University of Ghana http://ugspace.ug.edu.gh   UNIVERSITY OF GHANA COLLEGE OF HEALTH SCIENCES ASSOCIATION BETWEEN ARTERIAL STIFFNESS AND CIRCULATING ADIPOKINES IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS IN GHANA BY RICHARD NANA ABANKWAH OWUSU MENSAH (STUDENT NUMBER: 10552153) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL PHYSIOLOGY DEGREE JULY 2017   University of Ghana http://ugspace.ug.edu.gh DECLARATION The information presented are from a research I conducted myself. Information from other peoples’ research used in this write-up have been appropriately cited. I therefore declare this thesis to be an original work which has never been used to obtain any form of degree here or elsewhere. Signature: ………………………………… Date: ……………………………… Richard Nana Abankwah Owusu Mensah DECLARATION BY SUPERVISORS The practical work and presentation of this thesis were conducted under our supervision in accordance with guidelines on supervision of thesis in the University of Ghana. We therefore declare that this thesis is an original work by the student. Principal Supervisor: ……………………………………… Date: ……………………………. Daniel Ansong Antwi (PhD) Co-Supervisor ………………………………… Date: ………………………………. Kwame Yeboah (PhD)   ii   University of Ghana http://ugspace.ug.edu.gh DEDICATION To my loving sister, Adwubi   iii   University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENTS   “Now thank we all our God, with hearts and hands and voices, Who wondrous things hath done, In whom His world rejoices; Who, from our mother’s arms, Hath blessed us on our way, With countless gifts of love, And still is ours to-day”. (MHB 10, stanza 1). All thanks to God Almighty for His abundance of grace, gift of life and strength to this far. I am especially thankful to my wonderful supervisors, Dr. Daniel Ansong Antwi and Dr. Kwame Yeboah, I owe you lots of gratitude. I am especially grateful to Dr. Kwame Yeboah for his good words of encouragement and support in all dimensions, you have been my Mentor. My sincere gratitude also goes Dr. Dzifa Dei (Rheumatology Unit, Department of Medicine, KBTH), Rev. Dr. Antwi-Boasiako Charles, also to Eric and Kwadwo at the Immunology Department (Noguchi Memorial Institute for Medical Research-NMIMR), who assisted in the ELISA analysis, also to Patrick for the chemistry. I appreciate the support, encouragement and love provided by my family throughout this studies which helped in making my study a pleasant one. Also to, Ewurakua, Nancy Saana, Mr. Amponsah Asiamah (UCC) and Dr. Elvis Ofori Ameyaw (Vice Dean, SBAHS, UCC). Finally, I thank all my colleague graduate students, lecturers, technical staff of the Department of Physiology, University of Ghana, and Kennedy K. Dodam for their various instrumental roles they played. I am most grateful to all and sundry who in a way contributed to the successful completion of this research.   iv   University of Ghana http://ugspace.ug.edu.gh ABSTRACT   Background: Cardiovascular complication accounts for greater number of deaths that occur in lupus patients who have chronic inflammation. However, arterial stiffness is used to predict diseases of the cardiovascular system, characterized by persistent inflammation which affects adipose tissues, dysregulation of adipokines and hyperinsulinemia, tend to quicken hardening of arteries in patients with systemic lupus erythematosus (SLE). Aim: This study investigated association existing between arterial stiffness and adipokines in patients with SLE in Ghana. Methodology: The study was case-control designed. The cases were males and females with systemic lupus erythematosus (SLE) and were matched by age and gender with non-SLE individuals. Patients with SLE were recruited and examined at the Central Out-Patient Department (COPD), Korle-Bu Teaching Hospital. After obtaining informed consent, a data abstraction sheet was used to collect socio-demographic information, and recording of physical measurements were taken. Heavy-duty floor scale was used to measure the weights of the participants, body height measured with a stadiometer, and percentage body fat by Omron body fat monitor. Systolic BP and diastolic BP were assessed using semi-automated digital sphygmomanometer. Arterial stiffness was measured as CAVI together with haPWV by VaSera VS-1500N. Enzyme-linked immunosorbent Assay kit by R&G laboratories was used to analyze adipokines, insulin and C-reactive protein concentrations. Lipid profile and blood biochemistry were done with RT-900 Semi-auto chemical analyzer. Results: Compared to non-SLE controls, patients with SLE had higher levels of CAVI (7.3±1.1 vs 6.1±1, p<0.001), haPWV (7.7±1.3 vrs 6.5±0.8 m/s, p=<0.001), insulin [76.8 (45.9 – 184.8) vs 39.8 (22.9 – 86.3) pmol/ml, p=0.007], leptin [856.1 (364.8 – 1509.3) vrs 426.7 (426.8 (84.7 – 1178.7) ng/ml, p=0.039], adiponectin [1.1 (0.8 – 2.3) vrs 1.6 (1.3 – 2.6) ngml-1, p = 0.039]   v   University of Ghana http://ugspace.ug.edu.gh and CRP [1.6 (0.8 – 2.2) vrs 0.9 (0.6 – 1.2) mgml-1, p = 0.021]. In an adjustment in age and BMI for correlation, CAVI showed association with leptin (r=0.21, p=0.031), CRP (r=2.9, p<0.001) and insulin (r=0.18, p=0.04), but not adiponectin (r=-0.15, p=0.068).   Conclusion:   Patients who have systemic lupus erythematosus recorded higher arterial stiffness, which is associated to low-grade inflammation and deranged circulating adipokines levels   vi   University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS   DECLARATION .................................................................................................................... II DEDICATION ....................................................................................................................... III ACKNOWLEDGEMENTS .................................................................................................. IV ABSTRACT .............................................................................................................................. V TABLE OF CONTENTS .................................................................................................... VII LIST OF TABLES ............................................................................................................... XII LIST OF ABBREVIATIONS ............................................................................................ XIII CHAPTER ONE ...................................................................................................................... 1 1.0 INTRODUCTION ....................................................................................................... 1 1.1 BACKGROUND ................................................................................................................... 1 1.2 PROBLEM STATEMENT ................................................................................................ 4 1.3 JUSTIFICATION ............................................................................................................ 7 1.4 AIM ............................................................................................................................. 8 1.5 SPECIFIC OBJECTIVES ................................................................................................. 8 1.6 HYPOTHESES ..................................................................................................................... 8 CHAPTER TWO ..................................................................................................................... 9 2.0 LITERATURE REVIEW ........................................................................................... 9 2.1 SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) ....................................................................... 9 2.2 EPIDEMIOLOGY OF SLE, WORLD AND AFRICA ................................................................ 10 2.3 HYPERTENSION IN SLE PATIENTS ................................................................................... 12 2.4 CARDIOVASCULAR COMPLICATIONS OF SLE ................................................................... 13   vii   University of Ghana http://ugspace.ug.edu.gh 2.5 BASIC CONCEPTS OF ARTERIAL HEMODYNAMICS ............................................................. 15 2.6 HEMODYNAMIC CONSEQUENCES OF ARTERIAL STIFFNESS ............................................... 18 2.7 ELASTIC BEHAVIOUR OF ARTERIES .................................................................................. 19 2.8 STRESS – STRAIN RELATIONSHIP OF ARTERIES ................................................................. 20 2.9 PRESSURE–VOLUME AND PRESSURE–AREA RELATIONS ................................................... 22 2.10 ARTERIAL STIFFNESS ..................................................................................................... 23 2.11 INDICES OF ARTERIAL STIFFNESS ................................................................................... 26 2.12 STRUCTURAL COMPONENTS OF ARTERIAL STIFFENING .................................................. 27 2.12.1 Collagen fibers ..................................................................................................... 31 2.12.2 Elastin ................................................................................................................... 32 2.12.3 Advanced Glycation End-Products (AGEs) ........................................................ 32 2.12.4 Calcium ions ......................................................................................................... 33 2.12.5 Cellular modification ........................................................................................... 33 2.13 C-REACTIVE PROTEIN (CRP) ..................................................................................... 34 2.14 ADIPOKINES .................................................................................................................. 35 2.14.1 ADIPONECTIN ............................................................................................................. 36 2.14.2 LEPTIN ....................................................................................................................... 38 2.15 INSULIN ......................................................................................................................... 38 2.16 CARDIO-ANKLE VASCULAR INDEX AS A MEASURE OF ARTERIAL STIFFNESS ................. 39 CHAPTER THREE ............................................................................................................... 41 3.0 METHODOLOGY .......................................................................................................... 41 3.1 STUDY DESIGN AND POPULATION .................................................................................... 41 3.2 SETTING .......................................................................................................................... 41 3.3 ELIGIBILITY CRITERIA ............................................................................................... 41   viii   University of Ghana http://ugspace.ug.edu.gh Inclusion criteria .............................................................................................................. 41 Exclusion criteria ............................................................................................................. 41 3.4 SAMPLE SIZE ............................................................................................................. 42 3.5 PARTICIPANTS RECRUITMENT AND DATA COLLECTION ............................................. 42 3.6 BLOOD PRESSURE MEASUREMENT ............................................................................. 43 3.7 ANTHROPOMETRY ..................................................................................................... 43 3.8 VASERA .................................................................................................................... 45 3.9 BLOOD SAMPLE COLLECTION, PROCESSING AND STORAGE ........................................ 47 3.10 BIOCHEMISTRY ANALYSIS ......................................................................................... 47 3.10.1 Plasma lipid profile assay ............................................................................... 47 3.10.2 Adiponectin and leptin enzyme-linked immunosorbent assay ...................... 49 3.10.3 C-Reactive protein and Insulin enzyme-linked immunosorbent assay ......... 51 3.11 DATA HANDLING ...................................................................................................... 53 3.12 STATISTICAL ANALYSIS ............................................................................................. 53 3.13 ETHICAL APPROVAL .................................................................................................. 53 CHAPTER FOUR .................................................................................................................. 54 4.0 RESULTS ................................................................................................................... 54 4.1 GENERAL DESCRIPTION OF THE STUDY POPULATION ...................................................... 54 4.2 GENDER DISTRIBUTION OF PARTICIPANTS ....................................................................... 54 4.3 BIOCHEMICAL CHARACTERISTICS OF PARTICIPANTS ........................................................ 54 4.4 INFLAMMATION AND ADIPOKINES ................................................................................... 54 4.5 HAEMODYNAMIC AND ARTERIAL STIFFNESS INDICES ...................................................... 54 4.6 LEVELS OF ARTERIAL STIFFNESS ..................................................................................... 62 4.7 AGEING AND ARTERIAL STIFFNESS .................................................................................. 62   ix   University of Ghana http://ugspace.ug.edu.gh 4.8 BMI AND ARTERIAL STIFFNESS ....................................................................................... 62 4.9 ASSOCIATION BETWEEN ADIPOKINES AND ARTERIAL STIFFNESS INDICES ....................... 65 4.10 ASSOCIATION BETWEEN INSULIN LEVELS AND ARTERIAL STIFFNESS ............................. 65 4.11 ASSOCIATION BETWEEN INFLAMMATION AND ARTERIAL STIFFNESS .............................. 65 CHAPTER FIVE ................................................................................................................... 71 5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS ................................ 71 5.1 GENERAL FINDINGS ......................................................................................................... 71 5.2 GENERAL CHARACTERISTICS OF STUDY PARTICIPANTS ................................................... 71 5.3 ARTERIAL STIFFNESS AMONG STUDY POPULATION ......................................................... 72 5.4 HAEMODYNAMIC AND ARTERIAL STIFFNESS INDICES OF STUDY PARTICIPANTS ............... 73 5.5 ASSOCIATION BETWEEN ARTERIAL STIFFNESS AND ADIPOKINES: LEPTIN & ADIPONECTIN74 5.6 INFLAMMATION AND ARTERIAL STIFFNESS ...................................................................... 76 5.7 AGEING AND ARTERIAL STIFFNESS .................................................................................. 77 5.8 BODY COMPOSITION AND ARTERIAL STIFFNESS ............................................................... 78 5.9 CONCLUSION ................................................................................................................... 80 5.10 LIMITATIONS OF THE STUDY ......................................................................................... 80 5.11 RECOMMENDATIONS ..................................................................................................... 81 REFERENCES ....................................................................................................................... 82 APPENDIX 1: ETHICAL APPROVAL ............................................................................ 109 APPENDIX II: DATA CAPTURE TOOL ........................................................................ 110 APPENDIX III: CONSENT FORM .................................................................................. 112     x   University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES   Figure 1: Possible mechanisms through which inflammation induces changes in the structure of the walls of blood vessels to accelerate vascular hardening.. ..................................... 25 Figure 2: Cardio-ankle vascular index(CAVI) measured in patient lying in supine position.. 46 Figure3: Gender distribution of participants………………………………………………... 57 Figure 4: Comparison of degree of arterial stiffness among study groups…………………...61 Figure 5: Association between age and arterial stiffness in study groups…………………... 63 Figure 6: Association between BMI and arterial stiffness…………………………………... 64 Figure 7: Association between leptin levels and CAVI……………………………………... 66 Figure 8: Association between adiponectin and CAVI……………….……………………... 67 Figure 9: Association between CAVI and insulin levels……………………………………. 68 Figure 10: Association between CRP levels and CAVI………………………………..…… 69 Figure 11: A simplified schematic diagram showing the association of leptin and adiponectin to arterial stiffness as observed in this studies………………………………………….70   xi   University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 1: Anthropometric characteristics of Patients and Control. ........................................... 56 Table 2: Biochemical characteristics of SLE patients and non-SLE individuals. ................... 58 Table 3: Serological parameters measured in SLE patients and non-SLE individuals. ........... 59 Table 4: Haemodynamics and atherosclerotic indices of Patients and Control Groups. ......... 60       xii   University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS     aa Amino acid ABI Ankle-brachial index ACR American College of Rheumatology AdipoR Adiponectin receptor AFLAR African League of Associations in Rheumatology AGEs Advanced glycation end-products Aix Augmentation index ANA Antinuclear Antibody Anti-Sm Anti Stephanie Smith ATP Adenosine triphosphate BMI Body mass index BP Blood pressure CAD Coronary artery disease CAVI Cardio Ankle Vascular Index Cm centimeter CNS Central Nervous System CRP C-reactive protein CVDs/CVD Cardiovascular disease(s) DNA Deoxyribonucleic Acid dsDNA Double stranded deoxyribonucleic acid ECG Electrocardiogram ECM Extracellular matrix EDD Endothelium dependent dilation   xiii   University of Ghana http://ugspace.ug.edu.gh EDTA Ethylene di-amine tetra acetic acid EID Endothelium independent dilation ELISA Enzyme-linked immunosorbent assay eNOS Endothelial nitric oxide synthase ERK Extracellular signal-regulated kinase ESR Erythrocyte sedimentation rate ET-1 Endothelin-1 HAD Human autoimmune disease haPWV Heart-ankle pulse wave velocity HDL High density lipoprotein HF Heart failure HMW High molecular weight hsCRP High sensitivity C-reactive protein ICAM Intracellular adhesion molecule IL Interleukin kD KiloDalton LDL Low density lipoprotein M meter MetS metabolic syndrome Ml Milliliter mmHg Millimeter of Mercury mmol/l Millimole per Liter MMPs Matrix metalloproteases NCEP-ATP III National Cholesterol Education Programme – Adult Treatment Panel III   xiv   University of Ghana http://ugspace.ug.edu.gh NFB National Federation of the Blind ng/ml Nanogram per milliliter NIH National Institute of Health NO Nitric oxide Non-SLE Non-Systemic lupus erythematosus NSAIDs Non-steroidal anti-inflammatory drugs PDGF-BB Platelet derived growth factor – BB PP Pulse pressure pmol/ml Picomole per milliliter PW Pulse wave PWA Pulse Wave Analysis PWV Pulse Wave Velocity RAGE Receptor for advanced glycation end-product REASON Regression of Arterial Stiffness in Controlled double blind study Ro/SSA Anti-Sjögren’s-syndrome related antigen A ROS Reactive oxygen species RTGL Raised triglycerides SARAA South African Rheumatology and Arthritis Association SBP Systolic Blood Pressure SD Standard deviation SLE Systemic Lupus Erythematosus SLEDAI Systemic Lupus Erythematosus Disease Activity Index SLICC Systemic Lupus International Collaborating Clinics SPSS Statistical Package for the Social Sciences   xv   University of Ghana http://ugspace.ug.edu.gh TChol Total cholesterol TGF Transforming Growth Factor TIMMP Tissue inhibitor of matrix metalloproteases TNF Tumor necrosis factor Ug Microgram uL Microliter US$ United States Dollar VLDL/vLDL Very-Low-Density Lipoprotein VSMC Vascular smooth muscle cell WC Waist Circumference WHR Waist to Hip Ratio   xvi   University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1.0   INTRODUCTION 1.1 Background Systemic lupus erythematosus (SLE) is an inflammatory sickness initiated through autoimmune attack which indiscriminately damage various organs. Among these organ or tissue insult from SLE, cardiovascular complication has been shown to cause 50 % of all morbidity and mortalities in patients with SLE (Ben-Menachem, 2010). SLE has been found to be present in forty to two hundred per one hundred thousand, with black populations recording greater values (Jain & Halushka, 2009). Cardiovascular complication causes majority of deaths in SLE population as there has been an improvement in the treatment of other complications (Manger, Manger, et al., 2002). Arterial stiffness is found to predict cardiovascular morbidity and its related deaths, as well as a hypothetical target for treatment, and has an association with generalized inflammation of the body. Meanwhile, inflammation is known to be the primary pathophysiological cause of SLE (Mahmud & John, 2005). When the arteries become stiffened, they are not able to dilate to accommodate the blood that is pumped from the ventricles causing high pulsatile afterload which could lead to remodeling of the left ventricle resulting in its loss of function and eventually failing (Snigdha J. , Rohan, Vicente, Raymond, & Julio, 2014). Prevalence of atherosclerotic lesions have been found to be high in patients with SLE by an autopsy and angiographic studies but not clear whether it is peculiar to some patients with SLE only. Due to the existence of activated macrophages and abundance of T-cells in atherosclerotic lacerations and inflammation, assessed C-reactive proteins levels, atherosclerosis is described to be an inflammatory disease.   1   University of Ghana http://ugspace.ug.edu.gh Also significant amount of cytokines which are basically known to cause inflammation are usually in higher titre at atherosclerotic lesions (Frostegård, 2005). Therefore, enhanced inflammation in SLE patients would exacerbate arterial stiffness leading to increased prevalence of cardiovascular complications in these patients. The long term effect of large arteries stiffness may result in hypertension as well as possible hemorrhagic strokes as pulsatile pressure enters microvascular structures of the brain. Hypertension which is a leading risk factor of deaths in developing countries as well as developed countries (Yach, Hawkes, Gould, & Hofman, 2004) makes SLE more life threatening. Furthermore, various epidemiological studies that have been conducted in Europe and the Americas have shown significant increased risk of SLE and cardiovascular diseases in people of African origin than elsewhere. Epidemiological studies on SLE in Europe, the American continents, Africa, Australia and Asia have shown that although the disease is not common in Africa, it is still prevalent in African descendants around the world, this suggest some level of improper ascertainment of the disease in Africa. Meanwhile, the rate of occurrence and prevalence in people of Africa or of Asian background is twice to thrice higher than in white populations (Pons-Estel, Alarcón, Lacie, Leslie, & Glinda, 2008). The predominant risk factor for SLE is gender. Pons-Estel et al., (2008), reports that SLE occurs mostly in women than men as several studies have found out over 90 % of patients with SLE are females. The disease, though affects females than males, the onset is usually during the reproductive ages as childhood incidence and prevalence rates are found to be considerably lower than adult rates. In a study conducted by Huemer et al., (2001), in Austria, the annual incidence rate of SLE in children aged less than 16 years was less than 1 per 100, 000 (Huemer, Huemer, Dorner, Falger, Schacherl, & Bernecker, 2001).   2   University of Ghana http://ugspace.ug.edu.gh There is the suspicion that changes in reproductive hormones such as estrogen promotes SLE disease progression and flaring. According to Sanchez-Guerrero et al., (1997), exogenous estrogen has been found to exacerbate lupus or increases the risk of developing the disorder (Sanchez-Guerrero, Karlson, Liang, Hunter, Speizer, & Colditz, 1997). Although the mechanisms have not been understood, it can be hypothesized that the rapid increment of adiposity during the secondary sexual growth in females is likely to promote disease pathogenesis. This is due to the fact that the onset of the disease has been found to be at age 16 where there is high level of the production of reproductive hormones responsible for secondary sexual characteristics in females. During this time, there is increase in both peripheral adipose tissue accumulation as well as visceral adiposity which is highly inflammatory (Bosaeus, 2012) and may contribute to the flare of the disease. The assessment of the levels of C-reactive protein (CRP) - a good inflammatory marker, adipokines – involved in atherosclerosis pathway as biomarkers together with indices of arterial stiffness such as pulse wave velocity (PWV) (Townsend et al., 2015), augmentation index (AIx), and ankle-brachial index (ABI), (Urbina, Briton, Elkasabany, & Berenson, 2002; Williams, et al., 2006) would potentially establish a clear relationship between systemic lupus erythematosus and arterial stiffness. Cardiovascular complications of SLE involve all heart structures and the vascular systems (Brigden, Bywaters, & Lessof, 1960). These complications are as a result of a complex interplay of primary disease, traditional risk factors, and treatment related effects (Bulkley & Roberts, 1975). Cardiac complications of lupus affect most parts of the heart and its conduction systems (Jain & Halushka, 2009) with additional burden of vasculopathy, insulin resistance and   3   University of Ghana http://ugspace.ug.edu.gh nephritis (Chogle & Chakravaty, 2007). SLE is associated with an endogenous dyslipidemia, increased very low density lipoprotein (VLDL), triglycerides, low levels of high-density lipoprotein (HDL), and altered chylomicron metabolism (Borba, Bonfa, & Vingare, 2000), and creates the premise for atherosclerotic lesions as inflammation is initiated by the formation of foam cells (Chogle & Chakravaty, 2007). Inflammation has increasingly been established as the underlying factor for cardiovascular diseases in SLE where concentration of C-reactive protein has been a good biomarker for the assessment of the risk of CVDs in general population and patients with SLE (Abou-Raya & Abou-Raya, 2006). There is an accelerated and premature atherosclerosis in patients with systemic lupus erythematosus when compared to that of the general population as being reported (Bruce, Gladman, & Urowitz, 2000). Emerging reports suggest increase in prevalence of Systemic Lupus Erythematosus in Blacks resident in Africa. It is therefore important to understand various aspects of the disease pathophysiology and possible readily accessible diagnostic markers for early detection. This study therefore investigated how adipokines are associated with arterial stiffness in patients with systemic lupus erythematosus. 1.2  Problem statement Patients with SLE respond to treatment based on the traditional Systemic Lupus Erythematosus Disease Index (SLEDAI), American College of Rheumatology indices, and the criteria set by Systemic Lupus International Collaborating Clinics (SLICC), however, the mortality rate in these patients are increased with cardiovascular complications contributing to 50 % of all cases (Ben-Menachem, 2010).   4   University of Ghana http://ugspace.ug.edu.gh Systemic Lupus Erythematosus subjects have been shown in cohort study to have twice the hazards of all-cause mortality and composite death and new CVD events with 32% cardiovascular, 16 % renal failure with the rest being other factors as compared to control group (Bartels C. M., et al., 2104). Cardiovascular complications are major cause of deaths in this population as treatments of other complications have improved (Manger, et al., 2002). The traditional risk factors, based on which algorithm such as Framingham’s risk factors are computed do not completely predict CVDs in patients with SLE, meaning there are other factors that should be used to estimate cardiovascular risk in such patients. However, arterial stiffness has been established as a marker of cardiovascular morbidity and mortality, as well as potential therapeutic target, and is associated with systemic inflammation, which is also the primary pathophysiological cause of SLE (Mahmud & John, 2005). The chronic nature of SLE result in persistent inflammation leading to progressive premature aging of the large arteries. The endothelial-derived nitric oxide is reduced in half-life due to inactivation of nitric oxide (NO) by oxidative stress produced by inflammation and this induces endothelial cell dysfunction. Also, chronic inflammation is involved in the development and progression of atherosclerosis and arterial stiffness, at the same time inflammatory mediators promote leukocyte infiltration and activation of vascular smooth muscle cells and this in turn increase the expression and activity of MMPs to contribute to arterial stiffness (Xinkang, James, Allan, & Giora, 2008).   5   University of Ghana http://ugspace.ug.edu.gh Presently, clinicians manage Lupus disease with medicines such as non-steroidal anti- inflammatory drugs (NSAIDs), oral steroids, oral cyclophosphamide, corticosteroids, antimalarials and azathioprine (Marta, Guillermo, Munther, & Graham, 2001) for the management of pain and lupus nephritis (Ricard, et al., 2003). Meanwhile these drugs have been reported to cause transient and long term hypertension especially in young women (Curhan, Willet , Rosner, & Stampfer, 2002), this event is therefore suspected to worsen the course of patients with SLE because there could be potential double effect and during labour as stiffened arteries can contribute to eclampsia in these women especially when the prevalence of the disease is highest in women at their reproductive ages. Due to the knowledge gap on how SLE may hasten arterial stiffness, clinicians continue to prescribe those drugs, non-steroidal anti-inflammatory drugs (NSAIDs), oral steroids, oral cyclophosphamide, corticosteroids, antimalarials and azathioprine in an attempt to suppress pain and patients’ immune system in the management of the disease. It is critical and important that enough information is obtained to inform clinicians and drug developers to chat a new course for less contraindicated treatments regimen for such patients. Stiffened arteries cause reduction in arteries capacity to contain left-ventricular ejected blood which increases pulsatile afterload capable initiating remodeling of the ventricle, leading to poor functioning and eventually failing (Kawaguchi, Hay, Fetics, & Kass, 2003). Due to this phenomenon, when large arteries become stiff, there is a suspected promotion of damage to terminal organs like brain and kidney because of the direct penetration of high pulse energy into them (O'Rourke & Safar ME, 2005). Lack of proper investigation and establishment of novel diagnostic markers to monitor stiffening of arteries in SLE patients would continue increasing the risk of CVD casualties in such population.   6   University of Ghana http://ugspace.ug.edu.gh 1.3  Justification Adipokines which are useful in energy homeostasis, inflammation, angiogenesis and immune regulation is very important for consideration because of its role in Metabolic syndrome (MetS) frequently found in chronic inflammatory diseases like SLE. (Maria & Joao, 2009). Reduction in Leptin levels has been found to be in association with inflammation in models of autoimmune diseases (Lam & Lu, 2007). The rise in plasma C-reactive protein concentrations has also shown an association with leptin, calcification, vascular cell proliferation, and reduced distensibility of arteries. Also, leptin plays role in increase in blood pressure, and thus, likely contributes to the starting and further development of atherosclerosis (Beltowski, 2006; Beltowsku, 2006). A study has shown that leptin independently increases coronary artery disease risks in moderation (Wallace, et al., 2001). Maintenance of cells response to insulin and reduced inflammatory responses are centered on adiponectin levels. Furthermore, high levels of adiponectin prevent atherogenesis but reduced plasma adiponectin levels is an established risk factor that independently predicts deaths of cardiovascular diseases (Han, 2007). The association between adipokines, arterial stiffness, SLE, and eventually increment in the risk of cardiovascular complications needs to be thoroughly investigated as a potential independent maker for the diagnosis and prediction of increased CVD in patients with SLE. It is therefore vital that this study is conducted to clearly elucidate any association existing between adipokines and hardening of arteries in patients with SLE, in Ghana especially when literature provides little information on the disease in Ghana though clinic records show a rise in cases as well as increase in incidence in some countries in recent times (Pons-Estel et al., 2010).   7   University of Ghana http://ugspace.ug.edu.gh The results of this research would give more reliable information concerning the disease in Ghana which may settle present argument of involvement of the adipokines in the pathophysiology of the disease in other studies especially in indigenous black population resident in Africa. 1.4  Aim To investigate the relationship between arterial stiffness and levels of adipokines in systemic lupus erythematosus patients in Ghana. 1.5  Specific Objectives I.   To compare of arterial stiffness levels among SLE patients and non-SLE subjects of study population. II.   To examine arterial stiffness and the association between adipokines in SLE patients and non-SLE controls. III.   To compare the relationship between anthropometric indices and arterial stiffness in SLE patients. IV.   To compare severity of stiffening of arteries in patients with SLE and non-SLE individuals   1.6 Hypotheses I.   Compared to non-SLE individuals, SLE patients have high levels of arterial stiffness II.   Levels of adipokines correlate with degree of arterial stiffness. III.   Increasing body fat has an association with arterial stiffness. IV.   Increasing levels of C-reactive protein (a protein used to predict inflammation) is associated with stiffening of arteries   8   University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 2.0   LITERATURE REVIEW   2.1 Systemic Lupus Erythematosus (SLE) Autoimmunity is caused as a result of the body’s defense mechanisms attacking itself whereby endogenous components that would not be affected in normal physiological state. The pathophysiological states of autoimmune disease is due to that result from a list of self- intolerance and the resultant immune destruction of host tissues. Autoimmunity arise from different kinds of molecular and cellular activities and reactions. Progression of an autoimmune disease involves several linked processes where by lymphocytes identify internal antigens as foreign and starts destroying them which leads to the organ damage. In autoimmune diseases, the body’s own defense mechanism attacks other endogenous immune molecules which facilitate the progression of the disease. It may either involve only some specific tissues (such as thyroid glands, and beta cells of the pancreatic gland), where particular antigens that are specific to some tissues are involved, or can affect the whole body where many different tissues where almost all auto-antigens are included in the attack. (Von & Tan, 1995). As much as the disease concept may appear simplified, the actual mechanisms that are involved in the pathophysiology is very complex and this has made it difficult to find the exact underlying factors that interplay to bring about the disease. Because of the complex nature of autoimmunity, almost all aspect of organism is affected, from the genes and appearance to kinetics. In most cases the time between the onset of the disease to the time that any symptom is shown can be very long and the expression in same individual can vary.     9   University of Ghana http://ugspace.ug.edu.gh 2.2 Epidemiology of SLE, World and Africa Human autoimmune disease (HAD) can happen at any time (this affect in a whole, more than 5% of the the world population), and puts an appreciable burden of diseases and deaths in humans (Davidson & Diamond, 2006). It is reported that SLE has not been very common in Black Africans as have been seen in Black Americans and the Hispanics. Symmons, (1995), in his publication made a proposal that sort to suggest that the occurrence of SLE increased with the change of environment from Africa to Europe and North America (Symmons, 1995). Butcher, (1996) reported that, because malaria is endemic in Africa, it prevented the occurrence of autoimmune diseases like SLE among Black Africans (Butcher, 1994). Butcher’s postulate was based on the fact that the functional macrophage in immune process is prevented in malaria. Another report has stated that although the occurrence of SLE in people born and living in the central and southern parts of Africa has been increasing, SLE is still not common in those in western part of Africa (Ababio & Davis, 1994; McGill & Oyoo, 2002). That notwithstanding, there are still some studies that have reported an increasing cases of the disease in Black Africans, as follows, in South Africa by Dessein et al., 1988 and Tikly et al., 2008 (Dessein, Gledhill, & Rossouw, 1998; Tikly & Navarra, 2008); Adelowo et al., 2009 (Nigeria) (Adelowo & Oguntona, 2008); Ekwom et al., 2013 (Kenya) (Ekwom, 2013). The increased number of studies submitted as abstracts between 2007 and 2103 in a way validated the hypothesis of increasing cases of SLE in Africa. In 2007, there were only three abstract on SLE submitted during the 5th African League of Associations in Rheumatology (AFLAR) Congress held in Nairobi, Kenya, but the number increased exponentially to 15 abstracts during the 7th Congress organized in 2013 in Durban. It may be due to people being made aware of the disease.   10   University of Ghana http://ugspace.ug.edu.gh Some studies conducted in Cameroun, Zambia and Zimbabwe have reported similar results of increased prevalence of the disease in Black Africans (Duoalla, et al., 2013; Njogyu, et al., 2103) and Zimbabwe (Stein, Syoren, Davis, & Blankenberg, 1991). Problems such as lupus nephritis have also been reported to occur (Mody, Mody, & Haripersad, 2103; Adelewo, Olaosebikan, & Umezerike, 2013) and neuropsychiatric lupus (Adelewo, Ogunta, & Ojo, 2009). Other uncommon problems of SLE including digital gangrene has also bee documented (Adelowo, Olaosebikan, Ajani, & Omosebi, 2012). A study conducted in South Africa involving 36 cases of lupus nephritis found 14 Black children with SLE (Faller , Thompson, Kala, & Hahn, 2005). The female preponderance, as elsewhere, is common (Adelewo, Ojo, & Oduenyi, 2012) in other reports, example, Cameroun (Female: Male; 12:1); Nigeria (10:1) and Zambia (29:0). A study conducted in a rheumatology clinic in Nigeria by Adelewo, (2012), showed that SLE comprised 5.3% of a total of 1,250 cases that were seen (Adelewo, Ogunta, & Ojo, 2009). Serologic markers have also been seen to be present as found in Nigeria [antinuclear antibody (ANA) (95.7%); double stranded DNA (ds DNA) (54.4%); Anti- Sm (75.7%); Ro/SSA (69.7%) (Adelewo, Ojo, & Oduenyi, 2012). In South African blacks, ANA, ds DNA and Ro/SSA were (98.2%); (66.2%), (60.5%)] respectively. In Cameroon, ANA was (86%) (Tikly, Burgin, & Mohanla, 1996). There have been similar mean ages for reported cases in different countries such as South Africa was 35years in South Africa, 34 years in Kenya, 33 years in Nigeria and 38 years in Cameroun. SLE deaths have also been reported as high in Blacks by extensive studies among South African blacks (Budhoo, Mody, & Dubula, 2013). Most of the commonest symptoms of SLE such as skin rashes have not been mostly among Black population, however, polyarthritis and polyarthralgia have been found to be common, and also serositis.   11   University of Ghana http://ugspace.ug.edu.gh For instance, most of the patients in Nigeria presented with fever of unknown origin which had been variously diagnosed as malaria or typhoid fever. Various forms of alopecia locularis are quite common presentations as well as neuropsychiatric symptoms (Adelewo, Ogunta, & Ojo, 2009). 2.3 Hypertension in SLE patients Hypertension has very high prevalence of up to 65 % in SLE patients and partly account for the acceleration of atherosclerotic plaque formation and risk of cardiovascular diseases (Hashkes, Wexler, & Passo, 1997). One of the major clinical determinants for future cardiovascular events is blood pressure. Systolic hypertension has however found to be prevalent as one ages while good control of blood pressure at younger ages are suboptimal at ~50 % (Go, et al., 2014). Overcoming stiffening of the aorta could be an important intermediary to control resistant hypertension. When arteries hardened, it becomes difficult to control blood pressure, but when there is less hardening of the arteries, controlling of blood pressure is less difficult. (Protogerou, Blacher, Stergiou, Achimastos, & Safar, 2009). Additionally, some other studies have found out that, higher levels of aortic stiffness is likely to be in association with hypertension and other cardiovascular events (Mitchell, et al., 2010; Naijar, et al., 2008). Some studies have suggested that arterial stiffness regulate and also promote blood pressure, but others argue blood pressure rather contributes to arterial stiffness. But in all, enough evidence corroborate arterial stiffness involvement in hypertension related disease conditions. A study conducted in 2011, reported premature vascular ageing in young adults with hypertension which was characterized by stiff aorta (Kotsis V. , Stabouli, Karafi, & Nilson, 2011).   12   University of Ghana http://ugspace.ug.edu.gh The change in arterial stiffness occurring in hypertensive young adults is similar to that which occurs in old people without hypertension, therefore as BP reduces there is also a reduction in arterial stiffness in young people who have hypertension. This therefore suggests blood pressure contributes to making arteries stiffened at younger age (Harvey, Montezano, & Touyz, 2015). The two phenomenon has appeared to be in a negative feedback mechanism where each may moderate another. Hence blood pressure control could help control the rate of hardening of arteries and the risk of cardiovascular events. It has been suggested that, when the smooth muscles cells in the blood vessel wall are damaged, dilation of the vessel becomes difficult, there is an increment in constriction, more proliferation and migration as well as total difficulty in regulating the blood pressure and controlling arterial stiffness. 2.4 Cardiovascular complications of SLE The American College of Rheumatology does not consider cardiovascular complications as one of the criteria for diagnosis, rather the Systemic Lupus International Collaborating Clinics (SLICC) describe cardiovascular complications as a long-term damage. However, problems with the cardiac tissues start at early stage of SLE and can be improved when detected earlier and also help prevent other cardiovascular events. Myocardial infarction that lead to death in SLE is found to be thrice as high as in age and gender matched non-SLE (Esdaile, Abrahamowicz, & Grodzicky, 2001; Willerson & Ridker, 2004). Many studies have shown that deaths due to cardiovascular events are high in SLE. Several of the cardiac tissue pathologies in SLE such as pericardial, epicardial and endocardial diseases which are the commonest pathologies may be due to the autoimmune activities of SLE.   13   University of Ghana http://ugspace.ug.edu.gh It has wide range of clinical expressions ranging from patients without any symptoms to an acute left ventricular failure scenario which requires hemodynamic support (Recio-Mayoral, Mason, Kaski, Rubens, Harari, & Camici, 2009). A situation where blood supply to the heart tissues was blocked without any identifiable coronary artery disease (CAD) has been described in SLE. Research shows early death in patients with SLE due to cardiovascular diseases two years earlier before they are diagnosed of (Ishimori, Martin, Berman, Goykhman, Shaw, & Shufelt, 2011; Bartels C. M., Buhr, Goldberg, Bell, Visekruma, & Nekkanti, 2014), thereby supporting the assumptions that the inflammatory activity in SLE is a cardiovascular risk factor (Teixeira, Ferber, Vuilleumier, & Cutler, 2015; Elliott & Manzi, 2009; Arnaud, Mathian, Brucket, & Amoura, 2014; Bartels C. M., Buhr, Goldberg, Bell, Visekruma, & Nekkanti, 2014). A prevalence of 44 % cardiac MRI alterations assessed together with stress from perfusion of myocardium, with reduction in index of coronary perfusion reserves in addition to ordinary coronary angiography has been reported (Hashkes, Wexler, & Passo, 1997; Ishimori, Martin, Berman, Goykhman, Shaw, & Shufelt, 2011). The possible cause is likely to be due to shortfall in perfusion due to dysfunction of microvasculature of the coronary system (Recio-Mayoral, Mason, Kaski, Rubens, Harari, & Camici, 2009). Several studies have reported a prevalence of ischemic heart disease by clinical manifestations ranging between 8% and 16% ( Badui, Garcia-Rubi, & Robles, 1985; Gladman & Urowitz, 1987; Petri, Perez-Guttham, Spence, & Hochberg, 1992; Borchers, Keen, Shoenfeld, & Gershwin, 2004). When CAD is diagnosed early, the patients chances of living for longer time are higher and the prognosis of heart failure (HF) being improved. Clarke et al., (2014), estimates SLE management averagely cost over (US$10, 000) annually, more than what is spent in other connective tissue diseases (Clarke, Urowitz, Monga, & Hanly, 2014).   14   University of Ghana http://ugspace.ug.edu.gh Some other countries may have similar high expenditure but are not known because there hasn’t been any proper documentation. Together with the classical risk, CAD risk factors by Framingham ( Manzi, Meilahn , Rairie, Conte, Medsger, & Jansen-McWilliams, 1997; Esdaile, Abrahamowicz, Grodzicky, Li, Panaritis, & du Berger, 2001), the inflammatory activity of SLE further heightens coronary disease via the activities of systemic inflammation, loss of function of endothelial cells, and predisposition to thrombosis, and also adverse effects some of the administered drugs have on the cardiovascular system such as glucocorticoids (Elliott & Manzi, 2009; Arnaud, Mathian, Brucket, & Amoura, 2014; Teixeira, Ferber, Vuilleumier, & Cutler, 2015). Due to these factors, Framingham’s studies is not able to completely classify risk of cardiovascular diseases in SLE patients (Manzi, Meilahn , Rairie, Conte, Medsger, & Jansen- McWilliams, 1997; Esdaile, Abrahamowicz, & Grodzicky, 2001; Elliott & Manzi, 2009; Arnaud, Mathian, Brucket, & Amoura, 2014; Bartels C. M., Buhr, Goldberg, Bell, Visekruma, & Nekkanti, 2014; Teixeira, Ferber, Vuilleumier, & Cutler, 2015) Bartels et al., (2014) has revealed early deaths from CVD in patients 2 years earlier before they could be diagnosed of SLE (Bartels C. M., Buhr, Goldberg, Bell, Visekruma, & Nekkanti, 2014). It therefore support the argument on SLE inflammation being associated to risk of cardiovascular diseases, (Teixeira, Ferber, Vuilleumier, & Cutler, 2015) 2.5 Basic concepts of arterial hemodynamics The flow of blood is dependent on the pressure gradient, arterial diameter, hematocrit and peripheral resistance due to direction and orientation of individual blood vessels, these together defines the haemodynamics in each individual and may vary from person to person (Guyton &   15   University of Ghana http://ugspace.ug.edu.gh Hall, 2006). Despite these varying conditions, there is an established fundamental rules which blood flow adheres to (Guyton & Hall, 2006). Unlike regular water or oils that have constant viscosities and are described as Newtonian fluids, blood in living organisms has variable viscosity due to hematocrit of individuals. Also, the elasticity of the arteries contribute to flow and hence blood flow does not follow Newtonian fluid principles, erythrocyte deformation at capillary beds also account for variation in blood viscosity (Westerhof, Stergiopulos, & Noble, 2010). The in vivo characteristics such as pulsatility, arterial bends and branching affect blood flow and change in velocity distributions in the different layers. Under steady state, blood flows through long, smooth blood vessels in layers (lamina) such that there is a streamline flow of blood called laminar flow. In such state, the velocity of central laminar is greater while the peripheral (closer to the vessel wall) is lowest. This is caused by the adherence of cells to the vessel walls hence impeding flow. The subsequent layers towards the center of the vessel slip over their adjacent ones and wall-adherence-effect decreases toward the center of the vessel. This explains the high volume of blood that passes through larger arteries. In stiffened arteries, persistent laminar flow may facilitate atherosclerosis; the stacking of wall-adjacent blood traps VLDL and other cholesterols and may build up rapidly (Guyton & Hall, 2006). The flow of blood through vessels is universally determined by a simple Ohmic principle which states that the flow of blood is directly proportional to pressure difference and inversely proportional to peripheral resistance. The pressure difference in the arteries is particularly important since it determines the flow of blood. When there is stiffness in arteries, there is a possible flat pressure at all points in the artery and that would exacerbate peripheral resistance, this explains partially hypertension in patients with stiff arteries.   16   University of Ghana http://ugspace.ug.edu.gh When the left ventricle contracts, the ejected volume of blood is distributed in portions of 40 - 50 % to the peripheral tissues with majority of the volume maintaining aortic and large arteries pressure. That is, about 10 % pressure generated by the heart is dispensed in keeping constant the pressure in the arteries, this is to maintain constant and adequate pressure gradient (Nicholas & O'Rourke, 1998). At diastole, when the aortic valves are closed and pressure from the ventricles into the aorta has receded, the pulsatile flow of blood is transformed to a stable and continuous flow (Chirinos & Segers, 2010) in a peristaltic manner. The contracting and relaxing of the circular and longitudinal smooth muscle layers in the tunica media propel the blood downstream maintaining constant, the arterial pressure. This phenomenon requires energy, however, the lesser the required energy to dilate the aorta and central arteries at the time of ejection and recoil during filling/return, the more effective the dampening function (London & Pannier, 2010). In a normal (physiological) state, large arteries distend and close in response to this pressure dynamics, but in stiffened arteries there is a trans-mural pressure variation for every change in the vessel inner volume. Both diastolic and systolic pressures are affected by arterial stiffness in direct and indirect mechanisms (Nichols & O'Rourke, 2005). Stiffened arteries increase systolic blood pressure in direct mechanism. While normal arteries have compliance and distensibility to dampen the ventricular blood pressure in the aorta, stiffened arteries are not able to respond to such pressure-volume dynamics (Westerhof, Lankhaar, & Westerhof, 2009). As the ventricles contract to pump out blood, it generates a wave called pulse wave which moves along the arterial tree system at a given velocity, in stiffened arteries, the velocity of this wave is higher than in normal arteries, this has been found out to be affected by arterial stiffness levels, thus the stiffer the artery, the higher the pulse wave velocity (PWV).   17   University of Ghana http://ugspace.ug.edu.gh Pulse wave velocity is therefore used as indirect index of arterial stiffness (Quinn, Tomlinson, & Cockcroft, 2012). The value of of PWV is raised in stiffer arteries from the physiological value of 5 – 7 m/sec, wave moving along the pressure gradient reaches peripheral sites earlier than in normal arteries. The returned wave however proceeds to the heart during subsequent contraction rather than relaxation, this therefore leads to a lower diastolic blood pressure, higher systolic blood pressure and pulse pressure (Quinn, Tomlinson, & Cockcroft, 2012). 2.6 Hemodynamic consequences of arterial stiffness The proximal aortic segment is an elastic artery endowed with elastic fibers that give it extra distensibility and compliance. The distal arteries are muscular arteries with reduced distensibility. The pulse from the left ventricle ejection is dampened by this elastic artery reducing the pulse wave greatly. Beginning each cardiac cycle, the left ventricle initiates an onward pulse energy that causes an elevated pressure and anterograde flow within aortic root segment in systole onset. In a stiffened and/or narrowed lumen aorta, there is high characteristic proximal aortic impedance resulting in intensified pressure wave at each systole. This energy generated by the left ventricle which is the forward-travelling wave (incident wave) travels through the arterial walls and is partially reflected upon meeting points of bifurcation, change in tunica diameter, a turn or when there is an impedance mismatch. There is an association between the aortic PWV and aortic wall stiffness, (which is the root square of its elastic modulus) but is further affected by the positioning of the aorta (the length of the ascending and descending aortas, and aortic arch circumference) (Mitchell, 2009). When the aorta is stiffened, both the incident and reflected waves are conducted with greater velocity and hence reflected wave arriving at any given point after reflection regardless of the distance.   18   University of Ghana http://ugspace.ug.edu.gh Reflected wave magnitude therefore depends on the stiffness of the elastic and muscular arteries. When the reflected wave reaches the left ventricle too quick, it raises left ventricular preload, possibly leading to ventricular hypertrophy. The damaging effect of larger arterial stiffness include pulsatile penetration of narrow vessels at vascular beds in terminal organs like the brain, kidney and eyes (Mitchell, 2009; Kaess et al., 2012), causing microcirculation pathologies including hypertensive encephalopathy, hemorrhagic stroke, nephropathy (renal failure) and a plausible high intraocular pressure (Brunton, Parker, Blumenthal, & Buxton, 2008). High blood pressure may lead to the development of fulminant arteriopathy indicated by endothelial injury and notable intimal proliferation, ultimately blocking arteriolar perfusion and other life-threatening hypertension syndromes. In patients with isolated systolic hypertension, complex hemodynamics in stiff arterial system contribute to increased blood pressure, also some drugs may be mediated by changes in peripheral resistance as well as their effects on large artery stiffness (Brunton et al.,2008) 2.7 Elastic behaviour of arteries The adaptation of an artery is dependent on compliance and distensibility where compliance measures volume changes in response to blood pressure variation. As blood is pumped through the artery with high pressure, the arterial wall “opens up” to increase the diameter of the lumen. Distensibility on the other hand is the instantaneous opening of the luminal diameter to accommodate blood pumped by left ventricle, especially in larger arteries. The elastic and collagen arterial wall confers on the arteries this functional adaptation. As the gross elasticity, the direct inverse of stiffness is only a qualitative description, stiffness of arteries is best measured quantitatively by compliance and distensibility (Cecelja & Chowienczyk, 2012).   19   University of Ghana http://ugspace.ug.edu.gh Arterial stiffness is therefore an expression of the arterial wall to accommodate changes in pressure in respect of increase and decrease of luminal diameter. In recent times, measurement of arterial stiffness has become useful to predict cardiovascular complications (Gosling & Budge, 2003) and has made it very useful not only as a basic physiological tool but for clinical purposes as well. The prominence of this parameter is based on the fact that though various concepts including distensibility, compliance, PWV and elastic modulus have all been related to arterial stiffness but are not interchangeable (Nichols & O'Rourke, 2005) due to uniqueness of each concept and this makes the quantification of arterial stiffness complicated (Hughes, Dixon, & McVeigh, 2004). 2.8 Stress – strain relationship of arteries The application of arterial stiffness to predict cardiovascular diseases and deaths has been shown to be very useful in greater part of the population (Boutouyrie, Bussy, Lacolley, Girerd, Laloux, & Laurent, 1999), patients with hypertension (Paini, Boutouyrie, Calvet, Tropeano, Laloux, & Laurent, 2006; Bussy, Boutouyrie, Lacolley, Challande, & Laurent, 2000) atherosclerosis (Oliver & Webb, 2003) and myocardial infarction (Hirai, Sasayama, Kawazaki, & Yagi, 1989). Many studies have utilized relations in pressure and diameter in how arteries distend between one phase of systole and diastole, such as Peterson’s elastic modulus (Ep), stiffness index (b), compliance of arteries and arterial to propose carotid stiffness indices (Nichols & O'Rourke, 2005; Cheng, Tiwari, Baker, Morris, & Hamilton, 2002). There is a shortfall in those procedures since measurements taken from only one point is used for the generalizability occurrences in the whole arterial wall, however Young’s modulus is able to show every little change in all the various intimal (Claridge, Bate, Hoskins, Adam, Bradbury, & Wilmink, 2009).   20   University of Ghana http://ugspace.ug.edu.gh The contributions of the individual vascular wall layers to arterial stiffness is well established in the non-invasive in vivo Young’s modulus that estimates regional stress-strain relationships. (Danpinid, Luo, Vapou, Terdtoon, & Konofagou, 2010). Only a few studies have reported on the in vivo Young’s modulus measurement of the carotid artery based on the pressure–strain relationship ( Selzer, Mack, Lee, & Kwong-Fu, 2001; Claridge, Bate, Hoskins, Adam, Bradbury, & Wilmink, 2009; Arnett, Evans, & Riley, 1994), and also out of the gradient in the relationship between stress and strain (Nogata, Yokata, Kawamura, & Walsh, 2008) during one cardiac cycle. There isn’t any much studies on how the intimal layers interact mechanically within the body ( Cox, 1978; Nichols & O'Rourke, 2005). With a little increase in pressure, the elastic lamellae bulge while the collagen fibers stretches in length ( Fung, 1993; Humphrey, 2002)) to attain maximum stretchable length. The outer layer of the artery is therefore reinforced to prevent the artery from rapturing due to excessive stretching (Holzapfel, Gasser, & Ogden, 200). There is usually stiff arteries in older people because they have lower elastin and higher collagen (O'Rourke M. , 1990). The total stress on the circumference of the artery determines the spatial arrangement of arterial components and their elastic modulus. Ageing and hypertension have shown to increase the incremental Young’s modulus of carotid artery as function of blood pressure (Bussy, Boutouyrie, Lacolley, Challande, & Laurent, 2000). Carotid artery and abdominal arteries have both been share common characteristics in stiffening with ageing (Kawazaki, Sasayama, Yagi, Asakawa, & Hirai, 1987), however in young adults aged 20 years, carotid arteries had higher stiffness than the abdominal aorta (Ahlgren, Hansen, Sonesson, & Lanne, 1997), and in children (Martin, Hu, Gennser, & Norman, 2000).   21   University of Ghana http://ugspace.ug.edu.gh Both the aorta in the abdominal region and the carotid artery have varying levels of stiffness because each have different proportions of the structural proteins (Nagai et al., 1999). There is no validated association between carotid artery stiffness and aortic pulse wave velocity that predicts stiffening of arteries (Nagai, et al., 1998). 2.9 Pressure–volume and pressure–area relations Arterial stiffness plays significant role in the determination of risk of cardiovascular diseases (Protogerou, Blacher, Stergiou, Achimastos, & Safar, 2008), and several but complex methods such as assessment of local arterial blood flow and pulse wave velocity can be used to measure it. Pulse pressure at the periphery is also an alternative means to indirectly measure arterial stiffness and is able to independently predict cardiovascular-related deaths in normotensive and hypertensive patients (Benetos, et al., 1996). There is however, variation in pulse pressure along the length of the arterial tree due to difference in compliance at specific points as well as the process of wave reflection (Nichols & O'Rourke, 2005). It is also important to note that how much pulse pressure would be amplified depends on position of body, body activities and age of the individual (Nichols & O'Rourke, 2005). The pressure measured in the brachium alone is therefore not good enough to give trusted estimation of a central pulse pressure since central pressure is a determinant for workload of the left ventricle (Westerhof & O'Rourke, 1995). Central pressure waveform have shown an association with hypertrophy of the left ventricle which is able to independently predict mortality in normotensives (Saba, Roman, Pini, Spitzer, Ganau, & Devereux, 1993) and hypertensive individuals (Lakatta, 1991). Again, the central rather than peripheral pressure has an association with thickening of the intima-media which also predicts cardiovascular risks (Boutouyrie, Bussy, Lacolley, Girerd, Laloux, & Laurent, 1999).   22   University of Ghana http://ugspace.ug.edu.gh The waveform obtained from central arterial pressure can has adequate information that can be used to evaluate local and systemic arterial stiffness by the application of pulse wave analysis techniques (O'Rourke & Gallagher, 1996). Highly reliable waveforms are recorded from the radial artery by an applanation tonometry principles and mathematical transfer function with validation are used to derive corresponding central waveform (Karamanoglu, O'Rourke, Avolio, & Kelly, 1993; Takazawa, O'Rourke, & Fujita, 1996; Segers, Oasem, DeBacker, Carlier , Verdonck, & Avolio, 2001). Augmentation index is then calculated by analyzing the central waveform to deduce arterial determine arterial stiffness (O'Rourke & Gallagher, 1996) and the time it took the reflected pressure wave to travel back the ascending aorta used to evaluate aortic stiffness. 2.10 Arterial stiffness The pathophysiology of arterial stiffness involves physiological, cellular and molecular mechanisms. Also changes in structure and functions occur during stiffening of arteries and is as a result of the interactions between physiological and cellular/molecular events. The two scaffolding proteins, collagen and elastin determine how walls of arteries are resilient, compliant and stable. The production and degradation of these molecules are kept constant by a slowly but dynamic process to maintain optimum amounts at all times. When there is an inflammation, this balance is distorted leading to an overproduction of collagen and reduction in elastin thereby contributing to stiffness of (Johnson, Baugh, Wilson, & Burns, 2001). High pressure in the lumen of arteries, or hypertension, also stimulates unnecessary collagen production (Xu, Zarins, Pannaraj, Bassiouny, & Glagov, 2000). There supposed to be different ways through which inflammation leads to arterial stiffening.   23   University of Ghana http://ugspace.ug.edu.gh There could be an induction of rapid changes in large artery stiffening via effects of function on the cells of the endothelium or the cells of the vascular wall by some of the mechanisms. In autoimmune diseases, it is expected that there is long and progressive effect on these arterial tree leading to their indispensability. This is due to the fact that different processes could connect prolonged low-grade inflammation to changes in the structure of the arterial walls (Jain, Rohan, Vicente, Raymond, & Julio, 2014). This may be more related to the structural resistance which is more relevant to the structural disintegration which best describes stiffening of large arteries in chronic diseases such as SLE, although both the acute and chronic phases are interrelated to bring about arterial stiffness (Figure 1). As shown in Figure 1, the most possible mechanism for arterial stiffness in SLE is through the inflammatory pathway. Auto-immune attack which is the basic pathophysiology of SLE triggers inflammation and the subsequent downstream effects leads to accelerated arterial stiffness in patients with SLE. A very important factor that is used in controlling hypertension is aortic pressure. This was demonstrated by the REASON trial where increased arterial stiffness was found relating to poor control of blood pressure, whereas reduction of arterial stiffness contributed in the decrease and control of systolic blood pressure (Protogerou et al., 2009). Additionally, other investigations reported that, high aortic stiffness can be used to predict the occurrence of cardiovascular events. (Mitchell, et al., 2010; Naijar, et al., 2008). There are several arguments on the case of which precedes other, whether the presence of arterial stiffness result in the progression of high blood pressure or when there is a long-term untreated blood pressure, it eventually places stress on the vessel walls making them stiffer (Kotsis, Stabouli, Karafi, & Nilsson, 2011). However, there is no readily available technology to measure functions of smooth muscles in the aorta which is the major artery in the body.   24   University of Ghana http://ugspace.ug.edu.gh Figure 1:Possible mechanisms showing how inflammation initiate structural alterations in the vessel wall contributing to accelerated vascular stiffening.. Mφ: Macrophage, CRP: C reactive protein, ROS: Reactive oxygen species, O2: Superoxide, H2O2: Hydrogen peroxide, Akt/PKB: Serine threonine kinase/Protein kinase B SMC: Smooth muscle cell, MMP: Matrix metalloproteinases, TIMP: Tissue inhibitor of matrix metalloproteinases, BM: Basement membrane, ECM: Extracellular matrix, PO34: Phosphate, GAG: Glycosaminoglycan, TNF-a: Tumor necrosis factor a, IL-1: Interleukin-1. Adapted from Jain et al., (2014).         25   University of Ghana http://ugspace.ug.edu.gh 2.11 Indices of arterial stiffness In the circulation of blood, pulse wave which is a multifaceted physiological process is detected. As the left ventricle contracts, a particular volume of blood is pumped into the arteries due to the change in potential and kinetic energies at every portion of the flowing blood. At every point on the vessels where the pulse wave reaches the following three observations are made; flow of blood (flow pulse), blood pressure increment (pressure pulse) and transverse profile extension (profile of volume pulse). Numerous aggressive procedures exist for the detection of pulse wave. Geometry of the pulse waveform is determined by the physiology and pathophysiology, height and age of each organism and also differ at various sections of circulation route (Filipovsky, Svobodova, & Pecen, 2000), and also body fat and body mass index (Wykretowicz, Adamska, Guzik, Krauze, & Wysocki, 2007) belong to important physiological phenomena. The disease process in diabetes and arteriosclerosis have strong impact on elasticity of arteries. (Nicholas, 2005). In developed countries, arteriosclerotic-related cardiovascular diseases are the most prevalent, the arteries progressively lose elasticity and become non-distensible as grow from childhood to old age (Kelly, Hayward, Avolo, & O'Rourke, 1989; Nicholas & O'Rourke, 1998). Some metabolic syndromes have the propensity of hastening this process (Brooks, Molyneaux, & Yue, 1999). However, acceleration of arterial wall stiffening can be detected at the onset of arteriosclerosis course prior to any chage in the anatomy and clinical symptoms are seen. The increase in artery wall stiffness is noticeable from the beginning of the arteriosclerosis process, before anatomical changes and clinical manifestations are observed. Certain techniques assessing arterial stiffness make use of analysis of pulse waveform (Wilkinson, Maccallum, Flint, Cockroft, Newby, & Webb, 2000; Savage, Ferro, & Pinder, 2002).   26   University of Ghana http://ugspace.ug.edu.gh Presently, ankle-brachial index, augmentation index and pulse wave velocity application have been found out to identify vascular diseases at their early stages disease progression by assessment of arterial stiffness (Urbina, Brinton, Elkasabany, & Berenson, 2002; Williams, Lacy, & Thom, 2006). Pulse pressure increment has an association with arterial stiffness and negatively predicts risk of cardiovascular diseases (Madhavan, Ooi, Cohen, & Alderman, 1994; Fang, Madhavan, Cohen, & Alderman, 1995; Benetos, et al., 1997). Arterial stiffness has more effect on the pressure waveform of the proximal aorta, this waveform may provide adequate information together with pulse pressure. The central aortic pressure wave is composed of a forward- traveling wave generated by left ventricular ejection and a later-arriving reflected wave from the periphery (O'Rourke, Staessen, Vlachopoulos, Duprez, & Plante, 2002; Nicholas & Singh, 2002; Izzo, 2004; McVeigh, 2003). Velocity for wave transmission increases with increase increase in aortic and arterial stiffening, in such circumstances, there is an amplification of the pulse waves in both anterograde and retrograde movements. The reversing wave thereby gets to the central aorta to increase the pressure during late systole. This is because, the ventricle has to overcome both the end- diastolic volume pressure and the reflected pulse pressure at same time. Augmentation indices and augmented pressure are the absolute expressions for the reflected pulse wave pressure and is useful for the prediction of coronary artery diseases (Hayashi, Nakayama, Tsumura, Yoshimaru, & Ueda, 2002; Weber, et al., 2004; Imanishi, et al., 2004). 2.12 Structural components of arterial stiffening In an unprocessed samples of vascular tissues for pathological studies, the changes that occur in the molecules are seen to increase the thickness of the intima media two-three folds between the ages of 20 and 90 years (Nagai et al., 1998; O'Leary, et al., 1999) and also an overgrowth   27   University of Ghana http://ugspace.ug.edu.gh in the layers of smooth muscles of the vessels (Virmani et al., 1991). Histological examination of the intima of stiffened vessels reveals abnormal and disarrayed endothelial cells, increased collagen, frayed and broken elastin molecules, infiltration of vascular smooth muscle cells, macrophages and mononuclear cells, and increased matrix metalloproteinase, transforming growth factor (TGF), intracellular cell adhesion molecules (ICAM), and cytokines (Virmani, et al., 1991). In addition to vessel wall thickening, Lakatta et al., (2003), suggests ageing has relationship with steady increment of luminal diameter of central arteries, especially the ascending aorta, thus 9% in every 10 years from 20 to 60 years. (Lakatta, 2003; Watanabe, Sawai, Nagura, & Suyama, 1996). However, other latter researches have refuted that hypothesis (Mitchell, et al., 2003). Collagen and elastin are the structural proteins in vessel walls providing elasticity and structural integrity and are actively regulated by matrix metalloproteases, proteoglycans and glycoproteins forms the ground substance in which the structural proteins are embedded, together they form the ECM of vessel walls. (Jacob, 2003). Further, degradation of the basement membrane ECM and stimulation of chemotactic agents occur through gelatinase activation (MMP-2 and MMP-9) (Galis & Khatri, 2009; Li, Froehlich, & Galis, 1999). To regulate the activities enzymes, ROS, thrombin, plasmin and interaction of MMPs cleaves pro- MMP leading to post-translational activation and by augmentation in expression of genes (Dollery, McEwan, & Henny, 1995; Rajagopalan, Meng, Ramasamy, Harrison, & Galis, 1996; Visse & Nagase, 2003). The reaction is countered by TIMPs, regulation of remodeling is generally controlled by TIMPs. (Galis & Khatri, 2002). Accumulation of ground substance such as heparin and chondroitin sulfates, fibronectin and proteoglycans increases the thickness of the extracellular matrix of the arterial walls making it stiff. (Lakatta, 1993).   28   University of Ghana http://ugspace.ug.edu.gh Collagen molecules provide the tensile strength of the vessel wall and are enzymatically cross- linked soon after their formation to render them insoluble to hydrolytic enzymes (Reiser, McCormick, & Rucker, 1992). When the bonds between the molecules are broken, the collagen matrix is exposed, but because collagen has lower hydrolytic turnover, collagen is predisposed to the formation of cross-linkages without enzyme involvement. This increases a disorganized non-functional collagen fibers distribution. The formation of desmosine and its isoform from cross-linkage of elastin molecules make them stable. When those bonds are disrupted, the elastin matrix weakens and open up for minerals such as calcium and phosphorus to be deposited and increases arterial stiffness ( Spina & Garbin, 1976; Watanabe, Sawai, Nagura, & Suyama, 1996; Catell, Anderson, & Hasleton, 1996). Furthermore when metalloproteases and serine isoforms are activated, they damage elastin (Avalio, Jones, & Tafazzoli-Shadpour, 1998). Alterations in the production of elastin and some mechanisms for molecular repairs contribute loss of elastic properties of arteries ( Tokimitsu, Kato, Wachi, & Tajima, 1994; Robert, 1996; Bizbiz , Alperovitch, & Robert, 1997). The addition of carbohydrates to proteins after translation to form AGEs without enzymes involvement contributes to hardening of arteries by forming cross-linkages in long-lasting structural proteins like collagen (Lee & Cerami, 1992; Bailey, 2001). Collagen fibers with more AGEs are difficult to hydrolyze therefore accumulating non-functional collagen molecule (Verzijl, et al., 2000). Similarly, elastin molecules are susceptible to AGE crosslinking reducing the elastic matrix of the wall (Winlove, Parker, Avery, & Bailey, 1996; Konova, Baydanoff, Atanasova, & Velkova, 2004). AGE may also affect endothelial cell function by dowsing nitric activities of NO and causing rise in reactive oxygen species (Rojas, Romay, Gonzalez, Herrera, Delgado, & Otero, 2000).   29   University of Ghana http://ugspace.ug.edu.gh The AGE trigger stress signals, formation of reactive oxygen species, pro-inflammatory cytokines, vascular adhesion molecules and inflammation responses via their RAGE superfamily receptors. (Yan, et al., 1994; Throckmorton, Brogden, Min, Rasmussen, & Kashgarian, 1995). Those molecules are able to elevate vascular stiffness through matrix metalloproteases, (Kuzuya, Asai, Kanda, Maeda, Cheng, & Iguchi, 2001) contributing to dysfunction of the endothelium and increase smooth muscle tone, suppressing dilation mediated by endothelial flow and aggravate the reaction to vascular injury, disturbs angiogenesis and foster the formation of atherosclerotic plaques (Wendt, et al., 2002; Stern, Du, Fang, & Marie, 2002). When AGE interacts with its receptors, a pro-fibrotic response can occur aside the one caused by TGF pathway (Li, et al., 2004). There is no clear evidence that suggests that atherosclerosis and accumulation of lipids in the walls of arteries solely account for stiffening of arteries since people who are younger and have hypercholesterolemia in isolated cases present normal and highly compliant arteries (Lehmann, Watts, Fatemi-Langroudi, & Gosling, 1992). There is a negative correlation between low- density lipoprotein and arterial compliance as one ages because endothelial dysfunction becomes much dominant (Giannattasio, et al., 1996). There is no distinction between atherosclerosis and arterial stiffness due to their regular comorbidity. However, atherosclerosis pathophysiology involves similar stress/remodeling cascades mediated by oxidase, protease and inflammation that leads to reformation of vessels and alteration of the structures of collagen and elastin.   30   University of Ghana http://ugspace.ug.edu.gh 2.12.1 Collagen fibers Collagen fibers are structural proteins found in the large arteries which provide tensile strength. There are over 20 isoforms of collagen identified and are known to have effect on stiffening of arteries (Diez , 2007). Of all the 20 isoforms, type I and III are the most predominant with type I having the highest concentration in every part of the elastic artery (Tsamis, Krawiec, & Vorp, 2013). The predominance of type I and type III collagen isoforms is important physiological property of arteries. The type I provides tensile strength, and the type II which has some elastic properties enhances elasticity. The type I collagen in the three layers (the endothelium, smooth muscle and adventitial fibroblast) of the arteries increase as one ages. (Fleenor, 2013). The variation that exist in the change in composition of collagen I in each of the layers show its relevance of consideration in arterial stiffness. This was demonstrated in an experiment with collagenase-resistant mice where the inability to reduce collagen I produced higher mechanical stiffness (Vafaie, et al., 2014). Some studies have suspected that since collagen is particularly deposited on the abluminal layer of the arteries than the other layers with ageing, the adventitia may account for higher percentage as compared with the contributions of the other layers to the overall arterial stiffness. (Schulze-Bauer, Regitnig, & Holzapfel, 2002). One of the new forms of collagen that have been found to contribute to aortic stiffening in ageing is the type II collagen, which has been found to increase with ageing in both humans and rodents. (Jiang, et al., 2012). The type II collagen is a matrix protein which has been designed as a biomaterial that provide strength are is basically found in cartilage. Hence, increase in this protein (type II collagen) together with the type I have a potential of rapidly promoting arterial stiffness associated with ageing.   31   University of Ghana http://ugspace.ug.edu.gh 2.12.2 Elastin Blood vessels, especially arteries are able to distend and contract and have an elastic properties due to the presence of extracellular elastin protein which account for over 90 % composition of elastic fibers (Diez , 2007). The integrity of the protein is compromised in disease state and ageing thereby reducing the opening and recoiling abilities of the arteries and hence referred to them as being stiffened demonstrated in elastin haplo-insufficient model mice (Wagenseil, Nerukar, Knutsen, Okamoto, Li, & Mecham, 2005). Elastin fibers are present in all the arterial wall cells but highest in the intima media. (Fleenor, 2013). The amount of elastin reduces with ageing, children have more elastin in their arteries than the aged. Reduction of elastin has been found not to be reversible, once they are lost, it can not be replaced or increased by any known means, and hence very important to prevent its reduction in young people so as to avoid premature arterial stiffening when ageing. 2.12.3 Advanced Glycation End-Products (AGEs) Glycosylation, a post-translational modification of proteins, is also implicated in age and disease-induced aortic stiffening. Non-specific accumulation and cross- linking of extracellular proteins by AGEs has been shown to contribute to arterial stiffness in both aged rodents and adults (Fleenor, 2013; Samba, Najjar, Sun, Lakatta, & Ferrucci, 2009). AGEs have high intermolecular bonding properties resulting in elevated stiffness. Insofar this occurrence has been observed, accumulation of AGEs have been presumed to be because of one of the following mechanisms; high plasma glucose, increased oxidative stress in arteries and elevated signals of inflammation (Samba, Nicklett, & Ferrucci, 2010). These are thought to be the primary pathways for the formation of AGEs in arteries and therefore need further studies to unravel the mechanisms for each pathway. Both AGEs cross-linking and AGE cell signaling are considered the likely mechanisms through which arteries stiffen.   32   University of Ghana http://ugspace.ug.edu.gh 2.12.4 Calcium ions Accumulation of calcium within the walls of the vessels increased stiffness of the vessels in an in vivo experiment (Demer & Tintut, 2014). The increase in calcium levels with advancing in aging in the aorta correlates with that in the coronary arteries. (Collins, Munoz, Patel, Loukas, & Tubus, 2014). There was a correlation between vascular calcification and aortic stiffness among middle-aged paired group who did not have cardiovascular disease in the Framingham Heart study. (Tsao, et al., 2014). Increasing calcification of the aorta and hardening of the aorta is seen in early ages of forty years of in individuals (Sekikawa, et al., 2012). That is, the mechanism of calcification in important to be known since it leads to stiffening of the aorta. 2.12.5 Cellular modification The cumulative process of arterial stiffening is widely influenced by specific changes that occur in the smooth muscle cells and the cells of vascular (Qiu, et al., 2010; DeMarco, et al., 2015). High vascular smooth muscle cells stiffening has is known to be due to the alteration of actin, because there is reduction in VSMCs stiffening in aging when actin is reduced. (Qiu, et al., 2010). The linkages that occur between focal adhesions and actin to the extracellular matrix somehow explains why changes in the intracellular structure account for almost 50 % arterial stiffness is caused by VSMCs. (Gao, Saphirstein, Yamin, Suki, & Morgan, 2014). It seems both cytoskeleton of actin and focal linkages direct VSMCs role in overall stiffening of arteries. In an obese animal models, endothelial cells contributed to premature arterial stiffness. (DeMarco, et al., 2015). The observations were that, there was a 5-fold increment in the stiffness of endothelial cells analogous to that in VSMCs.   33   University of Ghana http://ugspace.ug.edu.gh A couple of other research have investigated how endothelial cells stiffens but do not clearly show the mechanisms by which this processes occur. In spite of this, it has been observed that, alpha actin and collagen I proteins of smooth muscle increased in their expression with aging in a model of senescent endothelial cells and so associate endothelial cells with arterial stiffness. The important factor is that TNF-alpha recapitulated the expression of collagen I and alpha actin of muscle cells in non-senescent endothelial cells (Fleenor, Seals, Zigler, & Sindler, 2012). For these reasons, stiffening of cells of the endothelium has become an evolving contributing factor that is suspected to be due to inflammation. This shows that the changes that occur in VSMCs and the endothelial cells highlights a new way of understanding hardening processes of arteries. To understand this processes better, there is a need for the determination of underlying mechanisms that cause VSMCs and cells of the endothelium to become stiffened when there is a disease or aging. 2.13 C-reactive protein (CRP) C-reactive protein comprises five subunits that are identical and has molecular mass of (110 – 140) kDa. The subunits are non-covalently arranged in a cyclic pentamer and is synthesized by the liver forming part of plasma and serum with very low concentration of 0.3 mg/dl. It has major role in bodies response to diseases. The concentrations rise within shortest time of general disease activities. In spite of this general relationship between the rise in CRP levels and different diseases, it has been a useful indicator of inflammation. Circulating C-reactive protein concentrations is a marker for inflammation and has shown direct correlation with coronary heart disease risks. Inflammation causes rearrangement of the structural components of arterial walls adversely causing loss of functional integrity of the vascular walls.   34   University of Ghana http://ugspace.ug.edu.gh This could be detected in early stages of vascular diseases, models of atherosclerosis have demonstrated atheroma formation by inflammation (Duprez, Somasundaram, Sigurdsson, Hoke, Florea, & Cohn, 2005). SLE disease progression has shown correlation with elevated serum CRP, extremely elevated in some patients (Williams, Harmaon, Burligame, & Du Clos, 2005). With association and as an inflammatory maker, CRP has been useful in the assessment of Cardiovascular risk in inflammatory diseases including Systemic Lupus Erythematosus (Nutall, Heaton, Piper, Martin, & Gordon, 2003). The elevated levels of CRP have been seen to be associated with SLE complications such as active renal involvement (Zuniga, Markowitz, Arkachaisri, Imperatore, D'Agati, & Salmon, 2003). Recent study by Mok et al., (2013) settles the long standing controversy on the association of CRP and hsCRP with disease progression and cardiovascular risk in SLE (Mok, Birmingham, Ho, Lee, & Rovin, 2013). 2.14 Adipokines Adipokines are hormones chiefly produced by adipocytes and these molecules participate in variety of physiological processes including food intake, insulin sensitivity, atherosclerosis, vasoactivity, immune cells regulation and modulation of inflammation. There is evidence that adipokines secretion is distorted in cardiovascular and immune system diseases (Marta, et al., 2013). Metabolism of adipocyte and levels of adipokines are modulated by generalized inflammation (Lago, Dieguez, Gomez-Reino, & Gualillo, 2007; Calabro, Limongelli, Pacileo, Di Salvo, Golino, & Calabro , 2008; Wang, Mariman, Renes, & Keijer, 2008).   35   University of Ghana http://ugspace.ug.edu.gh The various medicine for the management of SLE which are greatly steroids may be having effect insulin sensitivity, changes in plasma lipoproteins and some hormones which together alter functions and metabolism of adipocytes (Lago, Dieguez, Gomez-Reino, & Gualillo, 2007; Wang, Mariman, Renes, & Keijer, 2008; Calabro, Limongelli, Pacileo, Di Salvo, Golino, & Calabro , 2008). Juan et al., (2009), and 6 other papers have all reported reduction in leptin but elevation in visfatin and adiponectin (Juan, Mercedes, Nicolase, Jenny, & Liliana, 2009) while Garcia-Gonzalez et al., (2002) have observed increased leptin levels in SLE (Juan, Mercedes, Nicolase, Jenny, & Liliana, 2009). Meanwhile study by Marta et al (2011) indicates correlation between leptin levels and SLE activity and indicates higher values of leptin in SLE patients while adiponectin levels correlated with vascular strain (Marta, et al., 2013). 2.14.1 Adiponectin Adiponectin is an adipocytokine containing 244 aa, N-terminus-collagen-like domain, a C- terminus globular domain producing fat cells and a signaling peptide (Yoshihisa, Shinji , Tohru, Yuji, & Peter, 2006). The concentration of adiponectin in humans has been found to be 3-30 ug/ml which account for 0.01 % of total plasma protein (Arita, Kihara, & Ouchi, 2002). A study by Ouchi et al., (2001) showed reduction of intracellular cholesteryl ester content by suppressing expression of macrophage SR-As (class A scavenger receptors) in monocyte- derived macrophage human culture (Ouchi, Kihara, & Arita, 2001). Furthermore, adiponectin treated human macrophage inhibited phagocyte activities and lipopolysaccharide-induced tumor necrotic factor alpha (TNF alpha) production (Ouchi, Kihara, & Arita, 2001). Adiponectin may also favour plaque stability as it may increase secretion of IL-10 and following TIPM-1 (Kumada, Kihara, & Ouchi, 2004).   36   University of Ghana http://ugspace.ug.edu.gh When smooth muscle cells of blood vessel were cultured, adiponectin subdued the rapid division and movement by directly binding to growth factor-BB (PDGF-BB) that is obtained from platelets. It also inhibited extracellular-signal-regulated kinase (ERK) (Yoshihisa, Shinji , Tohru, Yuji, & Peter, 2006). Adiponectin, based on these findings by culture studies can be associated in modulating atherosclerosis progression by preventing cell proliferation and inflammation. A study conducted by Matsuda et al., (2002), demonstrated that mice models deficient of adiponectin had very thick intima and VSMCs aggregation after inducing mechanical arterial injury in them (Matsuda, Shimomura, & Sata, 2002). Same study showed reversal in neo- intimal thickening in adiponectin-deficient mice when supplemented with adiponectin through recombinant adenovirus (Matsuda, Shimomura, & Sata, 2002). Various in vivo mice-model studies have demonstrated adiponectin is able to suppress inflammation and atherogenesis (Yoshihisa, Shinji , Tohru, Yuji, & Peter, 2006). In humans, adiponectin has been found to decrease in CVD patients compared to healthy controls as reported by Giamila, (2008). Adiponectin production has been said to be inhibited by chronic inflammation and CVDs, meanwhile, the levels of adiponectin is reported to increase in inflammatory conditions that are not related to increased adipose tissue mass (Giamila, 2008). In SLE patients, increased levels of adiponectin has been observed, high levels of serum adiponectin in SLE has been observed with simultaneous high serum TNF alpha, which is rather one of the major inhibitors of adiponectin production (Sada, Yamasaki, Maruyama, Sugiyama, Yamamura, & Maeshima, 2006). There is also a negative correlation between adiponectin and insulin resistance in SLE patients although it is against the background of increased adiponectin levels with appreciable systemic inflammation (Giamila, 2008).   37   University of Ghana http://ugspace.ug.edu.gh 2.14.2 Leptin In the adipocytes, leptin is produced by ob genes and secreted by the adipose cells as hormones with molecular weight of 16 kDa. Leptin has receptors in the central nervous system which are cytokine-like receptors, which mediate metabolic activities and adipose tissue regulation. The release of leptin and its effect in the hypothalamus stimulates satiety centers thereby suppressing appetite, this makes leptin an anorexic (anorexigenic). Low fat corresponds to low levels of leptin in blood plasma of a healthy individual. In systemic lupus patients, the level of leptin is deregulated and has not been established yet to be associated with low or high fat deposition. The amount of leptin concentration in plasma usually reflects percentage body fat, also leptin acts through hypothalamic receptors and neurons to influence the perception of nutritional and energy status by the brain as well satiety. When the activity of leptin and its receptors are impaired, there is continuous appetite for food thereby leading to overeating and eventually resulting in obesity. The level of plasma leptin is reduced during fasting, hence could be a very important hormone for the control of obesity due to insulin resistance and hyperinsulinemia. 2.15 Insulin The beta cells in the islets of Langerhans located in the pancreas produce the hormone insulin as a 51 peptide residues. The metabolism of carbohydrates, lipids and proteins is actively regulated by insulin. Reduction in insulin concentration is signaled in the hypothalamus to stimulate another hormone in the pancreatic alpha cells to produce glucagon for the catabolism (breakdown) of glycogen. Uptake of amino acids and electrolytes by cells, triglycerides storage and release are affected by insulin as well as tone of small muscles in blood vessels. The secretion of adipokines by adipose tissues actively modulates several pathways that involve energy intake and expenditure, insulin-resistance and atherosclerosis.   38   University of Ghana http://ugspace.ug.edu.gh Insulin resistance occurs via two mechanisms, type A and type B. Type A has frequently been observed as disorders involving defects in the insulin receptors or in the post-receptor metabolic pathway (Yuuka, Yoshiro, & Yasuo, 2008). The type B is characterized by insulin receptor antibodies. SLE patients have been reported presenting with anti-insulin receptor antibodies (Arioglu, Andewelt, Diabo, Bell, Taylor, & Gorden, 2002; Moller, Ratner, Rosenstein, & Taylor, 1987). 2.16 Cardio-ankle Vascular Index as a measure of Arterial Stiffness Cardio-ankle vascular index (CAVI) is the derivation of resistance or compliance between the root of the aorta to the tibial artery at the ankle along the length or the arterial tree and is independent of arterial blood pressure. CAVI measures the level of arterial stiffness from the root of the aorta through the main descending length of the arterial tree including thoracic aorta, abdominal aorta, the common iliac artery, femoral, and tibial arteries. The CAVI is obtained by measuring the distance between aortic valve and ankle, as well as the duration from closing of aortic valve to detection of pressure change in the artery at the ankle (Yeboah, 2016). CAVI make use of pulse wave velocity (PWV), pressure from diastole and systole as well as pulse waveform of arteries are obtained from ECG, pressure cuffs and appropriate positions and PCG. (Sun, 2013). CAVI, unlike PWV measures organic stiffness (collagen, elastin and soft tissue matrix) of arteries and smooth muscle contracture in arteriosclerotic and atherosclerotic situation rather than blood pressure difference at different points. That is, CAVI does assess both functional and organic stiffness (Shirai, et al., 2013). This makes CAVI superior measure for the correct prediction of cardiovascular risk as it assesses progressive loss of compliance and distensibility in a chronic inflammatory disease state such as in systemic lupus erythematosus.   39   University of Ghana http://ugspace.ug.edu.gh Study by Shirai et al (2013) showed that a selective B1 receptor blocker that decreased both systolic and diastolic blood pressures for 6 hours did not change CAVI but baPWV decreased, indicating that CAVI is not influenced by blood pressure (Shirai, et al., 2013). On the other hand, Zarins et al (2010), has given an account of how alpha-1 adrenergic receptor blocker caused reduction in CAVI scores by relaxing the smooth muscles of peripheral arteries (Yeboah, 2016).   40   University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 3.0 METHODOLOGY   3.1 Study design and population The study was a case-control design. The cases were patients with systemic lupus erythematous (SLE) and the controls, age and gender matched non-SLE individuals. Due to the low prevalence of SLE among Ghanaians, the SLE patients were recruited conveniently as any consenting (Appendix III) adult who met the eligibility criteria, and the non-SLE participants were randomly sampled from staff and students of Department of Biomedical Sciences, University of Cape Coast. 3.2 Setting The study was conducted at the Out-Patient Department at the Rheumatology Unit, Department of Medicine and Therapeutics, Korle-Bu Teaching Hospital. The unit has temperature- controlled room that supports clinical examination and research. 3.3  Eligibility criteria Inclusion criteria i.   SLE patients and non-SLE individuals who are within the age range of 16 - 60 years were recruited as cases and controls respectively. ii.   Participants who clearly understood what the study is about and willingly agreed and gave informed consent. Exclusion criteria   i.   Participants with limb amputation and arterial reconstruction surgery. These individuals were identified based on their medical history records and physical appearance at presentation. ii.   Patients with diabetes and SLE co-morbidities.   41   University of Ghana http://ugspace.ug.edu.gh 3.4  Sample size The formula for prevalence below was used to calculate the sample size. % 
n   = $ & '(&% ) With confidence level of 95 % and prevalence of 20 %, estimated sample size would therefore be 1.962   𝑛 = 𝑥  0.2  (1−0.2)2 = 5.67'89  :.;  (:.6<) = :.8<8 = 67.6 0.1 :.:' :.:' The minimum number of SLE patients needed for this study is 68. A total of 78 SLE patients and 97 apparently healthy individuals were recruited for this study. 3.5  Participants recruitment and data collection Patients with SLE were recruited from the Rheumatology unit in the Department of Medicine by convenient sampling method. The controls were however recruited by simple random sampling method. The nature, rationale, procedure, risks and benefits of the study were explained to all the participants. They were allowed to ask questions and appropriate answers were provided by the researcher. After that, the researcher met privately with the invited individuals and addressed any personal concerns of the invitee, clarifying any doubts before recruiting them into the study by registration and signing or thumb-printing the informed consent form. The study participants were given a special identification number. Those who declined to participate in the study were thanked for their time and allowed to go for their normal clinical routine care. The controls were recruited from the general public who responded to the invitation to participate in the research.   42   University of Ghana http://ugspace.ug.edu.gh 3.6  Blood pressure measurement Systolic and diastolic blood pressures were measured using a semi-automated digital blood pressure monitor (Omron 991XL, Healthcare, Inc., Vernon Hills, IL). Before blood pressure measurement, the participants were asked the last time they passed out urine and instructed to empty their bladder if they had not passed out urine within the last 4 hours. The blood pressure cuff was placed on the right arm of the subject lying in a supine position; with the lower edge of the cuff about 2-3 cm above the elbow crease and the bladder was centered over the brachial artery. The arm was made to rest on a table and raised so that the cuff was at level with the heart. The subject was allowed to rest for at least 5 minutes to acclimatize with this condition. The blood pressure was measured three times; spacing with at least 60 seconds interval with the succeeding measurements. 3.7  Anthropometry Weight was measured in kilograms (kg) and reported to the nearest 0.1 kg, using a heavy-duty floor scale (Secca, Hamburg, Germany) with the participant standing upright on the platform, in light clothing with footwear and any heavy jewellery removed from the body; both feet directed forward and arm by the side of the body. Body height was measured in metres (m) and reported in 0.01 m, with a clinical measuring rule in a similar faction. Body mass index (BMI) was calculated by the ratio of the weight to square of the height. BMI categorised as 2 2 underweight (BMI < 18.50 kg/m ), normal weight (BMI: 18.50 – 24.99 kg/m ), overweight 2 2 (BMI: 25.00 – 29.99 kg/m ) and obese (BMI ≥ 30 kg/m ) (WHO, 2011).   43   University of Ghana http://ugspace.ug.edu.gh A stretch-resistance tape measure was used to measure the circumferences of the waists and hips, and values recorded in (0.1 cm). The tape was placed around the region between the midpoint from the lower margin of last palpable rib to the iliac crest. The tape was placed at the widest part of the hip around the peak of the gluteus maximum muscles to obtain the hip circumference. Waist and hip measurements were made with the tape held snugly, but not constricting, and at a level parallel to the floor (Yeboah, 2016) . Waist-to-hip ratio (WHR) was computed as waist girth/hip girth. Also, waist-to-stature ratio (WSR) was computed as waist girth/body height. The cut-off point for abnormal waist girth set at 90 cm for males and 88 cm for females (Levitt et al., 2006; Yeboah, 2016) The body composition was measured using the Omron Body Composition Monitor (BF- 506, Omron Healthcare, Inc., Vernon Hills, IL, USA). The participant’s data such as age, gender and height were entered into the equipment and the subject asked to stand upright (straight torso) on the platform in the same condition as the weight measurement. The subject then grabs the grip electrodes of the monitor by placing the palm of their hand on the top and the bottom of the electrodes while placing their thumbs up, resting on the top of the unit, and stretches the arms forward to approximately 90° to the axis of the body. The device works by transmitting a very low electric current usually 500mA and 50 kHz throughout the body which can not be felt or detected by the body and is able to measure quantity of fat tissues. When the electric signals are passed through the body, high water containing structures like muscles, bones and blood vessels are able to conduct the current whereas poor electricity conducting fat cells reflect the current back, the device then detect the impedance as fraction of body made up with fat. The proportion of fat in the body is calculated using five variables: electric resistance, height, weight, age and sex.   44   University of Ghana http://ugspace.ug.edu.gh 3.8  VaSera The VaSera VS-1500 (Fukuda-Denshi Company, Ltd, Tokyo, Japan) was used to measure cardio-ankle vascular index (CAVI) and heart-ankle PWV. After explaining to the participant the procedure involved in the assessment, they were allowed to rest in supine position for at least 10 minutes. Colour coded blood pressure cuffs were wrapped around both arms and ankles of the participant; lead I ECG electrodes was placed on both wrists and a phonocardiogram placed on the sternum to detect heart sound. The subject’s data such as unique identifier, initials, date of birth, height and weight entered into the equipment. The measurement starts with the four cuffs inflating simultaneously to 50 – 60 mmHg to detect brachial and ankle pulse waves. After a waiting time of 8 seconds, the cuffs are pressurized to measure the BPs of the right arm and ankle, followed by BP measurement of left arm and ankle 5 seconds afterwards. Pulse wave velocity was obtained by dividing the distance between the aortic valve and the ankle (L) by the time during which pulse waves travel that distance (T). T is difficult to obtain, because the exact time of the beginning of blood expulsion through the aortic valve is difficult to identify from the valve’s opening sound. T was therefore obtained by summing the time between the aortic valve’s closing sound and the notch of the brachial pulse wave (tb) and the time between the rise of the brachial pulse wave and rise of the ankle pulse wave (tba), in place of the time between the aortic valve’s opening sound and the rise of the brachial pulse wave (tb) and tba. Theoretically, tb and the time between the closing of aortic valve and the notch of the brachial pulse wave, tb, are equal (Figure 1) (Shirai et al., 2011; Yeboah, (2016). The scale conversion constants, ‘a’ and ‘b’, in are computed automatically in the equipment. After the measurements, the data obtained were analyzed using VaSera® Data Management Software, VSS-10 software (Fukuda Denshi, Tokyo, Japan), and the CAVI, PWV and ABI values of right and left sides calculated.   45   University of Ghana http://ugspace.ug.edu.gh Figure 2: Cardio-ankle vascular index(CAVI) being measured in supine patient. Adapted from Shirai et al (2011) cited in Yeboah (2016).   46   University of Ghana http://ugspace.ug.edu.gh 3.9  Blood sample collection, processing and storage An amount of 10 ml of venous blood sample were collected from the antecubital (medial cubital vein) into vacutainer tubes, using single-use disposable sterile syringe under aseptic conditions. The blood samples were collected into two vacutainer collection tubes: 5 ml into plain tube + with clotting activator (red-top) and 5 ml into sodium ethylenediaminetetraacetic acid (Na - EDTA) tubes (purple-top). The collection tubes, containing the blood sample, were immediately chilled on ice prior to centrifugation. Within 15 minutes of sample collection, the + collection tubes were centrifuged at 4000rpm; 15 minutes, for plain and Na -EDTA collection tubes. Plasma and serum samples were collected into Eppendorf tubes and stored at -80oC. 3.10   Biochemistry analysis 3.10.1   Plasma lipid profile assay Lipid profile of plasma was analyzed using Selectra Junior chemical auto analyzer from Namarka, using ELITech cholesterol SL, ELITech cholesterol HDL SL 2G and ELITech triglycerides Mono SL New reagents from ELITech clinical systems, France, following the manufacturer’s instructions. The total amount of cholesterol (TChol) in the plasma was assayed after enzymatic hydrolysis and oxidation. Briefly, cholesterol ester in the plasma was hydrolyzed by cholesterol esterase to form cholesterol and fatty acids. Cholesterol ester + H2O cholesterol ester cholesterol + fatty acids The cholesterol was oxidized afterward, by cholesterol oxidase to form cholestene-3-one and hydrogen peroxide. Cholesterol + O2 cholesterol oxidase cholestene-3-one + H2O2   47   University of Ghana http://ugspace.ug.edu.gh Plasma triglycerides (TG) were assayed after enzymatic hydrolysis with lipases. Triglycerides are hydrolyzed by lipases to form glycerol and fatty acids. Triglycerides + H2O lipases glycerol + fatty acids Phosphate is transferred from adenosine triphosphate (ATP) to glycerol, under catalysis of glycerolkinase (GK), to form glycerol-3-phosphate, which is oxidised by glycerol-3-phosphate oxidase (GPO) to form dihydroacetonephosphate and hydrogen peroxide. Glycerol + ATP GK glycerol-3-phosphate +ADP Glycerol-3-phosphate + O2 GPO dihydroacetonephosphate + H2O2 The hydrogen peroxide formed reacts, under the catalysis of peroxidase, with phenol and 4- aminophenazone to form a red-violet quinoneimine dye as indicator. 2H2O2 +4-aminophenazone +phenol POD quinoneimine + 4 H2O The concentration was determined by the equipment after reading the absorbance of the indicator at a wavelength of 500 nm. HDL cholesterol was assayed by the precipitation method. 500 µL of diluted precipitant solution, containing phosphotungstic acid in the presence of magnesium, was added to 200 µL of the plasma sample. The sample was allowed to sit for 10 minutes at room temperature and centrifuged afterwards at 4000g for 10 minutes to precipitate low density lipoproteins and chylomicrons. The HDL cholesterol was assayed from the supernatant solution at an absorbance of 500nm. The levels of LDL cholesterol were calculated from Frieldwald’s equation, LDL = TChol – (HDL+TG/2.2). The study subjects were further classified based on their lipid profile, FPG, BMI, WC and BP measurement into various CVD risk groups according to the NCEP-ATP III criteria of MetS in other to determine the prevalence of MetS.   48   University of Ghana http://ugspace.ug.edu.gh MetS was diagnosed using the NCEP ATP III criteria (NCEP, 2001) of 3 or more of the following: ∗   High fasting plasma glucose (FPG) ≥ 6.1mmol/L. ∗   High blood pressure (BP) ≥130/85mmHg. ∗   Decreased high density lipoprotein (DHDL) cholesterol (men ≤0.9mmol/L, women ≤1.01mmol/L). ∗   Raised Triglycerides (RTGL) ≥ 1.7mmol/L. ∗   Abdominal/ central obesity (high waist circumference): ≥ 102cm in men, ≥88cm in women (WHO, 2011) 3.10.2   Adiponectin and leptin enzyme-linked immunosorbent assay   Method described by Marta V et al., (2013) was used. Serum leptin and adiponectin levels were determined by commercial sandwich ELISA kits (Leptin sandwich ELISA kit EIA- 2395, DRG Instruments GmbH, Germany) with a 6.2% intra-assay precision, a 6.5% inter-assay accuracy and an analytical sensitivity of 1 ng/ml. Serum leptin and adiponectin were assayed using enzyme linked immunoassay (ELISA) method with duo set ELISA kits from R&D systems (United Kingdom). Sample Dilution For leptin, an amount of 1 µL of the serum samples was added to 499 µL of regent diluent to achieve 1:500 dilutions; and also for adiponectin, 1 uL of the serum was added to 499 of reagent diluents to achieve1: 500 dilutions. The samples were then stored in a refrigerator at a temperature of 2ºC and used for the assay within one week.   49   University of Ghana http://ugspace.ug.edu.gh Plate Preparation a.   Plate Coating with Capture Antibody: Before the assay of adiponectin and leptin, capture antibody was used to coat the wells in ELISA microplate overnight. For adiponectin, anti-human adiponectin was reconstituted with 1.0 mL of PBS to obtain a concentration of 720 µg/mL, which was further diluted in PBS to achieve a working concentration of 4 µg/mL. For leptin, anti-human leptin was reconstituted with 1.0 mL of PBS to achieve a concentration of 180 µg/mL, which was diluted further in PBS to a working concentration of 1.0 µg/mL. 100 µL of the diluted capture antibody solution was used to coat ten 96-well microplates (five for each hormone) overnight at room temperature. b.   Washing: After overnight incubation, each well was aspirated and washing was done by filing each well with 400 µL of wash buffer twice, for a total of three times, using an auto washer. Any remaining wash buffer was removed by blotting the microplate against paper towels. c.   The microplates were blocked by adding 300 µL of reagent diluents to each well and incubated for a minimum of 1 hour. d.   Washing: The washing process was repeated as in step ‘b’. Assay procedure e.   Sample and standard addition: In each well, 100 µL of sample or standard in reagent diluent was added, covered with adhesive strip and incubated for 2 hours at room temperature. f.   Washing: Washing process repeated as in step ‘b’. g.   Detection antibody addition: a volume of 100 µL of detection antibody (diluents with biotinylated mouse anti-human leptin detection antibody for leptin and also biotinylated mouse anti-human adiponectin detection antibody for adiponectin), diluted in reagent   50   University of Ghana http://ugspace.ug.edu.gh diluents, was added to each well. The microplates were covered with new adhesive strips and incubated for 2 hours at room temperature. h.   Washing: The washing process was repeated as in step ‘b’. i.   A volume of 100 µL of diluted Streptavidin-HRP was added to each well, covered and incubated for, at room temperature, for 20 minutes. The plates were not placed in direct light. j.   The washing process was repeated as in step ‘b’. k.   A volume of 100 µL of substrate solution was added to each well, incubated for 20 minutes at room temperature in darkness. l.   A volume of 50 µL of stop solution was added to each well. m.   The optical density was read at 450 nm using a microplate reader. The optical densities were converted into concentrations by using four-parameter logistic curve fit programme, Auditable Data Analysis and Management System for ELISA (ADAMSEL v1.1). 3.10.3   C-Reactive protein and Insulin enzyme-linked immunosorbent assay The conventional method for CRP quantitative assay was adopted as described by SIGMA – ALDRICH. The CRP ELISA kit is a solid phase direct sandwich method. The samples and anti- CRP-HRP conjugate are added to wells coated with MAb to CRP. CRP in the subjects’ serum binds to anti-CRP MAb on the well and the anti-CRP second antibody then binds to CRP. Unbound protein and HRP conjugate are washed off by wash buffer. Upon addition of the substrate, the intensity of color is proportional to the concentration of CRP in samples and read by a plate reader. For Insulin, Plasma insulin concentration method was used to measure sensitivity of insulin in participants.   51   University of Ghana http://ugspace.ug.edu.gh The product of fasting plasma insulin and fasting plasma glucose levels in addition to their ratios were used as proxies for insulin action. This method compared to the insulin clamp method has shown good correlation to insulin sensitivity (Ferrannini & Mari, 1998). To obtain optimal results, accurate and precise pipetting as well s adherence to protocol was ensured. i.   The reagents were allowed to stand at room temperature (18-26°C) prior to assay preparation. The following steps were followed ii.   The reagents were gently mixed iii.   The desired number (change after actual work) of coated strips were placed into a holder iv.   Samples and controls were diluted 100-fold by adding 5 µl of sample or control to 495 µl of sample diluent. v.   A volume of 10 µl of the standard, diluted samples and controls were picked into appropriate wells. vi.   Each of the wells were then filled with 100 ul of enzyme conjugate. Air bubbles were carefully removed from wells and mixture well mixed vii.   The mixture was incubated for 60 minutes at room temperature (18-26°C) viii.   The liquid was removed from all wells, and washed wells 3 times with 300 µl of 1x wash buffer. It was then blotted on absorbent paper towels. ix.   100 µl of TMB substrate was added to all wells. x.   It was then incubated for 15 minutes at room temperature 50 µl of stop solution was added to all wells and plate swirled gently to mix the solution after which absorbance was read on ELISA reader at 450 nm within 15 minutes after adding stop solution and the results recorded.   52   University of Ghana http://ugspace.ug.edu.gh 3.11   Data Handling Each participant was assigned a unique code which corresponded with all data obtained from such individual. The data obtained were computed into an Excel spreadsheet, cleaned before importing into SPSS database. A secret code was placed on the document to ensure adequate security while the hard copies of the questionnaire were locked under lock and key. 3.12   Statistical analysis Data obtained from this study was analyzed using Statistical Package for Social Sciences (SPSS) software, version 20. Normally distributed data were presented as mean ± standard deviation and compared by independent t-test. Categorical data were presented as frequency (percentage) and analyzed by Chi-square (χ2) test when necessary. Association between variables was analyzed using Pearson’s correlation for normally distributed data and Spearman’s correlation for non-normally distributed data. A 95% confidence interval was used and considered a value of p <0.05 as statistically significant. Independent t-test was used to analyze means of data with two predictors and non-normal distribution variables presented as median (interquartile range). 3.13   Ethical approval Ethical approval was granted (Appendix I) by the Ethics and Protocol Review Committee of the College of Health Sciences, University of Ghana (Protocol Identification number: CHS- Et/M.4 – P 4.2/2016-2017). Helsinki Declaration on Human Experimentation, 1964, with subsequent revisions, latest Seoul, October 2008 (Williams, 2008) was appropriately adhered to during the research. Only participants meeting the eligibility criteria were recruited for the study. All study participants were adequately informed of the purpose, nature, procedures, risks and hazards of the study. A written informed consent (Appendix III) was obtained from each of the participants.   53   University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4.0   RESULTS   4.1 General description of the Study Population The anthropometric characteristics of the participants are shown in Table 1. There was no difference in anthropometry characteristics between patients with SLE and non-SLE participants. 4.2 Gender distribution of participants For gender distribution, as expected, there was higher proportion (p<0.010) of female participants in both cases and control groups (Figure 3). 4.3 Biochemical characteristics of participants There were no significant differences in all the biochemical characteristics between the two study populations (Table 2). 4.4 Inflammation and adipokines The level of inflammation was assessed by the plasma concentration of C-reactive protein and adipokines by leptin and adiponectin. The results showed that, compared to non-SLE participants, SLE patients had higher levels of CRP, leptin and insulin. However, the levels of adiponectin were lower (p<0.04) in the cases as compared to the control as shown in Table 3. 4.5 Haemodynamic and arterial stiffness indices The haemodynamic characteristics measured includes systolic blood pressure, diastolic blood pressure, mean blood pressure and pulse blood pressure. While there were no significant   54   University of Ghana http://ugspace.ug.edu.gh differences in systolic, diastolic and mean blood pressures between the SLE patients and non- SLE participants, pulse blood pressure of the non-SLE participants was significantly higher than SLE patients (p<0.009). In the SLE group, compared to the non-SLE participants, ABI was lower in both legs (Table 4).   55   University of Ghana http://ugspace.ug.edu.gh Table 1 Anthropometric characteristics of Patients and Control Anthropometric Study subjects P parameter (mean±SD) SLE (n=78) Non-SLE (n=97) Age, years 31.6±8.5 28.9±10.4 0.058 Height, cm 165±7 163±8 0.286 Weight, kg 70±15 69.9±16.5 0.995 BMI, kg/m2 25.8±5.4 26.3±6.6 0.571 Data presented in mean±standard deviation and analyzed by t-test. BMI: body mass index, difference in means determined by independent t-test   56   University of Ghana http://ugspace.ug.edu.gh 120 Male 100 Female 80 60 40 20 0 SLE Non-SLE SLE status Figure3: Gender distribution of participants. Compared to males, the proportions of females were higher in SLE (2.5% vs 97.5%) and non-SLE (39.2% vs 60.8%, p<0.010).   57   Percent University of Ghana http://ugspace.ug.edu.gh Table 2 Biochemical characteristics of Patients and Control Parameter SLE (Mean±SD) Non-SLE P value (Mean±SD) Urea, mmol/l 3.0±1.8 2.7±1.6 0.257 Creatinine, mmol/l 79.4±29.7 78.8±19.1 0.884 Total Cholesterol, mmol/l 4.8±1.8 4.9±1.2 0.656 Triglyceride, mmol/l 0.97±0.47 1.0±0.34 0.569 HDL cholesterol, mmol/l 1.3±0.09 1.3±0.096 0.428 LDL cholesterol, mmol/l 3.11±1.69 3.2±1.18 0.72 vLDL cholesterol, mmol/l 0.47±0.32 0.46±0.15 0.753 Data presented as mean±standard deviations. Mean differences were analyzed by independent t-test.   58   University of Ghana http://ugspace.ug.edu.gh Table 3 Serological parameters of Patients and Control Parameter SLE Non-SLE P C-reactive protein, mg/l 1.6(0.8 – 2.2) 0.9(0.6 – 1.2) 0.021 Adiponectin, ng/ml 1.1(0.8 – 2.3) 1.6(1.3 – 2.6) 0.039 Leptin, ng/ml 856.1(364.8 – 1509.3) 426.8(84.7 – 1178.7) 0.039 Insulin, pmol/ml 76(45.9 – 184.8) 39.8 (22.9 – 86.3) 0.007 Data presented as median and inter quartile range. Mann-Whitney u-test was used to compare the medians.                                 59   University of Ghana http://ugspace.ug.edu.gh Table 4 Haemodynamics and atherosclerotic indices of Patients and Control Groups. Haemodynamic Subject’s Category P Parameter (mean±SD) SLE Non-SLE Systolic BP, mmHg 126±14.1 130±14 0.064 Diastolic BP, mmHg 79±11.9 80±10 0.684 Mean BP, mmHg 95±12 98±11 0.06 Pulse BP, mmHg 47±8 50±9 <0.009 Atherosclerotic indices right ABI 0.96±0.11 1.02±0.82 <0.001 left ABI 0.99±0.11 1.03±0.08 <0.01 Data presented in mean±standard deviation. Means compared by independent t-test             60   University of Ghana http://ugspace.ug.edu.gh SLE Non-SLE 9 8 7 6 5 4 3 2 1 0 CAVI haPWV (m/s) Arterial Stiffness indices Figure 4: Comparison of degree of arterial stiffness among study groups. CAVI (7.3±1.1 vs 6.1±1, p<0.001), haPWV (7.7 ± 1.3 vrs 6.5 ± 0.8 ms-1, p ≤ 0.001)     61   Degree of arterial stiffness University of Ghana http://ugspace.ug.edu.gh 4.6 Levels of Arterial stiffness Both cardio-ankle vascular indices (CAVI) and heart-ankle pulse wave velocity (haPWV) were higher in SLE patients, compared to non-SLE participants (p<0.001) (Figure 4).     4.7 Ageing and arterial stiffness The association between age and CAVI was strongly correlated as increase in age correlated with increase in CAVI values in SLE and non-SLE individuals. In the SLE patients, increase in age was associated with rapid increase in CAVI than in the non-SLE participants. The age in both groups were equally distributed, the gradient for the cases was higher due to higher arterial stiffness. Comparing the two graphs (Figure 5), SLE patients had higher CAVI than non-SLE participants when both groups are aged matched. 4.8 BMI and arterial stiffness Body mass index (BMI) correlated positively with CAVI in both SLE patients and non-SLE participants. However, there was a stronger correlation between BMI and CAVI of SLE patients, than in the non-SLE participants. Higher BMI associated with higher CAVI in the SLE patients, but increase in BMI moderately increased CAVI in the non-SLE participants (Figure 6).   62   University of Ghana http://ugspace.ug.edu.gh 11 SLE Non-­‐SLE 10 9 8 7 6 5 4 3 2 15 25 35 45 55 65 Age (yrs)   Figure 5: Association between age and arterial stiffness. For patients with SLE, r=0.41, p<0.001, and r=0.18, p=0.048 for non-SLE controls.         63   CAVI University of Ghana http://ugspace.ug.edu.gh 11 SLE Non-SLE 10 9 8 7 6 5 4 3 2 10 15 20 25 30 35 40 45 50 BMI  (kg/m2) Figure 6: Association between BMI and arterial stiffness. For patients with SLE, r=0.31, p<0.001, and for non-SLE controls, r=0.09, p=0.612.                   64   CAVI University of Ghana http://ugspace.ug.edu.gh 4.9 Association between Adipokines and Arterial stiffness indices In scatter plot analysis, leptin levels negatively correlated with CAVI in both study groups, however, the slope was steeper in the SLE patients than the non-SLE controls (Figure 7). Adiponectin correlated differently with arterial stiffness indices in SLE patients and non-SLE participants as shown in Figure 8. While there was a negative correlation between adiponectin and CAVI in the control, adiponectin and CAVI showed positive correlation in the case group. None of the control groups had a CAVI beyond the intersection of the two graphs, meanwhile that of the cases were still higher and beyond the point of intersection. This was shown by deranged pattern of adipokines in SLE patients as frequently reported with the mechanism described in 5.4 4.10 Association between Insulin levels and arterial stiffness There was a positive correlation between insulin levels and CAVI in SLE patients and non-SLE participants. CAVI increased steeply with increased levels of insulin in the SLE patients, but was very steady in the non-SLE participants. In the SLE patients, there was higher levels of insulin as compared to the non-SLE participants, as well as higher CAVI in same order and hence the positive correlation. 4.11 Association between inflammation and arterial stiffness The levels of C-reactive protein decreased with increased CAVI in SLE patients and non-SLE participants. The level of CRP was rather higher and barely reduced with increased CAVI which suggest chronic inflammation in the SLE patients as the levels of CRP remained fairly constant. Meanwhile in the non-SLE participants, CRP levels reduced with increase CAVI, meanwhile CAVI values for the non-SLE participants remained low as compared to that of the patients with SLE.   65   University of Ghana http://ugspace.ug.edu.gh 3000 SLE Non-SLE 2500 2000 1500 1000 500 0 3 4 5 6 7 8 9 10 11 CAVI Figure 7: Association between leptin levels and arterial stiffness index. For patients with SLE, r=-0.22, p=0.004, and r=-0.18, p=0.068 for non-SLE controls.               66   Leptin (ng/ml) University of Ghana http://ugspace.ug.edu.gh 4400 SLE Non-­‐SLE 3900 3400 2900 2400 1900 1400 900 400 3 5 7 9 11 CAVI Figure 8: Association between adiponectin and CAVI in study groups. For patients with SLE, r=0.34, p=0.001, and r=-0.21, p=0.02 for non-SLE controls   67   Adiponectin (ng/ml) University of Ghana http://ugspace.ug.edu.gh 450 SLE Non-­‐SLE 400 350 300 250 200 150 100 50 0 3 4 5 6 7 8 9 10 11 CAVI Figure 9: Arterial Stiffness and insulin levels. For patients with SLE, r=0.16, p=0.151, and r=0.07, p=0.705 for non-SLE controls.                     68   Insulin (pmol/ml) University of Ghana http://ugspace.ug.edu.gh     4000 SLE Non-­‐SLE 3500 3000 2500 2000 1500 1000 500 0 3 4 5 6 7 8 9 10 11 CAVI Figure 10: Association between C-reactive protein and arterial stiffness. For patients with SLE, r=-0.11, p=0.154, and r=0.08, p=0.652 for non-SLE controls                     69   C-reactive protein (mg/l) University of Ghana http://ugspace.ug.edu.gh       Figure 11: A simplified schematic diagram showing how adiponectin and leptin are in connection with arterial stiffness as observed in this studies. Leptin levels were high in SLE with arterial stiffness, while adiponectin levels were low (blue lines), in correlation with increase in arterial stiffness, leptin levels turned to reduce while adiponectin increased with increase in arterial stiffness (red arrows). These findings are proposed to be due to a compensatory/ negative feedback mechanism that may exist in patients with SLE to reduce arterial stiffness.   70   University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS   5.1 General findings This study has established that patients with systemic lupus erythematosus have arterial stiffness which was shown by higher levels of cardio-ankle vascular index (CAVI) as compared with age and gender-matched control subjects. Patients with systemic lupus erythematosus were found to have accelerated arterial stiffness with age. This was seen by a strong association between age and CAVI in the patients. Again, arterial stiffness increased with increased body mass index (BMI) in the patients with systemic lupus erythematosus. Increase in BMI was also associated with increased CAVI in non-SLE participants. As compared with the non-SLE participants, patients with SLE had subclinical inflammation which was indicated by elevated levels of C-reactive protein (CRP). This is the first study to investigate arterial stiffness in patients with systemic lupus erythematosus in Ghana, and one of the few studies that have considered a larger population in Africa. The study has established clear understanding on how adipokines and C-reactive proteins are associated with arterial stiffness. It has been discovered that the average age for patients with SLE in Ghana is lowest as compared with that of other African countries. However, this study is not able to emphasize on disease prognosis and arterial stiffness association since it was cross-sectional. Other adipokines were not also considered as done in some studies elsewhere.   5.2 General characteristics of study participants In this study, the results showed no differences in the biochemical characteristics of study population. The age and gender distribution among SLE and non-SLE participants showed   71   University of Ghana http://ugspace.ug.edu.gh higher proportion of females with SLE than males, and other studies have reported similar trend of a ratio of 9:1 female to male ratio (Soto, Vallejo, Guillen, Simon, Arena, & Reyes, 2004), and seen elsewhere in Africa; Cameroun (F:M – 12:1); Zambia (F:M – 29:0 and Nigeria (F:M – 10:1) (Adelewo, Ojo, & Oduenyi, 2012). In this study, of the 78 SLE patients, there were 2 males and 76 females giving a wider ratio between males and females as (F:M – 39:1). The age distribution of SLE patients showed similar trend like other studies conducted among people of African descent either outside or in Africa such as South Africans: 35 years; Kenyans: 34 years; Nigerians: 33 years; Cameroonians: 38 years (Adelewo, Ojo, & Oduenyi, 2012), the average age for the SLE patients in this study was found to be 32 years. 5.3 Arterial Stiffness among study Population Arterial stiffness was found to be higher in SLE patients as compared to the non-SLE participants. The CAVI for SLE was found to be 7.3±1.1 as against 6.1±1 at significant value of (p<0.001), haPWV was found to be at significance of (p<0.001) with 7.7±1.3 m/s as against 6.5±0.8 m/s in SLE and non-SLE groups respectively. Whereas the mean value for CAVI in SLE patients was found to be 7.3±1.1, the plot of CAVI against the measured adipokines and C-Reactive protein went beyond CAVI value of. However, according to the instruction of the manufacturer of the VaSera, a CAVI < 8.0: normal value less than 9.0 > CAVI ≥ 8.0: borderline. When the value of CAVI exceeds 9.0, there is a suspected arteriosclerosis and needs attention for management. Snigdha et al., (2014) reported that individuals with inflammatory diseases have stiffer arteries compared to normal control. While acute inflammation has widely been reported to be greatly linked with arterial stiffness (Wykretowicz, Guzik, & Kasinowski, 2005), Snigdha et al., (2014), have reported that sub-clinical chronic inflammation inflammation have some association with structural changes within intima-media through different mechanisms (Figure 1). This process leads to potentially permanent stiffened arteries.   72   University of Ghana http://ugspace.ug.edu.gh The result from this study was similar to those existing findings, and explains why there were higher arterial stiffness in the SLE patients than the non-SLE participants. Interestingly, while the result of this study is similar to a study by Selzer et al., (2001), it does not confirm their claim that, aortic arterial stiffness in premenopausal age is higher due to hypertension. (Selzer, Sutton-Tyrrell, Fitsgerald, Tracy, Kuller, & Manzi, 2001) 5.4 Haemodynamic and arterial stiffness indices of study participants There were no significant differences in the haemodynamic values among the SLE patients and the non-SLE participants except for the pulse pressure that was lower in SLE patients as compared to non-SLE participants at 47±8 and 50±9 in that order at significance of (p<0.001) (Table 4). The use of CAVI for the measurement of arterial stiffness has been reported to be blood pressure-independent by some other researchers (Cheuk-Kwan, 2013), and the results of this study showed similar characteristics. Some studies conducted in type 2 diabetes mellitus patients had arterial stiffness related to blood pressure (Yeboah, 2016). Schette et al., (2011), have also reported arterial stiffness independence of blood pressure. Yeboah, (2016) has reported of arterial stiffness being accelerated by higher systolic BP. Also due to structural and functional alterations in the central elastic arterial walls in chronically elevated distending pressures have been found to lead to stiffening of arteries (O'Rourke & Adji, 2102). In the Framingham Heart Study, the longitudinal increase in pulse pressure which is potentially useful in the assessment of arterial stiffness was found to be greater in subjects with higher baseline systolic blood pressure (Franklin, 1997). CAVI has theoretical postulation of BP independence at the time of measurement Sun, (2013); Shirai et al., (2011); Kim, Takada, Oka & Misaki, (2011) cited in Yeboah, (2106), meanwhile this was found in contrast in T2DM study by Yeboah, (2016), where there was an association between high blood pressure and increase in arterial stiffness.   73   University of Ghana http://ugspace.ug.edu.gh It is therefore noteworthy that the results of this study show that arterial stiffness in SLE patients is independent of blood pressure increase. SLE arterial stiffness pathophysiology differ significantly from that of other chronic metabolic diseases such as diabetes mellitus. In hypertensive patients, arterial stiffness may be due to shear stress of persistent increased systolic BP, therefore arterial stiffness may be indicative of high blood pressures. However, arterial stiffness in SLE is mainly due to chronic inflammation and independent of increased blood pressure and explains findings in this study where SLE patients with lower mean BP have higher CAVI than non-SLE participants who were found to have higher BP. 5.5 Association between arterial stiffness and adipokines: leptin & adiponectin In this study, the levels of adiponectin increased with increase in arterial stiffness (CAVI). Adiponectin levels increased with elevated arterial stiffness in SLE, in the non-SLE participants, adiponectin levels reduced with elevated in arterial stiffness. Serum concentration of adiponectin has been reported to be elevated in patients with chronic autoimmune diseases and could be explained as being a compensatory mechanism to suppress inflammation (a negative feedback effect) (Fantuzzi, 2008). In SLE, inflammation is higher and tends to remain constant with elevated arterial stiffness, increased levels of adiponectin explain why the level of inflammation indicated by CRP turn to reduce. This is due to the anti-inflammatory effect exerted by the increasing levels of adiponectin. As inflammation persists (plateau trend of CRP), the level of adiponectin increases steadily. Adiponectin suppresses the production of TNF-alpha and IL-6, which are known pro-inflammatory cytokines (Toussirot, Streit, & Wendling, 2007).   74   University of Ghana http://ugspace.ug.edu.gh High molecular weight multimer (HMW) adiponectin is an isoform of adiponectin and has been found to be both a predictor of future cardiovascular events as well as a marker for severity of coronary arterial disease (CAD) (Inoue, et al., 2007) . In the non-SLE participants, a different trend is observed as the absence of chronic inflammation accounts for the rapid decline of adiponectin levels with reduced arterial stiffening. Loss of weight has been connected to declining levels of leptin, whereas weight gaining of weight is also being linked to rise in leptin concentration. It is speculated that the changes in arterial stiffness that accompany the changes in weight could, in part, be also explained by leptin (Safar, Sébastien & Blacher, 2006). The results of this study are also in contrast with the report of (Safar et al., 2006) where hyperleptinemia and/or hypoadiponectemia were proposed as molecular mechanisms that link adiposity to arterial stiffness. This results of this study is similar with studies done among European and North American populace that have suggested that hyperleptinemia and hypoadiponectemia are associated with arterial stiffening (Bolanos-Garcia & Miguel, 2003) The potential mechanism to explain the speculated relationship between arterial stiffness and adipokines could be based on the presence receptors of adipokines on the smooth muscle and endothelial cells of human blood vessels which is suspected to trigger signaling for arterial stiffness. Beltowski, (2006) leptin affected endothelial cell nitric oxide production, while it facilitated growth of cells in the endothelium and increased angiogenesis thereby having possible effect on arterial stiffening cascade (Beltowski, 2006). The modification muscle tissues, elastin and collagen found in the walls of the vessel, genetic factors and endothelial malfunction could add up to the arterial stiffness pathogenesis (Laurent & Boutouyrie, 2007). Adiponectin has been observed to oppose endothelin-1-mediated vasoconstriction (Bussey, Kolka, Rattigan, & Richards, 2011), this is similar with the positive correlation between arterial stiffness and adiponectin levels in SLE patients found in this study. In this study, leptin levels   75   University of Ghana http://ugspace.ug.edu.gh were significantly higher (p<0.039) in SLE patients as compared to non-SLE participants. This study result is similar with the report that, plasma leptin concentration of patients with SLE are generally consistent and is indicative of elevated levels of leptin in serum as a probable contributor of systemic inflammation is SLE patients (Palmer & Gabay, 2003; Garcia- Gonzalez, Gonzalez-Lopez, Valera-Gonzalez, Cardona-Munoz, Salazar-Paramo, & Gonzalez- Ortiz, 2002) and has further been shown that serum levels was higher in women with SLE than in healthy controls (Garcia-Gonzalez, et al., 2002). The association between leptin and adiponectin and arterial stiffness based on this study results and other reports is schematically presented in Figure 11. 5.6 Inflammation and arterial stiffness Sub-clinical inflammation was observed in SLE patients with significantly elevated levels of C-reactive proteins as compared to the non-SLE participants. Meanwhile, SLE disease progression has shown correlation with elevated serum CRP, and has been reported to be elevated excessively in some patients (Williams, Harmaon, Burligame, & Du Clos, 2005). With association and as an inflammatory maker, CRP has been useful in the assessment of Cardiovascular risk in inflammatory diseases including Systemic Lupus Erythematosus (Nutall, Heaton, Piper, Martin, & Gordon, 2003). The elevated levels of CRP have been seen to be associated with SLE complications such as active renal involvement (Zuniga, Markowitz, Arkachaisri, Imperatore, D'Agati, & Salmon, 2003). Mok et al., (2013) has further placed emphasis on strong association between inflammation-assessed by CRP levels- and SLE disease activity and cardiovascular risk amid the standing controversy on the association of CRP and hsCRP with disease progression and cardiovascular risk in SLE patients (Mok, Birmingham, Ho, Lee, & Rovin, 2013).   76   University of Ghana http://ugspace.ug.edu.gh The results of this study has also shown elevated levels of CRP in SLE patients as compared to the non-SLE participants where there is association between arterial stiffness and CRP in SLE patients. However, increase in arterial stiffness correlate negatively with CRP levels which may be due a negative feedback mechanism that exist in SLE disease progression, arterial stiffness and inflammation-mediation molecules. In the general population, Snigdha et al., (2014) have suggested multiple potential mechanisms that inflammation may cause arterial stiffness, and have further suggested escalated levels of arterial stiffness in diseased patients, especially in chronic inflammatory diseases like SLE. This therefore support the results of this study where there is an elevated levels of CRP in SLE patients than in the non-SLE participants. The mechanism as explained by Snigdha et al., (2014) and with the import of this study is that, auto- immune attack in SLE leads to inflammation, and hence initiating a complex cascade of event which result in arterial stiffness (Figure 1). A recent study by Giannelou & Mavragni, (2017), showed that there is indeed heightened rates of CV events and subclinical atherosclerosis in SLE patients, however traditional CV factors did not fully explain the high rates of ischemic events which is reported prevalent in SLE (Giannelou & Mavragani, 2017). However, this study has shown that, chronic inflammation has a role to play in premature arterial stiffness in systemic lupus erythematosus. 5.7 Ageing and arterial stiffness There was no significant difference between the mean ages of SLE patients and non-SLE participants. Meanwhile, increase in age strongly correlated with higher CAVI in SLE patients. Arteries are known to stiffen with aging in the general population (Hickson, 2003; O'Rourke M. F., 2007; Lee & Oh, 2010). Effect of aging found in the study could be likened to reported studies by (Ezeala-Adikaibe, 2102; Hickson, 2003).   77   University of Ghana http://ugspace.ug.edu.gh Cardio ankle vascular index in known to increase with age in the general population and in disease state (Shirai K. , 2011; Shirai K. , 2013). A study by O’Rourke, (2007) reported that, as one ages, there is thickening of proximal ‘non-load bearing’ intima and media of the arterial system, mainly due to cellular hyperplasia (O'Rourke M. F., 2007). However, Hickson et al., (2003), has shown that though the media do not appreciably thicken with age, individual elastin lamellae rather shrink and become separated by increasing amounts of non-load-bearing materials while the intima and the media are indistinguishable in young subjects. Meanwhile in SLE, inflammation has been found to be chronic and as found in this study, chronic inflammation is suspected to leading to the production of MMT due to neutrophils and macrophage proliferation, whereas TIMP production is diminished by ROS production. This phenomenon has been linked with rapid degradation of elastin and collagen fibers where there is uncoiling of collagen and degradation of elastin (Snigdha et al., 2014). These events are likely to facility the easy separation and shrinking of the individual elastin lamellae as reported by Hickson et al., (2003). The stiffening of the aorta increases pulse wave velocity which has a devastating effect on terminal organs receiving the high-speed pulsatile waves (Nilsson, 2103). 5.8 Body composition and arterial stiffness In this study, SLE patients had lower mean BMI as compared to the non-SLE participants, whereas there was higher CAVI in SLE than non-SLE participants. A study of 33 SLE patients and 33 healthy control by Cacciapaglia et al., 2009 among Caucasian females showed no significant difference in BMI, as well as the percent of BMI > 25 kg/m2. However, despite the equivalent exposure to known cardiovascular risk factors, SLE patients had higher indices in all the arterial stiffness parameters that were employed (Cacciapaglia, et al., 2008).   78   University of Ghana http://ugspace.ug.edu.gh Increase in BMI in SLE patients had stronger association with increasing CAVI than in the non- SLE participants. BMI is found to independently predict cardiovascular diseases (CVDs) where obesity is documented to increase CVD risk through rise in BP in the general population. There has been a positive correlation between obesity and arterial stiffness index (PWV) (Yeboah, 2016). The plausible mechanism where obesity influences arterial stiffness is through the increase in production of circulating cytokines and leptin (Visser, Bouter, McQuillan, Wener, & Harris, 1999; Singhal, et al., 2002). There has been extensive documenting of correlation between high leptin levels and reduction arterial distensibility (Hickson, 2003). In this study, arterial stiffness was higher in SLE patients with high levels of leptin but smaller BMI. This study therefore introduces that, there is derangement of adipokines levels and metabolic activities in SLE patients (Tian-Ping, et al., 2016). However, this study shows stronger correlation between increase in BMI with elevated arterials stiffness. But on the other hand, leptin levels reduced marginally with rise in CAVI. Leptin levels increase obesity. Leptin acts through its hypothalamic receptors and smooth muscles of the blood vessels (Oda, Taniguchi, & Yokoyama, 2001; Sierra-Honigmann et al., 1998). However, leptin is found to exert receptor-mediated influence on vessel tone and growth and in cell culture, stimulate vascular smooth muscle proliferation and migration (Schafer, et al., 2004). In addition, leptin has been found to induce oxidative stress in endothelial cells leading to the trigger of transcription of oxidant-sensitive genes which participate in atherogenesis (Safar, Sébastien & Blacher, 2006). Furthermore, high leptin level has been associated with peripheral adipose deposition, whereas visceral obesity is rather linked more with arterial stiffness than BMI. In effect, the indifferent BMI, but higher arterial stiffness may have other complex interplay between obesity and inflammation.   79   University of Ghana http://ugspace.ug.edu.gh However, this study has shown that, obesity and inflammation synergistically increases arterial stiffness. The mechanism is that, inflammation causes intimal stiffness, while obesity (high BMI) increase leptin levels that have effect on VSMC proliferation and migration (Schafer, et al., 2004). 5.9 Conclusion Patients in Ghana with Systemic Lupus Erythematosus compared to non-SLE participants had arterial stiffness independent of age, gender, haemodynamics and BMI. There is elevated leptin levels and decreased levels of adiponectin. Patients with SLE have sub-clinical inflammation which was found by higher levels of C-reactive protein as compared to non-SLE participants. Meanwhile, leptin negatively correlated with arterial stiffness and positive correlation in adiponectin and arterial stiffness. While patients with SLE have lower levels of adiponectin, there is a rise in adiponectin levels with duration and degree of arterial stiffness as well as reduced levels of leptin with increasing arterial stiffness. Although, arterial stiffness in patients with SLE was found to be independent of age and BMI, increase in both parameters correlate positively with arterial stiffness. The average age of SLE patients in Ghana was found to be the lowest (32 years) compared with other studies.   5.10 Limitations of the Study The patients with systemic lupus erythematosus were undergoing treatment at the time of data collection and affected the arterial stiffness and CRP levels. The data was collected cross sectional and cannot infer causation, therefore limits predictive potential of arterial stiffness. Other vasoactive molecules which could affect arterial distensibility were not considered in this study.   80   University of Ghana http://ugspace.ug.edu.gh The study was conducted at the Rheumatology Referral Unit of Korle-Bu Teaching Hospital, therefore its generalizability to the Ghanaian population is limited.       5.11 Recommendations A cohort study could be designed to assess the impact of systemic lupus erythematosus with time. It is recommended that comorbidity of systemic lupus erythematosus and diabetes mellitus be conducted to establish the individual disease effect and their combined effect on the arteries of patients. Investigation of arterial stiffness and other vasoactive molecules such as nitric oxide, VEGF in patients with SLE could be designed, based on the results of this study. The validity of the various tools used to assess aortic stiffness and central pressure indices need to be determined in Ghanaians, taking into account age and other risk factors. 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Identification of IgG subclasses and C-reactive protein in lupus nephritis: the relationship between the composition of immune deposits and FCgamma receptor type IIA alleles. Arthritis Rheum , 48, 460-470.   108   University of Ghana http://ugspace.ug.edu.gh APPENDIX 1: ETHICAL APPROVAL   109   University of Ghana http://ugspace.ug.edu.gh APPENDIX II: Data capture tool   DATA ABSTRACTION SHEET Date of Evaluation: ____________________ ID. #. ____________ Name ______________________________ Age_____ Gender_____ (1 =M; 2=F) Anthropometry/Physical Measurements (a) Height in (cm) ___________ Weight (kg) ___________ (b) Waist circumference (cm) ___________ Hip circumference (cm) __________ BMI (kg/m2) __________ Body fat_____________ Visceral fat __________   Blood Pressures Doppler SBP Systolic BP Diastolic BP Heart rate Right arm 1st 2nd 3rd Left arm 1st 2nd 3rd Right Ankle: Dorsalis pedis artery 1st 2nd 3rd Right Ankle: Posterior tibia artery 1st 2nd 3rd   110   University of Ghana http://ugspace.ug.edu.gh Left Ankle: Dorsalis pedis artery 1st 2nd 3rd Left Ankle: Posterior tibia artery 1st 2nd 3rd     111   University of Ghana http://ugspace.ug.edu.gh APPENDIX III: Consent form       INFORMED CONSENT FORM PROTOCOL  ID:  CHS-­‐Et/M.4.2/2016-­‐2017     Participant ID Number: __________ Participant Name: _________________________ Study Title: Arterial stiffness and Circulating adipokines in patients with systemic lupus erythematosus in Ghana Systemic lupus erythematosus, often called lupus, is a disease in which the body produces some proteins that fight against the body itself and destroy some important organs such as heart and blood vessels. People who have lupus tend to have problems with their blood pressure as results of hardening of special blood vessels called arteries. Hardened arteries tend to be less elastic (unable to stretch and recoil) as blood passes through and this may lead to diseases of heart and blood vessels. We have not study the functions of blood vessels in patients with lupus in Ghana. In this study, we want to find out how hardened arteries are related to some chemicals in the blood, called leptin and adiponectin, produced by fats in the body adipose tissues. You are to understand that taking part in the research is entirely voluntary. You are further to note that you may refuse to take part or withdraw from the study at any time without anyone objecting. You are likely to spend the best part of the morning going through this study. For you to qualify to be part of this research, you should be between the ages of 16 to 50 years. We will ask you to provide information about yourself, family, alcohol intake and smoking information. You may feel uncomfortable providing such information. Also, your blood pressure, height, weight and amount of fat in your body will be measured. In addition, some special medical equipment that measures blood pressure and stiffness of the blood vessel will be applied to your arms and   112   University of Ghana http://ugspace.ug.edu.gh legs. These procedures are painless and might give slight tingling sensations for few seconds when the cuffs inflate. Also, some amount of blood equivalent to 3 teaspoonful (10ml) will be drawn to measure substances in the bloodstream. You are assured that this amount will not affect your health. All the tests we will do for you in connection with this research will be free of charge. Information we collect on you in this study will be kept confidential and secure. The information will only be available to the scientists conducting this study. You are further assured that if a report of this study is prepared for the scientific and medical community you will not be identified by name. You may experience a minor bruise and/or temporary discomfort at the site of taking the blood and this risk is not more than what you will normally be exposed to for having a blood drawn routinely at the hospital. We will reduce this discomfort happening by asking experienced staff to take the blood. The study will help us understand arterial stiffness and how this is associated with plasma leptin and adiponectin, body fat composition, in normal individuals and patients with SLE in Ghana. All your test results will be explained to you. You may through this study discover that you have bad fat in the blood, stiff arteries or hypertension. You will be advised professionally and/or referred appropriately if you should have any of these conditions on testing. If there is something that you do not understand or you have any questions or concerns about this Research or should you later wish to have any matter or question relating to this study clarified, do not hesitate to contact Richard Nana Abankwah Owusu Mensah, Department of Physiology, University of Ghana, P.O. Box 4236, Accra. (Tel number, 0249938660), the principal investigator for this study, to answer any questions you may have.   113   University of Ghana http://ugspace.ug.edu.gh I have fully explained to ____________________________ the nature and purpose of the above described research, its procedures, risks and benefits. I have allowed the participant to ask questions and have answered and will answer to the best of my ability, all questions relating to the study at any time. _______________ ____________________________ _______________ Signature Full Name of Investigator Date I ________________________, have read (or have had read to me in a language that I fully understand) the proposed study and that I have understood what is going to be done. Also, any concerns I have, have fully been addressed. My signature or thumbprint below indicates that I have understood what is going to be done and that I agree to take part in the study. ___________________________________ Date:__________________ (Signature/thumbprint of Subject) ___________________________________ Date: __________________ (Signature: Witness)   114   University of Ghana http://ugspace.ug.edu.gh