INVESTIGATING THE ROLE OF THE EXTRACELLULAR CALCIUM -SENSING RECEPTOR (CaSR) IN VASCULAR PATHOPHYSIOLOGY USING A NOVEL MOUSE MODEL OF SELECTIVE ABLATION OF CaSR FROM MOUSE VASCULAR SMOOTH MUSCLE CELLS DR. THOMAS DAVIES PHD THESIS 2013 SUMMARY The extracellular calcium-sensing receptor (CaSR) is a G-protein coupled receptor central to systemic Ca 2+ homeostasis in mammals. We have previously shown that CaSR is expressed in primary bovine aortic vascular smooth muscle cells (VSMCs). Furthermore, dominant-negative adenoviral knockdown of this receptor in bovine VSMCs in vitro causes enhanced calcification in the presence of culture conditions which mimic vascular calcification in vivo . Using mineralising conditions in vitro , we have also previously demonstrated that positive pharmacological allosteric activation of CaSR using the calcimimetic, R-568, significantly reduces calcification of bovine VSMCs. These findings now implicate the CaSR, not just in systemic Ca 2+ homeostasis, but also in potential protection against vascular calcification; a condition associated with increased blood pressure, left ventricular hypertrophy, chronic kidney disease and cardiovascular morbidity. Using a specific targeted deletion strategy, we have developed CaSR-specific knockout in VSMCs driven by the SM22 α promoter. Using in vitro, ex vivo and in vivo approaches, here, I have characterised the cardiovascular phenotype of this novel transgenic mouse model. In vivo data demonstrate that ablation of CaSR from VSMCs causes hyperkalaemia at 3 months of age and hypercalcaemia throughout life. 3 month old CaSR-VSMC Knockout (KO) mice also exhibit reduced bone mineral integrity and increased heart weight at the age of 18 months. Ex vivo analysis implicates the VSMC-CaSR as a modulator of blood vessel tone. CaSR-VSMC KO mice exhibit reduced luminal diameters of the aorta and mesenteric arteries. CaSR-VSMC KO also reduces contractile tone in addition to evoking Ca 2+ -dependent relaxation only, compared to CaSR-wild-type (WT) mice which exhibit both Ca 2+ -dependent contraction and relaxation of the aorta. In vitro analyses confirm that CaSR expression and activation reduces Ca 2+ -dependent proliferation and mineralisation. In conclusion, the VSMC CaSR is a modulator of vascular tone ex vivo and of VSMC proliferation and calcification in vitro . Page i ACKNOWLEDGMENTS I would firstly like to take this opportunity to extend my gratitude to both Cardiff University, for allowing me to carry out my research at this institution, and to my funders; the Biotechnology and Biological Sciences Research Council (BBSRC) and Amgen Limited. I would like to thank my supervisors Professor Daniela Riccardi and Professor Paul Kemp for their constant support and kindness during my study. I have deeply valued their input, comments and friendships over the past few years. I would also like to thank Martin Schepelmann, Dr. Sarah Brennan and Dr. Polina Iarova for their immense contributions to this project, and I am now extremely pleased to regard them as dear friends rather than colleagues. I also thank Dr. David Edwards for kindly providing me with the equipment necessary to carry out my research. I am also grateful to my friends within our group for the many enjoyable discussions and good memories during the last few years that I will not forget. I will always treasure your friendships, your laughs and endearing personalities: Aisha, Alex, Anna, Becky, Belinda, Bill, Birgitta, Brenda, Charlie, Cleo, Dave, Helen, Hsiu, Jess, João, Julia, Laurie, Leanne, Lydia, Mark, Marisol, Milica, Patrizio, Pawel, Rachel, Seva, Shuang and Stuart. To my good friends, my siblings and family who were there for me, who made the time for me whether I had any or not, I thank you all. And lastly, I thank my partner Nik for his ongoing and unending support throughout this challenging endeavour. Page ii ABBREVIATIONS 1,25-dihydroxyvitamin D 3 1,25(OH) 2D3 1,25-dihydroxyvitamin D 3 24-hydroxylase CYP24 25-hydroxyvitamin D 3 25(OH)D 3 Adenosine diphosphate ADP Adenosine triphosphate ATP Adenylyl cyclase AC Alkaline phosphatase ALP Aminoglycoside antibiotic AGA Annexin 5 Anx-5 Apolipoprotein B apoB Apolipoprotein E apoE Arachidonic acid AA Autoimmune hypoparathyroidism AH Autosomal dominant hypocalcaemia ADH Autosomal dominant hypophosphatemic rickets ADHR Bone morphogenetic proteins BMPs Bone morphogenetic protein 2 BMP-2 Bone morphogenetic protein 4 BMP-4 Bone morphogenetic protein 6 BMP-6 Bone morphogenetic protein 7 BMP-7 Breast arterial calcification BAC Calcific uremic arteriolopathy CUA Calcifying vascular cell CVC Calcium ion Ca 2+ 2+ Calcium ion (free ionized) Ca o Calmodulin CaM Cardiovascular Disease CVD Chronic kidney disease CKD Collagen type I Col1 Computerised tomography CT Concentration of calcium ion [Ca 2+ ] Connexin Cx Coronary arterial calcification CAC Cyclic 3’-5’adenosine monophosphate cAMP Cyclic 3’-5’guanosine monophosphate cGMP Diacylglycerol DAG Dulbecco’s Modified Eagle Medium DMEM End-stage renal disease ESRD Endoplasmic reticulum ER Endothelial cells EC Page iii Endothelial nitric oxide synthase eNOS Endothelium-derived hyperpolarisation EDH Endothelium-derived hyperpolarising factor EDHF Endothelium-derived relaxation factor EDRF Epoxyeicosatrienoic acids EET Extracellular matrix ECM Extracellular regulated protein kinase ERK Extracellular calcium-sensing receptor CaSR / CaS / CaR Familial hypocalciuric hypercalcaemia FHH Fibroblast growth factor 23 FGF23 Fibroblast growth factor receptor FGFR 2+ Free ionized extracellular calcium concentration [Ca ]o Fetal bovine serum FBS Generalized arterial calcification of infants GACI G protein-coupled receptor GPCR Guanosine diphosphate GDP Guanosine triphosphate GTP Guanylyl cyclase GC Heart disease HD Human embryonic kidney 293 cells HEK293 Hydrochloric acid HCl Hydrochloride HCl - Hydroxyapatite HA Inorganic phosphate Pi Inositol 1,4,5-trisphosphate IP 3 Inositol 1,4,5-trisphosphate receptor IP 3R Interferon-γ IFN-γ Internal elastic lamina IEL 2+ Intracellular free ionised calcium concentration [Ca ]i Jun amino-terminal kinase Jun Knockout KO 2+ + Large conductance Ca -activated K channels BK Ca 2+ L-type voltage-gated Ca channel LCa Low-density lipoprotein LDL Low-density lipoprotein receptor LDLR Matrix gla protein MGP Matrix metalloproteinase MMP Matrix vesicle MV Metabotropic glutamate receptors mGluRs Microcomputerised tomography µCT Mitogen-activated protein kinase MAPK Myoendothelial junction MEJ Page iv Myoendothelial projection MP Myosin light chain MLC Myosin light chain kinase MLCK Myosin heavy chain phosphatase MLCP National Centre for Health Services NCHS National Health Interview Survey NHIS Neonatal severe hyperparathyroidism NSHPT Nitric oxide NO Nitric oxide synthase NOS Nucleotide pyrophosphatase/phosphodiesterase-1 NPP1 Osteocalcin OC Osteoprotegrin OPG Osterix OSX Parathyroid cell PTC Parathyroid gland PTG Parathyroid hormone PTH Parathyroid hormone-related peptide PTHRP Sodium Phosphate co-transporter PiT-1 Phosphatidylinositol 3-kinase PI 3-K Phosphatidylinositol 4,5 bisphosphate PIP 2 Phosphatidylinositol-4 kinase PI4-K Phosphatidylinositol 4-phosphate PI4 Phospholipase A 2 PLA 2 Phospholipase B PLB/Akt Phospholipase C PLC Phospholipase D PLD Platelet-derived growth factor PDGF Polymerase chain reaction PCR Potassium chloride KCl Prostacyclin PGI 2 Protein kinase A PKA Protein kinase C PKC Protein kinase G PKG Pyrophosphate PPi Receptor activator of nuclear factor kappa-B RANK Receptor activator of nuclear factor kappa-B ligand RANKL Renin-angiotensin-aldosterone system RAAS Reverse transcriptase PCR RT-PCR Reverse transcription RT Ryanodine Receptor RyR Sarcoplasmic reticulum SR Sarcoplasmic reticulum Ca 2+ - ATPase SERCA Smooth muscle actin 22 α SM22 α Page v Standard error of mean SEM Store operated Ca 2+ entry SOCE Stress-activated protein kinase ERK kinase 1 SEK1 - Thiosulphate S2O3 Tissue non-specific alkaline phosphatase TNAP Transforming growth factor β TGF-β Transglutaminase 2 TG2 Transmembrane domain TMD 2+ T-type voltage-gated Ca channel TCa Tumour necrosis factor TNF University of California San Francisco UCSF Vascular calcification VC Vascular endothelial growth factor VEGF Vascular smooth muscle cells VSMC Venus flytrap domain VFT Vitamin D receptor VDR Vitamin D response elements VDRE Vitamin D 3 VD 3 Voltage-gated Ca 2+ channels VGCC Wild-type WT α1-adrenergic receptor α1-AR α2-Heremans Schmid glycoprotein Fetuin A α-Modified Eagle Medium αMEM β-glycerophosphate βGP γ-aminobutyric acid receptors GABA BRs Page vi TABLE OF CONTENTS SUMMARY ..................................................................................................................................... ACKNOWLEDGMENTS ................................................................................................................. ii ABBREVIATIONS ....................................................................................................................... iii LIST OF FIGURES ....................................................................................................................... xii LIST OF TABLES .................................................................................................................... xviii CHAPTER 1: ................................................................................................................................. 1 GENERAL INTRODUCTION ........................................................................................................... 1