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Sulfaphenazole treatment restores endothelium-dependent vasodilation in diabetic mice Elmi, Shahrzad 2008

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Sulfaphenazole treatment restores endothelium-dependent vasodilation in diabetic mice  by Shahrzad Elmi MSc, The University of British Columbia, 2008  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF  Master of Science in THE FACULTY OF GRADUATE STUDIES (Pharmacology and Therapeutics)  THE UNIVERSITY OF BRITISH COLUMBIA August 2008  © Shahrzad Elmi, 2008  Abstract  Vascular dysfunction is linked with increased free radical generation and is a major contributor to the high mortality rates observed in diabetes. Several probable sources of free radical generation have been suggested in diabetes, including cytochrome P450 (CYP) monooxygenase-dependent pathways. CYP-mediated superoxide production reduces nitric oxide (NO) bioavailability. In this study, we focus on the contribution of CYP monooxygenase enzyme-generated reactive oxygen species in vascular dysfunction in an experimental model of type II diabetes mellitus. The purpose of this study is to test the hypothesis that sulfaphenazole treatment can restore diabetic endothelial function in db/db mice.  Diabetic male mice (db/db strain) and their age-matched controls received daily intraperitoneal injections of either the CYP 2C inhibitor sulfaphenazole (5 mg/kg) or saline (vehicle control) for 8 weeks. Fasting plasma glucose levels were measured before starting, during, and after finishing the treatment. As well, plasma levels of 8-isoprostane (as a marker of oxidative stress) and nitrite levels of aortic tissue (as a marker of NO bioavailability) were determined.  Although sulfaphenazole did not change endothelium-dependent vasodilation in WT mice, it restored endothelial-mediated relaxation in treated db/db mice. We concluded that CYP 2C inhibition by sulfaphenazole reduces oxidative stress (measured as plasma levels of 8-  11  isoprostane), increases NO bioavailability (measured as NOj) and restores endothelial function in db/db mice without affecting plasma glucose levels.  Based on our findings, we speculate that inhibition of free radical generating CYP monooxygenase enzymes restores endothelium-dependent vasodilation induced by acetylcholine. In addition, it reduces oxidative stress and increases NO bioavailability.  111  Table of Contents  Abstract  .  ii  Table of contents  iv  List of tables  vii  List of figures  viii  Abbreviations  xi  1  2  Introduction  1  Increasing incidence of diabetes  1  The role of oxidative stress in diabetes  3  Cytochrome P450-dependent pathways in diabetes  6  Sulfaphenazole and inhibition of CYP 2C activity  10  Methods and materials  13  2.1  Drugs and solutions  13  2.2  Animals  13  2.3  Fasting plasma glucose (sugar) and weight measurements  14  2.4  Tissue prpreparation  15  iv  3  4  2.5  Isometric force measurements  .15  2.6  8-isoprostane enzyme immunoassay (ETA)  18  2.7  Nitric Oxide assay (measured as N0 2  19  2.8  Antioxidant activity of sulfaphenazole  20  2.9  Data analysis  20  Results  22  3.1  Fasting plasma glucose (sugar)  22  3.2  Endothelium-dependent and -independent vasodilation  25  3.3  Antioxidant activity of sulfaphenazole  42  3.4  Determination of 8-isoprostane  45  3.5  Determination of nitrite (N0 ) 2  47  Discussion  49  4.1  Summary  53  4.2  Future strategies  54  References  55  v  Appendix  .67  The effect of SOD treatment on ACh-induced vasodilation  68  vi  List of Tables  Table 3.2.1  50 and Emax values for ACh- induced endothelium- dependent EC vasodilation from control and treated groups  Table 3.2.2  50 and Emax values for SNP-induced endothelium- independent EC vasodilation in control and treated groups  Table App. 1  26  27  50 and Emax values for ACh- induced endothelium- dependent EC vasodilation from untreated and treated groups after incubation with SOD  74  vii  List of Figures  Fig 1.1  Oxidative stress and alteration of the balance between generation of ROS and antioxidant systems  11  Figure 1.2  Formation of ROS and the effect of antioxidants  12  Fig 2.5  A simplified illustration of wire myograph jaws  17  Figure 3.1  FPS values from control and treated groups  24  Figure 3.2.1  ACh-induced endothelium-dependent aortic vasodilation in db/db and their littermate WT mice  Figure 3.2.2  28  The effect of sulfaphenazole on ACh-induced endothelium- dependent aortic vasodilation in db/db and sulfaphenazole-treated db/db mice  Figure 3.2.3  The effect of sulfaphenazole on ACh-induced endothelium-dependent aortic vasodilation in WT and sulfaphenazole-treated WT mice  Figure 3.2.4  30  32  ACh-induced endothelium-dependent aortic vasodilation in db/db and WT and sulfaphenazole- treated db/db mice  viii  34  Figure 3.2.5  The effect of sulfaphenazole on SNP-induced endothelium-independent aortic vasodilation in db/db and WT mice  Figure 3.2.6  The effect of sulfaphenazole on SNP-induced endothelium-independent aortic vasodilation in db/db and sulfaphenazole-treated dbldb mice  Figure 3.2.7  44  Effect of sulfaphenazole on plasma 8-isoprostane level in control and treated groups  Figure 3.5  40  Comparison of intrinsic antioxidant activity among sulfaphenazole, vitamin E and tempol  Figure 3.4  38  The effect of sulfaphenazole on SNP-induced endothelium-independent aortic vasodilation in WT and sulfaphenazole-treated WT mice  Figure 3.3  36  46  Effect of sulfaphenzole on nitrite (NOj) levels in aortic tissue in control and treated groups  48  ix  Figures relating to appendix  Fig App. 1  The effect of SOD on ACh-induced, endothelium-dependent aortic vasodilaton in db/db mice  Fig App.2  70  The effect of SOD on ACh-induced, endothelium-dependent aortic vasodilation in sulfaphenazole-treated db/db mice  Fig App.3  The effect of SOD treatment on ACh-induced, endothelium-dependent aortic vasodilation in WT mice  Fig App.4  71  72  The effect of SOD treatment on ACh-mduced, endothelium-dependent aortic vasodilation in sulfaphenazole-treated WT mice  x  73  Abbreviations  Ab: antibody Ach: acetyicholine AChE: acetyicholinesterase ANOVA: analysis of variance ANCOVA: Analysis of covariance App: Appendix BHT: butylated hydroxy toluene Cat: catalase CNP: C-peptide natriuretic peptide CNP is one of the members of natriuretic peptide family which is produced by endothelial cells. COX: cyclooxygenase CYP: cytochrome P450 e-: electron : The concentration of the drug needed to produce 50% of maximum response 50 EC EDH1?: endothelium-derived hyperpolarizing factor EDHF is defined as a substrate released by endothelial cells that elicits the hyperpolarization of vascular smooth muscle cells. EDTA: Ethylenediaminetetraacetic acid EET: epoxyeicosatrienoic acid  xi  EETs are metabolite of arachidonic acid, which are formed by the action of cytochrome P 450 monooxygenases. EIA: enzyme immunoassay ELISA: enzyme-linked immunosorbent assay Emax : Emax (efficacy) refers to the magnitude of the maximum effect : ferrous ion 2 F? : ferric ion 3 F? Fig: figure FPS: fasting plasma sugar (glucose) Glu sys: glutathione system GSH-Px: glutathione peroxidase W: hydrogen ion HETE: hydroxyeicosatetraenoic acid hydroxyeicosatetraenoic acids are metabolites of arachidonic acid, which are formed by the action of cytochrome P-450 lipoxygenases. IgG: Immunoglobulin G K: potassium ion KC1: potassium chloride Kg: kilogram Log: logarithm M: mole/liter mg: milligram ji1’1: micromol/liter  xii  mmoIIL: millimole/liter NADPH oxidase: nicotinamide adenine dinucleotide phosphate-oxidase NADPH oxidase is a membrane-bound enzyme. It generates superoxide anions by transferring electrons from NADPH (nicotinamide adenine dinucleotide phosphate) to oxygen molecules. NOS: nitric oxide synthase NOS is an enzyme in the body that contributes to the generation of nitric oxide and regulation of vascular tone. pg/mi: pictogram! milliliter PIlE: phenylephrine PSS: physiologic saline solution ROS: reactive oxygen species ROS are ions and molecules of oxygen with an unpaired electron. ROS are highly reactive and can attack cellular structures. SOD: superoxide dismutase SOD is an important antioxidant enzyme that catalyzes the dismutation of superoxide anion into oxygen and hydrogen peroxide. SNP: sodium nitroprusside Tempol: 4-hydroxy-2,2,6,6-tetramethyl- 1-piperidinyloxy TPTZ: 2,4,6-tripyridyl-S- triazine vit: vitamin WHO: World Health Organization WT: wild type  xiii  1-Introduction  Increasing incidence of diabetes Diabetes mellitus is a prominent cause of death in the U.S. and Canada. Thirteen million Americans and over one million Canadians are currently diagnosed with diabetes. Type II diabetes constitutes 90% of all diagnosed cases and millions of people with the disease remain unaware. More than one million new diabetic cases are added in the U.S. each year. In Canada, 60,000 new diabetic cases are diagnosed per year. It is forecast that by the year 2050 the number of cases will increase to 3 and 29 million in Canada and the U.S., respectively. This might be due to the aging of the population and rising obesity rates. In this regard, worldwide rates are even more dramatic according WHO reports, it is -  estimated that about 180 million people all over the world have diabetes, and that this number will probably double by 2030. (Arias et al., 2003; Boyle et al., 2001; Cusick et al., 2005; Lucas et al., 2004; Morgan et al., 2000)  Diabetes is strongly associated with increased mortality rate. The WHO reports that approximately 2.9 million deaths per year are associated with diabetes. These reports show that people with diabetes have twice the risk of dying compared to their healthy peers, and that, without urgent action, this rate might increase to more than 50% within the next 10 years. Currently, about 80% of deaths attributable to diabetes occur in low- and middle income countries. It is predicted that between 2006 and 2015 more than 80% of diabetic deaths will occur in upper-middle income countries. In spite of being consistently underreported as a cause of death, diabetes has been cited as a maj or contributor to a  1  considerable number of deaths per year  —  e.g. 5700 in Canada and 71,000 in the United  States. It is the seventh leading cause of death in Canada. In the United States diabetes is the sixth major cause of death. In addition to the increased rate of mortality, diabetic patients are also prone to an elevated rate of morbidity from microvascular complications including nephropathy, neuropathy and retinopathy. For instance, in Canada, 40% of diabetic patients develop long-term complications which results in 25,000 person years of life lost before the age of 75. These dramatic statistics indicates an increased rate of mortality and reduced life expectancy in diabetic patients in comparison to healthy individuals and needs a serious approach regarding to the application of new therapeutic options (Arias et al., 2003).  Most deaths among diabetic are from cardiovascular and cerebrovascular disease, including myocardial infarction and stroke. Compelling evidence suggests that these vascular complications may be strongly correlated with the development of endothelial dysfunction. The vascular endothelium plays a cardinal role in homeostasis in the cardiovascular system of healthy individuals. It not only acts as a semipermeable barrier between the wall and lumen to regulate blood tissue exchange in the vasculature, but it also secretes several vasoactive mediators such as nitric oxide (NO) to regulate vascular tone and platelet aggregation, coagulation, fibrinolysis and smooth muscle proliferation. The regulation of vascular tone has been the most widely studied function of endothelium and NO has been implicated as a major contributor to endothelium-dependent relaxation in blood vessels especially in larger vessels such as conduit arteries. The term “endothelial dysfunction” refers to disruption in production of these protective mediators by vascular endothelial  2  -  cells, which can lead to the initiation and progression of vasospasm, thrombosis or vessel occlusion in experimental models and humans. Previous studies have demonstrated that individuals with cardiovascular risk factors may show abnormalities of their endothelial function before the development of overt cardiovascular disease. Likewise, other studies in human coronary, brachial, and forearm arteries demonstrated that a reduction in endothelium-dependent vasodilation may be strongly associated with life-threatening cardiovascular events. For instance, newly diagnosed hypertensive patients having a reduced flow-mediated vasodilation that does not show any improvement with follow-up have a sevenfold higher risk of hospitalization due to cardiovascular complications. Therefore, endothelial dysfunction has been implicated repeatedly as a major determinant in the pathogenesis of cardiovascular disease such as diabetes (Cusick et al., 2005; Rask Madsen and King 2007; Wheatcroft et al., 2003). ,  The role of oxidative stress in diabetes  It has been suggested that oxidative stress may play an important role in the development of endothelial dysfunction during conditions such as diabetes, atherosclerosis, hypercholesterolemia, and hypertension. Endothelial dysfunction may be triggered and propagated by reactive oxygen species. Oxidative stress represents an abnormal condition caused by the existence of products called free radicals and reactive oxygen species (Fig 1.1). Free radicals are atoms or groups of atoms possessing an odd number of unpaired electrons. They are highly reactive and affect different molecules by causing oxidative damage. Therefore, the prominent danger comes from damaging cellular components such  3  as DNA and cellular membranes. Most free radicals, such as, superoxide, hydroxyl radical and peroxides, in biological systems derive from oxygen and are called “reactive oxygen species” (ROS) (Fig 1.2) (Ceriello, 2003; Flora, 2007; Guerci et al., 2001; Lum and Roebuck, 2001).  Protection from free radical- and ROS-induced damage is necessary to maintain the integrity of endothelial function in biological systems. Antioxidants are molecules that are capable of inactivating ROS by accepting or donating an electron to neutralize the free radical molecule. Antioxidants can be divided into two major groups: enzymatic and non enzymatic. Antioxidant enzymes primarily contribute to the defensive intracellular antioxidant system by catalyzing the degradation of reactive oxygen species. Three major antioxidant enzymes are: superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (Cat). Most of these enzymes are widely distributed in various tissues and convert superoxide, or other reactive species, to less harmful intermediates. There are also a number of molecules distributed in biological systems that neutralize reactive oxygen species non-enzymatically- for instance, glutathione GSH, vitamins C, A, and E (Flora, 2007; Valko et al., 2007).  The generation of free radicals under normal conditions can be beneficial for living organisms. They are continuously being generated in the body and are involved in physiological processes such as in immune system- mediated host defence by neutrophils, macrophages and cell signalling including regulation of vascular tone, monitoring the  4  oxygen tension in the regulation of ventilation, erytbropoietin production (Droge, 2002; Flora, 2007).  In spite of the important physiologic roles of free radicals, their accelerated production under pathologic conditions such as diabetes can lead to cellular damage by mediating oxidative stress. Oxidative stress can originate from an overproduction of free radicals and their insufficient inactivation by antioxidant systems. Production of ROS can result in multiple lethal outcomes for the body. Reactive oxygen species can affect the activity of antioxidant systems, may inactivate the existing enzymes by glycation of their proteins, may oxidize various types of biomolecules and lead to cellular death by damaging DNA, or may stimulate the pathways involved in modulation of cellular apoptosis. Vascular walls located at the interface between blood and tissue are constantly exposed to oxidative stress that may lead to increased expression of adhesion molecules, activation of vascular remodelling processes, and an enhancement of smooth muscle growth that serves to alter the vessel integrity. The endothelium-derived nitric oxide (NO), the principal physiologic vosodilator acting on vascular smooth muscle, is usually inactivated by the superoxide anion radical. Reduction of NO bioavailability results an impaired endothelium-dependent vasodilation. Furthermore, it can lead to the promotion of smooth muscle cell migration, and to monocyte activation, adhesion, and migration  —  all of which are important  contributors in enhancing a thrombotic state in diabetic patients.  Increased oxidative stress is an important culprit in both types I and II diabetes mellitus. In fact, the production of ROS has been reported to increase in post-prandial periods in both  5  normal and diabetic individuals, but diabetic subjects, in contrast to healthy people, are unable to compensate for the increased ROS production. As well, oxidative stress in pre diabetic stages or glucose-intolerant individuals has been shown by increased isoprostane levels as a marker of oxidative stress (Droge, 2002; McQuaid and Keenan, 1997; Rask Madsen and King, 2007; Wiernsperger, 2003).  Cytochrome P450-dependent pathways in diabetes  There are several probable sources of free radical production in vasculature during diabetes, including NADPH oxidase, mitochondrial respiratory chain, nitric oxide synthase (NOS), xanthine oxidase, cytochrome P450 (CYP)-dependent pathways (Feletou and Vanhoutte, 2006; Triggle et al., 2005). In this study, we focus on the possible contribution of CYP monooxygenase enzymes as important sources of ROS generation in diabetes.  The CYP enzymes constitute a diverse superfamily of hemoproteins, which are available in both eukaryotes and in prokaryotes, such as bacteria. For instance, they have been identified in mammals, birds, fish, insects, worms, plants, sea squirts and sea urchins. In mammals, membrane-associated CYP enzymes are predominantly located in the mitochondrial inner membrane or endoplasmic reticulum. These enzymes were initially recognized in the liver and are involved in the metabolism of exogenous and endogenous substrates. Later, it was revealed that they are also present in extrahepatic tissues, including kidney, intestine, lung, heart, white blood cell, and vasculature. These enzymes catalyze the oxidation of various substrates and use (NADPH) as cofactors. In the catalytic cycle of  6  cytochrome P450 monooxygenase, the substrate binds to isoenzyme (Fe S) and one 3 electron of the heme iron is reduced 2 (Fe S ). Oxygen binds to this compound and forms “oxy-P450” (Fe O S). Reactive oxygen species such as superoxide anions, hydrogen 2 -  peroxide (H ) and hydroxyl radicals might be generated when the electrons for the O 2 reduction of iron in the central heme are transferred to the activated bound oxygen molecule (Coon et al., 1992; Fleming, 2001; Puntarulo and Cederbaum, 1998; Robin et al., 2005; Szczesna-Skorupa et al., 1998).  Although the possible use of arachidonic acid as a substrate in the monooxygenase reaction was noted as early as 1969, the role of CYP isoenzymes as an alternative pathway in oxidative metabolism of arachidonic acid received serious attention for the first time in 1981. Previously, it was known that cyclooxygenase (COX) and lipooxygenase enzymes metabolize arachidonic acid into hydroxyeicosatetraenoic acids (HETE5), prostaglandins, prostacyclins, thromboxane and leukoterines. It had been reported that arachidonic acid was metabolized in the liver and kidneys to epoxyeicosatrienoic acids (EET5). Subsequent studies demonstrated that some CYP enzymes are predominantly distributed in some extrahepatic tissues- including the heart and vasculature- and metabolize this substrate. Since many CYP isonezymes are capable of metabolizing arachidionic acid to biologically active products, they have been repeatedly referred to as the third pathway of arachidonic acid metabolism. (Capdevila et al., 2000; Fleming, 2001)  Studies have shown that CYP isoenzyme-derived EETs produced in response to vasoactive substances such as aceytyicholine have a vasodilatory effect. It also has been revealed that  7  there might be a link between the activity of CYP isoenzymes and the generation of EDHF, which is defined as a substrate released by endothelial cells that elicits the hyperpolarization of vascular smooth muscle cells. For instance, EETs have been reported to play an EDHF role in canine and pig coronary microcirculation. The same role for CYP 450-derived EET has been suggested in some human blood vessels, such as coronary, internal mammary, and subcutaneous arteries. Based on these and other similar studies, it was suggested that specific CYP isonenzymes located in endothelium and vascular smooth muscle cells, might contribute to this vasodilatory effect and to the regulation of vascular tone. Emerging evidence suggest that the major CYP isoenzymes contributing to vascular tone are members of 2 gene families in humans, pigs, and rats. Other studies in humans, pigs, cows, dogs, rats, and rabbits reported the existence of close similarities between the hyperpolarizing factor generated by coronary and renal arteries, and those from EETs generated by endothelial CYP monooxygenases. Based on these studies, EETs have been suggested as important candidates for EDHF-mediated vasodilation in multiple vascular beds. (Capdevila et al., 2000; Fleming, 2001; Feletou and Vanhoutte, 2004; Oltman et al., 1998)  So far, the most attention has been paid to the production of vasodilatory metabolites of arachidonic acid, and little has been focused on the generation of ROS during the formation of EETs, such as superoxide anions, hydrogen peroxide  (11202),  and hydroxyl radicals. This  occurs in the CYP reaction cycle during the reduction of iron in the central heme group when electrons are transferred to an oxygen molecule. As mentioned earlier, oxidizing species, including free radicals, are involved in signalling pathways that regulate vascular  8  tone under normal conditions. Regulation of vascular tone by CYP- derived products such as EET depends, to a great extent, on physiologic conditions. Under pathologic conditions, the detrimental impact of simultaneous increase of generating free radicals may overcome this vasodilator effect. Indeed, the continuous production of ROS by microsomal monooxygenases makes these enzymes an important potential source of generating oxygenderived free radicals. Considering that oxidative stress may play an important role in initiating the early stages of vascular disease and endothelial dysfunction, it is likely that endothelial CYP monooxygenases contribute to the generation of ROS in vascular beds.  Consistently, the CYP 2C isoenzymes of porcine coronary arteries have been reported to generate ROS in cultured and native endothelial cells. Studies on rat coronary arteries in an experimental model of ischemia-reperfusion injury where tissue damage occurs during reperfusion of an organ following a period of ischemia- have shown the same effect. As well, human studies have implicated the CYP 2C family as significant sources of ROS in patients with stable coronary artery disease or ischemia-reperfusion injury due to surgical treatments such as cardiac transplantation, balloon angioplasty and coronary bypass. (Chehal and Granville, 2006; Coon et al., 1992; Droge et al., 2002; Fichtlscherer et al., 2004; Hunter et al., 2005; Puntarulo and Cederbaum, 1998).  The expression or function of CYP monooxygenase in endothelial cells is affected by multiple factors: pharmacological, hemodynamic, hormonal stimuli and hypoxic conditions. In diabetes, genetics and nutritional factors, such as diets high in saturated fat or polyunsaturated fat, have also been suggested. It was reported that up-regulation of free  9  radical generating CYP isoenzymes occurs in hepatic and renal tissues of experimental diabetic animals and diabetic patients (Chen et al., 1996; Chen et a!., 2000; Coon et a!., 1992; Sindhu et al., 2006; Michaelis and Fleming, 2006; Murray et al., 2006).  Sulfaphenazole and inhibition of CYP 2C activity  Granville et a!. (2004) have previously reported that the application of su1faphenazo1e an agent that inhibits CYP 2C activity, can significantly reduce infarct size and restore post ischemic coronary flow following ischemia and reperfusion while decreasing the generation of superoxide anions. Subsequent studies revealed that sulfaphenazole can attenuate post ischemic vascular dysfunction and restore NO-mediated endothelium-dependent vasoclilatation. The latter studies are consistent with reports that sulfaphenazole can restore endothelium-dependent vasodilation in patients with coronary artery disease through the inhibition of CYP 2C activity. Considering these studies, and related evidence regarding the alteration of CYP monooxygenase activity in diabetes, we hypothesized that sulfaphenazole treatment would restore diabetic endothelial dysfunction. To this end, we treated diabetic (db/db) mice and their WT (non-diabetic genetic controls) littermate with this agent to investigate the role of the CYP monooxygenase pathway in diabetic endothelial dysfunction (Fichtlscherer et a!., 2004; Granville et al., 2004; Hunter et a!., 2005; Mono et a!., 2003; Thao and Ishizaki, 1997).  10  balance in health condition  r 0  02 OH  02-  0 2 H  oxidative stress  endothelial dysfunction  OH  -  02 02-  0 O2 2 H OH02-  0OH -  O2  0 2 H  00-  0Or 02O2  O2  O2  00OH  -  0 OH2 H  Fig 1.1- Oxidative stress and alteration of the balance between the generation of ROS and antioxidant systems. Under oxidative stress conditions, the balance shifts in favour of ROS.  11  Q  GSH -Px  C Figure 1.2- Formation of ROS and the effect of antioxidants. Supeoxide dismutase (SOD) plays a key antioxidant role in biological systems. Superoxide anions are dismutated to H 0 and 02 by SOD, thus cellular tissue is protected from their 2 detrimental effects.  12  2-Materials and Methods  2.1- Drugs and solutions  ACh, phenylephrine, sodium nitroprusside (SNP), alpha tocopherol (vitamin E), 2,4,6tripyridyl-S- triazine (TPTZ) and ferric chloride (FeC1 ,2 3 6H o ) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Sulfaphenazole was provided by Clinalfa (Weidenmattweg, Laufelfingen, Switzerland). Physiological saline solution (PSS) was prepared fresh daily and had the following composition (millimolar): NaCl 119, KC1 4.7, , 71120 1.17, NaHCO 4 MgSO 3 24, 2 CaCl 1.6, KH 4 P 2 O 1.18, glucose 11.1, EDTA 0.024. A solution rich in KC1 (80 mM KC1) was prepared by equimolar replacement of NaC1 with KCL The composition of Krebs solution was (millimolar): NaC1 110.8, NaHCO 3 25, KC1 5.9, CaCl 2.49, MgSO 4 1.07, NaH 2 P0 4 2.33, dextrosell.51.  2.2- Animals  Two groups of age-matched male mice were used in this study. Fourteen db/db mice (strain BKS.cg-m +1+ Leprdt)/J) and their matched wild type (WT) controls were purchased from Jackson Laboratories (Bar Harbor, MA). The db/db mouse is a monogenic model of type II diabetes. Considering that obesity and insulin resistance are important contributors to type II human diabetes, this model has been studied to get insights into the human diabetic pathophysiologic condition. Obese db/db are bred by heterozygous mating of diabetic mutants in C57BKSJ strain. The plasma insulin is increased from 10 days and reaches at  13  six to ten times normal by two to three months. Diabetic mice are obese, severely hyperglycaemic and diabetic when expressed on the C57BL/KsJ background. They develop hyperglycaemia since their 13-cells are unable to secrete the required high levels of insulin. The blood glucose can reach to over 22 mM until their death at five to eight months of age. Hyperglycaemia in db/db mice is severe and they show vascular abnormalities (McNeill, 1999; Rees and Alcolado, 2005).  Mice were housed under standard animal room conditions of a 12 hours/light cycle, room temperature 26°C and had free access to food and water. Diabetic and WT mice were randomly assigned to groups with equal numbers of members that received daily intraperitoneal (i.p.) injection of either sulfaphenazole (5 mg/kg which is consistent with the other studies- Pokreisz et a!., 2006; Zweers-Zeilmaker et al., 1997) or vehicle (saline) for a total of 8-weeks.  Mice were sacrificed when aged 14-16 weeks. All experimental protocols were designed and performed in accordance with the guidelines of the Animal Care Committee of the University of British Columbia.  2.3- Fasting plasma glucose and weight measurement  Fasting plasma glucose concentrations were measured through the caudal vein of db/db and  WT mice following 8 hours fasting using a one-touch glucometer. Samples of blood were  14  obtained at three time points: when the mice were aged 6 weeks (just before treatment), 10 and 14 weeks.  2.4- Tissue preparation  Mice were anaesthetized using sodium pentobarbital (50mg/Kg, i.p.) containing heparin (50U/Kg) on the day of experiment. Thoracic aortas were dissected from each animal and placed in ice-cold physiologic saline solution (PSS). The descending portion of the thoracic aorta was cleaned of adipose and connective tissue. Special care was taken to avoid damaging the endothelium.  2.5- Isometric force measurements  We used a wire myograph (Model 610 M, Multi myograph, Danish Myotech, Aarhus, Denmark) to measure isometric force of isolated small arteries in response to agonist stimulation. To ensure that the response is isometric, isolated ring segments of 200im were mounted on two fine stainless steel wires which were fastened at each end to a force transducer and a micrometer (Mulvany and Aalkjaer, 1990). The vasorelaxation response of aortic segments by ACh was calculated according to the following equation:  15  Relaxation percentage for each dose of ACh  =  Tension at maximum contraction by PHE  (phenylephrine) Tension at maximum relaxation for that dose I Tension at maximum -  contraction by PHE- Resting tension.  Individual ring segments with a length of 2 mm were mounted in the chambers of a wire myograph (Model 610 M, Multi myograph, Danish Myotech, Aarhus, Denmark). Artery segments were equilibrated at 37 °C during which a resting tension of 5 millinewtons was applied to each artery segment. Following the equilibration period, the PSS solution was substituted with 80 m’vI KC1 to examine tissue viability. After a maximal force was recorded, tissues were washed several times with fresh PSS to restore resting tension. The arteries were then contracted by addition of phenylephrine (106 M) to all chambers. Following reaching a stable contraction, cumulative concentrations of either acetyicholine (ACh; 10b0  --  i0 M) or sodium nitroprusside (SNP; 10°  rings.  16  --  i0 M) were added to artery  mounting hook  wire  Fig 2.5 A simplified illustration of wire myograph jaws. -  In this picture an aortic segment is mounted by means of two fine stainless steel wires.  17  2.6- 8-isoprostane enzyme immunoassay (EIA)  Plasma levels of 8-isoprostane (8-iso PGF2a) were measured using an ELISA kit (Cayman Chemical, Michigan) and used as marker of oxidative stress. The assay was performed as per the manufacturer’s instructions.  Isoprostanes, including 8-isoprostane, are prostaglandin-related compounds formed from arachidonic acid in a CYP 450-dependent manner through the modification of tissue membranes by free radicals (Möbert et al., 1997).  In this enzyme immunoassay, 8-isoprostane and the 8-isoprostane tracer (8-isoprostane AChE conjugate) compete for a limited number of 8-isoprostane- specific rabbit antiserumbinding sites. These complexes will bind to the rabbit IgG mouse monoclonal Ab which is already attached to the wells of a 96 well plate. Since the concentration of 8-isoprostane tracer is already known, the amount of tracer bound to the Ab is inversely proportional to the concentration of 8-isoprostane in the wells. Following incubation of the plate at room temperature for 18 hours and washing and removal of unbound reagents, Ellman’s reagent is added to develop the plate. This reagent includes a substrate for AChE and the resultant reaction will produce a yellow colour. The intensity of this colour can be measured spectrophotometrically at 412 nm, and is inversely proportional to the amount of free 8isoprostane in the wells.  18  Briefly, blood samples were collected from the inferior vena cava of each animal at the time of sacrifice and placed in Microtainer ® brand tubes (containing EDTA). Following centrifugation, the plasma portion was isolated from each sample and rapidly transferred to microeppendorff tubes containing butylated hydroxy toluene (BHT). Plasma samples were then stored at 8O0 C for later analysis of free 8-isoprostane levels.  2.7- Nitric Oxide assay (measured as N0 ) 2  The nitric oxide assay kit was purchased from Calbiochem (San Diego, California, USA) and used for determination of nitrite levels produced by the aorta according to the manufacturer’s instructions. The principle of this assay is the fact that nitric oxide is converted to nitrite in aqueous solution. Griess reagent is applied to determine the quantity of nitrite.  For this purpose, arteries from all groups were incubated in 0.5 ml Krebs-Henseleit solution for 30 minutes at 37°C and the solution was then replaced by 0.5 ml Krebs-Henseleit containing 10 iM ACh (37°C). After reaching equilibration for 5 minutes, 100 il of the perfusate was mixed with 100 p1 of Griess reagent. Spectrophotometric measurements were made using a microplate reader set at 540 nm.  19  2.8- Antioxidant activity of sulfaphenaziole  The Martinek procedure (Martinek, 1964) was used to determine the intrinsic antioxidant effects of sulfaphenazole. In this assay, vitamin E is used as a standard antioxidant, where fenic (Fe ) ion is reduced to ferrous (Fe 3 ) ion, which then reacts with 2,4,6-tripyridyl-S2 triazine (TPTZ). The reaction product can be measured spectrophotometrically at 600 nm. We compared the relative antioxidant activity of sulfaphenazole, tempol (4-hydroxy2,2,6,6-tetramethyl-1-piperidinyloxy), and vitamin E. Tempol is a well-described SOD mimetic which is able to scavenge free radicals and also improves diabetic endothelial function (Ceriello,2003; Nassar et al., 2002). Vitamin E is frequently used as a standard for antioxidant activity (Huang et al., 2002; Serafini, 2000) and already has been shown to reduce superoxide generation in diabetes (Chow,2001; Du et al., 2003).  For this purpose, various concentrations of sulfaphenazole, vitamin E and tempol were used and ranged from 25x i0 to 5x 102 jiM.  2.9- Data analysis  Results are expressed as the mean ± SE and comparisons are made using repeated measures ANOVA tests with multiple comparisons (Bonferroni’s test), one-way ANOVA, and ANCOVA, as required. The level of significance was considered less than 0.05 for  20  statistical tests. Analysis of dose-response curves was made using GraphPad Prism (version 3.02-2000).  21  3- Results  3.1- Fasting plasma glucose  Fasting plasma glucose values (Fig 1) were significantly higher in diabetic mice (at 6 weeks: db/db 13.63± 2.51 vs. WT: 7.13± 0.70 mmolJL, n=7 in each group). This pattern was maintained as the animals aged so that when mice were 10 weeks old, diabetic animals had fasting plasma glucose levels of 3 2.58± 3.78 mmolfL while in WT mice the values were 5.93± 0.23 mmolfL, (n=7 in each group). At the time of sacrifice (14 weeks), the db/db diabetic mice and their age-matched controls had plasma glucose levels of 31.70± 1.90 and 6.23± 0.64 mmolIL, respectively (n= 7). The db/db mice had significantly elevated fasting plasma glucose levels at all ages studied (repeated measures ANOVA, p<O.OO ) 1 . These plasma glucose levels are consistent with other studies indicating that in this diabetic model, a rise of blood sugar starts at 4-8 weeks and is followed by a sustained elevation in blood sugar.  Sulfaphenazole treatment did not affect the fasting plasma glucose level in diabetic and WT mice (at 10 weeks: 35.87± 2.06 in sulfaphenazole-treated db/db mice vs. 32.58± 3.78 mmol/L in untreated db/db mice; 5.07± 0.51 in sufaphenazole-treated WT vs. 5.93± 0.23 mmolJL in untreated WT). Similar trends were observed at the time of sacrifice (14 weeks) where the fasting plasma glucose values were 32.60 ± 2.04 in sulfaphenazole-treated vs.  22  31.70± 1.90 mmolIL in untreated db/db mice, and 5.24± 0.56 in sulfaphenazole-treated WT vs. 6.23±0.64 mmolIL in control WT, (n=7 in each group).  23  sulfaphenazole-treated db/db sulfaphenazole-treated WT control db/db control WT  J  0  E E  U, 0 ILli  iIIn 6 weeks  1.0 weeks  1.4 weeks  Age (weeks)  Fig 3.1- FPS (fasting plasma glucose) values from control (db/db and WT mice that did not receive sulfaphenazole) and treated groups (db/db and WT mice that received sulfaphenazole as a treatment). Fasting plasma glucose concentrations were measured after 8 hours fasting at three time points: 6 (just before treatment by sulfaphenazole), 10, and 14 weeks of age (repeated measures ANOVA, p>O.05).  24  3.2- Endothelium-dependent and -independent vasodilation  We compared the endothelium-dependent relaxation induced by ACh and endothelium independent relaxation induced by SNP in the thoracic aorta of sulfaphenazole treated and untreated db/db and WT mice. There was a pronounced impairment of endothelium dependent relaxation in aortic rings from untreated diabetic mice (Fig 2A). The sensitivity ) and maximal vasodilation (E) produced by ACh and SNP are summarized in 50 (EC Table 3.2.1 and 3.2.2. There was a significant impairment of ACh-mediated vasodilation in untreated db/db mice (p<O.OO1) with no changes in the sensitivity to ACh (Table 3.2.1). The maximal vasodilation and sensitivity to SNP were similar in untreated and treated db/db mice (Table 3.2.2).  Treatment of diabetic mice with sulfaphenazole restored the endothelium-mediated vasodilation produced by ACh (Fig 3.2.2 and Table 3.2.1). Sulfaphenazole significantly increased the maximal vasodilation produced by ACh without changing the sensitivity to ACh (Table 3.2.1). Sulfaphenazole treatment did not significantly alter ACh-induced relaxation in WT mice (Fig 3.2.3).  The endothelium-independent vasodilator SNP relaxed aortic rings in a concentration dependent manner. The resultant relaxations were similar in WT and diabetic groups. (Fig 3.2.6 and 3.2.7) As shown in Table 3.2.2, there were no significant differences in the .  maximal vasodilation either in diabetic or WT groups. Likewise, EC 50 values for SNP were similar in all groups (p >0.05).  25  ACh  control  /  sulfaphenazole-treated db/db  7  -6.9± 0.03  66.3± 1  controlWT  7  -7.5± 0.04  71.3± 1.1  sulfaphenazole-treated WT  7  -7.5  83.4± 1.1  .2± 0.6  *  **  Table 3.2.1 EC 50 and Emax values for Ach- induced, endothelium- dependent aortic -  vasodilation from control (db/db and WT mice that did not receive sulfaphenazole) and treated groups (db/db and WT mice which received sulfaphenazole as a treatment). *  = db/db vs. WT mice,  **  = sulfaphenazole-treated db/db vs. db/db mice; one-way  ANOVA, p<0.OO1; (n = the number of mice in each group)  26  J!. Group  50 Log EC  Euttax  I  control db/db  7  -7.7  84.1± 1  sulfaphenazole-treated db/db  7  -7.8  90.8± 2.3  controlWT  7  -7.8  88.8± 1.2  sulfaphenazole-treated WT  7  -7.9  93.9± 1.2  Table 3.2.2- EC 50 and Emax values for SNP-induced, endothelium-independent aortic vasodilation in control (db/db and WT mice that did not receive sulfaphenazole) and treated groups (db/db and WT mice that received sulfaphenazole as a treatment). n = number of mice in each group  27  100 0 C  II  controlWT controldb/db  -Jo Log [ACh] (M)  Fig 3.2.1- ACh-induced, endothelium-dependent aortic vasodilation in db/db (not receiving sulfaphenazole) and their WT littermates (not receiving sulfaphenazole).  Individual ring segments with a length of 2 mm were mounted in the chambers of a wire myograph (Model 610 M, Multi myograph, Danish Myotech, Aarhus, Denmark). Artery segments were equilibrated at 37 °C during which a resting tension of 5 millinewtons was applied to each artery segment. Following the equilibration period, the PSS solution was substituted with 80 mM KC1 to examine tissue viability. After a maximal force was recorded, tissues were washed several times with fresh PSS to restore resting tension. The  28  arteries were then contracted by addition of phenylephrine (106 M) to all chambers. After reaching a stable contraction, cumulative concentrations of either acetyicholine (ACh; 1010 --  iO M) were added to artery rings.  Relaxation percentage for each dose of ACh  Tension at maximum contraction by PIlE  (phenylephrine) Tension at maximum relaxation for that dose / Tension at maximum -  contraction by PHE- Resting tension  * =  db/db vs. wild type, repeated measures ANOVA, p<O.OO1; (n=7 mice in each group)  29  100-  control db/db • sulfaphenazole treated db/db  D  75-  1*  .—  50—  25-  -11  -10  -9  -8  -7  -6  -5  -4  Log [ACh] (M)  Fig  3.2.2- The effect of sulfaphenazole on ACh-induced, endothelium-dependent aortic  vasodilation in db/db (not receiving sulfaphenazole) and sulfaphenazole-treated dbldb mice.  Individual ring segments with a length of 2 mm were mounted in the chambers of a wire myograph (Model 610 M, Multi myograph, Danish Myotech, Aarhus, Denmark). Artery segments were equilibrated at 37 °C during which a resting tension of 5 millinewtons was applied to each artery segment. Following the equilibration period, the PSS solution was substituted with 80 mlvi KC1 to examine tissue viability. After a maximal force was recorded, tissues were washed several times with fresh PSS to restore resting tension. The  30  arteries were then contracted by addition of phenylephrine (106 M) to all chambers. After reaching a stable contraction, cumulative concentrations of either acetyicholine (ACh; 10 --  i0 M) were added to artery rings.  Relaxation percentage for each dose of ACh  =  Tension at maximum contraction by PHE  (phenylephrine) Tension at maximum relaxation for that dose / Tension at maximum -  contraction by PHE- Resting tension  * =  db/db vs. sulfaphenazole-treated db/db mice, repeated measures ANOVA, p<O.OOl;  (n=7 mice in each group)  31  • sulfaphenazole treated WT  a) 0  0  controlWT  fU —  -11  -10  -9  -8  -7  -6  -5  -4  Log [ACh] (M)  Fig 3.2.3- The effect of sulfaphenazole on ACh-induced, endothelium-dependent aortic vasodilation in WT (not receiving sulfaphenazole) and sulfaphenazole-treated WT mice.  Individual ring segments with a length of 2 mm were mounted in the chambers of a wire myograph (Model 610 M, Multi myograph, Danish Myotech, Aarhus, Denmark). Artery segments were equilibrated at 37 °C during which a resting tension of 5 millinewtons was applied to each artery segment. Following the equilibration period, the PSS solution was substituted with 80 mM KC1 to examine tissue viability. After a maximal force was recorded, tissues were washed several times with fresh PSS to restore resting tension. The arteries were then contracted by addition of phenylephrine (1 06 M) to all chambers. After  32  reaching a stable contraction, cumulative concentrations of either acetyicholine (ACh; 1010 --  5 M) were added to artery rings. i0  Relaxation percentage for each dose of ACh  Tension at maximum contraction by PHE  (phenylephrine) Tension at maximum relaxation for that dose / Tension at maximum -  contraction by PHE- Resting tension  p>O.O5, (n=7 mice in each group)  33  1  75.  o  controlWT  c  controldb/db  • sulfaphenazole= treated db/db  J 50 1 x’ —  o.  25  -11  -10  -9  -8  -7  -6  -5  -4  Log [ACh] (M)  Fig 3.2.4= ACh-induced, endothelium-dependent aortic vasodilation in db/db and WT (not receiving sulfaphenazole) and sulfaphenazole- treated db/db mice.  Individual ring segments with a length of 2 mm were mounted in the chambers of a wire myograph (Model 610 M, Multi myograph, Danish Myotech, Aarhus, Denmark). Artery segments were equilibrated at 37 °C during which a resting tension of 5 millinewtons was applied to each artery segment. Following the equilibration period, the PSS solution was substituted with 80 mlvi KC1 to examine tissue viability. After a maximal force was recorded, tissues were washed several times with fresh PSS to restore resting tension. The  34  arteries were then contracted by addition of phenylephrine (106 M) to all chambers. After reaching a stable contraction, cumulative concentrations of either acetylcholine (ACh; 1010 --  iO M) or sodium nitroprusside (SNP; 10b0  Relaxation percentage for each dose of ACh  =  --  5 M) were added to artery rings. i0  Tension at maximum contraction by PI-JE  (phenylepbrine) Tension at maximum relaxation for that dose / Tension at maximum -  contraction by PHE- Resting tension  * =  db/db vs. wild type,  * *  =  db/db vs. sulfaphenazole-treated db/db mice, repeated  measures ANOVA, p<O.OO1; (n=7 mice in each group)  35  1  o  controlWT contoldb/db  50 —  25  -11  -10  -9  -8  -7  -6  -5  -4  Log [SNP] (M)  Fig 3.2.5- The effect of sulfaphenazole on SNP-induced, endothelium-independent aortic vasodilation in db/db and WT (not receiving sulfaphenazole) mice.  Individual ring segments with a length of 2 mm were mounted in the chambers of a wire myograph (Model 610 M, Multi myograph, Danish Myotech, Aarhus, Denmark). Artery segments were equilibrated at 37 °C during which a resting tension of 5 millinewtons was applied to each artery segment. Following the equilibration period, the PSS solution was substituted with 80 mlvi KC1 to examine tissue viability. After a maximal force was recorded, tissues were washed several times with fresh PSS to restore resting tension. The arteries were then contracted by addition of phenylephrine (106 M) to all chambers. After reaching a stable contraction, cumulative concentrations of sodium nitroprusside (SNP; 10 10 --  i0 M) were added to artery rings.  36  Relaxation percentage for each dose of SNP = Tension at maximum contraction by PHE (phenylephrine) Tension at maximum relaxation for that dose I Tension at maximum -  contraction by PHE- Resting tension  p>O.O5, (n=7 mice in each group)  37  100• •  sulfaphenazole treated db/db controldb/db  Log [SNP] (M)  Fig 3.2.6- The effect of sulfaphenazole on SNP-induced, endothelium-independent aortic vasodilation in db/db (not receiving sulfaphenazole) and sulfaphenazole-treated db/db mice.  Individual ring segments with a length of 2 mm were mounted in the chambers of a wire myograph (Model 610 M, Multi myograph, Danish Myotech, Aarhus, Denmark). Artery segments were equilibrated at 37 °C during which a resting tension of 5 millinewtons was applied to each artery segment. Following the equilibration period, the PSS solution was substituted with 80 mM KC1 to examine tissue viability. After a maximal force was recorded, tissues were washed several times with fresh PSS to restore resting tension. The arteries were then contracted by addition of phenylephrine (106 M) to all chambers. After reaching a stable contraction, cumulative concentrations of sodium nitroprusside (SNP; 10 10 --  i0 M) were added to artery rings.  38  Relaxation percentage for each dose of SNP  =  Tension at maximum contraction by PHE  (phenylephrine) Tension at maximum relaxation for that dose I Tension at maximum -  contraction by PHE- Resting tension  p>O.O5, (n=7 mice in each group)  39  o  controlWT  •  sulfaphenazole treated WT  5(  -10  -9  -8  -7  -6  -5  -4  Log [SNP] (M)  Fig 3.2.7- The effect of sulfaphenazole on SNP-induced, endothelium-independent aortic vasodilation in WT (not receiving sulfaphenazole) and sulfaphenazole-treated WT mice.  Individual ring segments with a length of 2 mm were mounted in the chambers of a wire myograph (Model 610 M, Multi myograph, Danish Myotech, Aarhus, Denmark). Artery segments were equilibrated at 37 °C during which a resting tension of 5 millinewtons was applied to each artery segment. Following the equilibration period, the PSS solution was substituted with 80 miVi KC1 to examine tissue viability. After a maximal force was recorded, tissues were washed several times with fresh PSS to restore resting tension. The arteries were then contracted by addition of phenylephrine (106 M) to all chambers. After reaching a stable contraction, cumulative concentrations of sodium nitroprusside (SNP; 10 10 --  i0 M) were added to artery rings.  40  Relaxation percentage for each dose of SNP  =  Tension at maximum contraction by PHE  (phenylephrine) Tension at maximum relaxation for that dose / Tension at maximum -  contraction by PFIE- Resting tension  p>0.05. (n=7 mice in each group)  41  3.3.. Antioxidant activity of sulfaphenazole  Sulfaphenazole demonstrated weak intrinsic antioxidant capacity compared to that of either Vitamin E or tempol. The intrinsic antioxidant activity of tempol started at 25x i0 pM in contrast to vitamin E and sulfaphenazole which started at 5 pM. The slope of the linear regression line for sulfaphenazole (0.0036± 0.00008) was significantly different (p  <  0.000 1) from the slopes of either vitamin E (0.0 185± 0.00020) or tempol (0.0937± 0.00966), as shown in Fig 3.3. The intrinsic antioxidant activities of vitamin E and tempol are approximately 5 and 25 times more than that of sulfaphenazole.  42  A  2 1.8 1.6 1 .4  • sulfa phenazole  C 0  —Linear (sulfaphenazole)  0 I-C 04  O.0O36x+ 0.01 61  0  100  200  300  400  500  600  Concentration (pM)  B 2 1.8 1.6 1.4 C 0 ..;‘-  0  1.2  VitE  1  —Linear (Vit E)  08 0.6  4o  /.O185x 0.0138  04 0.2 0 0  50  100  Concentration (pM)  43  150  C  2.5  .1.  2  0  1 .5  • tempol (tern p01)  H- Linear  4.0 I-c  .00 0.1209  0.5  5  10  15  20  25  30  Concentration (pM)  Fig 3.3: Comparison of intrinsic antioxidant activities of sulfaphenazole, vitamin E and tempol.  A- The intrinsic antioxidant activity of sulfaphenazole. B- The intrinsic antioxidant activity of vitamin E. C- The intrinsic antioxidant activity of tempol.  The slope of linear regression line for sulfaphenazole is significantly different from the one for vitamin E (ANCOVA, p < 0.0001). The slope of linear regression line for sulfaphenazole is significantly different from the one for tempol (ANCOVA, p  <  0.000 1).  44  3.4- Determination of 8-isoprostane level  Treatment with sulfaphenazole reduced the plasma concentration of 8-isoprostane in db/db mice (Fig 3.4: 85.52±9.07 pg/mi in sulfaphenazoie-treated diabetic vs. 167.50 ±29.03 pg/mi in control diabetic, p <0.001). The piasma concentrations of 8-isoprostane in sulfaphenazoie-treated diabetic mice were similar to those found in WT mice (Fig 3.4: 85.52± 9.07, 73.56± 3.97, 70.26± 8.61 pg/mi in sulfaphenazoie-treated diabetic, control WT and sulfaphenazole-treated WT, respectiveiy; p>O.O5).  45  200*  175—  E  150125100-  t;  75-  0 0. 0  50-  ‘T  250-  C..  ‘C’  Fig 3.4- Effect of sulfaphenazole on plasma 8-isoprostane level in control (db/db and WT mice that did not receive sulfaphenazole) and treated groups (db/db and WT mice that received sulfaphenazole as a treatment). Spectrophotometric quantification of 8-iso PGF a was performed using 8-isoprostane 2 enzyme immunoassay in control and treated groups. * =  db/db vs. other groups, one-way ANOVA, p <0.001; (n=7 mice in each group)  46  3.5- Determination of nitrite (N0 ) level 2  The nitrite levels due to ACh stimulation of aortas from sulfaphenazole-treated diabetic mice were significantly higher than those from untreated db/db mice (Fig 3.5: 0.12 ± 0.01 tM/ dry weight of aorta (mg) in the sulfaphenazole-treated diabetic group vs. 0.06 ± 0.006 IIM in control db/db group). Sulfaphenazole increased the nitrite levels in aortas from diabetic mice to a level similar to WT groups (0.12 ± 0.01 tM/ dry weight of aorta (mg), 0.12 ± 0.01 and 0.14± 0.02 in sulfaphenazole-teated diabetic, untreated WT and sulfaphenazole-treated WT groups, respectively; p>O.O5).  47  ‘I  0.20 0.15 0.10. 0.05  0,4  Zo  0.00--  C.  Fig 3.5- Effect of sulfaphenazole on nitrite (N0 ) levels in aortic tissue in control (db/db 2 and WT mice that did not receive sulfaphenazole) and treated groups (db/db and WT mice that received sulfaphenazole as a treatment). Spectrophotometric quantification of nitrite (N0 ) was performed using a nitric oxide 2 assay in control and treated groups. * =  control db/db vs. other groups, one-way ANOVA, p.<O.05; (n=7 mice in each group)  48  4-Discussion  Intact endothelial function has been recognized as an important determinant of the health of vascular tissue. Endothelial dysfunction may represent a premature aging of vascular endothelium that is common in cardiovascular disease. Vascular complications may be caused by the acceleration of endothelial cell death and by reducing their regeneration. The statement “a man is as old as his endothelium” represents the view that a healthy endothelium is required to maintain the health of the cardiovascular system. Blunted endothelium-dependent relaxation is now recognized as an important indication of future development of vascular complications (Triggle et al., 2005).  Diabetic patients suffering from type II diabetes often have a high risk of endothelial dysfunction from the early stages of diabetes. Dysfunction in endothelium may even precede the incidence of overt diabetes and is commonly associated with an impairment of endothelium-dependent vasodilation in experimental models of diabetes and human type II diabetic patients. It is the diabetic vascular sequelae that contribute predominantly to major clinical problems in diabetic individuals- such as a 2 to 4 fold- increase in coronary artery disease (Ceriello et al., 2003; Hogikyan et a!., 1998; Matsumoto et al., 2004).  An overproduction of superoxide anions by endothelial cells during oxidative stress may be important in triggering or mediating diabetes-induced vascular complications. Our findings show a selective reduction in endothelium-dependent vasodilation in aortic rings from untreated diabetic mice without an alteration of endothelium-independent vasodilation.  49  They are in agreement with reports of an impaired ACh-induced vasodilation contrasted to an intact SNP-induced, endothelium-independent vasodilation in diabetic patients and experimental diabetic animals. It has became clear that, in patients with coronary artery disease or with its risk factors- including hypertension, atherosclerosis, hypercholesterolemia, diabetes, increasing age and tobacco smoking- the endothelium independent vasodilatory response to exogenous NO is preserved while endothelium dependent vasorelaxation in response to ACh is changed. As well, other studies have revealed the beneficial therapeutic potential of nitric oxide donors in these patients (Gladwin, 2006; Herman and Moncada, 2005; Pannirselvam et al., 2002).  Superoxide anions are reduced to the uncharged H 0 either spontaneously or 2 enzymatically by the enzyme superoxide dismutase (SOD). Then, H 0 is dismutated to 2 water and oxygen by catalase or glutathione peroxidase enzymes. Simply stated, SOD has a key antioxidant role in biological systems. Although SOD is the first line of physiologic defence against diabetic oxidative stress, the reaction of superoxide anions with NO is about 3 times (6.7x i0 9 mol/ 1 ‘/S’) faster than their reaction with SOD. Hence, in the presence of SOD, superoxides have a greater tendency to react with NO. Considering the fact that sulfaphenazole inhibits CYP 2C subfamily as a potential source of free radical gerneration, we used this agent to assess the contribution of CYP 2C subfamily-mediated superoxide generation in db/db mice (Feletou and Vanhoutte, 2006; Murphy et al., 1998)  50  Sulfaphenazole is an inhibitor of CYP 2C in humans and has been shown to inhibit putative homologs of CYP 2C9 in both rabbits and rats. Although the CYPs are not well characterized in mice, they do express CYP 2C29 that is considered the mouse homolog of human CYP 2C9. Sulfaphenazole has previously been shown to have a similar effect in mice as it does in humans and other animal models. Our results indicate that endothelium dependent, ACh-mediated vasodilation was restored when diabetic mice were treated with sulfaphenazole (Fichtlscherer et al., 2004; Granville et al., 2004; Hunter et al., 2005; Kobayashi et al., 2003; Pokreisz et al., 2006).  We show that, although sulfaphenazole has weaker intrinsic anti-oxidant activity than either vitamin E or tempol, it nonetheless is able to reverse diabetic vascular endothelial dysfunction with an increase in NO bioavailability and a reduction in oxidative stress. Likewise, previous studies have already shown that administration of vitamin E (Chow, 2001; De Young et al., 2004; Du et al., 2003; Neri et al., 2005) or tempol (Bender et al., 2007; Ceriello, 2003; Nassar et al, 2002; San MartIn et al., 2007) in diabetes significantly reduces superoxide generation while at the same time restoring endothelial function and increasing NO bioavailability.  There are several proposed mechanisms by which sulfaphenazole restores endothelial function, including increasing the sensitivity of ACh receptors or augmenting the sensitivity of vascular cells to NO. However, sulfaphenazole did not alter the sensitivity ) to either ACh or SNP. We then explored the possibility that sulfaphenazole 50 (EC increased the bioavailability of NO by reducing superoxide-mediated metabolism of NO.  51  Our data showed a significant increase in nitrite levels in sulfaphenazole-treated diabetic mice compared to untreated ones.  Another mechanistic possibility is blocking ACh-induced EDHF by sulfaphenazole in aortic tissue and its effect on ACh-induced vasodilation. First of all, while NO alone is the primary vasodilator in larger blood vessels such as the aorta, both NO and EDHF play vasodilatory roles in smaller arteries and arterioles. Previous studies have demonstrated the suppression of a relaxation response to ACh in the rings from conduit arteries of eNOS -Imice in contrast to WT mice, which may suggest a more prominent role for NO in these arteries in comparison to EDHE. Bradykinin did not produce any vasodilation in the same vessel rings from WT and eNOS -I- mice. Second, the beneficial effects of sulfaphenazole in the restoration of endothelial function have already been studied in cardiac vessels from cardiovascular patients, and in experimental models of ischemia-reperfusion injury and stable coronary artery disease patients. It is noted that coronary vessels are noticeably smaller than the aorta. Third, it is generally accepted that the identity of EDHF may actually be multiple and include different candidates, such as potassium (K), EETs, H , O 2 and C-peptide natriuretic peptide (CNP). Therefore, inhibition of the activity of CYP isoenzymes may not necessarily block the production of EDHF in treated mice. Consistently, it was reported that sulfaphenazole did not block the production of EDHF in mesenteric arteries in a rat model of diabetes type I, suggesting that pathways other than CYP monooxygenase may be involved in EDHF production in that model (Brandes et al., 2000; Feletou and Vanhoutte, 2004; Fichtlscherer et al., 2004; Granville et al., 2004; Yi Shi et al., 2006).  52  Our data demonstrate that sulfaphenazole does not affect plasma glucose levels. Thus, its improvement of diabetic endothelial dysfunction and increasing NO bioavailability is unlikely to be due to a metabolic action of the drug. As well, it lacked intrinsic antioxidant capacity, and so was unlikely to directly scavenge free radicals (Fig 3.3). These findings are in keeping with other studies suggesting sulfaphenazole does not have antioxidant properties. We next explored the possibility that sulfaphenazole restores endothelial function in db/db mice by reducing CYP-generated superoxide. For this purpose, we measured plasma levels of 8-isoprostane, a metabolite of arachidonic acid and a marker of oxidative stress. Generation of 8-isoprostane has already been shown to increase under oxidative stress conditions such as diabetes (Cracowski et al., 2000; Jiang et al., 2000; Hunter et al., 2005; Lawson et al., 1999; Mallat et a!., 1999; Mezzetti et al., 1999; Morrow, 2005). Our measurements indicated that the elevated levels of 8-isoprostane in diabetic mice were reduced by sulfaphenazole treatment to levels similar to those in control mice. In contrast, sulfaphenazole did not affect the levels of this marker in control mice.  4.1- Summary  The major findings of this study demonstrate that long-term, in vivo treatment of diabetic (db/db) mice with sulfaphenazole, an inhibitor of CYP 2C activity, restores endothelium dependent vasodilation without affecting plasma glucose concentrations, possibly by reducing superoxide levels.  53  According to previous studies, interventions that lead to an improvement in clinical complications do not necessarily result in restoration of endothelial function in cardiovascular patients. On the other hand, weight loss is a very difficult treatment option in diabetic patients. Considering this, and the fact that restoration of endothelial dysfunction may prevent the development and progression of manifest cardiovascular disease, targeting endothelial dysfunction through treatments such as sulfaphenazole might be of great importance (Rask-Madsen and King, 2007).  4.2-Future strategies  From a clinical point of view, more studies might help to clarify the possibility of application of sulfaphenazole in combination with euglycemic agents to achieve more effective diabetic treatments. As well, investigation of this agent in other diabetic experimental models will be required to determine the effect of sulfaphenazole on clinical complications in diabetes. Obviously, treatment of human subjects as the last step will clarify how clinical and research findings in animal experimental models and human beings are related.  54  References  Arias E, Anderson RN, Kung HC, Murphy SL, Kochanek KD, 2003. Deaths: final data for 2001. Nat! Vital Stat Rep, 52:1-115.  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Res Vet Sci 63: 269-272  66  Appendix  67  The effect of SOD treatment on ACh-induced vasodilation  Addition of SOD (150U/ml) restored the endothelium-dependent vasodilation induced by ACh in aortic rings from untreated diabetic mice. Addition of SOD did not further enhance the vasodilation produced by ACh in either sulfaphenazole-treated diabetic or in untreated wild type mice. The maximal vasodilation to ACh was not affected by SOD in sulfaphenazole-treated and untreated wild type mice (p>O.05). However, addition of SOD caused a moderate reduction of vasodilation produced by higher concentrations of ACh in sulfaphenazole-treated wild type mice, but the maximal relaxation was similar. The EC5Os for ACh in all groups were unaffected by SOD.  Incubation of aortic rings with SOD further supported our previous finding, since addition of SOD did not further enhance ACh-induced relaxations in blood vessels from sulfaphenazole-treated diabetic mice, implying that sulfaphenazole treatment limited the increased intrinsic free radical generation. We observed that SOD caused a modest attenuation of ACh-mediated vasodilation in arteries from sulfaphenazole-treated wild type mice SOD. This is due to superoxide and H O activation of guanylate cyclase under 2 physiologic conditions. Thus, those free radicals that are physiologically involved in the mediation of vascular smooth muscle cells (VSMC) relaxation may be inhibited by  68  sulfaphenazole or scavenged by SOD. This may lead to a moderate reduction of endothelium-dependent relaxation with higher concentrations of ACh in sulfaphenaozole treated wild type mice (Chehal and Granville, 2006; Droge, 2002).  69  1  OO .14.j  xlu  1*  —  ’ 4 a)  1  -10  -9  -8  -7  -6  -5  D  controldb/db  A  controldb/db after incubation with SOD  -4  Log [ACh] (M)  Fig App.1- The effect of SOD on ACh-induced, endothelium-dependent aortic vasodilation in db/db mice (not receiving sulfaphenazole). * =  db/db after addition of SOD vs. db/db, repeated measures ANOVA, p<O.OO1; (n=7  mice in each group)  Addition of SOD (l5OUIml) restored the endothelium dependent vasodilation induced by ACh in aortic rings from db/db mice (that did not receive sulfaphenazole).  70  • sulfaphenazole treated db/db OO •— (U  • sulfaphenazole treated db/db after incubation with SOD  (Uu  —  -10  -9  -8  -7  -6  -5  -4  Log [ACh] (M)  Fig App.2- The effect of SOD on ACh-induced, endothelium-dependent aortic vasodilation in sulfaphenazole-treated db/db mice. p>0.O5, (n=7 mice in each group)  Addition of SOD did not enhance the endothelium dependent vasodilation induced by ACh in aortic rings from diabetic mice treated with sulfaphenazole  71  1 0  controlWT controlWT after incubation with SOD  —  2  1  -10  -9  -8  -7  -6  -5  -4  Log [ACh] (M)  Fig App.3- The effect of SOD on ACh-induced, endothelium-dependent aortic vasodilation in WT mice (not receiving sulfaphenazole). p>O.O5, (n=7 mice in each group)  Addition of SOD did not enhance the endothelium-dependent vasodilation induced by ACh in aortic rings from WT mice.  72  • sulfaphenazole treated WT sulfaphenazole treated WT after incubation with SOD  Xg) —  -10  -9  -8  -7  -6  -5  -4  Log [ACh] (M)  Fig App.4 The effect of SOD on Ach-induced, endothelium dependent aortic vasodilation -  in sulfaphenazole-treated WT mice. p >0.05, (n=7 mice in each group)  Addition of SOD caused a moderate reduction of vasodilation produced by higher concentrations of ACh in sulfaphenazole-treated WT mice, but the maximal relaxation was similar (Table App.1).  73  ACh after incubation with SOD Group  a  Log EC  control dbldb  7  -6.S8±O.06  sulfaphenazole-treated db/db  7  -6.78±0.06  58. 95±1.6  control WT  7  -7.08±0.06  73.57±1.97  sulfaphenazole-treated Wi’  7  -6.84±0.05  79.67±1.78  52.65±1.41  50 and Em values for ACh- induced, endothelium- dependent Table App.1 EC -  vasodilation from non-treated (db/db and WT mice that not receiving sulfaphenazole) and treated groups (db/db and WT mice that received sulfaphenazole as a treatment) after incubation with SOD. *  (n  = control db/db vs. control db/db after incubatin with SOD, one-way ANOVA; p<O.OO1;  the number of mice in each group)  74  

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