University of Milano-Bicocca Department of medicine and surgery PhD PROGRAM IN TRANSLATIONAL AND MOLECULAR MEDICINE DIMET Circulating levels of soluble Receptor for Advanced Glycation End-products (sRAGE) decrease with aging and may predict age- related cardiac remodeling Coordinator: Prof. Andrea Biondi Tutor: Prof. Giulio Pompilio Co-tutor: Dr. Angela Raucci Dr. Filippo Zeni Matr N°: 787963 XXIX CYCLE Academic Year 2015-2016 Table of contents Chapter 1……………..……………………………………………………………….3 General Introduction ............................................................................. 4 Aging ............................................................................................................ 4 Molecular mechanisms of aging ................................................................ 5 DNA Damage and instability .............................................................................. 5 DNA damage and apoptosis ............................................................................... 6 Telomere shortening ............................................................................................. 7 Epigenetic Alterations .......................................................................................... 8 Mitochondrial dysfunction .............................................................................. 10 Stem cells exhaustion ......................................................................................... 11 Biomarkers of aging ........................................................................................... 12 Sex-Dependent Differences in aging ..................................................... 15 The heart: basic anatomy ................................................................... 18 Cardiac Aging .......................................................................................... 22 Vascular aging ............................................................................................... 23 Cardiac Changes During Aging ................................................................ 25 Morphologic changes ......................................................................................... 25 Systolic functions ................................................................................................. 28 Diastolic functions ............................................................................................... 29 Conduction system .............................................................................................. 30 Animal models ...................................................................................................... 32 Molecular changes ............................................................................................... 33 Altered Nutrient and Growth Signaling ............................................................... 33 Mitochondrial Oxidative Damage and Mitochondrial Dysfunction ......... 35 Adverse Extracellular Matrix (ECM) Remodeling ........................................... 36 Impaired Calcium Homeostasis ............................................................................... 38 Circulating biomarkers of cardiac aging .................................................... 40 MicroRNAs ........................................................................................................................ 40 Circulating factors ......................................................................................................... 41 Sex and age-related cardiac remodeling ............................................. 44 The Receptor for Advanced Glycation End Products (RAGE) 46 Splice Variants of RAGE .............................................................................. 51 1 Production of sRAGE via Proteolytic Cleavage of RAGE ................. 53 Modulation of sRAGE levels ...................................................................... 54 RAGE ligands .................................................................................................. 57 AGEs .......................................................................................................................... 57 High Mobility Group (HMG) Box-1 ............................................................... 59 S100 Proteins and RAGE................................................................................... 62 RAGE, aging and heart ................................................................................ 64 sRAGE, aging and heart .............................................................................. 66 Scope of the thesis ................................................................................. 70 References ............................................................................................... 72 Chapter 2 .................................................................................................. 91 Abstract .................................................................................................... 92 Introduction ............................................................................................ 95 Materials and Methods ........................................................................ 99 Human study .................................................................................................. 99 Study population .................................................................................................. 99 Human Elisa assays ............................................................................................. 99 Statistical analysis ............................................................................................ 100 Animal study ............................................................................................... 100 Animals ................................................................................................................. 100 Echocardiography ............................................................................................ 101 Western Blotting (WB) ................................................................................... 101 Immunoistochemistry (IHC) ........................................................................ 102 Quantitative RT-PCR (qRT-PCR) ................................................................ 103 Isolation of Mouse Cardiomyocytes .......................................................... 104 Evaluation of CMs cross sectional area ................................................... 105 Mouse ELISA assay ........................................................................................... 106 Statistical analysis ............................................................................................ 106 Gene expression analysis .............................................................................. 107 Results .................................................................................................... 111 Circulating levels of sRAGE decrease in healthy subjects during aging ............................................................................................................... 111 RAGE ligands behavior during human healthy aging................... 115 Circulating levels of sRAGE decrease with aging in mice............ 116 2 Serum levels of sRAGE correlate with age-associated cardiac changes ......................................................................................................... 118 Cardiac levels of RAGE isoforms during aging ................................ 121 Rage-/- mice show an increase of LV dimensions during aging 123 Rage-/- mice show enhanced cardiac fibrosis but not hypertrophy with aging .......................................................................... 125 Rage-/- mice show higher expression of heart failure markers .......................................................................................................................... 128 RAGE and aging influence LV genes expression ............................. 129 Differentially expressed genes correlates with cardiac mice phenotype .................................................................................................... 131 Discussion ............................................................................................. 134 References ............................................................................................ 144 Supplementary Information .......................................................... 149 Chapter 3 ............................................................................................... 154 Summary ............................................................................................... 154 Translational significance and future perspectives .............. 156 Publications ......................................................................................... 160 References ............................................................................................ 169 3 General Introduction Aging Population aging is accelerating rapidly worldwide, from 461 million people older than 65 years in 2004 to an estimated 2 billion people by 20501. The substantial gain of approximately 30 years in life expectancy in western world countries such as Spain and Italy and even larger gains in Japan, stands out as one of the most important achievement of the 20th century. The oldest-old group (aged >85 years) has been, during the last years, the