .................... 51 1.6 ADAMTS-7 ................................................................................................................ 56 1.6.1 Identification and protein domain organization ................................................ 56 5 1.6.2 Expression, localization and regulation ............................................................ 57 1.6.3 Activation ............................................................................................................ 60 1.6.4 Potential substrates of ADAMTS-7 ................................................................... 61 1.6.4.3 Alpha2-macroglobulin (A2M) ........................................................................ 66 1.6.5 ADAMTS-7 and bone and joint disease ........................................................... 67 1.6.6 ADAMTS-7 and cardiovascular disease ........................................................... 68 1.7 Hypothesis and Aims ................................................................................................. 70 1.7.1 Hypothesis ........................................................................................................... 70 1.7.2 Aims: ................................................................................................................... 71 Chapter 2 Materials and Methods................................................................................... 72 2.1 Cell culture ................................................................................................................. 72 2.1.1 Human vascular smooth muscle cells (VSMCs) isolation and culturing ........ 72 2.1.2 Human umbilical vein endothelial cells (HUVECs) isolation and culturing .. 73 2.1.3 Human Embryonic Kidney 293 (HEK293) cell line culturing ........................ 74 2.1.4 Preservation and recovery of cells ..................................................................... 75 2.2 Immunocytochemistry of VSMC marker and ADAMTS-7 .................................... 76 2.2.1 Verification of VSMCs by the specific marker alpha-actin ............................. 76 2.2.2 ADAMTS-7 staining in VSMCs ....................................................................... 77 2.3 Immunohistochemical staining of human atherosclerotic plaque ........................... 78 2.3.1 ADAMTS-7 and SMA double immunofluorescent staining in carotid atherosclerotic plaques ................................................................................................. 78 2.3.2 ADAMTS-7, TSP-5 and vWF single staining in carotid atherosclerotic plaques .......................................................................................................................... 79 2.3.3 ADAMTS-7 and SMA double staining in coronary atherosclerotic plaques . 80 2.4 Genotyping ................................................................................................................. 81 2.4.1 Genotyping by the KASPar method .................................................................. 81 2.4.2 VSMC Genotyping for rs3825807 by restriction enzymes .............................. 84 2.4.3 Genotyping by sequencing ................................................................................. 86 2.5 Cell proliferation, senescence and apoptosis assays ................................................ 87 2.5.1. Proliferation assay ............................................................................................. 87 2.5.2 VSMC senescence assay ...................................................................................