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Oxidative stress compromises vasomotor function of the thoracic aorta in Marfan Syndrome Yang, Huei-Hsin Clarice 2009

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OXIDATIVE STRESS COMPROMISES VASOMOTOR FUNCTION OF THE THORACIC AORTA IN MARFAN SYNDROME by HUEI-HSIN CLARICE YANG B.Sc., The University of British Columbia, 2006 A THESIS SUBMITTED iN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Pharmacology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2009 ©Huei-Hsin Clarice Yang, 2009 ABSTRACT Introduction: Marfan syndrome is an autosomal dominant connective tissue disorder that causes life-threatening cardiovascular complications such as thoracic aortic dilatation and aneurysm. We have demonstrated that Marfan syndrome compromises contractile function of the aorta and impairs nitric oxide-mediated relaxation. We hypothesize that oxidative stress impairs contractility and endothelium-dependent relaxation in the thoracic aorta of Marfan mice. Methods: Adrenergic contractions and cholinergic relaxations of thoracic aorta from mice heterozygous for FBN1 allele (Fbn1°390,n=40; age=3, 6, 9 months), a well-defined model of Marfan syndrome, were compared with those from control littermates (n=40). Results: At 3 and 6 months, oxidative stress, as indicated by the plasma 8-isoprostane level, was 50% greater in the Marfan group than in the control. In 9 months old Marfan mice, the depressed phenylephrine-induced contraction was normalized by the preincubation of superoxide dismutase (SOD) which increased the maximal contractile response (Emax) and pEC50 for phenylephrine-stimulated contraction by 91% and 2.75-fold. The compromised endothelial function was also restored by SOD which increased the sensitivity to acetylcholine by 10.7 and 12.3-fold at 3 and 6 months, respectively. Such improvement was absent in the controls. In 9 11 months old Marfan mice, the phenylephrine-contraction was potentiated 141% by 1400W, an inducible nitric oxide synthase (iNOS) inhibitor. The pEC50 was normalized by 1400W and allopurinol, an inhibitor of xanthine oxidase. In the same group, both Emax and pEC50 of acetyicholine was normalized by apocynin, an inhibitor ofNAD(P)H oxidase. Protein expression of SOD was decreased at 3 and 9 months in the Marfan group, whereas expression of xanthine oxidase, iNOS, gp9lphox, p47phox and p67phox, the subunits ofNAD(P)H oxidase, was all increased. Conclusions: The compromised vasomotor function in Marfan thoracic aorta could be associated with oxidative stress resulting from decreased expression of SOD and increased expression of iNOS, xanthine oxidase, and NAD(P)H oxidase. 111 TABLE OF CONTENTS ABSTRACT.ii TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xi ACKNOWLEDGEMENTS xiv CHAPTER 1. INTRODUCTION 1 1.1 Marfan Syndrome 1 1.1.1 History 1 1.1.2 Clinical manifestations & diagnostic criteria 3 1.1.3 Fibrillin, microfibrils & elastic fibers 7 1.1.4 Molecular genetics & pathophysiology 12 1.1.5 Mouse model of Marfan syndrome 17 1.1.6 Pharmacological treatment for Marfan syndrome 18 1.1.6.1 Current treatment 18 1.1.6.2 Potential alternative treatment: doxycycline 19 1.1.6.3 Newly investigated alternative treatment: losartan 21 iv 1.2 Oxidative Stress .25 1.2.1 Overview 25 1.2.2 Biomarkers for oxidative stress 26 1.2.3 Generation of oxidative stress 26 1.2.3.1 NAD(P)H oxidase 27 1.2.3.2 Xanthine oxidase 28 1.2.3.3 NOS 28 1.2.4 Removal of oxidative stress 29 1.3 Vasomotor Function 32 1.3.1 Structure of artery 32 1.3.2 Vascular smooth muscle contraction 33 1.3.3 Vascular relaxation 35 1.3.4 Marfan syndrome and vasomotor function 36 1.3.5 Impact of oxidative stress on vascular function 39 1.4 Thesis Hypotheses and Aims 44 1.4.1 Research rationale 44 1.4.2 Hypothesis & objectives 44 V CHAPTER 2. MATERIALS & METHODS .45 2.1 Animals & Anesthesia 45 2.2 Measurement of Isoprostanes (8-Isoprostane) 45 2.3 Tissue Preparation for Isometric Force Measurement 46 2.4 Measurement of Isometric Force 46 2.4.1 Inhibitor/enzyme pre-incubation 47 2.5 Western Immunoblotting 49 2.6 Drugs & Chemicals 50 2.7 Statistical Analysis 50 CHAPTER 3. RESULTS 52 3.1 Plasma Isoprostane 8-epi-PGF2 Levels 52 3.2 Preincubation with SOD and Catalase 53 3.3 Blockade of NAD(P)H Oxidase with Apocynin 57 3.4 Blockade of Xanthine Oxidase with Allopurinol 59 3.5 Blockade of iNOS with 1400W 61 3.6 Protein Expression of Superoxide-Generating and -Degrading Enzymes 65 3.6.1 SOD 65 3.6.2 NAD(P)H oxidase 66 vi 3.6.3 Xanthineoxidase.68 3.6.4 iNOS 69 CHAPTER 4. DISCUSSION 70 4.1 Level of Oxidative Stress 70 4.2 SOD and Catalase 72 4.3 NAD(P)H Oxidase 74 4.4 Xanthine Oxidase 75 4.5 iNOS 76 4.6 Contributor to Oxidative Stress in Disease Progression 77 CHAPTER 5. CONCLUSION 79 CHAPTER 6. FUTURE DIRECTIONS 80 6.1 Antioxidants 80 6.2 Losartan 80 6.2.1 Losartan treatment and vasomotor function 81 6.2.2 Losartan & oxidative stress 83 REFERENCES 84 APPENDIX 95 VII LIST OF TABLES Table 3.1 Summary of Emax and pEC50 of PE-induced contraction 63 Table 3.2 Summary of Emax and pEC50of ACh-induced relaxation 64 viii LIST OF FIGURES Figure 1.1 Structure of fibrillin- 1 protein 8 Figure 1.2 Main assembly steps from fibrillin- 1 to microfibrils and elastic fiber 11 Figure 1.3 Binding of large latent complex of TGFI3 to fibrillin-1 15 Figure 1.4 Pathophysiology of the most common form of Marfan syndrome 16 Figure 1.5 Mouse model of Marfan syndrome 18 Figure 1.6 Aortae of control and Marfan mice 18 Figure 1.7 Angiotensin II and oxidative stress 22 Figure 1.8 Current and alternative treatments for MFS 24 Figure 1.9 Oxidative stress 31 Figure 1.10 Ion channels and receptors responsible for regulating intracellular Ca2 34 Figure 1.11 Endothelium-mediated and agonist-induced vaso-contraction and relaxation 36 Figure 1.12 Cross-sectional area of control and Marfan mouse aorta 37 Figure 1.13 Processes leading to aortic aneurysm in MFS 39 Figure 1.14 Effects of reactive oxygen species on Ca2 signaling and vasoconstriction 41 Figure 1.15 Processes leading to decreased vasodilatation 43 Figure 2.1 Superoxide-generating and —neutralizating enzymes and their inhibitors 48 Figure 3.1 Plasma level of isoprostane 8-epi-PGF in control and Marfan aorta 52 ix Figure 3.2 Effect of SOD and SOD-plus-catalase on PE-stimulated contraction 54 Figure 3.3 Effect of SOD and SOD-plus-catalase on ACh-induced relaxation 56 Figure 3.4 Effect of apocynin (NAD(P)H oxidase inhibitor) on contraction and relaxation 58 Figure 3.5 Effect of allopurinol (xanthine oxidase inhibitor) on contraction and relaxation 60 Figure 3.6 Effect of 1400W (iNOS inhibitor) on contraction and relaxation 62 Figure 3.7 Protein expression of SOD-i and SOD-2 65 Figure 3.8 Protein expression of enzymatic subunits of NAD(P)H oxidase 67 Figure 3.9 Protein expression of xanthine oxidase 68 Figure 3.10 Protein expression of iNOS 69 Figure 6.1 Effect of chronic losartan treatment on PE-stimulated contraction 82 Figure 6.2 Effect of chronic losartan treatment on ACh-induced relaxation 82 x LIST OF ABBREVIATIONS AAA Abdominal aortic aneurysm ACE Angiotensin converting enzyme ACh Acetyicholine ANG II Angiotensin II ARB Angiotensin receptor blocker AT1 Angiotensin II type 1 receptor 13-blocker 3-adrenoceptor antagonist Ca2 Calcium ions [Ca2] Intracellular calcium concentration cbEGF Calcium-binding epidermal growth factor EC Endothelial cell ECM Extracellular matrix EC50 Potency/sensitivity Emax Efficacy/maximal response EGF Epidermal growth factor eNOS Endothelial nitric oxide synthase ET- 1 Endothelin- 1 xi Fbnl Fibrillin-1 GPCR G-protein coupled receptor H20 Hydrogen peroxide iNOS Inducible nitric oxide synthase 1P3 Inositol trisphosphate IP3R 1P3 receptor LAP Latency-associated protein LTBP Latent TGFI3 binding protein MFS Marfan syndrome MLCK Myosin light chain kinase MMP Matrix metalloproteinase mRNA Messenger ribonucleic acid NADH Nicotinanñde adenine dinucleotide NADPH Nicotinamide adenine dinucleotide phosphate NCX Sodium calcium exchanger NO Nitric oxide NOS Nitric oxide synthase Oj Superxoxide anion xl’ 0N00 Peroxynitrite PE Phenylephrine PM Plasma membrane ROS Reactive oxygen species SERCA Sarco-endoplasmic reticulumCa2-ATP se SMC Smooth muscle cell SOD Superoxide dismutase SR Sarcoplasmic reticulum TGF-3 Transforming growth factor- 43 VGCC Voltage-gated Ca2 channel VSMC Vascular smooth muscle cell xlii ACKNOWLEDGEMENTS I would first like to offer my gratitude to my co-supervisors, Drs. Cornelis van Breemen and Kuo-Hsing Kuo, for their guidance and support in both the scientific field as well as in the pursuit of future career. Sincere appreciation is extended to my supervisory committee members, Drs. Pascal Bematchez and Hong-Lin Luo, for offering invaluable suggestions and insights for my project. I am very grateful for the two graduate student advisors, Drs. David V. Godin and Darryl Knight, for the counsel they provided to all graduate students and for always being there for us. I sincerely thank the graduate secretary, Mrs. Wynne Leung, who always keeps her door open and offers her timely help. To Dr. Ada Chung, I owe special thanks, for sharing her extensive knowledge, offering constant technical assistance and moral support, and providng close guidance in the last two years. I am grateful for my labmates, graduate student Harley Syyong and undergraduate student Karen Au Yeung, for their friendship and assistance. This work was supported by the operating grant from Canadian Institutes of Health Research. I would also like to thank the Natural Sciences and Engineering Research Council of Canada and Michael Smith Foundation of Health Research for their generous support. xiv Special gratitude is owed to my loving and supportive family who has always been there to share my happiness and help me get through difficult times. xv CHAPTER 1 INTRODUCTION 1.1 Marfan Syndrome Marfan syndrome (MFS) is a genetic disorder that affects connective tissues. Due to the structural importance of connective tissue in all organ systems, the mutation in the FBN- 1 gene results in a multisystem disorder which not only involves the cardiovascular, skeletal and ocular system, but also the skin, lungs, and muscle tissues (Chaffins, 2007; Judge & Dietz, 2005; Judge & Dietz, 2008; Pyeritz, 2000). The prevalence of the disorder is around 2-3 in 10,000 individuals (Judge & Dietz, 2005). Without diagnosis and treatment, the mean lifespan of affected individuals is 44 years for men and 47 years for women, with the main cause of death (over 90% of cases) being cardiovascular (mainly aortic dissection) (Judge & Dietz, 2005; Williams et al., 2008). However, with recent improvement in the recognition and management of the disorder, the life expectancy of the patients with MFS is approaching normal. 1.1.1 History The clinical signs of Marfan syndrome were first described by physician E. Williams in 1875 when he reported a family (a father and two children) with large body habitus, loose joint, and lens dislocation. However, without the inclusion of photographs and further comments, his description went unnoticed (Chaffins, 2007). 1 Marfan syndrome was named for pediatrician Antoine-Bernard Marfan who evaluated a 5-year-old girl, Gabrielle P. in 1896. He described the disproportionately long, thin limbs, narrow skull (dolichocephaly), tall stature, and long, slender digits which he termed “pattes d’araignee” or “spider fingers,” and accompanied his description with drawings (Chaffins, 2007; Judge & Dietz, 2005; Judge & Dietz, 2008; Pyeritz, 2000). Tn the subsequent forty years, Dr. Marfan reviewed over 150 similar cases and recognized Mendelian inheritance with cosegregating malfunction of mitral valve, congenital displacement of the lens (ectopia lentis) and dolichostenomelia, a term he preferred for excessively long limbs (Chaffins, 2007; Judge & Dietz, 2005; Judge & Dietz, 2008; Pyeritz, 2000). Due to his contribution, Marfan’s name was associated with this condition by early 20th century, and the term “syndrome” was used due to the frequent occurrence of certain physical signs (Chaffins, 2007). The involvement of the aorta was first described in 1943, and in 1955, the extent of cardiovascular abnormality was documented by Victor McKusick who reported dilatation and dissection of the aorta and aortic valve regurgitation in MFS (Judge & Dietz, 2008). Moreover, McKusick also correctly speculated the disorder to be a heritable disorder of connective tissue (Judge & Dietz, 2008; Pyeritz, 2000). 2 In 1986, Sakai et al discovered the major component of the microfibril, which they named fibrillin (Chaffins, 2007). Fibrillin, a 350kD connective tissue glycoprotein, was found to be widely distributed in connective tissue matrices of skin, lung, kidney, vasculature, cartilage, tendon, muscle, cornea, and ciliary zonule (Sakai et al., 1986), most of which are later found to be affected in patients with MFS. Five years later in 1991, a mutation in the gene FBN1, which encodes fibrillin- 1 (Fbn 1) protein, was discovered to be responsible for the classic Marfan syndrome by Dietz and colleagues (Chaffins, 2007; Dietz et aL, 1991; Pyeritz, 2000). 1.1.2 Clinical manifestations & diagnostic criteria Marfan syndrome is a pleiotropic disorder, with diverse manifestations in different organ systems resulting from one single mutation. The clinical presentations and severity of the disorder are different depending on the location of the mutation and expressivity in each individual. Due to the lack of genetic heterogeneity, diagnosis of MFS is based on clinical features rather than molecular testing (Judge & Dietz, 2005; Judge & Dietz, 2008; Pyeritz, 2000). The first standard (Berlin nosology) for the diagnosis of MFS was proposed in 1986, and the criteria focused on the three most prominent organ systems: the skeleton, eyes, and heart and aorta (Chaffins, 2007; Judge & Dietz, 2005; Pyeritz, 2000). In 1995, a revision (Ghent nosology) 3 was proposed which recognized the positive family history, included other organ systems (e.g. skin, fascia, lungs, skeletal muscle and adipose tissue), and placed greater emphasis on the skeletal findings (Chaffins, 2007). Moreover, Ghent nosology contains classifications with more stringent and explicit criteria, thus solving the problem of overdiagnosis or misdiagnosis with Berlin nosology (Chaffins, 2007; Judge & Dietz, 2005). The skeletal features, mainly caused by the disproportionate overgrowth of the long bones, are the most striking and immediately evident manifestations of MFS. The overgrowth gives rise to tall stature (above 97th percentile), arachnodactyly, dolichocephaly, and elongation of limbs which leads to an arm span greater than 1.05 times the height (Gray & Davies, 1996; Judge & Dietz, 2005). Another prominent feature of MFS, which occurs in 30-60% of patients, is scoliosis/kyphoscoliosis caused by vertebral deformities (Gray & Davies, 1996). On the ventral side, anterior chest deformity can result from overgrowth of the ribs, pushing the sternum anteriorly or posteriorly (Judge & Dietz, 2005). The combination of long fingers and loose joints leads to the characteristic Walker-Murdochlwrist sign and Steinberg/thumb sign (Judge & Dietz, 2005). For the wrist sign, the distal phalanges of the thumb and fifth finger need to fully overlap when wrapped around the wrist of the other arm. With regard to the thumb sign, the distal phalanx of the thumb would be fully extended beyond the border of the hand when folded across 4 the palm (Judge & Dietz, 2005). The main manifestations in the ocular system are early and severe myopia and ectopia lentis (lens dislocation), the latter of which happens in approximately 60% of the patients. In the pulmonary system, the most frequently occurring presentation (4-15%) is spontaneous pneumothorax caused by widening of the distal air spaces (Pyeritz, 2000). Restrictive lung disease may become life-threatening in people with severe chest deformity (Pyeritz, 2000). In two thirds of the patients, the skin can also be affected, with the presentation of striae astrophicae or “stretch marks” (Gray & Davies, 1996; Judge & Dietz, 2005). Another common manifestation which is present in 63-92% of individuals with MFS is dural ectasia, an abnormal protrusion of dural membranes (Gray & Davies, 1996; Judge & Dietz, 2005). Cardiovascular complications are the major cause of morbidity and mortality in patients with MFS (Ammash, Sundt, & Connolly, 2008). In the heart, the atrioventricular valves, especially mitral valves, are often affected, and indeed mitral valve disturbances are one of the earliest clinical presentations in patients (Judge & Dietz, 2005; Pyeritz, 2000). In young children with MFS, insufficiency of mitral valve, which may lead to congestive heart failure and pulmonary 5 hypertension, is the leading cause of death. In around 80% of the patients, mitral valve abnormality progresses into mitral valve prolapse which in some cases leads to mitral valve regurgitation (Gray & Davies, 1996). The most life-threatening manifestations of MFS are aortic dilatation and aneurysm which could result in aortic rupture (Judge & Dietz, 2005; Pyeritz, 2000). Unfortunately, the prevalence of this manifestation is high where some degree of dilatation is observed in 50% of children and 70 to 80% of adults with MFS (Gray & Davies, 1996). Due to their proximity to the heart, aortic root and ascending aorta withstand the highest hemodynamic stress and thus are the main areas where dissection and dilatation occur (Gray & Davies, 1996). However, dilatation or dissection of the descending thoracic aorta is also diagnosed in patients less than the age of 50 (Ammash et al., 2008). The changes in the walls of the elastic arteries (e.g. fragmentation and disarray of elastic fibers, a paucity of smooth muscle cells, separation of muscle fibers by collagen and mucopolysaccaride) are primarily in the media layer and result in the observed decrease in distensibility and increase in stiffness in the aorta (Pyeritz, 2000). In the clinical settings, pulse wave velocity (PWV), which is increased in patients with MFS, is a well-established parameter to measure aortic wall 6 stiffness; furthermore, an increase in stiffness is a marker for aortic dilatation and susceptibility to aortic rupture (Hirata et al., 1991; Marque et al., 2001; Vitarelli et al., 2006). Though the various clinical manifestations may appear unrelated in this multi-systemic disorder, all the tissues affected have a common principal component in the extracellular matrix: fibrillin- 1. 1.1.3 Fibrilhin. microfibrils & elastic fibers FBN-l is a 110kb gene with 56 exons and 10kb of coding sequence (Gray & Davies, 1996). The fibrillin proteins are mainly composed of calcium-binding epidermal growth factor-like (cbEGF) domains interspersed with domains with homology to transforming growth factor-13 (TGF-13) binding proteins or unique cysteine-rich EGF-TGF hybrid domains (Figure 1.1) (Arteaga-Solis et al., 2000; Kielty & Shuttleworth, 1995; Kielty et al., 2002). The cbEGF repeats have six crucial cysteine residues that are vital for disulphide bonding to form stable 13-sheets (Gray & Davies, 1996). Furthermore, the calcium binding sites within the cbEGF are also important for stabilizing cbEGFs into a linearly rigid structure, mediating fibrillin monomer interactions and lateral packing of microfibrils, and organizing the macroaggregates and protecting them against proteolysis (Arteaga-Solis et al., 2000; Kielty & Shuttleworth, 1995; Ramirez et al., 1999). In the 7 most conmion form of Marfan syndrome, the mutation occurs in the cbEGF domain and thus reduces theCa2-binding affinity of fibrillin- 1. The deficientCa2-binding thereby results in microfibril instability and decreased susceptibility to proteolytic degradation by proteases such as matrix metalloproteinase (MMP), elatase, and thrombin (Kielty & Shuttleworth, 1995; Ramirez et al., 1999; Williams et al., 2008). Figure 1.1 Structure of fibrillin- 1 protein (Boileau et al., 2005). Microfibril is the product of the head-to-tail polymerization of fibrillin molecules with the addition of other proteins (Arteaga-Solis et al., 2000; Ramirez & Dietz, 2007). Microfibrils, without association with elastin, form fibrous aggregates to link different constituents of the extracellular matrix (ECM) and hold tissue components in place (Figure 1.2) (Ramirez et al., 1999). The head-to-tail polymerization gives rise to the bead-to-bead structure with extendibility and flexibility (Figure 1.2). For instance, the microfibrils in the ciliary zonule of the eye can 1II:n un :11101: :ciini ciiuiciNH2 COOlI [1 Epidermal growth factor (EGF)-like domain TOF- 8binding protein-like domain I Calcium-binding EGF-like domain EGF-TGF hybnd domain 8 anchor the lens as well as adjust the thickness of the lens by conducting tension from the muscular movement of the ciliary body (Ramirez & Dietz, 2007). In addition to microfibrillar aggregates, the self-assembled fibrillin-rich microfibrils also participate in the highly regulated elastic fibrillogenesis by acting as a template upon which tropoelastin (precursor of mature elastin) is deposited (Figure 1.2) (Kielty et al., 2002). With the elastic and stretchable outer microfibrillar mantle and inner cross-linked elastin core (Figure 1.2), mature elastic fibers are organized into tissue-specific structures that reflect the mechanical demands of each system (Kielty et al., 2002; Ramirez & Dietz, 2007). In the skin, the loosely organized network of microfibrils and elastic fibers confers pliability (Ramirez & Dietz, 2007). In the aortic wall, elastic fibers form thick concentric lamellae that separate individual vascular smooth muscle cell (VSMC) layers in the tunica media (Arteaga-Solis et al., 2000; Ramirez & Dietz, 2007). In addition, microfibrils further stabilize the tissue by connecting lamellar rings to one another, to VSMC, and to the subendothelial basement membrane (Ramirez & Dietz, 2007). With the composition of microfibrils and elastin, human aorta is able to keep their elastic properties up to 140% extension (Kielty et al., 2002). On the other hand, since the elastic fiber contributes to 50-55% of the dry weight of the aorta (Rosenbloom et al., 1993), its disruption would result in severe consequences. In the case of MFS, the defect in the fibrillin- 1 protein, one 9 of the main scaffolding components of the aorta, leads to the most life-threatening MFS complication. 10 Fibrillin-1 protein E 1111 11II 11111111111111111111110111111111— (‘(NiH polymerization Beaded structure 4 Fbn-1 (lOOnM) Microfibrils +elastjn Elastin core Microfibrfl —+-—--‘Z ‘Z”— mantle Function • Confers elasticity to tissues Figure 1.2 Schematic representation of the main assembly steps from fibrillin- 1 protein to microfibrils and elastic fiber (Fbn- 1, fibrillin- 1; Byers, 2004; Kielty et al., 2002; Ramirez et aL, 1999). Elastic Fiber Elastin-free Microfibrils cD Functions • Connect elastic fibers • Anchor cells to matrix • Hold organ in place 11 1.1.4 Molecular genetics & pathophysiology Marfan syndrome is an autosomal dominant disorder with high penetrance but variable expressivity (Dietz et al., 1991). For classic MFS, linkage analyses have mapped the locus to chromosome 1 5q2 1.1, where the gene encoding fibrillin- 1 is located (Boileau et al., 2005; Dietz et al., 1991; Judge & Dietz, 2005; Pyeritz, 2000). Approximately 66 to 75% of people with MFS inherited the disorder from their parents; however, 25% of the patients have de novo mutations (Chaffins, 2007). Currently, over 600 genetic mutations have been identified (Williams et al., 2008), and they can be divided into two mutation classes: nonsense and missense mutation. Nonsense mutation (38.6% of the mutations) results in premature termination codon and shortened fibrillin-1 molecule. With the mutant mRNA being preferentially degraded by nonsense-mediated mRNA decay, the severity of the disorder is determined by the quantity of mutant mRNA transcripts and the percentage of truncated proteins incorporated into microfibrils (Boileau et a!., 2005). The second class, missense mutation, is more common and accounts for 60.3% of the mutations (Boileau et al., 2005). Moreover, 78% of the point mutation locates in the cbEGF modules and affects mostly the cysteine residues or amino acids involved in calcium binding (Figure 1.4) 12 (Boileau et al., 2005). Even with the lack of focus heterogeneity for Marfan syndrome, 12% of mutations are recurrent and affect a mutation hotspot, CpG., for a cysteine residue (Boileau et al., 2005; Gray & Davies, 1996). This type of mutation is the basis of the mouse model used in this project. Both haploinsufficiency and dominant negative mechanism, in which abnormal protein interacts and interferes with normal proteins, have been shown to be responsible for disease pathogenesis (Judge et al., 2004; Judge & Dietz, 2008). Haploinsufficiency for wildtype protein could bring the amount of fibrillin- 1 down to the threshold, resulting in phenotypic consequences (Ramirez & Dietz, 2007). Furthermore, the mutant fibrillin-1 with dominant negative potential or the recruitment of inflammatory cells by fibrillin-1 degradation products can promote progressive loss of fibrillin-1 with increased proteolytic clearance (Ramirez & Dietz, 2007; Judge et al., 2004). All the above-mentioned processes could lead to the loss of fibrillin- 1 in the ECM and thus a breakdown of tissue integrity (Figure 1.4). Consequently, in response to stress, such as hemodynamic forces in the proximal aorta, this structural insufficiency may lead to accelerated degeneration (Judge & Dietz, 2008). Though this traditional explanation could account for some of the clinical presentations, many are left unexplained. Recently, an upregulation of TGF-13 signaffing has been shown to be closely associated with disease progression and responsible for 13 the pathogenesis of MFS (Figure 1.4) (Habashi et al., 2006; Judge & Dietz, 2005). Microfibrils containing fibrillin- 1 have been shown to not only have structural function but also regulate the signalling pathway of TGFI3, a cytokine that regulates cellular performance (i.e. proliferation, migration, synthetic repertoire, death) and tissue development and homeostasis (Judge & Dietz, 2005; Lacro et al., 2007). TGFI3 is synthesized and secreted as a large latent complex which is composed of three proteins: the latent TGFI3 binding protein (LTBP), the active form of TGFI3 and the latency-associated protein (LAP) (Matt Ct al., 2008). The latter two are associated and sequestered by LTBP which provides safe harbour for TGFf3 before its release (Byers, 2004). Fibrillin-1 shares a high degree of homology with the LTBP, and indeed, the LTBP of the large latent complex of TGFI3 localizes to the microfibrils and interacts directly with fibrillin- 1 (Figure 1.3) (Judge & Dietz, 2005; Lacro et al., 2007). Therefore, it has been suggested that some clinical manifestations of MFS (e.g. aortic, pulmonary, skeletal, skin complications) may result from the failure of latent complex sequestration and subsequent excessive TGFI3 release and downstream signalling (Byers, 2004; Lacro et al., 2007). 14 Figure 1.3 The direct binding of the large latent complex (LLC) of TGFI3 to fibrillin- 1. The LLC is composed of the latent TGFI3 binding protein (LTBP), the active form of TGFI3 and its latency-associated protein (LAP) (N, N-terminus; C, C-terminus; Byers, 2004). LLC TGF9 LAP Fibrillin-1 15 Missense mutation on FBN1 gene (15q21 1) Abnormal fibrillin-1 protein (Ca2 -binding EGF domain) 1i 1’ microfibril — 4’ Ca2 binding in fibrillin proteolysi s Abnormal microfibrils ‘ TGFj3 signaling N / Abnormal arterial function ( distensibility, j’ stiffness, Impaired contractile/endothelial function) Hemodynamic jrstress Aortic dilatation 7 Aortic dissection Aortic rupture Figure 1.4 Pathophysiology of the most common form of Marfan syndrome (Ca2,calcium; EGF, epidermal growth factor; TGFI3, transforming growth factor-13; Williams et al., 2008). 16 1.1.5 Animal model of Marfan syndrome The animal model used was heterozygous for a cysteine substitution (C1039G) in the cbEGF-like domain in Fbnl (Fbn1C39G+), the most common class of mutation observed in classic MFS (Habashi et a!., 2006; Judge et aL, 2004; Ng et aL, 2004). This mutant transgene harbors a naturally occurring human mutation (C1663R). In a patient with the missense mutation, normal synthesis of fibrillin- 1 was observed; however, fibrillin- 1 deposition was impaired (Judge et a!., 2004). Similarly, in murine cells heterozygous for the mutation (C1039G), histological examination also consistently demonstrated a reduction in the deposition of microfibrils (Judge et al., 2004). In addition to histological similarities, the Fbnf’°39mouse model also demonstrated the clinical manifestations common in Marfan patients. Beginning at 2 months of age, there was progressive deterioration of the aortic wall with elastic fiber fragmentation and disarray of vascular smooth muscle cells (VSMC) which eventually led to aortic dilatation (Figure 1.6) (Judge et al., 2004). Skeletal features, including kyphosis and overgrowth of the ribs, were also observed (Figure 1.5). 17 Wildtype Heterozygous (Fbnl clo39GI+) “Control mice” “Marfàn mice” Figure 1.5 Mouse model of Marfan syndrome. The heterozygous (Fbnfo39G’+) mouse, the mouse model used in this study, has phenotypes (e.g. kyphoscoliosis) similar to those of the patients with Marfan syndrome. Control mouse Marfan mouse Figure 1.6 The aortae of control and Marfan mice. The mouse carrying the FbnlC39Gl’+ mutation (Marfan mouse) developed an aneurysm in the aortic arch which was absent in the control. 1.1.6 Pharmacological treatment for Marfan syndrome 1.1.6.1 Current treatment The pharmacological treatments for MFS have been focusing on the aortic complications. Since over three decades ago, 3-adrenoceptor blockers (13-blockers) have been widely considered the standard of care to decrease the progression of aortic dilatation in patients with MFS (Judge & Dietz, 2005; Judge & Dietz, 2008; Williams et al., 2008). Its rationale relies on the reduction of 18 hemodynamic stress on the aorta due to its negative chronotropic and ionotropic effects (Figure 1.8) (Williams et al., 2008). However, controversial results have been obtained in clinical trials with regard to the effectiveness of f3-blockers (Williams et al., 2008). Moreover, 10-20% of patients are intolerant toi3-blockers due to its side effects (Matt et a!., 2008). The other two classes of drugs currently used clinically are calcium channel blockers and angiotensin converting enzyme (ACE) inhibitors. Calcium channel blockers (e.g verapamil), like 13-blockers, have negative ionotropic and chronotropic effects. Moreover, they also cause generalized arterial and arteriolar dilatation (Figure 1.8) (Williams et aL, 2008). They are sometimes prescribed to patients who are intolerant to 13-blockers (Judge & Dietz, 2008). ACE inhibitors are used due to their ability to increase aortic distensibility, reduce central arterial pressure and conduit arterial stiffness (Figure 1.8) (Judge & Dietz, 2008; Williams et al., 2008). However, so far a very limited number of studies have been conducted to assess the effectiveness of calcium channel blockers and ACE inhibitors on patients with MFS (Williams et al., 2008). 1.1.6.2 Potential alternative treatment: doxycycline In the mouse model of MFS, Chung et at have demonstrated that MMP-2 and -9 were upregulated in the aortic wall, and the upregulation was concomitant with extensive degradation 19 of elastic fibers, endothelial dysfunction, and reduction of smooth muscle contractility (Chung et al., 2007a; Chung et at, 2007b; Chung et al., 2007c; Chung et al., 2008a). Therefore, inhibition of MMP-2 and -9 may be a potential strategy for slowing down the deterioration of aortic function in MFS. Doxycycline, a tetracycline-class antibiotic, inhibits a broad spectrum of MMPs at a subantimicrobial dose. In animal models of abdominal aortic aneurysm (AAA), doxycycline has been shown to reduce the activation of MMPs, stabilize elastic lamellae and aortic wall, and suppress aneurysm development (Chung et al., 2008a; Manning et al., 2003; Prall et al., 2002). In patients with AAA, the expression of MMP-2 and -9 was also reported to be decreased by doxycycline (Thompson & Baxter, 1999); moreover, in a recent meta-analysis for the pharmacological treatments for AAA, doxycycline was suggested to have beneficial effects with regard to suppression of aneurysm formation (Guessous et al., 2008). In the Marfan mouse model, we compared doxycycline with a f3-blocker, atenolol, and found that doxycycline was significantly more effective than atenolol in ameliorating thoracic aortic aneurysm. Doxycycline normalized the level of MMP-2 and -9 in the aortic wall, and thereby preserved elastic fiber integrity, contractile capacity, endothelial function, and prevented vessel 20 weakening (Chung et aL, 2008a). With the established tolerability of patients to doxycycline (Baxter et al., 2002), doxcycline may prove to be a potential alternative to the current treatments of MFS (Figure 1.8). 1.1.6.3 Newly investigated alternative treatment: losartan With the recent discovery of the association between fibrillin- 1 abnormality and TGFf3signaling activation, angiotensin II type 1 (AT 1) receptor antagonists have caught much attention as an alternative treatment for MFS. Angiotensin receptor blockers (ARB) for AT1 receptors (e.g. losartan) have multiple effects that may help alleviate the cardiovascular complications in MFS patients. Angiotensin II (ANG II) may cause arterial stiffening through stimulating collagen formation, triggering matrix remodeling and vascular hypertrophy, decreasing nitric oxide (NO) signaling, reducing elastin synthesis, and increasing inflammatory response (Williams et al., 2008). In addition to reducing arterial stiffness, ARBs are also found to antagonize TGFj3 receptors (Figure 1.8) (Williams et al., 2008). The mechanism through which AT1 receptor blockade antagonizes TGFI3 signalling is still unclear. However, AT1 receptor activation has been shown to increase the expression of TGFI3 ligands and receptors and induce the activation of thrombospondin- 1, a 21 potent activator of TGFf3 (Habashi et aL, 2006; Williams et al., 2008). In a mouse model of Marfan syndrome, Habashi et at showed that losartan blunted TGFI3 signaling, prevented elastic fiber fragmentation, decreased aortic root growth rate, and reversed alveolar septation (Habashi et al., 2006). Angiotensin II has also been shown to cause cardiovascular diseases by augmenting oxidative stress. Angiotensin II is a potent activator of NAD(P)H oxidase, which in turn increases nitric oxide synthase (NOS) uncoupling and xanthine oxidase activation (Figure 1.7) (Hitomi, Kiyomoto, & Nishiyama, 2007). As will be discussed in the next section on “oxidative stress,” NAD(P)H oxidase, NOS and xanthine oxidase are all important contributors to oxidative stress. Figure 1.7 Angiotensin II and oxidative stress. Activation of AT1 receptors may cause oxidative stress through multiple and inter-related pathways (ANG II, angiotensin II; AT 1, angiotensin II type 1 receptor; NOS, nitric oxide synthase; 02, superoxide anion). 22 With the various potential beneficial effects of the ARBs for the protection of vasculature, losartan can be an attractive candidate as an alternative treatment for patients with MFS. A randomized clinical trial is underway to compare atenolol with losartan for the management of clinical manifestations of MFS (Lacro et al., 2007). The primary aim of the study is to examine aortic root size and the rate of aortic root growth, which are considered the best predictors of the risk of aortic dissection, over a period of 3 years. The secondary end points include: incidence of aortic dissection, aortic root surgery, progression of aortic and mitral valve regurgitation, left ventricular size and function, central aortic stiffness, skeletal growth, and death (Lacro et al., 2007). 23 Missense mutation on FBNI gene I Abnormal fibrillin-1 protein (Ca2 -binding EGF domain) ________________ t microfibril Doxycycline __.] proteolysis 4 Ca2 binding in fibrillin(e.g. MMPs) // ________ Abnormal microfibrils t TGF$ signaling[— 1 ARB $-blockers CCB Abnormal arterial function $-blockers(t stiffness, 4 distensibility, ACE inhibitors Cardiac notropyl Impaired contractile/endothelial function) ARB inotropy Central arterial pressures - Aortic dilatation CCB ACE inhibitors Figure 1.8 Current and alternative treatment for MFS (f3-blockers, 3-adrenoceptor antagonist; CCB, calcium channel blockers; ACE, angiotensin converting enzyme; ARB, angiotensin receptor blocker; Williams et al., 2008). 24 1.2 Oxidative Stress 1.2.1 Definition & overview Oxidative stress refers to the condition in which excessive production of reactive oxygen species (ROS) outstrips endogenous antioxidant defense mechanisms. (Cal & Harrison, 2000). Many ROS are free radicals with unpaired electrons, and some examples are superxoxide anions (02), hydroxyl radical (H0), nitric oxide (N0), and lipid radicals. Others which are not free radicals, such as hydrogen peroxide (H20), peroxynitrite (ONOO), have oxidizing effects that contribute to oxidative stress (Cal & Harrison, 2000). In the vasculature, all cell types (i.e. endothelial cells, VSMC, underlying tissues) contain enzymes that generate ROS (Gutterman et al., 2005; Schulz et al., 2004). At low concentrations, 02, 11202, or other ROS act as mediators for vascular functions, such as vascular tone, proliferation, and cell signaling (Anozie et al., 2007; Droge, 2002; Faraci & Didion, 2004). However, excess ROS contributes to vascular dysfunction and has been associated with the pathogenesis of cardiovascular diseases including hypertension, atherosclerosis, diabetes, heart failure, chronic kidney disease, and aneurysm (Figure 1.9) (Cal & Harrison, 2000; Ejiri et al., 2003; Faraci & Didion, 2004). 25 1.2.2 Biomarkers for oxidative stress Oxidative stress is usually measured indirectly using techniques that detect oxidative damages to DNA, proteins, and lipids (Tsukahara, 2007). Some examples of the most frequently used biomarkers include: 8-hydroxydeoxyguanosin for DNA damage, 3-nitrotyrosine for protein damage, malondialdehyde (MDA), triphenyiphosphine andF2-isoprostanes for lipid peroxidation (Rosen et aL, 2001). In particular, isoprostanes are used clinically to investigate the pathophysiological role of lipid peroxidation in cardiovascular diseases (e.g. atherosclerosis, ischemia-reperfusion), and strong linkage between these has been found (Cracowski & Durand, 2006). 1.2.3 Generation of oxidative stress In mammalian cells, potential pro-oxidant enzymes include mitochondrial respiration, arachidonic acid pathway enzymes (i.e. lipoxygenase and cyclooxygenase), cytochrome P450s, xanthine oxidase, NAD(P)H oxidase, NOS, peroxidases, and others (Cai & Harrison, 2000). In the vasculature, NAD(P)H oxidase, xanthine oxidase, and NOS have been intensively studied and are believed to play predominant roles in vascular diseases (Figure 1.9) (Landmesser & Harrison, 2001). 26 1.2.3.1 NAD(P)H oxidase NAD(P)H oxidase, an enzyme which catalyzes reduction of oxygen with electrons from NADH or NADPH, is believed to be the major contributor of superoxide anions in the vasculature (Jiang et al., 2004; Matsumoto et al., 2006; Yokoyama et al., 2000). The enzyme, which is found on the membranes of vascular endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts, is made up of multiple components (Forstermann, 2008). The catalytic unit consists of a Nox protein (NADPH oxidase) and p22phox subunit (phox is derived from pagocyte idase). Among the five Nox protein isoforms, Nox2 (also known as gp9lphox) is expressed in endothelial and adventitial cells in large vessels and in VSMC in small vessels whereas Nox4 is constitutively expressed and active in VSMC and EC (Rojas et al., 2006). The p22phox subunit, a docking protein for other subunits, acts to stabilize the Nox proteins (Rojas et al., 2006). The activity of NAD(P)I-T oxidase is regulated by the cytosolic regulatory subunits (i.e. p40phox, p47phox, p67phox, and Rac) in cardiovascular diseases (Rojas et al., 2006; Szasz et al., 2007). The common stimulators of NAD(P)H oxidase include ANG II, thrombin, platelet-derived growth factor, and tumor necrosis factor-a. The activity of the enzyme is also regulated by cytokines, hormones and mechanical forces that are associated with the pathogenesis of vascular diseases (Yung et al., 2006). Activation of NAD(P)H oxidase in the vasculature has been 27 reported in animal models of diseases such as hypertension, diabetes, hypercholesterolemia, and atherosclerosis (Forstermann, 2008). 1.2.3.2 Xanthine oxidase Xanthine oxidase, which catalyses the oxidation of hypoxanthine, xanthine, and NADH during purine metabolism, donates electrons to molecular oxygen to produce 02 and H20 (Cai & Harrison, 2000; Rojas et al., 2006). Xanthine oxidase is mainly present in vascular EC and its expression and activity are modulated by ANG II and NAD(P)H oxidase (Lee & Griendling, 2008). The activity of the enzyme is shown to be altered in the animal models of the diseases such as hypertension, heart failure, and myocardial infarction (Yung et al., 2006). Moreover, in humans, xanthine oxidase is reported to be involved in coronary artery disease and atherosclerotic processes (Yung et al., 2006). 1.2.3.3 NOS Nitric oxide synthases are cytochrome P450 reductase-like enzymes that catalyze flavin-mediated electron transport from NADPH to the heme group in the oxygenase domain of the enzyme (Forstermann, 2008; Landmesser & Harrison, 2001; Li & Forstermann, 2000). There are three isoforms of NOS: neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS 28 (eNOS). While eNOS, which is mainly found in endothelial cells, is constitutively expressed and active, iNOS, which can be found in both VSMC and EC, needs to be activated (Rang et al, 2003). In physiological conditions, NOS uses L-arginine as substrate to produce L-citrullin and NO. Nitric oxide is a major vasodilator, and it also affords protection to the vessels with its anti-atherosclerotic and anti-proliferative effects (Li & Forstermann, 2000). However, in conditions where electron flow within NOS is disturbed, oxygen reduction and NO generation are uncoupled and 02 and 11202 are generated (Cai & Harrison, 2000; Forstermann, 2008). The flow of electrons can be disrupted by the absence of either L-arginine or tetrahydrobiopterin (BH4,a NOS cofactor important for regulating NOS function by coupling reduction of 02 to oxidation of L-arginine) (Cal & Harrison, 2000; Rojas et al., 2006). The mechanism through which eNOS uncoupling occurs is still unclear. However, it has been suggested that it may occur through oxidation of BH4by peroxynitrite (Cai & Harrison, 2000). 1.2.4 Removal of oxidative stress Some examples of the antioxidant systems are superoxide dismutase (SOD), catalase, glutathione redox cycle, and ROS scavengers (Szasz et al., 2007). Among them, SOD is believed to be the 29 main endogenous antioxidant responsible for superoxide removal (Didion et al., 2002). Superoxide dismutase breaks down superoxide to produce H20.Hydrogen peroxide is in turn converted to water and oxygen by catalase (Figure 1.9), a homotetrameric heme-containing enzyme (Szasz et al., 2007; Yung et al., 2006). There are three isoforms of SOD: cytosolic or copper-zinc SOD (CuZnSOD or SOD-i), manganese SOD (Mn-SOD or SOD-2) in mitochondria, and extracellular CuZnSOD (EC-SOD or SOD-3). In the mouse aorta, SOD-i and SOD-2 are the predominant isoforms expressed (Faraci & Didion, 2004). The expression of SOD-i can be increased in the endothelium by shear stress and exercise and decreased by disturbed blood flow in the vasculature. On the other hand, SOD-2 is responsive and upregulated by oxidative stress (Faraci & Didion, 2004). 30 ANG II, ET-1, mechanical stretch, Ml, ischemic heart disease, hypertension, diabetes, aneurysm, etc. 1r ROS Generation Xanthine NAD(P)H Uncoupled oxidase oxidase NOS ROS Neutralization H202 1 _____ - J ROS generation0,I tCataIas I SOD ROS neutralization _________ j NO Oxidative Stress StressLDL —. oxidation RO_— 1’ O 0N00, peroxides Signalling • NO bloavailability Lipid N MMP peroxidation/ / activation Remodeling VSMC Endothe hal Platelet Inflammation growth dysfunction aggregation I _ _________---- —CARbI OVASC U LAR - Z!fYNCTION& D,1SEA Figure 1.9 Oxidative stress: mechanisms through which oxidative stress is induced and leads to cardiovascular pathology (ANG II, angiotensin II; ET-1, endothelin-1; MI, myocardial infarction; ROS, reactive oxygen species; NOS, nitric oxidase synthase; NO, nitric oxide; Cai & Harrison, 2000; Landmesser & Harrison, 2001; Pacher et al., 2007). 31 1.3 Vasomotor Function In MFS, all the clinical manifestations in the cardiovascular system pertain only to the heart and the artery but not the vein. Moreover, due to its proximity to the heart and high content of elastic fiber in the composition, aorta is the most severely affected in the vasculature. Therefore, the main focus of discussion will be on the aortic function. 1.3.1 Structure of artery Based on their composition, arteries can be divided into three layers from the innermost to the outermost: tunica intima, tunica media, and tunica externa. Tunica intima (often called intima) is in direct contact with the blood flow, and is mainly composed of endothelial cells. Tunica media (also known as media) consists predominantly of elastic fibers and smooth muscle. In the mouse aorta, five to seven layers of smooth muscles alternate with layers of elastic fiber which is present in considerable amount to allow for elastic recoil and maintenance of blood pressure. Tunica externa (formerly known as tunica adventitia) is made up of nerves and connect tissue (Widmaier et a!., 2003). The endothelium and smooth muscle are the main players in vascular contraction and relaxation. Vasoconstriction results when the smooth muscle cells, which are long when relaxed, contract 32 and shorten. Endothelial cells, on the other hand, play a crucial role in regulating vascular tone by producing potent chemical mediators (Rang et al., 2003; Widmaier et al., 2003). 1.3.2 Vascular smooth muscle contraction Vasoconstriction is mainly generated by calcium-dependent mechanisms. The calcium ion (Ca2) is a central messenger of SMC function and regulates not only tone, but also proliferation, migration and apoptosis. The regulation of intracellular Ca2 concentration ([Ca2])involves the interplay of various Ca2transport proteins on the plasma membrane (PM) and sarcoplasmic reticulum (SR). On the PM, voltage-gated Ca2 channels (VGCC) and non-selective ion channels allow the influx of Ca2 upon stimulation, whereas the PMCa2-ATP se (PMCA) and the sodium calcium exchanger (NCX) are responsible for the efflux to restore the [Ca2j, to the resting state (Figure 1.10). The SR membranes contain sarco-endoplasmic reticulumCa2-ATP se (SERCA) for uptake and two types of channels, ryanodine receptors (RyR) and 1P3 receptors (JP3R), for Ca2 release (Figure 1.10) (Rang et al., 2003). 33 PM SR Figure 1.10 Ion channels and receptors responsible for regulating intracellular calcium ion (Ca2) concentration in smooth muscle cells (PM, plasma membrane; SR, sarcoplasmic reticulum; Na, sodium ion; VGCC, voltage-gated calcium channel; NCX, sodium calcium exchanger; P3R, 1P3 receptor; RyR, ryanodine receptor; SERCA, sarco-endoplasmic reticulumCa2-ATPase). The increase in [Ca2hij enables the binding of Ca2to calmodulin, which in turn binds to and activates myosin light chain kinase (MLCK). The activated MLCK then phosphorylates myosin and activates myosin ATPase involved in cycling of the actin-myosin crossbridges that power SMC contraction and vasoconstriction (Widmaier et al., 2003). Vasoconstriction can be induced by various agonists, such as endothelin-1 (ET-1), ANG II, and a1-adrenoceptor agonists (e.g. phenylephrine). After the agonist (e.g. phenylephrine) binds to the G-protein coupled receptor (GPCR; e.g. ai-adrenoceptor) on the VSMC, the associated G 34 protein is activated which in turn activates phospholipase C. Phospholipase C is an enzyme that cleaves phosphatidylinositol biophosphate (PIP2)to form diacyiglycerol (DAG) and inositol trisphosphate (1P3). 1P3 thereby causes Ca2 release from the SR by binding to 1P3 receptors. The increase in [Ca2i1consequently causes contraction of the smooth muscle cell (Figure 1.11) (Rang et al., 2003). 1.3.3 Vascular relaxation Vasodilatation results when the SMCs are relaxed, and it is predominantly regulated by the mediators released by the ECs. After an agonist (e.g. acetyicholine) binds to the GPCR (e.g. muscurinic receptor type 3, M3) on the EC, 1P3 is produced, and the [Ca2]increases. The increase in [Ca2]1causes the release of the vasodilating mediators, the most important of which is NO. Nitric oxide thereby enters the VSMC and increases the level of cGMP which consequently leads to a decrease in [Ca2]1and hence smooth muscle relaxation (Figure 1.11) (Rang et al., 2003). 35 Agonist (e.g. ACh) Ca2 SMC Figure 1.11 Endothelium-mediated and agonist-induced vascular smooth muscle contraction and relaxation (EC, endothelial cell; SMC, smooth muscle cell; SR, sarcoplasmic reticulum; ER, endoplasmic reticulum; ACh, acetylcholine; PE, phenylephrine; GPCR, G-protein coupled receptor; PLC, phospholipase C; IP3R, 1P3 receptor; TXA2,thomboxane A2; NO, nitric oxide; PGI2,prostacyclin; EDHF, endothelium-derived hyperpolarizing factor; [Ca2], calcium concentration; Rang et al., 2003). 1.3.4 Marfan syndrome and vasomotor function Previously, in the thoracic aorta of the Marfan mouse model, we have reported impairment in the SMC contractility in response to membrane depolarization and vaso-constricting agonists (i.e. phenylephrine) (Chung et al., 2007b). In addition, Marfan syndrome also causes endothelial Ca2 36 dysfunction by disrupting NO production signaling (Chung et al., 2007a). The functional abnormality in the aorta may be a consequence of the lack of attachment of cells to the elastic laminae and/or the malfunction of the cells themselves (Figure 1.13). To examine the processes that lead to the lethal aortic complications, Bunton et al used ultrastructural analysis and discovered the first abnormality which they believed initiates the destructive processes in the aorta of mice expressing low levels of fibrillin (Bunton et al., 2001). In the fibrillin- 1 deficient mice, the cross-sectional area of the aorta presents an unusually smooth surface of elastic laniinae, an observation which was also made in the Fbnf’°39°mouse model (Figure 1.12b) (Chung et al., 2007b). The smoothness of the surface manifests in the loss of cell attachments (Figure 1.13) (Bunton et al., 2001). 0 y Control mouse Marfan mouse model (C57BL/6) (Fbn1ClO39) Figure 1.12 Cross-sectional area of(a) control and (b) Marfan mouse aorta (Movat’s staining). The elastic fibers (dark purple lines) of the Marfan mice (b) are more discontinuous, broken, and smoother as compared with those of the control littermates (a). 37 The lack of attachment of the vascular endothelial and smooth muscle cells to the elastic laminae may result in functional abnormalities causing an alteration in vasomotor function (Figure 1.13). Fibriffin-containing microfibrils have been found in abundance immediately subjacent to the arterial ECs, and it has been suggested that the close association between the EC and fibrillin may play a functional and structural role (Wilson et al., 1999). The lack of anchorage of the EC to the connective tissue may lead to disruption in the NO production pathway. Similarly, the deficient attachment of the VSMC to the elastic fiber layer may result in compromised contractility. Furthermore, the contractile capacity of arteries has been reported to be associated with aortic dilatation (Chew et al., 2004). In abdominal aortic aneurysm, the inhibition of active and tonic contraction of vascular smooth muscle was believed to reduce the ability of the aortic wall to withstand the pulsatile hemodynamic force generated during systole, and consequently resulting in progressive aortic dilatation and aneurysm (Figure 1.13) (Chew et al., 2004). New therapeutic approach aimed at preserving the vascular function (i.e. contraction and relaxation) of the aorta may be developed to slow down the progression of the life-threatening vascular manifestations of MFS. 38 Mutation on FBNI gene Abnormal fibrillin-1 protein (Ca2 -binding EGF domain) I Abnormal microfibrils Smooth surface of elastic Iaminae I Lack of cell attachment ____ (e.g EC, VSMC) Endothelial dysfunction N4. Ability to withstand Impaired contractile hemodynamic force response V Aortic aneurysm Figure 1.13 Processes leading to aortic aneurysm in Marfan syndrome (EC, endothelial cell; VSMC, vascular smooth muscle cell). 1.3.5 Impact of oxidative stress on vascular function Vasomotor function is tightly regulated by ROS (Gutterman et al., 2005; Landmesser & Harrison, 2001; Lee & Griendling, 2008; Lounsbury et al., 2000). Whether ROS causes vasoconstriction or dilatation depends on the vascular bed and amount of ROS present (Yung et al., 2006). For example, H20has been suggested to be an endothelium-dependent hyperpolarizing factor (EDHF), causing vasodilatation in pulmonary, coronary and mesenteric arteries by membrane depolarization (Cai, 2005; Yung et al., 2006). On the other hand, ROS (e.g. metabolites of arachidonic acid) can also cause vasoconstriction by acting directly on the VSMC (Yung et al., 2006). 39 In addition to the direct effects ROS has on vasomotor responses, they have also been shown to affect the Ca2 signalling pathway. In the EC, superoxide enhances agonist-stimulated Ca2 signaling by increasing both the intracellular Ca2release from the endoplasmic reticulum as well as extracellular Ca2influx. On the other hand, peroxides attenuate Ca2 signalling by inhibiting the entry of Ca2into the cytoplasm from both sources (Lounsbury et al., 2000). In the VSMC, both superoxide and peroxides disrupt Ca2 homeostasis by inactivating SERCA (Figure 1.14) (Elmoselhi et al., 1996; Lounsbury et al., 2000; Walia et al., 2000). The effect of ROS on contractile function has not been well studied (Lee & Griendling, 2008; Lyle & Griendling, 2006). However, the changes caused by ROS on Ca2 signaling in vascular EC and SMC have been suggested to play a role (Elmoseihi et al., 1996; Lounsbury et al., 2000; Walia et a!., 2000), and the impairment of the Ca2 signaling pathway may consequently lead to an alteration in vascular reactivity (Sener et al., 2004). Another way that ROS (e.g. superoxide, H2O)may cause a decrease in contraction is by the activation ofCa2—activated potassium channels (Ka) on the membrane of the VSMC and hence hyperpolarization (Figure 1.14) (Gutterman et al., 2005). Previously, we have found that in the aorta of Marfan mice, the expression of cyclooxygenase (COX)-2 was upregulated and the level of prostacyclin was increased (Chung et a!., 2007c). Prostacyclin causes vasodilatation, and thus its upregulation can 40 be another explanation for the reduction in aortic contractility. [4_i ER Ca2 t [Ca2] NO 2+ HO/ Ca / 22: K’ Hyperpolarization N )SMC I [C&4] Contraction 1 Ca2 Figure 1.14 Effects of reactive oxygen species on calcium signaling and vasoconstriction (EC, endothelial cell; SMC, smooth muscle cell; SR, sarcoplasmic reticulum; ER, endoplasmic reticulum; 02, superoxide anion; H20,hydrogen peroxide; SERCA, sarco-endoplasmic reticulumCa-ATPase). It has been well-recognized that oxidative stress is associated with endothelial dysfunction in cardiac and vascular diseases (Johnstone et al., 1993; Panza et al., 1995; Payne et al., 2003). Endothelial dysfunction is commonly described as the impairment of endothelium-dependent vasorelaxation caused by a loss of NO bioactivity in the vessel wall (Cai & Harrison, 2000; Li & Forstermann, 2000). Oxidative stress may cause endothelial dysfunction through several direct EC 41 and indirect pathways, the most well-known of which is the scavenging of NO by superoxide. Superoxide radicals bind to NO at a rate three times faster than they bind to SOD; therefore, excess superoxide production would increase the rate of NO degradation (Cai & Harrison, 2000; Harrison, 1997; Schulz et al., 2004). The reduced NO half-life consequently decreases the activation of the downstream guanylyl cyclase/cGMP pathway, thus resulting in the impairment of endothelium-dependent relaxation (Schulz et al., 2004). In addition, peroxynitnte, the product of the reaction between O2 and NO, could cause the uncoupling of eNOS by oxidizing BH4.The uncoupled eNOS can lead to endothelial dysfunction via at least three mechanisms: reduce the production of NO, produce superoxide thus further exacerbating oxidative stress, generate more peroxynitrite as machinery for both 02 and NO production (Figure 1.15) (Cal & Harrison, 2000; Schulz et al., 2004). 42 Xanthine NAD(P)H oxidase oxidase \ // (eNOS) NO / / ONOO- 1’UncoupledN L__—-- ‘eNQS) 8 8 .1. NO bloavailability 4. NO production .1. Vasodilatation Figure 1.15 Processes leading to decreased vasodilatation (02, superoxide anion; NO, nitric oxide; 0N00, peroxynitrite; eNOS, endothelial nitric oxide synthase). 43 1.4 Hypothesis and Aims 1.4.1 Research rationale Although it is well-established that oxidative stress has a profound influence on vascular function, the effect of oxidative stress on the aortic vasomotor response in the progression of Marfan syndrome has never been investigated. 1.4.2 Hypothesis & objectives We hypothesize that in the thoracic aorta of a mouse model of Marfan syndrome, the aberration in vasomotor function is caused by oxidative stress; therefore, by increasing superoxide removal or decreasing superoxide generation in vitro, vascular function can be improved. The three objectives are: 1. To determine whether oxidative stress contributes to the reduction in contractility and relaxation in Marfan aorta 2. To study the mechanisms via which oxidative stress is elevated 3. To measure SOD and pro-oxidant enzyme levels in Marfan aorta 44 CHAPTER 2 MATERIALS & METHODS 2.1 Animals & Anesthesia Heterozygous (FbnfAo39Gh’+) mice were mated to C57BL16 mice to produce equal numbers of Fbn1CO39Gl‘Marfan’ subjects (n=40) and wild-type ‘control’ (n=40) (Chung et al., 2007a; Chung et aL, 2007b). Both strains were housed in the institutional animal facility (The University of British Columbia, Child and Family Research Institute) under standard animal room conditions, and all animal procedures were approved by the institutional Animal Ethics Board. Mice at age 3 (n=30), 6 (n=30) and 9 (n=20) months were anaesthetized with a mixture of ketamine hydrochloride (80mg kg’) and xylazine hydrochloride (12mg kg’) intraperitoneally. The state of anesthesia was checked with toe-pinching. 2.2 Measurement of Isoprostanes (8-Isoprostane) After the pain reflex was completely abolished with deep anesthesia, whole blood was collected via cardiac puncture. Plasma was separated by centrifugation. 8-Isoprostane (8-epi-PGF2)levels were determined in plasma using an enzyme immunoassay kit according to the manufacture’s procedures. 45 2.3 Tissue Preparation for Isometric Force Measurement After the pain reflex was abolished and whole blood was drawn, the animals were sacrificed by cutting the throat and spinal cord. The rib cage was then removed, and the descending thoracic aorta, the straight section between the aortic arch and the diaphragm, was carefully isolated and cleaned of fat and connective tissue in cold oxygenated Krebs solution (6.95g NaCl, 2.09g NaHCO3,2.Og glucose, O.350g KC1, O.161g KH2PO4,O.141g MgSO4,O.0086g EDTA, 1.6mL CaC12 (1M) for 1 L of solution; pH 7.4-7.45). The descending thoracic aorta was cut into 2mm segments, and two one-inch-long wires (4Ojim in diameter) were inserted in the lumen. The aortic segments were then mounted isometrically in a multi wire myograph system (A/S Danish Myotechnology, Aarhus N, Denmark) for force generation measurement (Chung et al., 2007a; Chung et al., 2007b). The temperature of the bath solution (Krebs) in the chambers was maintained at 37°C, and the tissues were oxygenated with carbogen (95% 02, 5% C02). 2.4 Measurement of Isometric Force Aortic segments were allowed to equilibrate for 30 minutes before stretched to the resting tension (=6.0 mN). After the tension stabilized, the tissue was challenged twice with 60mM KC1 (5.55g NaC1, 4.lOg KC1, 1.80 g glucose, 1.19g Hepes, 0.0952g MgCl2,0.17Mg CaC12.2H0for 1 L of solution; pH 7.6) before experiments were continued. 46 To assess agonist-induced contraction, the concentration response curves for phenylephrine (PE) were constructed by cumulative addition of PE from mM to 3M. After sustained pre-contraction was obtained with PE (3tM), cumulative concentrations of acetyicholine (ACh; io to 104M) were added to examine endothelium-dependent relaxation. In previous studies (Chung et al., 2007a; Chung et al., 2007b; Chung et aL, 2008a), the contractile function of the Marfan aorta was observed to be impaired at and after 9 months while the relaxation function was compromised at younger ages, i.e. 3 and 6 months. Therefore, in this study, the effect of oxidative stress was studied at 9 months for PE-induced contraction and at 3 and 6 months for ACh-mediated relaxation. 2.4.1 Inhibitor/enzyme pre-incubation To assess whether removal of superoxide affected contractile function, aortic segments were pre-incubated with SOD (l50units mL’) or SOD plus catalase (l000umts mIJ’) for 30 minutes before the addition of phenylephrine (1nM-3iM). After sustained PE pre-contraction was obtained, concentration response curves of ACh (10 to 104M) were constructed to determine the effect of oxidative stress on endothelium-dependent relaxation. 47 To decipher the mechanisms that contribute to the elevation of oxidative stress, aortic segments were pre-incubated with three inhibitors that block the potential superoxide-generating enzymes (Figure 2.1): the NAD(P)H oxidase inhibitor apocynin (1O0M), the xanthine oxidase inhibitor allopurinol (3001M), and the iNOS inhibitor 1400W (1!IM). Aortic segments were incubated with the inhibitors for 30 minutes before concentration response curves for PE and ACh were constructed. Xant [ INOS] flNAD(P)H Oxidase A1Iopurino\\\ H20 [Catalase 02+1-120 Figure 2.1 Flow chart showing the enzymes responsible for superoxide-generation and -neutralization and the inhibitors of the enzymes (enzymes are shown in rounded rectanglar boxes). 48 2.5 Western Immunoblotting Because of the limited sample from each mouse, thoracic aortic segments were pooled in groups with the same strain and age. To homogenize the aortic tissue samples, flash-frozen aortic segments were grounded with a stainless-steel motor and pestle. The tissue powder was well mixed with ice-cold cell-lysis buffer and protease inhibitor, and after centrifugation at 14000g for 20 minutes at 4°C, the supernatant was collected (Chung et al., 2007a). Protein samples (40tg) were separated on 6% (for iNOS and xanthine oxidase), 9% (for p67phox and gp9lphox) or 13% (for SOD-i, SOD-2, and p47phox) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinyldifluoride membranes. Membranes were incubated with primary antibodies (dilution 1:200-1:800) for 2 hours at room temperature, then 1 hour at room temperature with IgG peroxidase-conjugated secondary antibodies (dilution 1:2500). Inimunoreactive proteins were detected and visualized using the ECL western blotting detection kit. To ensure equal protein loading, membranes for 13% SDS-PAGE were stripped and re-probed with anti-13-actin antibody (1:5000) (Chung et al., 2007a). 49 2.6 Drugs & Chemicals Ketamine hydrochloride and xylazine hydrochloride (Research Biochemicals International, Natick, MA); phenylephrine, acetyicholine, catalase, allopurinol, 1400W, potassium chloride, chemicals for preparing Krebs solution, rabbit anti-SOD 1 and SOD-2 primary antibodies, anti-rabbit and mouse IgG peroxidase-conjugated secondary antibodies (Sigma-Aldrich, Oakville, ON); apocynin and SOD (Calbiochem, San Diego, CA); rabbit anti-xanthine oxidase, anti-p47phox, anti-p67phox, and mouse anti-iNOS primary antibody (Santa Cruz, Santa Cruz, CA); mouse anti-gp9lphox primary antibody (BD Biosciences, Mississauga, ON); ECL western blotting detection kit (Amersham Life Sciences, Arlington Heights, IL); 8-Isoprostane enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). 2.7 Statistical Analysis Percent of maximum contraction was determined by normalizing the increase in force from baseline at each concentration of PE with the maximal contractile force (Em). Percent relaxation was calculated as the percent decrease in force with respect to the initial PE-induced pre-contraction, and the percent relaxation was used to construct the concentration response curves of ACh-induced relaxation. To compare the sensitivities of the vessels to the agonists in the presence of different inhibitors, the negative logarithm (pD2) of the concentration of PE or 50 ACh giving 50% of maximum response (pEC50)was determined by linear interpolation on the semilogarithm concentration response curve [pD2= -Log(EC50)j.Maximal contraction and relaxation responses as well as pEC50 values were compared by student’s un-paired t-test. Statistical significance was defined as p<O.05. Data were reported as mean ± s.e.mean from 4-9 independent experiments. Statistical analysis and construction of concentration response curves were performed using GraphPad Prism software (San Diego, CA). For western immunoblotting, aortic samples were pooled from 10-15 mice for each group; therefore, the error bars for the desitometric analysis were not available. 51 CHAPTER 3 RESULTS 3.1 Plasma Isoprostane 8-epi-PGF2Levels The plasma isoprostane 8-epi-PGF2 level, a systemic marker of oxidative stress, increased with age in both groups. The plasma isoprostane levels in Marfan mice 3 and 6 months of age were 50% higher than those in the normal controls (p < 0.001). This difference disappeared at 9 months (Figure 3.1). E 1I0, 0. Age (m) Figure 3.1. Bar graph presenting the plasma level of isoprostane 8-epi-PGF2 in pg/mi in the control and Marfan groups at 3, 6 and 9 months of age (n=4, *p<0.05 as compared with age-matched control). *Control — * I 52 3.2 Preincubation with SOD and Catalase To reveal the influence of superoxide on the agonist-induced contractile response, the aortic segments were preincubated with SOD, an enzyme that converts superoxide to H20,before the addition of PE. We previously showed that control and Marfan aorta demonstrated a difference in the PE-contraction only at 9 months of age (Chung et aL, 2007b), and thus the effect of superoxide removal on contraction was examined in this age group. Pretreatment of SOD potentiated PE-induced contraction in the Marfan aorta and increased the Emax from 3.72±0.63 to 7.10±1.l7mN (Figure 3.2a) and the pEC50 by 2.75-fold (Figure 3.2b; absence of SOD: 6.76±0.07; presence of SOD: 7.20±0.07), returning both values to the control level (Emax: 6.47±0.88mN; pEC50:7.26±0.07). In contrast, SOD had no effect on PE-induced contraction in the control aorta (Figure 3.2; Table 3.1). The participation ofH2O,produced by SOD, in the alteration of PE response was further assessed with the co-incubation of SOD and catalase. In physiological conditions, SOD converts superoxide to H20,which is subsequently converted to oxygen and water by catalase. In Marfan aorta, the effect of SOD plus catalase was not different from that of SOD alone; like SOD, the combination treatment also significantly improved the Emax and pEC50 of PE in the Marfan aorta as compared with no treatment (Figure 3.2; Table 3.1). 53 a b I — 7z C, ILl E w2_5 CoiErd I I No Treatment With SOD With SOD & catalase Figure 3.2. Effect of superoxide dismutase (SOD, 150 units m1’) and SOD-plus-catalase (1000 units mf1) on phenylephrine-stimulated contraction in thoracic aorta from control and Marfan mice. Bar graphs showed (a) Emax and (b) pEC50 in response to phenylephrine in the presence and absence of SOD or SOD-plus-catalase preincubation at the age of 9 months (n=5-8, *p<0•05 as compared with control in the absence of the enzymes, #p<O.O5 as compared with the responses in the absence of the enzymes in their respective groups. SOD, superoxide dismutase; Cat, catalase). fan Coithd 54 Impaired endothelium-dependent ACh-stimulated relaxation was previously observed in the Marfan aorta at 3 and 6 months of age (Chung et aL, 2007a); therefore, in this study, the effect of oxidative stress on endothelium-dependent relaxation was assessed at these two ages. The SOD and SOD-plus-catalase pretreatment affected ACh-induced relaxation in both control and Marfan aorta, and the effects were age-dependent (Figure 3.3; Table 3.2). At 3 months, neither SOD nor the SOD-plus-catalase combination altered the maximal relaxation response and sensitivity in the control aorta (Figure 3.3a, b). Nevertheless, in the Marfan aorta, both SOD and combination treatment restored the ACh-maximal relaxation to the control level (Figure 4a). SOD alone also increased the ACh-pEC50by 10.7-fold (absence of SOD: 6.56±0.36; presence of SOD: 7.59±0.22); however, the increase in sensitivity in the presence of SOD and catalase did not reach significance (Figure 3.3b). At 6 months of age, SOD treatment significantly decreased the AChEmax by 17.6% (Figure 3.3c) but increased the pEC50 by 14.1-fold (Figure 3.3d; absence of SOD: 7.29±0.18; presence of SOD: 8.44±0.39) in the control aorta. However, these effects disappeared upon the addition of catalase. In the Marfan aorta, SOD alone did not cause a significant effect on the Em; however, the combination of SOD and catalase increased AChEmax by 3 1.5% (Figure 3.3c). Moreover, SOD and SOD-plus-catalase increased the ACh-pEC50value by 12.3 and 19.1-fold, respectively (Figure 3.3d; absence of enzymes: 5.85±0.22; presence of SOD: 6.94±0.18; presence of SOD and catalase: 7.13±0.18). 55 Emaxa b 0 I 9, ID.. 0 w 7. pEC50 ConoI Uai d 9. = 0 . ‘U 6 5. C 1II 0 Th rbn I I No Treatment With SOD .-....‘ With SOD & catalase Figure 3.3. Effect of superoxide dismutase (SOD) and SOD plus catalase on acetyicholine (ACh)-induced relaxation in thoracic aorta from control and Marfan mice. Bar graphs showed (a) Emax at 3 months, (b) pEC50 at 3 months, (c) Emax at 6 months, (d) pEC50 at 6 months in response to ACh in the presence and absence of SOD or SOD plus catalase (n=5-8, *p<O.05 as compared with control in the absence of the enzymes, #p<O.05 as compared with the responses in the absence of the enzymes in their respective groups. SOD, superoxide dismutase; Cat, catalase). 56 3.3 Blockade of NAD(P)H Oxidase with Apocynin Apocynin, an NAD(P)H oxidase inhibitor, did not cause significant changes in P&Emax and pEC50 at 9 months old in either control or Marfan aorta (Figure 3.4a, b). For the ACh-stimulated relaxation, apocynin did not have an effect on either Emax or pEC50 at 3 months in both groups (Table 3.2). However, at 6 months, apocynin increased the Em from 52.7 to 82.6% and pEC50 by 37.2-fold (Figure 3.4d; Table 3.2) in the Marfan aorta whereas in the control aorta, no significant changes were observed (Figure 3.4c). 57 o (-)Apocynin • (+)Apocynin Figure 3.4. Effects of apocynin (100FtM) on phenylephrine-.induced contraction and ACh-mediated relaxation. Phenylephrine-contraction was not affected by apocynin at 9 months in both (a) control and (b) Marfan aorta. (c) The ACh-induced responses in the thoracic aorta were not altered by apocynin in 6 months control mice. (d) Both the ACh-Emax and pEC50 were improved in the 6 months Marfan aorta (n=4-6; *p<O.05 vs. no treatment). MarfanControl I a 1100 C 0 e a I -Lc4PE (Mfl a a b C 0 0 a a C 0 0 E E ‘C a E d C 0 N ‘C0 S 1 -LoqIPE (MI S 5 }* 98765 -Log [ItCh (Mfl 43 10 9 8 7 6 5 4 3 -Log [ACh (Mfl 58 3.4 Blockade of Xanthine Oxidase with Allopurinol Allopurinol, a xanthine oxidase inhibitor, potentiated PE-contraction in the Marfan aorta at 9 months of age. Though no significant difference was observed in the maximum response to PE (Table 3.1), the pEC50 value was increased from 6.76±0.07 to 7.13±0.12 (Figure 3.5b), normalizing it to the control value (7.26±0.07). With regard to ACh-mediated relaxation, allopurinol, like apocynin, had no effect on either control or Marfan aorta at 3 months (Table 3.2). At 6 months old, allopurinol had no effect on the maximal response of ACh in the Marfan group; however, it increased the sensitivity by 58.9-fold (Figure 3.5d; absence of allopurinol, 5.85±0.22; presence of allopurinol, 7.62±0.44). Allopurinol did not cause significant changes in either the PE-contraction (Figure 3.5a) or ACh-relaxation (Figure 3.5c) in the control aorta. 59 o (-)Allopurinol • (+) Allopurinol Figure 3.5. Effects of allopurinol (3OOiM) on PE-mediated contraction and ACh-induced relaxation. At 9 months of age, PE-sensitivity was increased significantly in Marfan aorta (b), but not in the control (a). Similarly, an improvement in ACh-pEC50was observed only in Marfan (d) and not in control (c) at 6 months (n=4-8; pO.O5). Control 75 25 a I Marfan J b 1 75 C 0 0 E f 25 ‘C a E d a C 0 4- 0 a I 4- C 0 0 E E ‘C a E C C 0 I x S 0 I I I * -LO!IPE (Mfl a a I I -LA4PE (Mfl a a C 0 S S 0 a • 765 -Log[ACh(M)] toqACh(M] 60 3.5 Blockade of iNOS with 1400W The specific iNOS inhibitor, 1400W, altered PE and ACh responses in both control and Marfan aorta. At 9 months of age, 1400W increased PEEmax in both control and Marfan aorta by 49.9 and 133%, respectively (Figure 3.6a, b; Table 3.1); however, the sensitivity was only significantly increased in Marfan but not in the control (Table 3.1). With respect to ACh-induced relaxation, 1400W had no effect on control aorta at 3 months but increased the Em from 54.8 to 90.1% in the Marfan aorta (Table 3.2). At 6 months, the AChEmax was decreased from 89.2 to 64.9% in the control (Figure 3.6c) whereas ACh-sensitivity was increased by 11.0-fold in the Marfan (Figure 3.6d; Table 3.2). 61 a4) C.) I 0 LI a 4) C.) I 0 C 0 ‘C(U 4’ . (+) 1400W Marlan pEC50 Figure 3.6. Effects of 1400W (lp.M) on PE-induced contraction and ACh-mediated relaxation. At 9 months of age, maximal responses (Emn) to PE were increased significantly in both (a) control and (b) Marfan aorta. (c) In 6-month control mice, 1400W significantly decreased AChEmn, while (d) in Marfan mice, 1400W significantly increased ACh-sensitivity (n=5-8, *p<005 vs. no treatment). Control max a b }* Th1* C -Log [PE (NI)] 9 8 d 7 6 -Log [PE (NI)] 5 C 0 (C ‘C(C 4) I 10 9 8 7 6 5 4 3 -Log [ACh (M)] 10 9 8 7 6 5 -Log [ACh (M)] 0 (-) 1400W 4 3 62 Table 3.1 Summary of Em (mN) and pEC50 values of PE-induced contraction in the absence or presence of enzymes or inhibitors in the thoracic aorta from control and Marfan mice at 9 months of age. (mN): Strain Control Marfan PE 6.47±0.88 3.72±0.63* PE+SOD 7.92±1.06 7.10±1.17# Drug PE + SOD + Cat 6.46±1.06 6.90±0.74# treatment PE + Allopurinol 4.89±2.54 5.92±1.62 PE+Apocynin 6.21±0.18 5.26±1.26 PP + 1400W 9.70±0.75# 8.68±0.55# pEC50: PE 7.26±0.07 6.76±0.07* PE + SOD 7.19±0.06 7.20±0.07# Drug PE + SOD + Cat 7.20±0.06 7.15±0.05# treatment PE + Allopurinol 7.20±0.06 7.13±0. 12# PE + Apocynin 7.17±0.06 6.97±0.07 PE÷1400W 7.26±0.07 7.23±0.04# Abbreviations: PE, phenylephrine SOD, superoxide dismutase; Cat, catalase. *p.<005, vs. age-matched control. # p<O.O5, vs. without pharmacological treatment in respective control or Marfan group. 63 Table 3.2 Summary of Emax and pEC50 values of acetylcholine-induced relaxation (% of precontraction) in the absence or presence of enzymes or inhibitors in the thoracic aorta from control and Marfan mice at 3 and 6 months of age. Strain Control Marfan (mN): Age (Month) 3 6 3 6 ACh 78.0±9.5 89.2±2.0 54.8±9.6* 52.7±6.8* ACh + SOD 75.3±11.9 71.6±10.2# 76.3±6.3 1 65.7±10.4 Drug ACh + SOD + Cat 81.5±13.0 78.8±7.9 83.1±2.99 84.2±4.4# treatment ACh+Allopurinol 30.0±13.9 72.1±11.5 69.1±8.9 71.2±15.8 ACh + Apocynin 61.4±14.4 68.6±12.9 72.5±11.8 82.6±8.8# ACh + 1400W 49.8±12.4 64.9±9.6# 90.1±3.1# 58.0±16.7 pE( ACh 7.06±0.31 7.29±0.18 6.56±0.36 5.85±0.22* ACh + SOD 7.15±0.64 8.44±0.39# 7.59±0.22# 6.94±0.18# Drug ACh + SOD + Cat 7.33±0.32 7.48±0.24 7.23±0.17 7.13±0.18# treatment ACh + Allopurinol 6.96±0.54 7.85±0.39 7.06±0.26 7.62±0.44# ACh + Apocynin 7.86±0.48 7.96±0.39 7.82±0.43 7.42±0.33# ACh + 1400W 7.48±0.44 7.34±0.26 7.53±0. 10 6.89±0.40# Abbreviations: ACh, acetyicholine; SOD, superoxide dismutase; Cat, catalase. *p<005, vs. age-matched control. # p<O.O5, vs. without pharmacological treatment in respective control or Marfan group. 64 3.6 Protein Expression of Superoxide-Generating and -Degrading Enzymes 3.6.1 SOD The expression of SOD-i (CuZnSOD or cytosolic SOD) in the Marfan aorta was comparable to that of the age-matched control at 3 and 9 months, but was less in the Marfan at 6 months of age. SOD-2 (MnSOD or mitochondrial SOD) expression was similar between groups at 6 months, but was lower in the Marfan aorta at 3 and 9 months (Figure 3.7). Marfan Control Age(m): 3 6 9 3 6 MW(kDa) SOD-I iirZ1*i 24 SOD-2 24 fi -actin L t 42 ‘ ‘SOD-i SOD-2 0 Cl) 0 . 6M 6M Marlan Control Figure 3.7 Western immunoblots showing the protein expression of SOD-i, SOD-2 and 13-actin as well as bar graphs showing the ratio of SOD to J3-actin in the Marfan and control aorta during aging (3, 6, and 9 months old). Aortic samples are pooled from 10-15 mice in each group. 65 3.6.2 NAD(P)H oxidase The gp9lphox, a catalytic subunit of NAD(P)H oxidase, was increased in the Marfan mice at all ages, especially at 6 and 9 months old. With regard to the regulatory subunits, the expression of p47phox was elevated at 3 months and that of p67phox was elevated at 3 and 6 months in the Marfan aorta. However, at 9 months, the expression of both regulatory subunits was higher in the control (Figure 3.8). 66 Marfan Control Age(m): 3 6 9 3 6 9(i) gp9lphox 1 58 P47 phox rz.z — 47 P67 phox r 67 fl-actin — 42 1.5 I ‘gp9lphox p47phox p67phox .$1.o. E C O.5• 0 0.0• - 3M 6M 9M 3M 6M 9M Martan Control Figure 3.8 Western immunoblots showing the protein expression of gp9 iphox, p47phox, and p67phox subunits of NAD(P)H oxidase and j3-actin, and bar graphs showing the ratio of the enzymatic subunits to f3-actin in the Marfan and control aorta during aging (3, 6, and 9 months old). Aortic samples are pooled from 10-15 mice in each group. 67 3.6.3 Xanthine oxidase The xanthine oxidase expression was similar in the control and Marfan groups at 3 and 9 months of age. However, at 6 months, the enzyme expression was doubled in the Marfan aorta as compared with the control (Figure 3.9). Marfan Control Age(m): 3 6 9 3 6 9() Xanthine oxidase 150 $-actin 4ê ____ 42 1.00 4- 0 0.75 0.25 4- 0.00 —F- - —F• - —r i ,— 3M 6M 9M 3M 6M 9M Martan Control Figure 3.9. Western immunoblots showing the protein expression of xanthine oxidase and f3-actin, as well as bar graphs showing the ratio of xanthine oxidase to f3-actin in the Marfan and control aorta during aging (3, 6, and 9 months old). Aortic samples are pooled from 10-15 mice in each group. 68 3.6.4 iNOS In the Marfan aorta, iNOS protein expression was 260, 107, and 168% higher than that of the control at 3, 6, and 9 months, respectively (Figure 3.10). Marfan Control Age(m): 3 6 9 3 6 9() NOS [i 130 $ -actin I: — — — _‘9 42 0.45 0.40’ 0.3& 0.30’ 0.25’ 0.20’ 0.15 .2 0.10- 0.05’ 0.00’ —,— - —I— - —I- . 3M 6M 9M 3M 6M 9M Martan Control Figure 3.10. Western immunoblots showing the protein expression of iNOS and j3-actin and bar graph showing the ratio of iNOS to 13-actin in the Marfan and control aorta during aging (3, 6, and 9 months old). Aortic samples are pooled from 10-15 mice in each group. 69 CHAPTER 4 DISCUSSION The current study is the first to show the elevation of oxidative stress in the thoracic aorta of a mouse model of Marfan syndrome. The major findings are that oxidative stress is present in the thoracic aorta of the Marfan mice as indicated by the elevated 8-isoprostraglandin F2a level. Therefore, vascular function of the Marfan aorta could be improved by pre-incubation of the superoxide-eliminating enzymes (i.e. SOD, catalase) and the blockade of the superoxide-generating enzymes (i.e. apocynin, allopurinol, 1400W). The presence of oxidative stress may be attributed to the downregulation of SOD and upregulation of superoxide-producing enzymes (i.e. iNOS, xanthine oxidase, NAD(P)H oxidase). Therefore, vascular dysfunction associated with Marfan syndrome could be attributed to oxidative stress. 4.1 Level of Oxidative Stress The isoprostane 8-epi-PGF2 assay is a well-established method to provide a reliable and accurate assessment of oxidative injury in vivo (Chiabrando et a!., 1999; Delanty et al., 1996; Hoffman et a!., 1996; Milne et al., 2008; Patrono & FitzGerald, 1997). Oxidative stress has been hypothesized to be responsible for the pathogenesis of diseases due to damage of the biomolecules such as lipids. Under such conditions, isoprostanes (e.g. isoprostane 8-epi-PGF2) are the products generated in abundance in vitro and in vivo as lipids are peroxidized by ROS 70 (Milne et al., 2008). Indeed, increased formation of F2 isoprostanes has been reported to be associated with vascular diseases such as vascular reperfusion, diabetes mellitus and hypercholesterolemia (Delanty et al., 1996; Patrono & FitzGerald, 1997), all of which have strong links to oxidative stress (Cai & Harrison, 2000; Faraci & Didion, 2004). Similarly, in the plasma of Marfan mice, a significantly increased level of isoprostane 8-epi-PGF2was observed at 3 and 6 months as compared with the control. Therefore, the higher level of isoprostane 8-epi-PGF2cin the plasma of Marfan mice indicated that oxidative stress was indeed elevated by the disorder. The difference in the isoprostane 8-epi-PGF2level between control and Marfan disappeared at 9 months of age, which may be explained by the impact of ageing on the control aorta. It has been well-established that oxidative stress is associated with ageing in the vasculature (Donato et al., 2007; Hamilton et al., 2001; Newaz et al., 2006; Taddei et al., 2001). Moreover, in healthy people, it was found that endothelial dysfunction and increased oxidative stress biomarkers in the aged subjects could be linked to increased expression of NAD(P)H oxidase (Donato et al., 2007). In the control mice, we also observed a significant increase in the expression of NAD(P)H oxidase subunits (Figure 3.8), which may contribute to the increased isoprostane 8-epi-PGF2 level at 9 months. 71 4.2 SOD and Catalase We have shown that Marfan syndrome and the progression of thoracic aortic aneurysm were accompanied by a pronounced impairment of aortic contractile function (Chung et al., 2007b; Chung et aL, 2008a). In the present study, the pre-incubation of SOD alone or a combination of SOD and catalase normalized the contractile response of Marfan aorta to the control level (Figure 3.2). This further confirmed the presence of oxidative stress in the Marfan aorta, and also demonstrated the impact of excess superoxide on vasoconstriction in Marfan syndrome. Oxidative stress has been reported to be involved in the pathogenesis of various cardiovascular diseases. Moreover, SOD treatment was shown to reverse the hypersensitivity of the arteries in diabetic and hypertensive animal models and normalize the agonist-induced contraction to that of the conUol animals (Alvarez et al., 2008; Kanie & Kamata, 2000). In the rat model of chronic renal failure, similar to the present study, phenylephrine-induced contraction was compromised in the aorta but could be restored to the control level with an antioxidant, melatonin (Sener et a!., 2004). Although the mechanism of action of ROS on smooth muscle cell contractility is still unclear (Lyle & Griendling, 2006), ROS has been reported to impede calcium signaling, which may consequently lead to a reduction in vascular contractility (Lounsbury et al., 2000; Sener et al., 2004). Therefore, the removal of superoxide with SOD and catalase may restore calcium signaling and thereby the contractile responses. 72 In the Marfan aorta, reduced endothelium-dependent relaxation could also be reversed by addition of SOD or SOD-plus-catalase. In the presence of oxidative stress, the excess superoxide anion can cause endothelial dysfunction by reducing NO bioavailability (Harrison, 1997). Indeed, in hypercholesterolemia, diabetes, heait failure, hypertension and chronic renal disease, endothelial function has been shown to be improved by antioxidants, such as SOD, melatonin, and vitamins C and E (Miyagawa et al., 2007; Schulz et al., 2004; Sener et al., 2004). Therefore, in addition to reduced NO production caused by downregulation of eNOS and reduction of Akt phosphorylation (Chung et al., 2007a), the present study indicated that oxidative stress may be another contributor to endothelial dysfunction in Marfan syndrome. In the control aorta at 6 months of age, SOD pre-incubation increased the sensitivity to ACh. This could be explained by the fact that SOD converts superoxide anions to H20,which has been suggested to be an endothelium-derived hyperpolarizing factor in rodent aorta and certain human vascular beds (Bharadwaj & Prasad, 1995; Hatoum et al., 2005). However, in the Marfan aorta, the similarity of effects caused by SOD treatment alone or the combination of SOD and catalase suggested that the improvement of endothelial function was not due to the vaso-relaxant effect of the H2O,but rather the removal of superoxide anions (Figure 3.3). 73 Oxidative stress could be associated with reduced superoxide neutralization, which could result from the downregulation of SOD-i and -2 (Figure 3.7). SOD is one of the most important endogenous anti-oxidant enzymes in the vasculature (Didion et aL, 2002; Lounsbury et al., 2000). In the mouse aorta, among the three isoforms of SOD, SOD-i and SOD-2 account for 50-80% and 2-12% of SOD composition, respectively (Faraci & Didion, 2004). Therefore, the decreased expression of these two isoforms would likely cause a reduction in superoxide removal. Indeed, in the streptozotocin-induced diabetic rat, decreased SOD expression has been shown to be associated with vascular dysfunction caused by superoxide anion (Kamata & Kobayashi, 1996). Moreover, in the mouse models of both SOD-i and SOD-2 deficient mice, basal superoxide level in the vasculature was elevated and ACh-mediated relaxation was impaired (Brown et al., 2007; Faraci & Didion, 2004). Therefore, the impairment of ACh-induced relaxation and phenylephrine-mediated contraction in the Marfan aorta may be attributed to the downregulation of SOD-i and -2. 4.3 NAD(P)H Oxidase NAD(P)H oxidase is believed to be the major contributor of superoxide anions in the vasculature (Jiang et al., 2004; Matsumoto et al., 2006; Yokoyama et al., 2000). Moreover, upregulation of NAD(P)H oxidase is found to be associated with various animal models of hypertension and to 74 cause impairment of NO-dependent vaso-dilatation in patients with coronary artery disease (Jiang et al., 2004). Apocynin, an NAD(P)H oxidase inhibitor, normalized ACh-mediated relaxation in the Marfan aorta at 6 months of age (Figure 3.4), but had no effect on contraction at 9 months. This can be explained by the protein expression of the subunits of NAD(P)H oxidase. The gp9lphox, a catalytic subunit of NAD(P)H oxidase, was upregulated at 6 months accompanied by the higher expression of the regulatory subunits, p47phox and p67phox. However, at 9 months, the expression of the regulatory subunits in the control aorta was significantly higher than that in the Marfan (Figure 3.8). 4.4 Xanthine Oxidase Inhibition of the xanthine oxidase with allopurinol improved both contraction and relaxation (Figure 3.5). In patients with hypercholesterolemia and chronic heart failure, similar observations have been made where the administration of oxypurinol and allopurinol reduced oxidative stress and improved endothelial function (Cardillo et al., 1997; George et aL, 2006). The apparent upregulation of xanthine oxidase in the Marfan aorta at 6 months (Figure 3.9) can contribute to endothelial dysfunction which can be partially restored by allopurinol. 75 4.5 iNOS The selective iNOS inhibitor, 1400W, differentially influenced the vasomotor function responses in control and Marfan aorta. In the control aorta, 1400W increased the contraction and decreased the relaxation, both of which can be explained by the blockade of NO production. However, in the Marfan aorta, the sensitivity to phenylephrine was improved with 1400W as with SOD incubation. Furthermore, rather than exerting inhibitory effects as seen in the control aorta, 1400W improved and normalized ACh-relaxation in the Marfan aorta (Figure 3.6d). It is believed that induction of iNOS produces excessive NO as well as ROS (Liu et al., 2005); thus, the upregulated iNOS expression in the Marfan aorta (Figure 3.10), which may be a compensatory mechanism for the reduced NO (Vaziri et al., 2000), may be another contributor to oxidative stress. Therefore, the normalization of vasomotor function by the preincubation with 1400W can be explained by the observation that iNOS expression was increased in the Marfan aorta at all ages. Previously, Chung et al has demonstrated that the endothelial dysfunction in the Marfan aorta was accompanied by downregulation of active eNOS (eNOS5’77)(Chung et al., 2007a). The upregulation of iNOS may be a compensatory mechanism for the decreased eNOS activity. However, the reaction between NO and 02 leads to the production of peroxynitrite, which is 76 suggested to cause NOS uncoupling (Cai & Harrison, 2000). Moreover, the upregulated NAD(P)H oxidase may also contribute to the uncoupling of NOS (Hitomi et al., 2007). The combination of decreased NO production caused by eNOS downregulation and NOS uncoupling may then lead to elevation of oxidative stress in the Marfan aorta. 4.6 Contributor to Oxidative Stress in Disease Progression Considering the 3 and 6 month-old data from ACh-induced relaxation, 9 month-old data from phenylephrine-mediated contraction, and the protein expression of the enzymes at all three ages, the contribution of each enzyme to the augmentation of oxidative stress in the Marfan aorta may be deduced. The removal of superoxide with SOD improved vascular function at all ages, which can be explained two ways: 1) The SOD expression andlor activity in the wall of the Marfan aorta was reduced, which was supported by the results of western imrnunoblotting. 2) The rate of superoxide production exceeded that of supreoxide neutralization. This is supported by the observation that the inhibitors of the superoxide-producing enzymes also improved vascular function. The contribution of the pro-oxidant enzymes to the increase in superoxide production may be different during the progression of the disease. At 3 months old, 1400W was the only inhibitor 77 that restored endothelial function in the Marfan aorta, suggesting that iNOS was the main contributor to oxidative stress in endothelial cells. At 6 months, NAD(P)H oxidase could be the major contributor while iNOS and xanthine oxidase played a minor role; however, the contribution of NAD(P)H oxidase completely disappeared at 9 months when the elevation in oxidative stress was attributed more to iNOS than to xanthine oxidase. 78 CHAPTERS CONCLUSION Oxidative stress has been shown to be present in the thoracic aorta of the Marfan mice at all ages and in the aorta of the control mice at 9 months of age. However, with various pharmacological agonists and inhibitors, we were able to restore the vasomotor function impaired by the excess superoxide. The elevated oxidative stress could be due to reduced expression of SOD and increased expression of iNOS, NAD(P)H oxidase, and xanthine oxidase at different ages. Since aneurysm formation may in part be caused by vascular cell dysfunction (Chew et al., 2004) and is found to be associated with oxidative stress (McCormick et al., 2007), the observed normalization of vasoconstriction and dilatation with the use of antioxidants could suggest a novel phannacological strategy in the treatment of thoracic aortic aneurysm in Marfan syndrome. 79 CHAPTER 6 FUTURE DIRECTIONS This new evidence indicating that oxidative stress contributes to the vascular dysfunction in MFS could lead to novel therapy for patients with Marfan syndrome. 6.1 Antioxidants The most direct way to alleviate oxidative stress would be the use of antioxidants such as SOD and vitamin C. The optimal doses of antioxidants can be calculated and administered chronically to Marfan mice starting at week 6 when aortic dilatation begins to develop. By the end of 3, 6, and 9 months, contractile function and endothelium-dependent relaxation in the aorta will be examined with myograph to assess the effectiveness of the treatments on the progression of the disorder. 6.2 Losartan With the link between oxidative stress, angiotensin II, and cardiovascular diseases established (Hitomi et aL, 2007), losartan can be another potential candidate to lower ROS level. The clinical trial for losartan is currently underway for MFS patients. The launch of this trial relies mainly on the theoretical evidence (i.e. losartan reduces TGFI3 signalling whose activation by the mutation in fibrillin- 1 is believed to cause many of the clinical manifestations) and the study reported by 80 Habashi et al (Habashi et al., 2006). It is important to further understand the mechanisms through which losartan affords its beneficial effects and to provide more evidence to support the use of losartan as an alternative treatment for MFS. One of the mechanisms would be its effect on oxidative stress. 6.2.1 Losartan treatment and vasomotor function Preliminary experiments have been done to examine the effect of losartan on vasomotor function. Three groups of mice were used: control (C57BL16), untreated Marfan mice (Fbnl 0390k) and Marfan mice with chronic oral administration of losartari (6gIL). The treatment was started at 6 weeks of age, and the vascular function of the descending thoracic aorta was examined at 3 (n=20), 6 (n20), and 9 (n=20) months. The maximal contractile response to phenylephrine in Marfan aorta at 9 months was normalized by losartan treatment, and the sensitivity to phenylephrine is also partially preserved (Figure 6.1). At both 3 and 6 months, the sensitivity to ACh, which is decreased in the Marfan aorta, was restored to the control level after treatment. The AChEmax is also normalized at the age of 3 months. However, the treatment was unable to preserve the endothelial function at 6 months where the maximal relaxation response was comparable before and after treatment (Figure 6.2). 81 a b 0 I E w Control Marfan Losartan Figure 6.2 Effect of chronic losartan treatment on acetyicholine-induced relaxation in thoracic aorta from control and Marfan mice. Bar graphs showing (a) Emax and (b) pEC50 in response to acetyicholine at 3, 6, and 9 months of age (n=6-8, *p<O.05 vs. control; #p<O.05 vs. Marfan). z E w 0 U- *# IF Age (Months) Age (Months) EEJ Control — Marfan Losartan Figure 6.1 Effect of chronic losartan treatment on phenylephrine-stimulated contraction in thoracic aorta from control and Marfan mice. Bar graphs showing (a) Emax and (b) pEC50 in response to phenylephrine at 3, 6, and 9 months of age (n=6-8, *p<OO5 vs. control; #p<O.05 vs. Marfan). a b I’, C.) w 0. * * Age (Months) Age (Months) 82 6.2.2 Losartan & oxidative stress To further understand the mechanisms of action of losartan, with a focus on its anti-oxidant effect, on the aortic function, the following experiments are proposed: 1. Measure oxidative stress in mice treated with losartan 2. Determine the shift in the effect of pro-oxidant and anti-oxidant enzymes on vasomotor function after the treatment with the use of pharmacological agents 3. Measure protein expressions of ROS-generating and —neutralizing enzymes in the aorta of the treated mice 83 REFERENCES Alvarez Y, Briones AM, Hernanz R, Perez-Giron JV, Alonso MJ, Salaices M. 2008. Role of NADPH oxidase and iNOS in vasoconstrictor responses of vessels from hypertensive and normotensive rats. Br J Pharmacol 153(5):926-35. Ammash NM, Sundt TM, Connolly HM. 2008. 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