Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Thesis for doctoral degree (Ph.D.) 2012 Pleiotropic Effects of HMG-CoA Reductase Inhibitors Thesis for doctoral degree (Ph.D.) 2012 – studies in vitro and in vivo Pleiotropic Effects of HMG-CoA Reductase Inhibitors –studies in vitro and in vivo Cristine Skogastierna Cristine Skogastierna From the Department of Laboratory Medicine, Division of Clinical Pharmacology Karolinska Institutet, Stockholm, Sweden PLEIOTROPIC EFFECTS OF HMG-COA REDUCTASE INHIBITORS – STUDIES IN VITRO AND IN VIVO Cristine Skogastierna Stockholm 2012 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by US-AB © Cristine Skogastierna, 2012 ISBN 978-91-7457-818-8 Live as you were to die tomorrow. Learn as if you were to live forever. - Mahatma Gandhi ABSTRACT Cardiovascular disease (CVD) is the prime cause of death in industrialized countries, resulting from the progression of atherosclerotic lesions. An increased level of plasma cholesterol, particularly within low-density lipoproteins, is a principle risk factor, but also elevated triglycerides and low levels of high-density lipoprotein cholesterol. Recognition of dyslipidemia as a risk factor for CVD has led to an international consensus that anti-hyperlipidemic drug therapy is essential for the primary and secondary prevention in risk patients. HMG-CoA reductase inhibitors, i.e. statins, are the most common drugs used for this purpose, inhibiting the rate-limiting step in cholesterol biosynthesis. The medical significance of HMG-CoA reductase inhibitors have in recent years been expanded to include cholesterol-independent or "pleiotropic" vascular effects, including improvement of vascular endothelial function, inhibition of vascular smooth muscle cell proliferation and migration, anti-inflammatory actions, anti-oxidative effects or stabilization of vulnerable plaques. These effects have potential in the treatment of coronary artery disease in various settings, such as prevention of its onset as well as its progression. The major aim of this thesis was to contribute to the general knowledge about the pleiotropic effects of statins, particularly in relation to the vascular endothelium and to carcinogenesis. We found that fluvastatin at clinically relevant doses up-regulates transcription and translation of prostacyclin synthase and endothelial nitric oxide synthase in human vascular endothelial cells, associated with an increase in cellular production of prostacyclin (PGI2) and nitric oxide (NO), and induces rapid dilatation in isolated human arteries via contribution of endothelium derived factors like NO and PGI2. These results suggest beneficial effects of fluvastatin on endothelial maintenance in vivo and a protective role on the cardiovascular system, particularly at the level of vascular endothelium. We also show that simvastatin induces a potentially negative alteration of vascular endothelial cells both in vivo and in vitro. Exposure of clinically comparable concentrations of simvastatin led to decreased production and release of PGI2 in endothelial cells and to significantly increased urinary thromboxane A2 (TXA2) levels in healthy volunteers, possibly resulting in a shift of the balance between PGI2 and TXA2, favoring the thromboxane pathway. Fluvastatin and simvastatin were also shown to have different impact on vascular endothelial oxidative stress status, both in cultivated endothelial cells and in healthy volunteers. Compared to simvastatin, fluvastatin seems beneficial both regarding expression of anti-oxidative stress enzymes and reactive oxygen species activity in vitro and anti-oxidative capacity in vivo. The suggested anti-carcinogenic properties of statins in relation to expression of the redoxactive enzymes thioredoxin reductases (TrxR) were investigated in paper IV. We found that statin treatment decreases the hepatic expression of TrxR1 in humans and rats. In addition, the decreased TrxR1-levels were correlated with inhibited carcinogenesis in rat liver. The effect on TrxR1 levels might explain some of the anti-carcinogenic effects of statins. Overall, our results add to the conjecture that the impact on production of vascular active substances may be significantly different among the statins, and thus the consequences of statin exposure may not be drug class related but rather compound specific, highlighting the importance of more comparative studies where several statins are included. These findings add novel insight in the field of pleiotropic effects of statins and provide some insight for functional differences among statins. LIST OF PUBLICATIONS This thesis is based on the following publications, which will be referred to by their Roman numerals as indicated below: I. II. Skogastierna C, Luksha L, Kublickiene K, Eliasson E, Rane A, Ekström L. Beneficial vasoactive endothelial effects of fluvastatin: focus on prostacyclin and nitric oxide. Heart Vessels. 2011 Nov;26(6):628-36. Skogastierna C, Björkhem-Bergman L, Bergman P, Eliasson E, Rane A, Ekström L. Influence of simvastatin on the thromboxane and prostacyclin pathways, in vitro and in vivo. Accepted in Journal of Cardiovascular Pharmacology 2012. III. Skogastierna C, Björkhem Bergman L, Rane A, Ekström L. Anti-oxidative effects of statins on vascular endothelium. Manuscript. IV. Skogastierna C*, Johansson M*, Parini P, Eriksson M, Eriksson LC, Ekström L, Björkhem-Bergman L. Statins inhibit expression of thioredoxin reductase 1 in rat and human liver and reduce tumour development. Biochem Biophys Res Commun. 2012 Jan 20;417(3):1046-51. *Equal contribution ADDITIONAL PUBLICATIONS Garevik N, Skogastierna C, Rane A, Ekstrom L. Single dose testosterone increases total cholesterol levels and induces the expression of HMG CoA Reductase. Subst Abuse Treat Prev Policy. 2012 Mar 20;7(1):12. Bogason A, Masquelier M, Lafolie P, Skogastierna C, Paul C, Gruber A, Vitols S. Daunorubicin metabolism in leukemic cells isolated from patients with acute myeloid leukemia. Drug Metab Lett. 2010 Dec;4(4):228-32. Aomori T, Yamamoto K, Oguchi-Katayama A, Kawai Y, Ishidao T, Mitani Y, Kogo Y, Lezhava A, Fujita Y, Obayashi K, Nakamura K, Kohnke H, Wadelius M, Ekström L, Skogastierna C, Rane A, Kurabayashi M, Murakami M, Cizdziel PE, Hayashizaki Y, Horiuchi R. Rapid singlenucleotide polymorphism detection of cytochrome P450 (CYP2C9) and vitamin K epoxide reductase (VKORC1) genes for the warfarin dose adjustment by the SMart-amplification process version 2. Clin Chem. 2009 Apr;55(4):804-12. Olsson M, Gustafsson O, Skogastierna C, Tolf A, Rietz BD, Morfin R, Rane A, Ekström L. Regulation and expression of human CYP7B1 in prostate: overexpression of CYP7B1 during progression of prostatic adenocarcinoma. Prostate. 2007 Sep 15;67(13):1439-46. CONTENTS 1 2 3 4 5 Populärvetenskaplig sammanfattning ................................................................ 1 Introduction ........................................................................................................ 3 2.1 The history of statins ................................................................................ 3 2.2 Cholesterol ................................................................................................ 5 2.2.1 Biosynthesis of cholesterol .......................................................... 5 2.2.2 Cholesterol and cardiovascular disease ....................................... 7 2.3 Pharmacology of statins ........................................................................... 8 2.3.1 Function and molecular properties .............................................. 8 2.3.2 Pharmacokinetics ....................................................................... 12 2.3.3 Clinical applications ................................................................... 12 2.4 Pleiotropic effects of statins ................................................................... 15 2.4.1 Definition.................................................................................... 15 2.4.2 Statins and the vascular endothelium ........................................ 15 2.4.3 Prostanoids ................................................................................. 20 2.4.4 Nitric Oxide ................................................................................ 24 Aims.................................................................................................................. 25 Methodological considerations ........................................................................ 26 4.1 Experimental systems ............................................................................. 26 4.1.1 Cell culture systems ................................................................... 26 4.2 Study subjects ......................................................................................... 27 4.2.1 Human ........................................................................................ 27 4.2.2 Animals ...................................................................................... 27 4.3 Experimental methods ............................................................................ 28 4.3.1 Quantitative real time PCR ........................................................ 28 4.3.2 Promoter studies ......................................................................... 28 4.3.3 Enzyme Immunoassay ............................................................... 31 4.3.4 Western Blotting ........................................................................ 33 4.3.5 Colorimetric assay – detection of NO production..................... 34 4.3.6 LC-MS/MS ................................................................................. 34 4.3.7 Myographic readings ................................................................. 35 Results and discussion...................................................................................... 36 5.1 General comments .................................................................................. 36 5.2 Paper I: Beneficial vasoactive endothelial effects of fluvastatin: .............. focus on prostacyclin and nitric oxide ................................................... 37 5.3 Paper II: Influence of simvastatin on the thromboxane and ..................... prostacyclin pathways, in vitro and in vivo ........................................... 38 5.4 Paper III: Anti-oxidative effects of statins on vascular endothelium ... 39 5.5 Paper IV: Statins inhibit expression of thioredoxin reductase 1 in ........... rat and human liver and reduce tumour development ........................... 40 5.6 Undisclosed and unpublished results ..................................................... 41 5.6.1 Atorvastatin and rosuvastatin vs. expression of COX, PGIS ....... and eNOS ................................................................................... 41 5.6.2 Atorvastatin and rosuvastatin vs. PTGIS promoter activity ..... 42 5.6.3 6 7 8 Atorvastatin and rosuvastatin vs. anti-oxidative stress ................ enzyme genes.............................................................................. 43 Conclusions....................................................................................................... 45 Acknowledgements .......................................................................................... 46 References......................................................................................................... 48 LIST OF ABBREVIATIONS CYP PGI2 TXA2 PCR HMG-CoA HMGR CVD MI ROS AA TXA2 PGIS NO eNOS EC HUVEC HAEC PTGIS PGE2 TXB2 EIA NE L-NAME Indo COX TXAS CYP IL TNFα TrxR Cat SOD GPx LDL ADR Cytochrome P450 Prostacyclin Thromboxane A2 Polymerase chain reaction 3-hydroxy-3-methylglutaryl-CoA HMG-CoA reductase Cardiovascular disease Myocardial infarction Reactive oxygen species Arachidonic acid Thromboxane A2 Prostacyclin synthase (enzyme) Nitric oxide Endothelial nitric oxide synthase Endothelial cell Human umbilical vein endothelial cell Human aortic endothelial cell Prostacyclin synthase (gene) Prostaglandin E2 Thromboxane B2 Enzyme immunoassay Norepinephrine L-nitro-arginine methyl ester Indomethacin Cyclooxygenase Thromboxane synthase Cytochrome P450 Interleukin Tumour necrosis factor α. Thioredoxin reductase Catalase Superoxid dismutase Glutathion peroxidase Low density lipoprotein Adverse drug reaction 1 POPULÄRVETENSKAPLIG SAMMANFATTNING Kardiovaskulära sjukdomar är den främsta orsaken till för tidig död i västvärlden. Förhöjda och/eller obalanserade nivåer av blodfetter är viktiga riskfaktorer för utvecklandet av sjukdomar i hjärta och kärl, till exempel åderförkalkning (ateroskleros), som leder till förtjockning och minskad rörlighet av kärlväggarna. Den vanligaste typen av läkemedel för att behandla eller förhindra återkommande symtom är HMG-CoA reduktas hämmare, de som vi i vardagligt tal kallar för statiner. Utvecklingen av statiner för att minska konsekvenserna av aterosklerotiska kärlsjukdomar, främst hjärtinfarkt, är ett av de största medicinska framstegen de senaste decennierna. Många hjärt-kärl sjuka får positiva effekter av statiner utöver den primära effekten gällande korrektion av lipidnivåerna. Syftet med denna avhandling var att utöka kunskapen om hur några av de vanligaste statinerna interagerar med blodkärlens innersta celler, det vaskulära endotelet, och dess produktion av ämnen som har betydels för kärlens funktion samt hur statiner kan motverka cancerutveckling. Kväveoxid och prostacyklin är viktiga substanser som vidgar kärlen och bidrar till ett hälsosamt vaskulärt endotel. Vi visar i delarbete I att fluvastatin påverkar metabolismen av dessa substanser positivt och att deras bildande i humana kärlendotelceller ökar. Vi visar också att fluvastatin akut påverkar blodkärlens förmåga att vidga sig. Detta betyder att fluvastatinbehandling kan ge visst skydd för kärlväggen både akut genom att minska vaskulär tonus och över längre tid genom att bromsa åderförkalkningsprocessen. I delarbete II studerade vi hur en annan vanlig statin, simvastatin, påverkar bildning och balans av två av de viktigaste ämnena för endotelfunktion i cirkulationen, prostacyklin och tromboxan A2. Dessa två ämnen är funktionella antagonister, det vill säga de har motsatt funktion och måste vara i balans. Vi fann att hos friska frivilliga försökspersoner minskades utsöndringen av en viktig prostacyklinnedbrytningsprodukt medan tromboxan A2-utsöndringen höjdes efter simvastatinintag. I isolerade vaskulära endotelceller som behandlades med simvastatin, minskade produktionen av prostacyklin med så mycket som 40 %. Resultaten indikerar att simvastatin rubbar den viktiga jämvikten mellan tromboxan A2 och prostacyklin till fördel för tromboxan A2, vilket kan ha negativ inverkan på den vaskulära hälsan. Oxidativ stress och kronisk inflammation i kärlväggarna är ett kännetecken för kardiovaskulär sjukdom. Syftet med delarbete III var att jämföra de två statiner som studerats i delarbete I och II i relation till oxidativ stresspåverkan. Studiens resultat visar att fluvastatin verkar vara mer fördelaktig än simvastatin, då simvastatin hämmade genuttrycket av flera försvarsenzymer i endotelceller. Försök på friska försökspersoner visade att de som fått fluvastatin hade bättre försvar mot oxidativ stress. Dessa resultat tyder på att statinernas effekt kan vara ämnesspecifika och kan påverka den oxidativa stresstatusen i vaskulärt endotel. 1 C. Skogastierna I delarbete IV undersöktes mekanismerna bakom statiners anticarcinogena effekter i leverceller. Thioredoxin reduktaser är proteiner som har stor betydelse för cancerutveckling och har vistats vara förhöjda i celler med onormal tillväxt. Statinbehandling visades minska levercellsuttrycket av thioredoxin reduktas 1 i människa och råtta. Denna hämning kan bidra till att förklara potentiella anticancereffekter av statiner. Sammanfattningsvis så bidrar våra resultat till ökad kunskap och förståelse kring statiners verkan i kroppen, samt medverkar till ökad insikt om funktionella ämnesspecifika skillnader mellan statiner. 2 2 INTRODUCTION 2.1 THE HISTORY OF STATINS In the second half of the 19th century, the German pathologist Rudolf Virchow discovered that artery walls of patients dying from occlusive vascular disease were often thickened and irregular. He also observed that they also contained a fatty, yellowish substance later identified as cholesterol [1]. This pathological condition was termed atheroma, the Greek word for porridge [2]. In the 1950s scientists revealed a link between high blood cholesterol levels and cardiovascular disease, known as the lipid hypothesis [3]. Treatment was limited essentially to dietary changes (reductions in saturated fats and cholesterol), the bileacid sequestrants (cholestyramine and colestipol), nicotinic acid (niacin), the fibrates and probucol. Unfortunately, all of these treatments have limited efficacy or tolerability, or both. Novel and more effective ways to lower blood cholesterol levels without modifying the diet and lifestyle of subjects suffering with elevated blood cholesterol levels became high priority [2]. HMG-CoA reductase was found to be the rate-limiting enzyme in the cholesterol biosynthetic pathway (Figure 1) and hence became a natural target for drug development. Figure 1: The biosynthetic pathway of cholesterol 3 C. Skogastierna In contrast to other late-stage intermediates there are alternative metabolic pathways for the breakdown of hydroxymethylglutarate when HMG-CoA reductase is inhibited, rendering no build-up of potentially toxic precursors. In 1971, Akira Endo, a Japanese biochemist, in the search for a antimicrobial agent discovered the natural cholesterol-lowering substance mevastatin (ML-236B) [4] produced by the fungus Penicillium citrinum. Endo and his team believed that certain microorganisms may produce inhibitors of the enzyme HMG-CoA reductase to defend themselves against other organisms, as the product, mevalonate, is a precursor of many substances required by organisms for the maintenance of their cell wall (ergosterol) or cytoskeleton (isoprenoids) [5]. Mevastatin was however never marketed, because of its adverse effects with tumors, muscle deterioration, and sometimes death in laboratory dogs. By 1978, Alfred Alberts and colleagues at Merck Research Laboratories isolated another natural product in a fermentation broth from the fungus Aspergillus terreus. This compound was called mevinolin (MK803), later known as lovastatin and first marketed in 1987 as Mevacor. When lovastatin (Mevacor, Altocor, Altoprev) became available for prescription use, physicians were able, for the first time, to easily obtain large reductions in plasma cholesterol. Lovastatin at its maximal recommended dose of 80 mg daily produced a mean reduction in LDL cholesterol (low density lipoprotein cholesterol, or the “bad cholesterol”1) of 40 %, a far greater reduction than could be obtained with any of the treatments available at the time. Equally important, the drug produced very few adverse effects, and with once- or twice-daily dosing, was easy for patients to take. For these reasons, lovastatin was rapidly accepted by prescribers and patients. The second entrant, simvastatin (Zocord, Lipex), which differs from lovastatin only in that it has an additional side chain methyl group, was initially approved for marketing in Sweden in 1988 and subsequently worldwide. Pravastatin (Pravachol, Selektine, Lipostat) followed in 1991, fluvastatin (Lescol) in 1994, atorvastatin (Lipitor, Torvast) in 1997, cerivastatin in 1998, rosuvastatin (Crestor) in 2003, pitavastatin in 2003 (Livalo, Pitava). Atorvastatin became by 2003 the best-selling pharmaceutical in history. Some types of statins are naturally occurring, and can be found in such foods as oyster mushrooms and red yeast rice. Lovastatin is a fermentation product, simvastatin is a semisynthetic derivative of lovastatin and pravastatin is derived from the natural product compactin by biotransformation2. All other HMG-CoA reductase inhibitors 1 Low-density lipoprotein, or LDL, is one of the five major groups of lipoproteins that enable transport of multiple different fat molecules, including cholesterol, within the watery extracellular matrix and within the water-based blood stream. Higher levels of a certain types of LDL particles have been coupled to promotion of health problems and cardiovascular disease, and are therefore often informally termed the bad cholesterol, as opposed to HDL particles, which are frequently referred to as good cholesterol or healthy cholesterol particles. 2 Biotransformation is the chemical modification (or modifications) made by an organism of a chemical compound such as (but not limited to) nutrients, amino acids, toxins and drugs in the body, rendering polar compounds to be excreted in the urine. 4 are fully synthetic products. The structures of these drugs are shown in Figure 5. The generic names for all HMG-CoA reductase inhibitors end with ‘statin’, and the members of this class are today often referred to as ‘statins’, as opposed to the formal, although rather cumbersome, class name ‘HMG-CoA reductase inhibitors’. All inhibitors of HMG-CoA reductase produce a qualitatively similar effect on the lipid profile [6]. The mean reduction in LDL cholesterol attainable with the maximal recommended dose of different statins ranges from 35 to 55 %. 2.2 CHOLESTEROL Cholesterol is a waxy steroid of fat and an essential structural component of mammalian cell membranes, required for establishing proper membrane permeability and fluidity through the interaction with the phospholipid fatty acid chains [7, 8]. Within the cell membrane, cholesterol also functions in intracellular transport, cell signaling and nerve conduction. It is essential for the structure and function of invaginated caveolae and clathrin-coated pits, including caveola-dependent and clathrin-dependent endocytosis [9]. Within cells, cholesterol is the precursor molecule in several biochemical pathways, for example the production of bile salts in the liver hepatocytes, for the synthesis of vitamin D and the steroid hormones, including the adrenal gland hormones cortisol and aldosterone, as well as the sex hormones progesterone, estrogens and testosterone, and their derivatives. Mammalian cells retrieve cholesterol primarily from two sources; from outside the cell by receptor mediated endocytosis of LDL or by new synthesis from acetyl-CoA. All cells in the body are able to produce cholesterol, but with relative production rates varying with cell type and organ function. It is a complex 37 step process, involving more than 30 enzymes starting with the intracellular protein enzyme HMG-CoA reductase. Cholesterol is the principal sterol synthesized by vertebras, formed predominantly in the liver. Other sites of higher synthesis rates include the intestines, adrenal glands, and reproductive organs. Cholesterol is extensively recycled; upon excretion by the liver via the bile into the digestive tract, typically about 50 % of the excreted cholesterol is reabsorbed by the small bowel back into the bloodstream, where it is carried around by different lipoproteins. For a person of about 68 kg, the average de novo synthesis of cholesterol in the body is about 1 g per day. The total body content of the same person is about 35 g, primarily located within the membranes of all the cells of the body. Typical daily dietary intake of additional cholesterol, in the United States, is 200 – 300 mg [10]. Small quantities are synthesized in other cellular organisms (eukaryotes) such as plants and fungi, but it is almost completely absent among prokaryotes, i.e. bacteria. 2.2.1 Biosynthesis of cholesterol HMG-CoA reductase is a resident glycoprotein of the endoplasmic reticulum (ER) that consists of two distinct domains; a hydrophobic N-terminal domain with eight 5 C. Skogastierna membrane-spanning regions and a hydrophilic C-terminal domain that projects into the cytosol, which exerts the catalytic activity [11]. The N-terminal domain of the enzyme, which anchors the protein in membrane of the ER, contains sites for binding of regulatory proteins (Insig) and sterol-regulated ubiquitination (Figure 2) [12-14], sensing signals for its degradation. The enzyme activity can also be reduced by phosphorylation by an AMP-activated protein kinase, leading to halted cholesterol synthesis when ATP levels are low [15]. As a key enzyme in this pathway, HMG-CoA reductase is regulated by feedback mechanisms operating at multiple levels: transcription, translation, posttranslational modification and protein degradation. Figure 2: The HMG-CoA Reductase enzyme with its two distinct domains; the hydrophobic Nterminal domain which anchors the protein in membrane of the ER and the hydrophilic, catalytically active C-terminal domain that projects into the cytosol. Biosynthesis of cholesterol is directly regulated by the cholesterol levels present through classic end product feedback inhibition. When cells are depleted of sterols, membrane-bound transcription factors called sterol regulatory element binding proteins, SREBPs, together with another protein, SCAP (SREBP-cleavage-activating protein), dissociate from the membrane to the Golgi apparatus. Two proteases, S1P and S2P, activated by SCAP, cleave SREBP to a water soluble N-terminal domain that subsequently is translocated to the nucleus. The activated SREBP bind to specific sterol regulatory element DNA sequences, thus upregulating the synthesis of proteins involved in sterol biosynthesis, including the LDL receptor and HMG-CoA reductase (Figure 3). In sterol-overloaded cells, SREBP is inactive and transcription of these genes falls, synthesis of additional sterols is reduced and sterol levels returns to normal [16-18]. These homeostatic mechanisms involved are only partly understood. 6 Figure 3: Low intracellular levels of cholesterol stimulate the expression of proteins involved in sterol synthesis, including HMG-CoA reductase and LDL-receptor proteins. Although cholesterol is important and necessary for human health, high levels of cholesterol in the blood is associated with damage to arteries and cardiovascular disease, which leads us to the next topic, unbalanced cholesterol levels and what that can do to your blood vessels. 2.2.2 Cholesterol and cardiovascular disease The term ‘cardiovascular disease’ can be confusing. Commonly, it centers on atherosclerosis, focusing on cholesterol levels, plaque formation, and the narrowing and hardening of the arteries. From here, it is necessarily linked to the other diseases that lead to and result from these changes, including hypercholesterolemia, diabetes, hypertension and stroke. This broadens the scope of the relevant pathophysiology tremendously. Atherosclerosis can be further linked with metabolic syndrome, which is a group of risk factors (obesity, insulin resistance, aging, genetic predisposition) that increase the risk for coronary artery disease, stroke and type 2 diabetes. Cardiovascular disease, in this broad sense then, is not so much a single disease as an extensive web of interconnected diseases. 2.2.2.1 Cholesterol affecting endothelial cells A functional arterial blood vessel is to a large extent dependent on endothelial cells to release vasoactive agents, such as nitric oxide and prostacyclin. In atherosclerotic and high LDL-cholesterol vessels, this endothelium-dependent relaxation is lessened. Several studies have shown that patients with increased cholesterol levels (mainly oxidized LDL) have reduced ability to respond with vasodilation to acetylcholine, also 7 C. Skogastierna in vessels without any visibly apparent atherosclerosis. Hypercholesterolemic patients also lack the ability to release nitric oxide. Lipid lowering therapy with statins improves and normalizes the ability to relax and dilate the blood vessels [19, 20]. 2.3 PHARMACOLOGY OF STATINS 2.3.1 Function and molecular properties HMG-CoA reductase is the rate-limiting step in cholesterol biosynthesis. The liver is the main target for HMG-CoA reductase inhibition since most of the circulating cholesterol comes from internal liver production rather than the diet. Statins are competitive antagonists of HMG - CoA, as they directly compete with the endogenous substrate for the active site cavity of HMG-CoA reductase. By blocking this enzyme, the first committed enzyme of the HMG-CoA reductase pathway, they reduce the rate by which the enzyme is able to produce mevalonate, the consecutive molecule in the cascade that eventually produces cholesterol (see Figure 1). This ultimately reduces cholesterol as well as a number of other compounds, including the isoprenoids (required for production of dolichol3), tRNA isopentenylation, heme A, ubiquinone 4, and prenylated5 proteins such as Ras [15]. Products of the isoprenoid pathway are also required for proper germ cell migration during development [21]. When HMG-CoA reductase is inhibited by statins, intracellular sensors detect low cholesterol levels and stimulate endogenous production by the HMG-CoA reductase pathway, as well as increasing lipoprotein uptake by up-regulating the LDL-receptor. The LDL receptor then relocates to the liver cell membrane and binds passing LDL and VLDL particles. LDL and VLDL (very low density lipoprotein) extracted from the circulation into the liver where the cholesterol is reprocessed into excretable bile salts. The end result is lower LDL, TG (Triglycerides) and total cholesterol levels, as well as increased HDL (High Density Lipoprotein) levels in serum [2, 4]. 2.3.1.1 Structure All statins have the same pharmacophore, a HMG-like moiety, which may be present in an inactive lactone form. In vivo, these prodrugs are enzymatically hydrolyzed to their active hydroxy-acid forms [22, 23]. The inhibitors differ from each other and from the natural enzyme substrate in the rigid, hydrophobic structures covalently linked to the HMG-like moiety (Figure 4), which also accounts for most of their difference in 3 Dolichols are long-chain mostly unsaturated organic compounds, involved in many processes in the body; for example the co-translational modification of proteins known as N-glycosylation, the posttranslational modifications of proteins and they can function as a membrane anchor of certain oligosaccharides. 4 Ubiquinone is a component of the electron transport chain and participates in aerobic cellular respiration, generating energy in the form of ATP. 5 Prenylation is the addition of hydrophobic molecules to a protein. 8 pharmacodynamic6 effects, such as affinity for the active site of the HMG-CoA reductase, rates of entry into hepatic and non-hepatic tissues, availability in the systemic circulation for uptake into non-hepatic tissues and routes and modes of metabolic transformation and elimination. The activity of each statin is dependent on the binding affinity of the compound at the substrate site and the length of time it binds to the site [24]. HMG-CoA Reductase inhibitor HMG-CoA Figure 4: The pharmacophore of statins (HMG-CoA reductase inhibitor). Shown for reference is also the structure of HMG-CoA. The statins can be classified as type I or type II statins due to their structural relationship. The type I are nonsynthetic inhibitors, such as lovastatin, pravastatin and simvastatin, with a decalin ring structure linked to the HMG-like moiety. Type II statins are synthetic inhibitors featuring larger fluorophenyl groups linked to the HMGlike moiety and include fluvastatin, atorvastatin, cerivastatin and rosuvastatin (Figure 5). These additional groups are responsible for additional polar interactions that cause tighter binding to the HMG-CoA reductase enzyme and range in character from very hydrophobic (eg, cerivastatin) to partly hydrophobic (eg. rosuvastatin) [22, 25, 26]. 6 Pharmacodynamics is the study of the biochemical and physiological effects of drugs at its site of action, the mechanisms of drug action and the relationship between drug concentration and effect. 9 C. Skogastierna Type 1 Butyryl group of Butyryl group of type 1 statins type 1 statins pravastatin simvastatin lovastatin Type 2 rosuvastatin fluvastatin atorvastatin Figure 5: The chemical structures of statins. Type 1 statins, e.g. lovastatin, simvastatin and pravastatin, are partially reduced napthylene ring structures with a butyryl group. Type 2 statins all exist in their active hydroxy-acid forms. Fluvastatin has an indole ring structure, while atorvastatin and rosuvastatin have pyrrole and pyrimidine based ring structure respectively. 10 2.3.1.2 Comparative pharmacology of statins The statins differ in their absorption, plasma protein binding, excretion and solubility (Table 1), and exhibit variable dose-related efficacy in reducing LDL-C [27, 28]. They are often divided into two groups depending on origin; naturally occurring or synthetic (Table II). Table I. Comparative efficiency and pharmacology of statins. Drug Reductio n in LDL (%) Increase in HDL (%) Reduction in TG (%) Metabolism Protein binding (%) T1/2 (h) Hydrophilic IC50 (nM) FLU 22 – 36 3 – 11 12 – 25 CYP2C9 98 0,5 – 3,0 No 28 SIM 26 – 47 8 – 16 12 – 34 CYP3A4 95 – 98 1–3 No 11 ATV 26 – 60 5 – 13 17 – 53 CYP3A4 98 13–30 No 8 ROS 45 – 63 8 – 14 10 – 35 CYP2C9 88 19 Yes 5 LOV 21 – 42 2 – 10 6 – 27 CYP3A4 >95 2–4 No NA PRA 22 – 34 2 – 12 15 – 24 Sulfation 43 – 67 2–3 Yes 44 TC indicates total cholesterol; TG, triglycerides; T1/2, half-life. FLU indicates fluvastatin; SIM , simvastatin; ATV, atorvastatin; ROS, rosuvastatin; LOV, lovastatin; PRA, pravastatin. Table II. The statins can be divided into two groups: fermentation-derived and synthetic. Presentation of some of the most common statins along with brand names, which may vary between countries. Statin Fluvastatin Simvastatin Brand name Lescol, Lescol XL Zocor, Lipex Atorvastatin Lipitor, Torvast Rosuvastatin Crestor Mevacor, Altocor, Lovastatin Altoprev Pravastatin Pravachol, Selektine, Lipostat Derivation Synthetic Fermentation-derived (synthetic derivate of a fermentation product of Aspergillus terreus). Synthetic Synthetic Fermentation-derived. Naturally occurring compound (oyster mushrooms and red yeast rice). Fermentation-derived (of bacterium Nocardia autotrophica). 11 C. Skogastierna 2.3.2 Pharmacokinetics Statins have different pharmacokinetic7 profiles that are associated with their physicochemical properties. Most of the statins are metabolized by the liver before reaching the systemic circulation, which causes their relatively low systemic bioavailability [29]. Lovastatin and simvastatin are administered in their lactone forms, which are more lipophilic than their free acid forms, and therefore they have to be activated by hydrolysis to the active anionic carboxylate form [29]. Cytochrome P450 (CYP) isoenzymes are involved in the oxidative metabolism of the statins, with CYP3A4 and CYP2C9 isoenzymes being the most dominant. CYP3A4 isoenzyme is the most predominant isoform involved in metabolism of lovastatin, simvastatin, atorvastatin and cerivastatin [30]. CYP2C9 isoenzyme is the most predominant isoform involved in metabolism of fluvastatin, but CYP3A4 and CYP2C8 isoenzymes also contribute to the metabolism of fluvastatin [29]. Rosuvastatin is metabolized to a small degree by CYP2C9 and to a lesser extent by CYP2C19 isoenzymes [30, 31]. Pravastatin and pitavastatin undergo little metabolism. Their plasma clearances are governed by the transporters involved in the hepatic uptake and biliary excretion. Also for other statins, which are orally administered as open acid forms (i.e. fluvastatin, cerivastatin and atorvastatin), hepatic uptake transporter(s) play important roles in their clearances. The statins that have the ability to be metabolized by multiple CYP isoenzymes may avoid drug accumulation when one of the pathways is inhibited by co-administered drugs. Polymorphisms in transporter genes can have profound effects on statin pharmacokinetics, providing a rational basis for the individualization of lipid-lowering therapy according to some researchers [32]. The hepatic uptake of many statins are reduced by a common genetic variant of the organic anion-transporting polypeptide 1B1, increasing the risk of statin-induced myopathy [33]. Similarly, genetically impaired efflux activity of adenosine triphosphate (ATP)-binding cassette G2 transporter results in a marked increase in systemic exposure to various statins [34]. Importantly, the effects of these genetic polymorphisms differ depending on the specific statin that is used [35-37]. 2.3.3 Clinical applications Statins are lipid-lowering agents, primarily prescribed to patients with hypercholesterolemia or hyperlipidemia. In Sweden 2010, about 1/10 of the total population were prescribed statins at least once (http://www.socialstyrelsen.se). Since the big breakthrough for statin treatment with the 4S study [38] the treatment recommendations have been made through monitoring goal LDL-levels, where the target concentrations have been defined through consensus and gradually lowered with 7 Pharmacokinetics is the determination of the fate of substances administered externally to a living organism. It includes the study of the mechanisms of absorption and distribution of an administered drug, the rate at which a drug action begins and the duration of the effect, the chemical changes of the substance in the body and the effects and routes of excretion of the metabolites of the drug and /or the drug itself. 12 strengthened evidence for beneficial effects of statins. Clinical practice guidelines generally recommend that patients have tried "lifestyle modification", including a cholesterol-lowering diet and physical exercise, before statin use is considered; statins or other pharmacologic agents may then be recommended for those who do not meet their lipid-lowering goals through diet and lifestyle approaches [23, 31]. Treatment with statins for atherosclerotic disease is recommended at total cholesterol levels over 4.5 or LDL cholesterol over 2.5, and is given the highest priority in the guidelines from The Swedish National Board of Health and Welfare (http://www.socialstyrelsen.se/nationellariktlinjerforhjartsjukvard/sokiriktlinjerna/atero sklerotiskkarlsjukdomochk1). Statins are effective in decreasing mortality in patients with preexisting cardiovascular disease [39]. They are also currently advocated for use in patients at high risk of developing heart disease. On average, statins can lower LDL cholesterol by 1.8 mmol/l (70 mg/dl), which translates into a 60 % decrease in the number of cardiac events (heart attack, sudden cardiac death) and a 17 % reduced risk of stroke after long-term treatment [40]. The benefit of statins in secondary prevention8 for treating cardiovascular disease (CVD) is convincingly shown, but their value in primary prevention9 remains controversial. A recent meta-analysis on the placebo controlled studies with statins performed by Cholesterol Treatment Trialists’ (CTT) Collaboration, comparing low dose statin treatment with high dose, it was demonstrated that high dose statin treatment reduces the risk even more in relation to the decrease in LDLcholesterol that the high dose treatment resulted in. The CTT authors suggest that patients with high risk of cardiovascular events would benefit from intensive statin therapy even if baseline LDL cholesterol is below 2.0 mmol/L [39, 41]. The Scandinavian IDEAL trial also indicates that health benefits could be obtained with statin therapy resulting in LDL cholesterol below 2.0 mmol/L compared with those above this level. This turned some researchers into “the lower the better” advocates, being convinced that some of the guidelines of today are far too conservative. However, another recent meta-analysis did not find a mortality benefit in those at highrisk but without prior cardiovascular disease [42], while other reviews concluded that there is a mortality and morbidity benefit, but concerns regarding the quality of the evidence were raised [6, 43]. Some researchers conclude that with respect to quality of life there is limited evidence of improvement when statins are used for primary prevention [44]. Statins exhibit variable dose-related efficacy in reducing LDL-C [27], in general a reduction by an additional 7 % with each doubling of the statin dose [45]. Higher doses also give additional CVD improvement effects but at the same time increase the risk of adverse drug effects, hence in primary prevention the initial risk of the patient should be decisive if treatment will be recommended. Other researchers opine that statins are overly used since their use has included groups who do not benefit from treatment or where evidence for treatment is not clarified [46, 47]. There are also 8 Secondary prevention are methods to diagnose and treat existent disease in early stages before it causes significant morbidity. 9 Primary prevention are methods to avoid occurrence of disease. 13 C. Skogastierna investigators that claim that elevated cholesterol levels cannot be coupled to CVD and that statins thus are not effective nor safe [48]. As a class, the statins have been shown to measurably reduce the burden of atherosclerotic illness. However, muscle- and, more recently, nerve-related toxicity has emerged as potential complications contributing to treatment withdrawal [49]. The adverse effects are relatively rare but severe, and they are thought to a large extent being dose dependent. This is for example illustrated in the recent SEARCH study, where 80 mg simvastatin gave more cases of myopathy10 than treatment with 20 mg simvastatin [50, 51]. Generally, the myopathic signs and symptoms of tenderness, myalgias, cramping and elevated serum creatine kinase activity are fully reversible after drug discontinuation. Pathophysiologic clues regarding the potential causes of statin myopathy with or without neuropathy are discussed with particular attention paid to the implications of disrupted mevalonate metabolism. For example, secondary defects in isoprenoid biosynthesis are expected to impair the production of a variety of intermediaries such as dolichols (crucial for N-linked glycosylation), farnesylpyrophosphate (facilitates the endoproteolytic cleavage and maturation of prelamin A, and modifies B-type lamins and G-proteins) and geranylgeranyl pyrophosphate (necessary for coenzyme Q10 and G-protein synthesis). Also isopentenylpyrophosphate, involved in a nucleoside modification of selenocysteinyltRNA and thus indirectly related to the synthesis of all selenoproteins is proposed to be affected [49]. The beneficial effects of statins are indeed devaluated by some serious adverse reactions (ADRs) in the muscular system, liver and kidneys. The ADR frequency varies in different studies, although often reported to be of low risk. However, the clinical impression is that muscular side effects are considerably more common as a cause of termination of statin treatment. Independent studies on the safety and tolerabilty of this treatment are lacking. There is a poor implementation of statin treatment, in all probability adverse reactions contribute this, leading to under-treatment [52] and low adherence to treatment with lipid-lowering drugs [53-56]. Considering that it takes at least 18 to 24 months to observe an effect of statins on coronary heart disease [38] these reports are worrying since a large proportion of patients at risk may be untreated. An alternative way of translating the evidence to treatment guidelines has recently been presented [57]. The authors advocate that treatment shall be adjusted to the risk of each individual patient, where low dose of statins would be recommended for individuals at moderate risk for CVD, and high dose for those at high risk, primarily as secondary prevention. While no direct comparison exists, all statins appear equally effective on reducing cardiovascular morbidity and mortality regardless of potency or degree of cholesterol reduction [6]. A comparison of three statins; atorvastatin, pravastatin and simvastatin at commonly prescribed doses, found that there are no statistically 10 Myopathy is a muscular condition in which the muscle fibers do not function, resulting in muscular weakness. 14 significant differences in morbidity and mortality reduction of CVD based on their effectiveness against placebo [29]. 2.4 PLEIOTROPIC EFFECTS OF STATINS 2.4.1 Definition In pharmacology, pleiotropy refers to the action of a drug, often unanticipated, other than those for which the agent was specifically developed [58]. It may include adverse effects which are detrimental, but is often used to denote additional beneficial effects, illustrated in Figure 6 [59]. HMG-CoA reductase inhibitors (statins) Cholesterol lowering (LDL, HDL) Rho GTPases T-Cell activation MCH II expression chemokine secretion tissue factor Acute coronary syndrome Heart failure anti-inflammatory action anti-oxidative effects NO synthesis/ endothelial function immunomodulatory effects anti-cancer Sepsis Stroke Multiple sclerosis Chronic kidney disease Figure 6: Suggested pleiotropic effects of statins. 2.4.2 Statins and the vascular endothelium The endothelium is the thin layer of cells that lines the interior surface of blood vessels and lymphatic vessels, forming an interface between circulating blood or lymph in the lumen and the rest of the vessel wall. The cells that form the endothelium are called endothelial cells (ECs), and the endothelial cells in direct contact with blood are called vascular endothelial cells. Vascular endothelial cells line the entire circulatory system, from the heart to the smallest capillaries. These cells have very distinct and unique functions that are paramount to vascular biology, including fluid filtration, such as in the glomeruli of the kidney, blood vessel tone, hemostasis, lipid transport, neutrophil recruitment and hormone trafficking [60, 61]. Although the exact role of the 15 C. Skogastierna endothelium in the vascular diseases in vivo remains unclear, accumulating evidence indicates that endothelial dysfunction is pivotal in the development of atherosclerosis and its complications [62, 63]. The effectiveness and rapidity of statin-induced decreases in coronary events and evidence for restoration of endothelial function before significant reduction in serum cholesterol levels, have led to a continuous expansion of the medical significance of HMG-CoA reductase in recent years [20, 64-66]. Many of the observed cholesterolindependent or "pleiotropic" vascular effects of statins appear to especially target the concept of ‘vascular failure’, including the improvement of vascular endothelial function, inhibition of vascular smooth muscle cell proliferation and migration, antiinflammatory actions, anti-oxidative effects or stabilization of vulnerable plaques [65, 67]. These effects have potential in the treatment of coronary artery disease in various settings, such as prevention of its onset as well as its progression. Endothelial dysfunction can be broadly defined as an imbalance between vasodilating and vasoconstricting substances produced by, and/or acting on the endothelium. It has been proposed to be of prognostic significance in predicting vascular events including stroke and heart attacks and to be a useful marker of early cardiovascular diseases. A dysfunctional endothelium may result from and/or contribute to increased production of reactive oxygen species (ROS), decreased synthesis, release or activity of endothelial derived nitric oxide (NO) [68] and /or modified expression/ activity of anti-oxidative enzymes. 2.4.2.1 Vascular inflammation Inflammation plays important roles in the genesis, progression and complications of atherosclerosis and thrombosis [69, 70]. For example, clinical studies have stressed that in as much as 80 % of all sudden cardiac deaths inflammation is the underlying cause [71]. The inflamed vascular wall produces exaggerated levels of chemotactic proteins and increased expression of adhesion molecules, leading to monocytic adhesion, extravasion and formation of fatty streaks [72]. Other inflammatory mediators, including mast cells and activated T cells, also attach to endothelial cells and contribute to the progression of the atheromatous lesion. In addition, smooth muscle cells secrete a number of factors in this chemically modified environment that contribute to an increase in the inflammatory response. Some of the inflammatory cytokines, including IL-6, IL1β and TNFα, induce the release of C reactive protein (CRP) by the liver, which in turn together with modified LDL, superoxide anions and certain interleukins may decrease the NO availability, contributing to endothelial dysfunction [73]. Inflammatory cells and molecules augment the risk of plaque rupture and exposure of subendothelial tissue to blood end platelets through contribution of plaque instability via the release of chemokines, interleukines and metalloproteinases that degrade the collagen of the fibrous cap. Proinflammatory eicosanoid production is an important hallmark of CVD [74]. Statins have been shown to modulate the expression of 16 eicosanoids in a favourable way, and to modulate other factors, reducing inflammation of diseased blood vessels [75]. Both experimental and clinical outcome data support the hypothesis that statins, in addition to being potent LDL-lowering agents, also attenuate plaque inflammation and influence plaque stability. Simvastatin, fluvastatin and atorvastatin appear to reduce intimal inflammation [76] and suppress the expression of tissue factor and matrix metalloproteinases both in vivo and in vitro [77, 78], whereas pravastatin and cerivastatin can reduce macrophage content within experimental atherosclerotic plaques [79, 80]. Statins may also inhibit expression of adhesion molecules critical for monocyte attachment and adhesion to the vascular endothelium [81]. 2.4.2.2 Oxidative stress In all aerobic cells, being dependent on oxygen for energy production in the respiratory chain, ROS (reactive oxygen species) are formed. Thus, all aerobic cells are dependent on efficient machineries for detoxification of ROS. When excessive oxidative load risk to damage the cells, these natural efficient defense systems are activated and an intracellular reducing environment is created due to the presence of anti-oxidative stress enzymes like these studied in Paper III (Figure 7). The condition called oxidative stress occurs when redox homeostasis within the cell is altered, due to either an overproduction of ROS or a deficiency in antioxidant defense mechanisms, leading to up regulation of these systems. If these systems do not work properly, excess reactive oxygen species can be deleterious. They can for example inhibit endotheliumdependent vasodilator pathways (i.e. the NO pathway and the endothelium-derived hyperpolarizing factor (EDHF) pathways) and shift the balance in eicosanoids action from vasodilatation and antithrombosis towards endothelial dysfunction, vasoconstriction and thrombosis. Accumulating data suggest that several proatherogenic stimuli lead to increased production of ROS within the endothelial microenvironment and the resultant excessive oxidative stress plays a key role in mediating the pathologic manifestations of EC dysfunction associated with cardiovascular diseases such as hypertension, atherosclerosis or vascular diabetic complications [82]. It is important to remember, that although ROS often are mentioned in negative context, they also serve as regulators for crucial cellular functions, including the regulation of many important proteins in signal transduction, such as protein kinase C and tyrosine kinases [83] as well as the regulation of redox-sensitive transcription factor like Nuclear Factor k (NFk) and activator protein-1 (AP-1) [84]. In addition, activated phagocytes in the immune system produce ROS to kill bacteria [85]. Antioxidants in mammals include the lipid soluble vitamin E and ubiquinol, the water soluble vitamin C, glutathione (GSH) and thioredoxin (Trx). Of major importance are also the enzymatic antioxidants and antioxidant regenerating systems including the superoxide dismutase (SOD), catalases (Cat), thioredoxin system (Trx system), glutathione peroxidase (GPx) and the glutathione system [86]. 17 C. Skogastierna Figure 7: Reactive oxygen species and antioxidant systems in the vasculature. Highlighted in black are the most important ROS of the vascular cells. Oxidases, such as NADPH oxidase or xanthine oxidase, transfer an electron to oxygen, which is converted to superoxide (O2−). Superoxide dismutase (SOD) converts superoxide to hydrogen peroxide (H2O2), which can be converted to H2O by either catalase or glutathione peroxidase. H2O2 may also be converted to a hydroxyl radical (OH) in the presence of reduced iron (Fe2+). Superoxide may also react with nitric oxide (NO) to form peroxynitrite (ONOO−), which is unstable at a pH lower than 8, and therefore rapidly decomposes to OH. There is competition between NO and SOD for O2−. 2.4.2.3 The thioredoxin system The thioredoxin system comprises thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH (Figure 8) [87]. The system exists in all living cells and is essential proteins for regulating cellular redox balance and mitigating the damage caused by reactive oxygen species. The system also participates in redox signaling using molecules like hydrogen peroxide and nitric oxide [88] and in the regeneration of antioxidants, including ascorbic acid [89] and lipoic acid [90] as well as ubiquinone. Up-regulation of Trx and TrxR in several forms of cancer as well as during the carcinogenetic process have been reported [91-93]. Figure 8: The thioredoxin system which includes thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH. 18 Thioredoxins are a family of 12-kD redox active proteins ubiquitously expressed in both prokaryotic and eukaryotic cells [94, 95] that have been implicated in a variety of physiological processes and biological pathways. Trx1 is mainly found in the cytosol but can be translocated to the nucleus. In addition of being an electron donor for ribonucleotide reductase, Trx1 participates in the intracellular defence against oxidative stress by restoring intracellular thiols11 lost by oxidation and regenerates other antioxidant enzymes such as peroxiredoxins [86, 87]. Trx1 is also involved in signal transduction and regulation of transcription factors, for example NF-Upon oxidation of NF-in the nucleus Trx1 restores the DNA-binding activity, but in the cytosol Trx1 is being inhibiting [96]. Since ROS are involved in the regulation of intracellular signalling pathways, Trx1 may indirectly influence this regulation by being a ROS scavenger. In addition, Trx1 contributes to the regulation of apoptosis by binding to ASK-1 in a redox dependent manner and regulating the activity of p53 in several ways. Trx is present in human plasma [97] and certain cells in the immune system as well as various tumor cells have been shown to secret Trx extracellulary [98100]. Extracellulary, Trx has growth factor-like effects in an autocrine manner as well as proinflammatory effects, inducing chemotaxis of neutrophils, monocytes and T-cells [101]. TrxR, redox-active selenoenzymes, reduce Trx in a NADPH-dependent fashion [102], and is a central component in the thioredoxin system [103]. Humans express three thioredoxin reductase isozymes; TrxR1, TrxR2 and TrxR3. TrxR belongs to a family of NADPH-dependent pyridine nucleotide-disulfide oxidoreductases, to which glutathione reductase, lipoamide dehydrogenase and mercuric ion reductase also belong [104]. The mammalian TrxR has an exceptionally broad substrate specificity in comparison to the other enzymes of this family [105]. Mammalian TrxR is a homodimer composed of two identical 58 kDa subunits, with a FAD-domain and a NADPH-binding site in each subunit [106]. Three different isoforms of the mammalian enzyme have been described so far. The classical TrxR1 is found in the cytosol, TrxR2 is localised in the inner mitochondria membrane and is structurally and biochemically very similar to TrxR1 [107, 108] and the third TrxR isoenzyme, TGR, is expressed only in testis. TrxR has been shown to be secreted extracellulary both by neoplastic and normal cells and is present in human plasma [109]. 2.4.2.4 Carcinogenesis due to chemical modification Chemical alterations in DNA may occur due to chemical modifications of DNA-bases caused by for example ROS or binding of chemical compounds to DNA, possibly leading to errors during the replication process. Such DNA lesions are usually rapidly discovered and eliminated by the DNA repair systems, but if cell division occurs before DNA repair is complete, mutated daughter cells appear that might bear dysfunction in 11 A thiol is an organosulfur compound containing a carbon-bonded sulfhydryl (–C–SH or R–SH) group (where R represents an alkane, alkene, or other carbon-containing group of atoms). Thiols are the sulfur analogue of alcohols (that is, sulfur takes the place of oxygen in the hydroxyl group of an alcohol). The – SH functional group itself is referred to as either a thiol group or a sulfhydryl group. 19 C. Skogastierna their growth regulation, including defect contact inhibition and dysfunction in their apoptotic machinery, possibly developing into a tumor. Such a mutated cell that shows a growth advantage under the influence of a selective pressure during promotion compared to the surrounding, normal cells, is called an initiated cell. The initiated cells can respond to growth stimulatory signals appearing during regeneration after cell injury and are able to grow under circumstances where growth is inhibited in normal cells. As a consequence, focal proliferation may occur, generating clones of altered preneoplastic cells [110]. Genetic instability facilitates further mutations and after several steps of genetic alteration and selection due to growth advantages, malignant tumor cells develop. The different cells within the same tumor can show great and progressive genetic variability although they all, from the very beginning of the carcinogenesis, originate from one single cell [110-112]. In experimental models the preneoplastic as well as the neoplastic12 cells are characterised by changes in the expression of drug metabolising enzymes, upregulation of antioxidants and antioxidant regenerating systems and a capacity to avoid apoptosis [113-115]. The carcinogenetic process can be described as a chronic selection of cells with a drug resistant phenotype, at least for tumors that develop as a consequence of environmental exposure and chronic cell injury. During recent years it has been proposed that statins have anticarcinogenic effects, both in liver [116, 117] and in other tissues such as the prostate, lung and pancreas [118121]. 2.4.3 Prostanoids The biosynthesis of prostanoids is initiated by the activation of phospholipase A2 (PLA2) and subsequent release of arachidonic acid (AA) from cell membranes. The AA is then transformed by cyclooxygenases (COX) and specific prostaglandin (PG) synthases. There are two major COX enzymes, COX-1 and COX-2, which differ in structure, tissue distribution, subcellular localization and function. COX-1 is commonly described as ubiquitous and constitutively expressed. COX-2 is on the other hand normally absent from most cell types. Its expression is induced at sites of inflammation and vascular injury by inflammatory mediators like TNF-α and IL-1β, and its mRNA as well as protein have short half-lives. Both enzymes are membrane-bound homodimers, found predominantly on the perinuclear membranes producing the intermediate PGH2, which is then converted to the final prostanoid products by tissue specific, terminal enzymes. For example, the predominant prostaglandin formed in endothelial cells after 12 Neoplasm is an abnormal mass of tissue as a result of neoplasia ("new growth" in Greek), the abnormal proliferation of cells. Preneoplastic cells, such as metaplasia (cell type conversion) or dysplasia (maturation abnormality) often undergo an abnormal pattern of growth but do not always progress to neoplasia. The growth of neoplastic cells exceeds and is not coordinated with that of the normal tissues around it. The growth persists in the same excessive manner even after cessation of the stimuli, usually causing a lump or tumor. Neoplasms may be benign, pre-malignant (carcinoma in situ) or malignant (cancer). 20 PLA2 activation is prostacyclin (PGI2) due to high expression of prostacyclin synthase (PGIS, also known as CYP8A1). PGIS is a single-pass membrane-bound protein, situated in the endoplasmic reticulum and isomerizes PGH2 to PGI2. It is also abundant in the ovary, heart, skeletal muscle, lung, and prostate. Unlike most P450 enzymes, PGIS does not require molecular oxygen (O2), instead it uses its heme cofactor to catalyze the isomerization of prostaglandin H2 to prostacyclin. 2.4.3.1 Prostacyclin PGI2 is released by healthy endothelial cells and performs its function through a paracrine signaling cascade that involves G protein-coupled receptors on nearby platelets, endothelial cells and smooth muscle cells; regulating homeostasis, hemostasis, smooth muscle function, preventing inflammation, dilating vessels, inhibiting platelet aggregation and also separating existing aggregates in vitro. PGI2 is therefore considered to play an important role in vasoprotection and its deficiency may lead to thrombosis and other vascular lesions [122, 123]. While the expression of PGIS is constitutive, the production of PGI2 can be dramatically increased by the induced expression of COX-2, particularly in vascular cells and macrophages. PGI2 is rapidly secreted from source cells, presumably through an ATP-binding cassette (ABC) transporter, activating neighboring cells which bear the specific I-prostanoid (IP) receptor, a G-protein coupled receptor (GPCR). The PGI2/IP interaction signals through the Gαs subunit to stimulate G protein-coupled increase in cAMP and protein kinase A (Figure 9, modified from www.caymanchem.com), resulting in decreased [Ca2+]i. It could also cause inhibition of Rho kinase, leading to vascular and bronchial smooth muscle relaxation. In addition, PGI2 intracrine signaling may target nuclear peroxisome proliferator-activated receptors and regulate gene transcription, suppressing the production of proinflammatory cytokines, angiogenic cytokines, and mediators of extracellular matrix remodeling (e.g., IL-1β IL-6, VEGF, TGF-α, thromboxane). The transcriptional regulation of the human PTGIS gene is poorly understood. The core PTGIS promoter contains several presumed cis-acting elements for different transcription factors like Sp1, NFB and AP-2, which have been shown to be linked to induction of other genes after treatment with statins [124, 125]. The PTGIS promoter activity has been shown to be regulated by epigenetic mechanisms in human cancer cells [122, 126] but the genetic regulation in normal cells is not well studied. 21 C. Skogastierna Figure 9: Prostacyclin (PGI2) is synthesized from arachidonic acid (AA) in endothelial cells by the COX pathway via prostacyclin synthase (PGIS). Secreted PGI2 activates the IP receptor on neighbouring smooth muscle cells, which induces cAMP synthesis from ATP. 2.4.3.2 Thromboxane Thromboxanes are a group of prostaglandins named for their role in clot formation (thrombosis). Thromboxane A2 (TXA2), one of the major forms, is in the cardiovascular system predominantly derived from platelet COX-1. The interaction with the G-protein-coupled TXA2 receptor, TP, elicits not only platelet aggregation and smooth muscle contraction but also the expression of adhesion molecules and the adhesion and infiltration of monocytes/macrophages [127]. Vasoconstriction and various pro-inflammatory effects exerted by TXA2 on tissue microvasculature is considered to be strong contributing factors to why TXA2 is pathogenic in various diseases, such as ischemia-reperfusion injury, hepatic inflammatory processes, acute hepatotoxicity etc. [128, 129]. 2.4.3.3 Prostacyclin vs. Thromboxane In the circulatory system, TXA2 is in homeostatic balance with PGI2 [74]. The balance of the oppositely-acting COX-derived prostanoids influences many processes throughout the body, such as blood pressure regulation, clotting, and inflammation. The 22 PGI2/TXA2 ratio is of particular interest in vivo, with the corresponding synthases shown to be differentially regulated in a variety of disease states [130, 131]. In many cardiovascular diseases, the widespread vascular and organ inflammation and the associated oxidative stress, enhance the production of eicosanoids and shift their production/effects from vasodilatation and anti-thrombosis to vasoconstriction, prothrombosis and further inflammation [132, 133], representing the functional characteristics of endothelial dysfunction. An imbalance towards TXA2 seems to promote atherosclerosis and increase risk for cardiovascular events, while an imbalance towards PGI2 likely inhibits the development of CVD [134-136]. The widely used drug aspirin13 acts by irreversibly binding to COX and thus inhibits the synthesis of the precursors of thromboxane within platelets. This anticoagulant property makes aspirin useful for reducing the incidence of heart attacks, strokes, and blood clot formation in people at high risk of developing cardiovascular diseases. Aspirin irreversibly inhibits COX-1 and modifies the enzymatic activity of COX-2. High analgetic doses of aspirin inhibits COX-2, disturbing the synthesis of PGI2 and thus the protective anti-coagulative effect of PGI2 is decreased, increasing the risk of thrombus and associated heart attacks and other circulatory problems. Newer NSAID drugs, COX-2 inhibitors (coxibs), have been developed to inhibit only COX-2, with the intent to reduce the incidence of gastrointestinal side-effects but [137] several of the new COX-2 inhibitors have been withdrawn recently, after evidence emerged that inhibition of COX-2 increases the risk of heart attack and stroke due to prostacyclin down-regulation relative to thromboxane levels, as COX-1 in platelets is unaffected [138]. Thus, the protective anticoagulative effect of PGI2 is removed, increasing the risk of thrombus and associated heart attacks and other circulatory problems. Prostacyclin synthase and thromboxane synthase signaling via arachidonic acid metabolism affects a number of tumor cell survival pathways such as cell proliferation, apoptosis, tumor cell invasion and metastasis, and angiogenesis. However, the effects of these respective synthases differ considerably with respect to the pathways described. While prostacyclin synthase expression generally is believed to be antitumor, a pro-carcinogenic role for thromboxane synthase has been implicated in a variety of cancers [139-142]. In contrast, increased prostacyclin synthase expression and activity has been suggested protect against tumor development [143-145]. 13 Aspirin, also known as acetylsalicylic acid (abbreviated ASA), is a salicylate drug often used as an analgesic to relieve minor aches and pains, as an antipyretic to reduce fever and as an antiinflammatory medication. Aspirin acts as an acetylating agent whereby an acetyl group is covalently attached to a serine residue in the active site of the COX enzyme, making aspirin different from other NSAIDs such as diclofenac and ibuprofen, which are reversible inhibitors. Aspirin is today one of the most widely used medications in the world. 23 C. Skogastierna 2.4.4 Nitric Oxide Nitric oxide (NO) is synthesized by the enzyme Nitric Oxide Synthase (NOS) in biological systems. NOS is a remarkably complex enzyme which acts on molecular oxygen, arginine and NADPH to produce NO, citrulline and NADPH+. This process requires five additional cofactors; FMN, FAD, Heme, calmodulin and tetrahydrobiopterin, and two divalent cations; calcium and heme iron. Three different isoforms of NOS have been identified; neuronal, endothelial and inducible NOS. NO is produced in trace quantities by neurons, endothelial cells, platelets and neutrophils in response to homeostatic stimuli [146, 147] and scavenged rapidly, having a half-life of about four seconds. NO acts in a paracrine fashion to transduce cellular signals through interaction with guanylate cyclase, leading to activation of the enzyme and subsequently increased cGMP levels. Other cells also produce NO, e.g. macrophages, fibroblasts, and hepatocytes, in response to inflammatory or mitogenic stimuli, providing a defense mechanism against pathogens through oxidative toxicity. These high NO levels lead to the formation of peroxynitrite, destruction of iron-sulfur clusters, nitration of protein tyrosine residues and thiol nitrosation. Thus, the amount of NO produced in different biological systems can vary over several orders of magnitude and its subsequent chemical reactivity is diverse. 24 3 AIMS In the work included in this thesis, I have studied some of the commonly prescribed HMG-CoA reductase inhibitors with respect to the vascular endothelium. More specifically the aims were to: Investigate the influence in vitro of clinically relevant fluvastatin concentrations on the metabolism and production of eicosanoids and NO in human endothelial cells. Study the impact on vascular function after fluvastatin exposure. Study how clinically relevant simvastatin concentrations affect the production and balance of two of the most important vascular active prostaglandins, prostacyclin and thromboxane A2, in vitro as well as in vivo. Compare fluvastatin and simvastatin with regard to impact on oxidative stress in human vascular endothelium in vitro and in vivo. Evaluate the suggested anti-carcinogenic properties of statins in relation to expression of the redox-active enzymes TrxR, by investigating if statin treatment is associated with alterations in the hepatic expression of TrxR. 25 C. Skogastierna 4 METHODOLOGICAL CONSIDERATIONS The purpose of this section is to provide a methodological overview, considerations and limitations to some of the techniques used in the studies of this thesis. More detailed descriptions of the separate methods can be found in the Material and Methods section of paper I-IV. 4.1 EXPERIMENTAL SYSTEMS 4.1.1 Cell culture systems Endothelial cells are situated at the interface between the circulating blood and the vessel wall. They serve as a sensor and transducer of signals within the circulatory microenvironment and is integral in maintaining the homeostatic balance of the vessel through the production of factors that regulate vessel tone, coagulation state, lipid transport, cellular proliferative response and leukocyte trafficking [60, 61]. Cultivated endothelial cells isolated from human vessels are commonly used for physiological and pharmacological investigations. Endothelial cells vary in morphology and functions according to the type and size of the associated vessel. The work presented in this thesis involves therefore endothelial cells from two different locations in the body; aorta and umbilical cord. Furthermore, only primary cells were used. Primary cells are taken directly in vivo (e.g. biopsy material) and established for growth in vitro. These cells have undergone very few population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous (tumor or artificially immortalized) cell lines, thus representing a more representative model of the in vivo state. With the exception of cells derived from tumors, most cell cultures have limited lifespan. After a certain number of population doublings (called the Hayflick limit), cells undergo the process of senescence and stop dividing, while generally retaining viability. The cells used in our work were well within the limit of population doublings recommended by the manufacturer. In general, human endothelial cells are isolated from normal human tissue, in this case large blood vessels. Human aortic endothelial cells (HAEC) are isolated from the human ascending (thoracic) and descending (abdominal) aorta and human umbilical vein endothelial cells (HUVEC) are isolated from the vein of the umbilical cord. These cells are commercially available; we used pooled cells isolated from five different donors, provided by PromoCell (Heidelberg, Germany). Shortly after isolation, cells are cryopreserved in liquid nitrogen at passage one or passage two until experiments are carried out. Rigid quality control tests are performed for each lot of cells. They are tested for cell morphology, adherence rate and cell viability, cell-type specific markers, e.g. von Willebrand Factor and CD31, as well as Dil-Ac-LDL uptake assays. Growth performance is tested through multiple passages up to 15 population doublings under culture conditions without antibiotics and antimycotics. In addition, 26 all cells have been tested for the absence of HIV-1, HBV, HCV and microbial contaminants (fungi, bacteria, and mycoplasma). 4.2 STUDY SUBJECTS 4.2.1 Human For our human pilot studies in Paper II and III, five healthy volunteers were recruited. The subjects were two men and three women, age 30-45, taking no other medication or naturopathic drugs, not having any disease or infection, not being pregnant or nursing or with known sensitivity to statins. Each subject was given a single dose of 80 mg simvastatin and 40 mg fluvastatin, at two separate time points with a two week washout period in between. Serum was collected immediately before the tablets were taken and two hours after the dose (time of Cmax for simvastatin) [148]. Urine was collected for 24 hours before and for 24 hours after administration and thereafter aliquoted and kept at -80 °C until urinalysis. For Paper IV we used patient samples from two different cohorts. Cohort 1included stored RNA from liver biopsies from normocholesterolemic gallstone patients treated with either fluvastatin 20 mg/day (N=5) or atorvastatin 80 mg/day (N=6) or placebo (N=8) for two weeks. Fluvastatin 20 mg/day was used to achieve a low cholesterol synthesis inhibition and atorvastatin 80 mg/day to achieve a high cholesterol synthesis inhibition. From each subject stored plasma was used for the lathosterol och LDLmeasurements [149]. Cohort 2 included cDNA from 18 human livers belonging to the human donor liver bank established at the Division of Clinical Pharmacology, Karolinska University Hospital, Huddinge. In this cohort three subjects were treated with statins and 15 had no statin-treatment according to the medical records. We had no information about which statin or which dose the subjects were using. 4.2.2 Animals A strain, in reference to rodents, is a group in which all members are as nearly as possible genetically identical. In rats, this is accomplished through inbreeding. By having this kind of population, it is possible to conduct experiments on the roles of genes, or conduct experiments that exclude variations in genetics as a factor. The Fischer 344 (F344) rat is one of the most commonly used rat strain in carcinogenesis because of its genetically in-bred status, relatively small size, ease of maintenance and sensitivity to various carcinogens and its relatively low spontaneous tumor rate except for testicular Leydig cell tumors [150]. The rat model was originally described by Solt and Farber [151] and was called “the resistant hepatocyte model” implementing the appearance and selection of resistant hepatocytes with up-regulated cellular defence. In Paper IV we used rat liver tissue from a study conducted earlier in our laboratory, where male rats had been treated according to the chemical induced hepatocarcinogenesis protocol described previously [38] and 5 rats had been given 27 C. Skogastierna lovastatin in the diet for 5 weeks (dose approximate 13 mg/kg bodyweight/ day) and 5 rats standard chow diet (R36) for 5 weeks. Since rodents have a much quicker metabolism of statins compared to humans this is likely to be an adequate statin dose with an exposure similar to or less than that in humans. Similar or higher doses have been used in previous works [152, 153]. 4.3 EXPERIMENTAL METHODS 4.3.1 Quantitative real time PCR Real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (Q-PCR/qRT-PCR) or kinetic polymerase chain reaction (KPCR), is a powerful technique which is used to amplify and simultaneously quantify a targeted DNA molecule as the reaction progresses in real time. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes. Prior to the PCR step, reverse transcription was performed to obtain the first strand DNA, then TaqMan Probe-based assays were used in the expression experiments in Papers I-IV. Briefly, the assays use a fluorogeniclabeled oligonucleotide probe to enable the detection of a specific PCR product as it accumulates during PCR cycles. A reporter dye is incorporated on the 5´end and a quencher dye on the 3´end of the probe. As long as the probe is intact, the proximity of the quencher greatly reduces the reporter dye emitted fluorescence. If the target sequence however is present, the probe anneals between primer sites and is cleaved by the 5´nuclease activity of the DNA polymerase during the extension phase of the PCR reaction. The reporter dye is then separated from the quencher, which increases the reporter dye signal and also allows the primer extension to continue to the end of the template strand. With each cycle additional reporter dye molecules are cleaved, resulting in increased fluorescence intensity proportional to the amount of amplicon produced. Thus the higher the starting copy number of the target sequence, the sooner significant increase in fluorescence is observed, i.e. when the fluorescence increases above the baseline and crosses the threshold [154]. The cycle at which this occurs is called the Ct value and can be employed for quantification [155]. 4.3.2 Promoter studies In Paper I and II, to assess the genetic background for the variation observed in statin induced regulation of PGIS mRNA in endothelial cells, we transfected HUVECs with the PTGIS core promoter incorporated into a reporter construct. Briefly, the method works as follows (see also Figure 10): 1. Amplification by PCR is carried out. We amplified PGIS promoter fragments, including the cis-acting elements required for basal transcriptional activity (approximately 300 bp), using human genomic DNA as template. The PCR products were verified on an agarose gel by electrophoresis. 28 2. Next step is subcloning. The PCR product is cloned into a vector14 (here supplied with single 3´-thymidine (T) overhangs, allowing PCR inserts to ligate efficiently with the vector. The plasmid also contains topoisomerase I covalently bound to the vector - referred to as "activated" vector) by cleaving the plasmid and the PCR product from step 1, using restriction enzymes15, and then ligate them together. 3. We are now ready to perform transformation16 into competent17 bacteria and expand them overnight on selective agar plates, on which only successfully transformed bacteria are able to grow. 4. Isolated clones are selected for further expansion and subsequent purification of plasmids. The plasmids are then cut by restriction enzymes and desired fragments isolated by gel-purification. We also analyzed purified fragments from each clone for verification of insert by sequence analysis and compared with PGIS official sequence using BLAST18. 5. A second round of subcloning can now be executed, but this time we put our promoter sequence into a construct containing an expression reporter downstream of our insert. This results in a luciferase19-gene containing expression vector construct driven by the PGIS promoter insert. 6. The complete vector is then expanded in bacteria through transformation, purified and analysed as before. 7. Human umbilical endothelial cells (HUVECs) could then be transfected20 with our expanded and purified reporter constructs. As expression control, renilla21-containing plasmids were co-transfected into the HUVECs. 8. Eight hours after the transfection, statin or vehicle was added and cells were let grow for 24 hours before harvest and analysis. 14 Vector: used in genetic engineering and are commonly used by molecular biologists to deliver genetic material into cells. A vector is a type of plasmid, a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA usually occurring naturally in bacteria, but is sometimes also found in eukaryotic organisms. 15 Restriction enzymes: enzymes that cut DNA at specific recognition nucleotide known as restriction sites. 16 Transformation: bacterial transformation may be referred to as a stable genetic change due to the uptake of naked DNA (DNA without associated cells or proteins). Transformation occurs naturally in some species of bacteria, but it can also be performed by artificial means in other cells. 17 Competent bacteria: refers to the state of being able to take up exogenous DNA from the environment. 18 BLAST: Basic Local Alignment Search Tool, an algorithm used in bioinformatics. 19 Luciferase: class of oxidative enzymes used in bioluminescence. 20 Transfection: the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells. 21 Renilla luciferase control reporter vectors constitutively express Renilla luciferase, cloned from the anthozoan coelenterate Renilla reniformis (sea pansy), and thus provide an internal control value to which expression of the experimental firefly reporter gene may be normalized. 29 C. Skogastierna 9. Luciferase activity was measured by the Dual-Luciferase Reporter Assay System where the ratio between the PTGIS firefly luciferase signal and the control renilla luciferase signal for each statin exposed sample was compared to the ratios for each vehicle treated sample. 8. ”Experiment” 9. Measure Luciferase activity Figure 10: Schematic principle of the transfection procedure. See text for details. 30 4.3.3 Enzyme Immunoassay Enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) is a powerful type analytic biochemistry technique used for detecting and quantifying the presence of an antigen in tissue homogenates, plasma or other body fluids [156, 157]. There are many variants including indirect, sandwich, reverse and competitive EIA. The work in Papers I, II and III was performed using the competitive EIA method. The principal protocol for competitive EIA (see Figure 11 and www.caymanchem.com) utilizes an immunoplate, pre-coated with a secondary antibody and the non-specific binding sites blocked. Sample containing an unknown amount of antigen is added to the wells of the immunoplate, together with tracer (molecules of the analyte each covalently attached to a molecule of acetylcholinesterase (AChE22 )) and antiserum (primary antibody with a Fc fragment to which the secondary antibody can bind, and a Fab fragment specific to the analyte). Any unbound reagents are washed away after incubation and the plate is developed with Ellman’s Reagent which contains the substrate of AChE. The product of this enzyme reaction is a distinct yellow color, which absorbs strongly at 412 nm. The intensity of the color determined spectrophotometrically, is proportional to the amount of analyte Tracer (labeled antigen) bound to the well, which is inversely proportional to the amount of free analyte (sample antigen) in the well during the incubation, i.e. in the sample added. In other words, the labeled antigen now competes for primary antibody binding sites with the sample antigen. The more antigen in the sample the less labeled antigen is retained in the well and the weaker the signal. Figure 11: Schematic principle of the EIA method. 22 AChE: a stable enzyme isolated from the electric eel, Electrophorus electricus, with a high turnover for the hydrolysis of acetylthiocholine. The reaction produces thiocholine, which after non-enzymatic convertion to 5-thio-2-nitrobenzoic acid has a strong absorbance at 412 nm. 31 C. Skogastierna Interpretation of the signal produced in an immunoassay requires reference to a calibrator that mimics the characteristics of the sample medium. For qualitative assays the calibrators consist of a negative sample with no analyte and a positive sample having the lowest concentration of the analyte that is considered detectable. For quantitative measures additional calibrators with known analyte concentrations are also included. Comparison of the assay response of a real sample to the assay responses produced by the calibrators makes it possible to interpret the signal strength in terms of the presence or concentration of analyte in the sample. The main limitation with EIA is that due to the very small volume, even a small deviation of each reagent can have a compounded effect. Therefore, samples should ideally be run in triplicates. Other parameters should also be taken into account when using EIA; accuracy of the measurement, detection limit and detectability range of the EIA and the specificity of the antibodies [158]. Accuracy is especially crucial when determining concentration of the samples. In the studies included in this thesis we therefore chose to use relative concentrations, i.e. changes in detection between untreated controls and the samples. 4.3.3.1 Enzyme Immunoassay - PGI2 PGI2 is very short-lived. In the body it is quickly non-enzymatically hydrated to 6-keto PGF1α and then converted to the major metabolite, 2,3-dinor-6-keto PGF1α (t1/2 = 30 minutes) [159-161], which is why we measured the contents of both metabolites in the urine. Although 6-keto PGF1α is commonly measured in plasma and urine as an estimate of prostacyclin synthesis, it should be noted that there may be more than one source of PGI2 in these samples [162]. Urinary concentrations of 6-keto PGF1α are confounded by the fact that some plasma prostacyclin (~14 %) is excreted into urine as 6-keto PGF1α and the remainder is of renal origin [162, 163]. These problems are circumvented by measuring urinary 2,3-dinor-6-keto PGF1α as an indicator of systemic PGI2 production. Moreover, in our studies we only use the relative changes and not absolute values when interpreting and presenting the data. The EIAs for these prostacyclin metabolites typically displays an IC50 (50 % B/B0) of about 40 pg/ml (6keto PGF1α) and 400 pg/ml (2,3-dinor-6-keto PGF1α), and a detection limit (80 % B/B0) of approximately 6 pg/ml (6-keto PGF1α) and 100 pg/ml (2,3-dinor-6-keto PGF1α). 4.3.3.2 EIA – Thromboxane A2 In the body, the produced thromboxane A2 is rapidly hydrolyzed non-enzymatically to form thromboxane B2 (TXB2). Most of the TXB2 measured in the plasma or urine of healthy individuals is however due to ex vivo platelet activation or intra-renal production [159]. Normal concentrations of circulating TXB2 are extremely low (1-2 pg/ml) and highly transient (t1/2 = 5-7 minutes), which is why we chose to assay a metabolite of TXB2, 11-dehydro TXB2, not formed by platelets or the kidney and with a longer circulating half-life of 45 minutes [164, 165]. In Paper II a competitive EIA assay was used for quantification of 11-dehydro TXB2 in urine. Its measurement in plasma or urine will give a time-integrated indication of TXA2 production and is 32 recommended to estimate TXA2 levels to circumvent measurement complications associated with TXB2. The EIA typically displays an IC50 (50 % B/B0) value of 120 pg/ml and a detection limit (80 % B/B0) of approximately 34 pg/ml. 4.3.4 Western Blotting The Western blot is an analytical technique used to detect specific proteins in a sample. The technique uses gel electrophoresis to separate native or denatured proteins by the size of the polypeptide or by the 3-D structure of the protein. Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples. The general protocol (Figure 12) starts with separation of the proteins and biotinylated molecular weight markers by gel electrophoresis, usually SDS-PAGE23. The proteins are then transferred to a sheet of nitrocellulose membrane, retaining the same pattern of separation they had on the gel. Figure 12: Schematic principle of the Western Blot procedure. See text for detailed description. 23 SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis separates proteins according to their electrophoretic mobility, which is a function of the length of a polypeptide chain and its charge and no other physical feature. SDS is an anionic detergent applied to protein sample to linearize proteins and to impart a negative charge to linearized proteins. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. 33 C. Skogastierna The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose and block unspecific binding sites. A primary antibody specific to the target protein is then added to the solution, let bind during incubation and unbound antibody is then washed away. We then used a chemiluminescent detection method based on a HRP-linked (horseradish peroxidase) secondary antibody, which forms a complex with the primary antibody. The location of the antibody complex is revealed by incubating it with specific substrate to generate a light signal and the image is analysed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density. HRP conjugates have a very high turnover rate, yielding good sensitivity with short reaction times. 4.3.5 Colorimetric assay – detection of NO production In biological fluids, NO undergoes a series of reactions with several molecules, the end-products being nitrite (NO2-) and nitrate (NO3-). The relative proportion of these two is variable and cannot be predicted with certainty, thus the best index of total NO production is the sum of NO2- and NO3-. The fluorometric assay used in Paper I is a convenient and accurate method for measurement of total NO2- and NO3- concentration by firstly converse all nitrate to nitrite utilizing nitrate reductase, followed by 2,3diaminonaphthalene (DAN) and NaOH. DAN is then converted to the fluorescent product, 1(H)-naphthotriazole. Measurement of this compound determines the NO2concentration [166]. 4.3.6 LC-MS/MS Liquid chromatography – mass spectrometry (LC-MS) is a technique in analytical chemistry that combines the physical separation capabilities of liquid chromatography (or HPLC24) with the mass analysis capabilities of mass spectrometry (MS25). LC-MS is a powerful technique used for many applications, having very high sensitivity and selectivity, and is generally used for the detection and potential identification of compounds in a complex mixture in the presence of other chemicals. The major advantage MS has is the use of tandem MS-MS, also known as MS/MS or MS2, where the detector may be programmed to select certain ions to fragment and further separate [167]. Sample concentrations of simvastatin, simvastatin-acid (SIM-OH) and fluvastatin in serum from the healthy volunteers and from simvastatin or fluvastatin incubated HAEC in Paper III, were measured by a standard LC-MS/MS method previously developed for at our department. Briefly, sample preparation was based on pH-controlled solid 24 HPLC is a common chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture. 25 MS is a powerful technique where the sample molecules are ionized and the mass-to-charge ratio of charged particles or molecule fragments is measured. 34 phase extraction followed by evaporation under nitrogen and subsequent reconstitution. Subsequent analysis was performed on a reversed-phase column with a triple quadrupole mass spectrometer as detector and quantification was calculated on analyte/internal standard peak area ratios. The limit of detection was 0.02 ng/mL. 4.3.7 Myographic readings A myograph is any device used to measure the force produced by a muscle when under contraction and can be used to study the velocity and intensity of muscular contraction. In Paper I ex vivo experiments were conducted on vessels isolated from fat biopsies obtained from ten healthy normotensive pregnant women (two nulliparous26). Using a stereomicroscope, one or more subcutaneous arteries of similar size, internal diameter of 203±8 µm, were dissected and immediately placed in ice cold physiological salt solution. The arterial vessel rings were mounted on two parallel stainless steel wires, 25 m in diameter, in organ baths of a four-channel wire myograph filled with KrebsRinger physiological salt solution continuously gassed with 95 % O2 / 5 % CO2 at a temperature of 37 °C. One pin was connected to a force transducer and the other to a micrometer screw for length adjustment. The vessels were preconstricted with norepinephrine, NE, a catecholamine with multiple roles but here acting through α-adrenergic receptor activation of the vascular smoth muscle cells. The concentration added was sufficient to evoke a sustained, steady contraction before adding cumulatively concentrations of fluvastatin (10-7 - 10-4 mol/L, log unit steps). Failure to respond to NE in the freshly isolated vessels excluded the preparations from further experimentation. Concentration-response curves were in this way obtained prior to and after incubation with either N-nitro-L-arginine methyl ester (L-NAME) or indomethacin. L-NAME is an inhibitor of the enzyme endothelial NO synthase, eNOS, and thus reduces the cellular formation of cGMP. Indomethacin is a non-steroidal anti-inflammatory drug, nonselectively inhibiting COX-1 and COX-2, which are enzymes responsible for formation of the prostacyclin precursor PGH2 in endothelial cells. In order to figure out the relative contribution of eNOS and COX products to the fluvastatin-induced relaxation, their respective inhibitors were added alone or in combination. The force (mN) developed per length (mm) of artery segment during application of each concentration of vasoactive compound was calculated. All absolute measurements were corrected for the baseline force developed by the arteries. The relaxation to fluvastatin was calculated as percentage of reduction of the active force at the stable plateau level. The contractions and concentration-response curves were then compared before and after incubation with the two different pharmacological inhibitors. 26 Nulliparous refers to a woman who has never completed a pregnancy beyond 20 weeks and never given birth. 35 C. Skogastierna 5 RESULTS AND DISCUSSION 5.1 GENERAL COMMENTS Notably, many previous studies in vitro claiming mechanistic insights behind pleiotropic effects of statins have used concentrations 1000-fold higher than those detected in human plasma [168]. In the studies of this thesis much lower, clinically relevant concentrations of simvastatin and fluvastatin, were used in our in vitro experiments. Also, the intracellular concentrations, analyzed by means of LC-MS/MS, of statins in our cultivated cells were in the same range as the plasma concentrations of statins of our human subjects (reported in Paper III). Further, when testing cell viability for concentrations higher than used in this work, 10 µM, the cells showed signs of decreased membrane integrity by retaining dye in the viability test, which was another reason for us to excluded concentrations above 1.0 µM. Another intention with this work was to use time points that are clinically relevant. Since statins are usually therapeutically administrated every 24 h, we chose this as time point for our studies. One can argue that when doing transcriptional studies, the information on the time profile is of interest and by only testing for 24 h we may miss the acute effects on the transcription. But since we here not only assess the genetic effects but also the phenotype consequences (i.e. effect on prostacylin release) we decided to only include 24 h. I have also received the question why we have not extended and/or verified the effects of the statins on PGI2 generation by ECs using different models, i.e. laminar shear stress that would mimic the flow of blood in the vessels and/or in response to an inflammatory stimulator like IL1-β. I cannot but agree, these two situations would be very valuable to add to the studies. The models are commonly used to assess the protective effects of compounds and to stimulate an atherosclerotic (inflammatory) environment. But the ultimate model is of course to study humans, and since we confirmed our findings in healthy volunteers, we considered these extra in vitro models not to be required in this work. I pass this task on to my successor PhD student, and the exciting job to study these effects in statin patients. Regarding the choice of different doses of simvastatin (80 mg) and fluvastatin (40 mg) in the in vivo study, the rationale behind this was that we aimed to study both a commonly given maximum dose of statin and a commonly given medium dose of statin. 80 mg fluvastatin is approved by the Swedish Medical Products Agency (Läkemedelsverket) to be prescribed but is extremely rare. 36 5.2 PAPER I: BENEFICIAL VASOACTIVE ENDOTHELIAL EFFECTS OF FLUVASTATIN: FOCUS ON PROSTACYCLIN AND NITRIC OXIDE We show in Study I that fluvastatin is a modulator of vascular tone in human small arteries, most likely via acute and possibly also prolonged increased formation and release of endothelium-derived relaxing factors such as NO and PGI2, since inhibitors of both eNOS and COX significantly decreased relaxation in response to fluvastatin. A robust concentration dependent relaxation was produced with fluvastatin, reaching a maximum within three minutes. Fluvastatin-induced relaxation was reproducible and did not deteriorate after repeated application. Moreover, we showed that fluvastatin induces significant transcriptional and translational up-regulation of PGIS and eNOS, with increased cellular production of PGI2 and NO in EC cultures, suggesting that this compound may have beneficial effects on endothelial maintenance in vivo. Relaxation due to fluvastatin administration in the resistance vasculature may associate with reduced peripheral resistance and increased blood flow to target organs. The tone of resistance arteries is considered to be critical for blood pressure control. To our knowledge this is a first study that reports an acute relaxant effect of a statin – fluvastatin - in isolated small human arteries. Interestingly, preliminary data using the same technique, protocol and same type of material as in Paper I, but incubating with atorvastatin, show no effect on vascular reactivity. As discussed in the methodological considerations section, endothelial cells vary in morphology and functions according to the type and size of the associated vessel. The works presented in this thesis therefore involve endothelial cells from two different locations in the body; aorta (HAEC) and umbilical cord (HUVEC), both cell types being commonly used for studies of the vascular endothelium. It is important to note that although HUVECs have venous features, the umbilical cord vein has the physiological role of conveying oxygenated, nutrient-rich “arterial blood” from the placenta to the foetus. In fact, umbilical cord veins have long been used in clinical arterial transplantation [169]. Regarding patient group for the ex vivo experiments, one might question the rationale for choosing pregnant women and how to approach effects of pregnancy sex hormones - would it not be more accurate to use samples from men having abdominal surgery? Indeed, other patients groups are of interest, however at the time for Study I it was not possible to get such biopsies from e.g. males with normal or altered lipid profile. Furthermore, normal pregnancy is associated with significant changes in serum lipids (a shift towards small, dense LDL subfractions, raised triacylglycerols and cholesterol) and by the third trimester most women have a lipid profile which would be considered highly atherogenic in the non-pregnant state. This undoubtedly represents a transient disturbance which reverts to normal after delivery, and is related to the maintenance of nutrient fuel to the mother and fetus. Therefore, it might be anticipated that vessels from normal pregnant women might represent a good model to test the direct effects of fluvastatin on vascular function. It is well known that sex hormones can affect vascular reactivity, but since we have worked with isolated arteries in conditions when tissue was kept out from organism's environment, 37 C. Skogastierna ex vivo, for several hours the acute modifying effects of hormones can be neglected. Also, in Study I, the aim was to show the primary vascular effect of fluvastatin with subsequent clarification of the contribution of endothelium-derived factors to fluvastatin-induced responses of resistance arteries. With such an aim in mind we withdrew our attention from possible modifying effects of pregnancy hormones. 5.3 PAPER II: INFLUENCE OF SIMVASTATIN ON THE THROMBOXANE AND PROSTACYCLIN PATHWAYS, IN VITRO AND IN VIVO In contrast to Paper I which concerns fluvastatin, the HMG-CoA reductase inhibitor simvastatin, studied in Paper II, shows a potentially unfavorable alteration of the vascular endothelium both in vivo and in vitro. After treating endothelial cells at clinically relevant concentrations of simvastatin, the cells were shown to decrease their production and release of PGI2. Addition of mevalonic acid lactone, a precursor of cholesterol, reverted the effects of simvastatin on PGI2 release in ECs, indicating that this effect is mevalonate dependent. Also, simvastatin administrated as a single dose to healthy volunteers was shown to significantly increase urinary thromboxane A2 (TXA2) levels, which may result in a shift of the balance between PGI2 and TXA2, favoring the thromboxane pathway. Uncoupling of this balance could have pathophysiological implications by promoting a prothrombotic state in the blood vessels as discussed in Paper II. These findings add novel insight in the field of pleiotropic effects of statins and provide some insight for functional differences among statins. As a drug class, statins are considered by clinicians to be useful to their patients but these data may question the effects of statins on health and raise the question of thorough comparative studies on secondary vascular effects of statins. One of the aims with Paper II was to compare the effect on PGI2 metabolism after exposure to fluvastatin, studied in Paper I, with exposure to the in Europe more commonly prescribed simvastatin. The majority of the in vitro experiments in these two papers are directly comparable with each other. This is for example why we in Paper II chose to include the PTGIS promoter activity studies performed in HUVEC only, although the transcriptional effect on PTGIS seemed more pronounced in HAEC. Unfortunately, the transfection method was for unknown reasons not successful in the HAEC. Being primary cells make them more similar to the in vivo situation, but it also renders them more sensitive to chemical modifications than cell lines. On the subject of presenting and discussing the data from the human subjects, the TXA2/PGI2 biosynthesis ratio is commonly used in the literature to visualize balance changes of these two important metabolites. An abnormal TXA2/PGI2 ratio may predispose affected patients to for example thrombosis. Also, the TXA2/PGI2 balance is particularly critical in the regulation of maternal and fetal vascular function during pregnancy and in the newborn. An increase in the ratio in the maternal, fetal and neonatal circulation may contribute to preeclampsia, intrauterine growth restriction and 38 persistent pulmonary hypertension of the newborn, respectively [170]. On the other hand, increased PGI2 activity may contribute to patent ductus arteriosus27 and intraventricular hemorrhage in premature newborns [171]. Recent studies have shown that urinary metabolites of TXA2 and PGI2, constitute unique markers of the activation and/or interaction between platelets and vascular cells. Such an approach reflects the in vivo production of these mediators but has the advantage of circumventing the artifactual production of eicosanoids occurring during blood or tissue sampling. There are other arachidonic acid metabolites of interest that are active and affected in the vasculature, but these are two of the most important, being abundant, potent and having contrasting effects. Also worth highlighting is the seeming dependence on the mevalonate - HMG-CoA reductase pathway for the actions of simvastatin reported in study II, since addition of mevalonate abolished the reduction of PGI2 synthesis in HAECs. Mounting experimental evidence suggests that inhibition of the isoprenoids by statins might explain the clinical observations that statins improve cardiovascular outcomes, even in subjects with atherosclerosis and normal cholesterol levels [172]. However, these basic observations have not been fully translated into humans and may be challenged by our findings of a simvastatin mediated perturbation of the balance between the vasoregulatory TXA2 and PGI2 pathways with a potentially negative effect on the vascular homeostasis. It should also be noted that statins usually in the clinical situation are used in combination with low doses of acetylsalicylic acid, which could influence the levels of TXA2, as described in section 2.4.3.3, and perhaps the weight of our findings. 5.4 PAPER III: ANTI-OXIDATIVE EFFECTS OF STATINS ON VASCULAR ENDOTHELIUM Although statins in the last years have been acknowledged for their range of often beneficial pleiotropic effects, particularly relevant to cardiovascular disease including improved endothelial function and reduced oxidative stress, very sparse data on how statins affect the anti-oxidative status of the vascular endothelium are to be found. Our aim with Paper III is thus to provide additional information and hopefully to shed some light on the complex processes of cellular oxidative stress. The same cellular model systems as in Paper I and II was used, cells were exposed in the same way to either fluvastatin or simvastatin, and the expression pattern of central oxidative stress defence enzyme genes (SOD-1, TXN, TrxR1, GPx4 and Cat) and the generation of ROS in human vascular ECs was assessed. However, in order to estimate a time for maximum ROS generation, which does not necessarily parallel the expression of the enzymes included in the study, three different time points were studied. Moreover, the effect on oxidative capacity in vivo was analysed in urine after statin administration, a single dose of simvastatin (80mg) or fluvastatin (40mg), to healthy volunteers. 27 Ductus arteriosus ia a congenital disorder in the heart wherein a neonate's ductus arteriosus, a blood vessel connecting the pulmonary artery to the aortic arch, fails to close after birth. 39 C. Skogastierna The results from Study III indicate that the effects of statins may also here be compound specific and could have major impact on the vascular endothelial oxidative stress status. Analogous to Study I and II, the two statins were shown to have different impact on the mRNA expression of the investigated genes. Simvastatin significantly down-regulated the expression of all genes tested, whereas for fluvastatin no clear trend was observed. Also the levels of intracellular reactive oxygen species activity in vitro decreased after exposure to fluvastatin but not to the same extent for simvastatin. A small but significant increase in anti-oxidative capacity was observed in urine after fluvastatin but not simvastatin administration compared to before statin intake, which is in accordance with our in vitro results, i.e. that FLU seems to have a less undesirable effect on the expression on the anti-oxidative enzymes. As discussed more thoroughly in Paper III, the antioxidative capacity of fluvastatin has been reported by others in other model systems, such as human aortic smooth muscle cells [173] and on the plasma lipids of hyperlipidemic subjects [174]. This is proposed to be a consequence of its unique structure compared to other statins, giving it a free radical-scavenging activity against the superoxide anion and hydroxyl radical [175, 176] that is independent of its HMG-CoA reductase inhibition capacity [177]. 5.5 PAPER IV: STATINS INHIBIT EXPRESSION OF THIOREDOXIN REDUCTASE 1 IN RAT AND HUMAN LIVER AND REDUCE TUMOUR DEVELOPMENT In Paper III we concluded that statins have an impact on the expression of the TrxR1 enzyme. Since this enzyme has been suggested to be a significant player in carcinogenesis, we set out to expand our studies of this enzyme in relation to cancer and statins, Paper IV. We show in this article that statin treatment decreases the hepatic expression of TrxR1 in humans and rats. Further, a clear correlation between inhibition of carcinogenesis and decreased TrxR1-mRNA levels in a rat model for liver cancer was observed, making us postulate that a possible causal relationship between statin-induced decrease in TrxR1expression and inhibition of carcinogenesis seems to exist. Since the statins display a clear inhibition of TrxR1 independent of carcinogenesis in our human material, we conclude that the reduced TrxR activity is caused directly by the statins and not to a statin-induced reduction of nodule size. These findings add insight to the field of possible anti-carcinogenic effects of statins, as well as other side effects of statins. I also hope that these findings may invite larger studies involving human subjects to further clarify the relationship between statins, TrxR1 and carcinogenesis in man. 40 5.6 UNDISCLOSED AND UNPUBLISHED RESULTS An initial aim with the thesis was to include all four of the most commonly prescribed statins around the world, something I could not fully proceed with due to unforeseen circumstances and the nature of science. This however means that there are some results that are not finalized for publication but that I still mean to share. 5.6.1 Atorvastatin and rosuvastatin vs. expression of COX, PGIS and eNOS Similar effects on transcription and translation of PGIS were observed after exposure to the other two statins, i.e. both atorvastatin and rosuvastatin increase the mRNA expression of PGIS in HUVEC (Figure 13A). The mRNA expression however, did not correlate with the PGI2 production (Figure 13B), probably due to inhibition of COX activity. When assaying the activity of the COX enzymes in EC cultures, we found that simvastatin, atorvastatin and rosuvastatin all interfered with the enzymes, inhibiting approximately 90 % of the total COX activity. Possibly this could decrease the production of PGH2 and depleting the cells of the substrate for PGIS, resulting in a decrease in prostacyclin production. This might partly explain why the increase in mRNA and protein does not correlate with product release. A 41 C. Skogastierna B Relative change in 6KPG1Fα 2,50 2,00 1,50 ATV - MEV ATV + MEV 1,00 ROS - MEV ROS + MEV 0,50 0,00 0,0 0.01 (µM) 1.0 Figure 13: Additional HUVEC and HAEC analyses. A) mRNA expression in HUVEC exposed to either rosuvastatin for 24 hours or 48 hours (ROS 24 and ROS 48, respectively) or atorvastatin for 24 hours or 48 hours (ATV 24 and ATV 48, respectively). B) 6-keto-PG1Fα (6KPG1Fα) release in HAEC cell medium after treatment with rosuvastatin (ROS) or atorvastatin (ATV), alone (-MEV) or with mevalonate 100 µM (+MEV). 5.6.2 Atorvastatin and rosuvastatin vs. PTGIS promoter activity Apart from fluvastatin and simvastatin, we have also looked at the influence on PTGIS promoter activity in PGIS core promoter transfected HUVECs exposed to 0.1 µM atorvastatin, rosuvastatin or corresponding vehicle for 24 hours, using the same cloning and transfection procedure as in Papers I and II. Atorvastatin exposed cells displayed a significant twofold increase in promoter activity (P = 0.001), while exposure to rosuvastatin indicated a slightly increased promoter activity although not significant (Figure 14). 42 control activity, luciferase of control fold of activity,fold Luciferase 4 3 2 1 0 ATV ROS SIM FLU M annactivity Whi in tney test Figure 14: PTGIS promoter PGIS core promoter transfected HUVECs exposed to 0.1 P val ue 0.0011 µM atorvastatin (ATV), rosuvastatin (ROS) or corresponding vehicle for 24 hours. The bars Exact or approxi m ate P val ue? Gaussi an Approxi m a represent mean ± S.E.M., N=15. The luciferase expression was normalised to the transfection efficiency P valexpression ue sum m ary by the control renilla luciferase in each sample and normalised against the** mean of respective control, set as 1. P val ue 0.3183 Exact or approxi m ate P val ue? P val ue sum m ary Gaussi an Approxi m a ns P val ue 0.0304 Atorvastatin and rosuvastatin vs. anti-oxidative stress enzyme Exact or approxi m ate P val ue? Gaussi an Approxi m a genes val ue sum m ary In addition to studyPhow fluvastatin and simvastatin affects the mRNA* expression of P val in ue 0.0034 several enzymes involved the defence against oxidative stress (Paper III), the HAEC Exact or approxi m ate P val ue? Gaussi cells were also exposed to atorvastatin and rosuvastatin for 24 hours (figure 14). an In Approxi m a P val ue sum m ary ** opposite to the other statins, rosuvastatin exert a significant increase in GPx4. In agreement with fluvastatin and simvastatin, atorvastatin and rosuvastatin also inhibits mRNA expression of catalase. Neither rosuvastatin or atorvastatin inhibits the mRNA expression of TXN in HAEC. In fact simvastatin appears to be the only statin that significantly inhibit the TXN mRNA level in HAEC. This is in agreement with our now ongoing studies that are being performed in human hepatoma (HepG2) cells to further assess the molecular mechanisms behind the statin induced inhibition on TrxR1 transcription. Preliminary results in HepG2 cells indicate that the statins inhibit TrxR1 to different extent, and that simvastatin exert highest inhibitory effects. The in vitro findings in HAEC and HepG2 cells are not agreement with our in vivo results in Paper IV, i.e. that atorvastatin inhibits the hepatic expression of TrxR1 in patients administrated with atorvastatin for four weeks. The reason for this discrepancy may be explained by the fact that the human subjects had been taking statins for a long period time, whereas in the in vitro experiments, the cells have been exposed to a single dose of statins. 5.6.3 43 C. Skogastierna Figure 15: mRNA expression of oxidative stress enzyme genes in HAEC cells exposed to increasing concentration (0.01-1 uM) of rosuvastatin (ROS) and atorvastatin (ATV) for 24 hours. 44 6 CONCLUSIONS The main results obtained from these studies can be summarized as follows: Fluvastatin is proposed a protective role of on the cardiovascular system, particularly at the level of vascular endothelium since it up-regulates transcription and translation of PGIS and eNOS in human vascular endothelial cells, associated with an increase in cellular production of PGI2 and NO, and induces rapid dilatation in isolated human arteries via contribution of endothelium derived factors like NO and PGI2. Simvastatin induces a potentially unfavorable alteration of the vascular endothelial cells both in vivo and in vitro. Exposure to clinically comparable concentrations of simvastatin decreased the production and release of PGI2 in cultivated endothelial cells. Simvastatin was further shown to significantly increase urinary TXA2 levels in vivo, which may result in a negative alteration of the balance between PGI2 and TXA2. Fluvastatin and simvastatin have different impact on vascular endothelial oxidative stress status. Compared to simvastatin, fluvastatin seems beneficial both regarding expression of anti-oxidative stress enzymes and reactive oxygen species activity in vitro and anti-oxidative capacity in vivo. Statin treatment decreases the hepatic expression of TrxR1 in humans and rats which is relevant for suggested anti-carcinogenic effects of statins. Overall, our results add to the conjecture that the impact on production of vascular active substances may be significantly different among the statins, and thus the consequences of statin exposure may not be drug class related but rather compound specific, highlighting the importance of more comparative studies where several statins are included. 45 C. Skogastierna 7 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to the following people, without whom I could not have completed my PhD: Lena Ekström, my main supervisor. We have had some fun during this time, haven´t we? Our Japan trip still makes me laugh! Thank you for accepting me, first for a master thesis project, giving me the first real glimpses of science, and later as a PhD student. Under your supervision I have learned countless valuable things about research and molecular biology. From the bottom of my heart, thank you for always having time for me, always being supportive and for being that creative “doer”. My co-supervisors Anders Rane, Linda Bergman-Björkhem and Erik Eliasson, thank you for being great advisors, for sharing your experiences and knowledge along the way, and being supportive. Anders, for you extensive knowledge in clinical pharmacology, your excellent inputs and good advice. I highly appreciate that you have always taken time for detailed proof-reading of my material. Linda, you have truly been that spark of energy that made this project take its final form! I am truly grateful that you came into the picture and agreed to be a part of this work. Thank you for always being positive and helpful, and being incredible fast with feedback. Erik, for you good advice and for being the “sensible one”, always thinking two rounds and coming with invaluable thoughts and comments. Janeric Seidegård, for being the helpful and supportive connection to the industry. Thank you for the fun meetings and chats. I hope our collaboration will not stop here! My gratitude to all co-authors for our successful collaboration. Margit Ekström, I would not have been able to finish this work without you, you are truly the goddess of paperwork! Thank you for always taking time when I have been in need and for all the fun conversations and stories. Clin Pharm has not been the same since you left. Birgitta Ask, what would I have done without you? Thank you for your patience with me in the lab and for sharing your valuable experience. My roomies, (present) Emmanuel Strahm, Jenny Schulze, Maria Johansson, Eleni Aklillu; (past) Roza Ghotbi, Sara Karlsson, Annika Alqvist and Maja Hotzen, for all the non-scientific discussions, endless coffees, dinners, drinks and fun! Yassin Ahmed and Jonas Lundmark, I count you into this category as well. Thanks for being great friends and for being a blast! Other past and present members of “plan 8”, the power ladies Lilleba Boman, Jolanta Wiren and Margarita Mahindi, for being sweet and helpful. 46 To all you on “plan 6”, esprecially Tobias Bäckström (thanks for great music tips and training talk, keep it up!), Jonatan Lindh, Marine Andersson, Annika Asplund, Fadiea Al-Aieshy, Staffan Olsson, Nina Gårevik, Camilla Linder and Karin Nilsson, for nice company during and off working hours. Lars L Gustavsson, for your enthusiasm. It was fun and a good experience to start up and run the journal club with you. I hope it will survive for many years! My friends at Fysiologen, especially Thomas Gustavsson and Anna Strömberg, for all the nice chats and laughs. Good luck with research and life my future colleauges! My wonderful friends in science and in life, Karin Larsson, love you to pieces! I will always and forever be grateful that “forskarskolan” brought us together Can´t believe that we made it! Carolina Gustavsson, Britta Stensson and Kylie Foo, for the awesome time and all the adventures in Tokyo! Carolina, my ENDOMET board buddy, we were great representatives, weren´t we? You made some of my travels, both scientific and non-scientific, really into something extra! Kylie, I will see you in NY! Soon! I wish you all the best and hope our paths will keep on crossing in life and in research. My dear and lovely friends outside KI, Jessie Dahlström, Louise Brattström-Stolt, Linda Kylberg, Frida Olofsson, Stina Erhardsson, Martin Krook, you have all kept me updated with the real world, without you life would indeed be a much duller place! You are the best! Jessie, for being a great friend from day one at Astra Zeneca! I really look forward to all the adventures still to experience with you. Louise,for your warmth, all “fikas” and for encouraging me to apply for Med School. Linda, the #1 bundle of energy! Thanks for introducing me to the world of house music and real clubbing! Frida, this journey really started with you, in Lund 2004. It would definitely not have been the same without you! You are truly the strongest person I know. Stina, for always being there with smiles and laughter. You brought lovely knasighet in my life for real! Martin, you have made my time in Umeå. The best in so many ways! “Inga begränsningar, tid och pengar är allt vi har!” Magnus Hellsten, for always being there. You and your family mean a lot to me. To my big and warm family, Sven-Bertil Skogastierna,Tina, Pontus, Diana, Rickard; Janet Andersson, Lennart, Filip, Hanna. Although my research has not always been very clear to you, you have always been supportive and cheered me on. You are the best! 47 C. Skogastierna 8 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 48 Virchow R (1956) Standpoints in Scientific Medicine, 1877. Bull Hist Med 30:537-43 Tobert JA (2003) Lovastatin and beyond: the history of the HMG-CoA reductase inhibitors. Nat Rev Drug Discov 2:517-26 Duff GL and Mc MG (1951) Pathology of atherosclerosis. Am J Med 11:92108 Endo A (1992) The discovery and development of HMG-CoA reductase inhibitors. J Lipid Res 33:1569-82 Endo A, Kuroda M and Tsujita Y (1976) ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinium. J Antibiot (Tokyo) 29:1346-8 Tonelli M, Lloyd A, Clement F, Conly J, Husereau D, Hemmelgarn B, Klarenbach S, McAlister FA, Wiebe N and Manns B (2011) Efficacy of statins for primary prevention in people at low cardiovascular risk: a meta-analysis. CMAJ 183:E1189-202 Yeagle PL (1991) Modulation of membrane function by cholesterol. Biochimie 73:1303-10 Haines TH (2001) Do sterols reduce proton and sodium leaks through lipid bilayers? Prog Lipid Res 40:299-324 Incardona JP and Eaton S (2000) Cholesterol in signal transduction. Curr Opin Cell Biol 12:193-203 Intake of Calories and Selected Nutrients for the United States Population, 1999-2000, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Centers for Disease Control and Prevention National Center for Health Statistics. Liscum L, Finer-Moore J, Stroud RM, Luskey KL, Brown MS and Goldstein JL (1985) Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum. J Biol Chem 260:52230 Hua X, Nohturfft A, Goldstein JL and Brown MS (1996) Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein. Cell 87:415-26 Yabe D, Brown MS and Goldstein JL (2002) Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins. Proc Natl Acad Sci U S A 99:12753-8 Yang T, Espenshade PJ, Wright ME, Yabe D, Gong Y, Aebersold R, Goldstein JL and Brown MS (2002) Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell 110:489-500 Goldstein JL and Brown MS (1990) Regulation of the mevalonate pathway. Nature 343:425-30 Wang X, Sato R, Brown MS, Hua X and Goldstein JL (1994) SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell 77:53-62 Gasic GP (1994) Basic-helix-loop-helix transcription factor and sterol sensor in a single membrane-bound molecule. Cell 77:17-9 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. Espenshade PJ and Hughes AL (2007) Regulation of sterol synthesis in eukaryotes. Annu Rev Genet 41:401-27 Vita JA, Yeung AC, Winniford M, Hodgson JM, Treasure CB, Klein JL, Werns S, Kern M, Plotkin D, Shih WJ, Mitchel Y and Ganz P (2000) Effect of cholesterol-lowering therapy on coronary endothelial vasomotor function in patients with coronary artery disease. Circulation 102:846-51 Treasure CB, Klein JL, Weintraub WS, Talley JD, Stillabower ME, Kosinski AS, Zhang J, Boccuzzi SJ, Cedarholm JC and Alexander RW (1995) Beneficial effects of cholesterol-lowering therapy on the coronary endothelium in patients with coronary artery disease. N Engl J Med 332:481-7 Santos AC and Lehmann R (2004) Germ cell specification and migration in Drosophila and beyond. Curr Biol 14:R578-89 Istvan ES and Deisenhofer J (2001) Structural mechanism for statin inhibition of HMG-CoA reductase. Science 292:1160-4 Hamelin BA and Turgeon J (1998) Hydrophilicity/lipophilicity: relevance for the pharmacology and clinical effects of HMG-CoA reductase inhibitors. Trends Pharmacol Sci 19:26-37 White CM (2002) A review of the pharmacologic and pharmacokinetic aspects of rosuvastatin. J Clin Pharmacol 42:963-70 McTaggart F, Buckett L, Davidson R, Holdgate G, McCormick A, Schneck D, Smith G and Warwick M (2001) Preclinical and clinical pharmacology of Rosuvastatin, a new 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. Am J Cardiol 87:28B-32B Holdgate GA, Ward WH and McTaggart F (2003) Molecular mechanism for inhibition of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase by rosuvastatin. Biochem Soc Trans 31:528-31 Igel M, Sudhop T and von Bergmann K (2002) Pharmacology of 3-hydroxy-3methylglutaryl-coenzyme A reductase inhibitors (statins), including rosuvastatin and pitavastatin. J Clin Pharmacol 42:835-45 Vaughan CJ and Gotto AM, Jr. (2004) Update on statins: 2003. Circulation 110:886-92 Corsini A, Bellosta S, Baetta R, Fumagalli R, Paoletti R and Bernini F (1999) New insights into the pharmacodynamic and pharmacokinetic properties of statins. Pharmacol Ther 84:413-28 Malmstrom RE, Ostergren J, Jorgensen L and Hjemdahl P (2009) Influence of statin treatment on platelet inhibition by clopidogrel - a randomized comparison of rosuvastatin, atorvastatin and simvastatin co-treatment. J Intern Med 266:457-66 McTaggart F (2003) Comparative pharmacology of rosuvastatin. Atheroscler Suppl 4:9-14 Niemi M (2010) Transporter pharmacogenetics and statin toxicity. Clin Pharmacol Ther 87:130-3 Link E, Parish S, Armitage J, Bowman L, Heath S, Matsuda F, Gut I, Lathrop M and Collins R (2008) SLCO1B1 variants and statin-induced myopathy--a genomewide study. N Engl J Med 359:789-99 Robey RW, To KK, Polgar O, Dohse M, Fetsch P, Dean M and Bates SE (2009) ABCG2: a perspective. Adv Drug Deliv Rev 61:3-13 Lee E, Ryan S, Birmingham B, Zalikowski J, March R, Ambrose H, Moore R, Lee C, Chen Y and Schneck D (2005) Rosuvastatin pharmacokinetics and pharmacogenetics in white and Asian subjects residing in the same environment. Clin Pharmacol Ther 78:330-41 49 C. Skogastierna 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 50 Ieiri I, Suwannakul S, Maeda K, Uchimaru H, Hashimoto K, Kimura M, Fujino H, Hirano M, Kusuhara H, Irie S, Higuchi S and Sugiyama Y (2007) SLCO1B1 (OATP1B1, an uptake transporter) and ABCG2 (BCRP, an efflux transporter) variant alleles and pharmacokinetics of pitavastatin in healthy volunteers. Clin Pharmacol Ther 82:541-7 Keskitalo JE, Pasanen MK, Neuvonen PJ and Niemi M (2009) Different effects of the ABCG2 c.421C>A SNP on the pharmacokinetics of fluvastatin, pravastatin and simvastatin. Pharmacogenomics 10:1617-24 (1994) Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 344:1383-9 Baigent C, Keech A, Kearney PM, Blackwell L, Buck G, Pollicino C, Kirby A, Sourjina T, Peto R, Collins R and Simes R (2005) Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins. Lancet 366:1267-78 Pfefferkorn JA, Song Y, Sun KL, Miller SR, Trivedi BK, Choi C, Sorenson RJ, Bratton LD, Unangst PC, Larsen SD, Poel TJ, Cheng XM, Lee C, Erasga N, Auerbach B, Askew V, Dillon L, Hanselman JC, Lin Z, Lu G, Robertson A, Olsen K, Mertz T, Sekerke C, Pavlovsky A, Harris MS, Bainbridge G, Caspers N, Chen H and Eberstadt M (2007) Design and synthesis of hepatoselective, pyrrole-based HMG-CoA reductase inhibitors. Bioorg Med Chem Lett 17:4538-44 Baigent C, Blackwell L, Emberson J, Holland LE, Reith C, Bhala N, Peto R, Barnes EH, Keech A, Simes J and Collins R (2010) Efficacy and safety of more intensive lowering of LDL cholesterol: a meta-analysis of data from 170,000 participants in 26 randomised trials. Lancet 376:1670-81 Ray KK, Seshasai SR, Erqou S, Sever P, Jukema JW, Ford I and Sattar N (2010) Statins and all-cause mortality in high-risk primary prevention: a metaanalysis of 11 randomized controlled trials involving 65,229 participants. Arch Intern Med 170:1024-31 Mills EJ, O'Regan C, Eyawo O, Wu P, Mills F, Berwanger O and Briel M (2011) Intensive statin therapy compared with moderate dosing for prevention of cardiovascular events: a meta-analysis of >40 000 patients. Eur Heart J 32:1409-15 Taylor F, Ward K, Moore TH, Burke M, Davey Smith G, Casas JP and Ebrahim S (2011) Statins for the primary prevention of cardiovascular disease. Cochrane Database Syst Rev CD004816 Roberts WC (1997) The rule of 5 and the rule of 7 in lipid-lowering by statin drugs. Am J Cardiol 80:106-7 Abramson J and Wright JM (2007) Are lipid-lowering guidelines evidencebased? Lancet 369:168-9 Hippisley-Cox J and Coupland C (2010) Unintended effects of statins in men and women in England and Wales: population based cohort study using the QResearch database. BMJ 340:c2197 Ravnskov U, Rosch PJ, Sutter MC and Houston MC (2006) Should we lower cholesterol as much as possible? BMJ 332:1330-2 Baker SK and Tarnopolsky MA (2005) Statin-associated neuromyotoxicity. Drugs Today (Barc) 41:267-93 Pedersen TR, Faergeman O, Kastelein JJ, Olsson AG, Tikkanen MJ, Holme I, Larsen ML, Bendiksen FS, Lindahl C, Szarek M and Tsai J (2005) High-dose atorvastatin vs usual-dose simvastatin for secondary prevention after 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. myocardial infarction: the IDEAL study: a randomized controlled trial. JAMA 294:2437-45 Armitage J, Bowman L, Wallendszus K, Bulbulia R, Rahimi K, Haynes R, Parish S, Peto R and Collins R (2010) Intensive lowering of LDL cholesterol with 80 mg versus 20 mg simvastatin daily in 12,064 survivors of myocardial infarction: a double-blind randomised trial. Lancet 376:1658-69 Wettermark B and Hjemdahl P (2001) [Can we afford good cholesterol lowering therapy? Budgeting of statin costs versus medical needs in the county of Stockholm]. Lakartidningen 98:5472-3, 5476-8, 5481-3 Andrade SE, Walker AM, Gottlieb LK, Hollenberg NK, Testa MA, Saperia GM and Platt R (1995) Discontinuation of antihyperlipidemic drugs--do rates reported in clinical trials reflect rates in primary care settings? N Engl J Med 332:1125-31 Ando H, Tsuruoka S, Yamamoto H, Takamura T, Kaneko S and Fujimura A (2004) Effects of pravastatin on the expression of ATP-binding cassette transporter A1. J Pharmacol Exp Ther 311:420-5 Bergman U, Anderssen M, Vaccheri A, Larsen J and Montanaro N (1999) Marked differences in lipid-lowering drug use in Bologna, Italy and Funen, Denmark. Eur Heart J 20:1135 Larsen J, Vaccheri A, Andersen M, Montanaro N and Bergman U (2000) Lack of adherence to lipid-lowering drug treatment. A comparison of utilization patterns in defined populations in Funen, Denmark and Bologna, Italy. Br J Clin Pharmacol 49:463-71 Hayward RA, Krumholz HM, Zulman DM, Timbie JW and Vijan S (2010) Optimizing statin treatment for primary prevention of coronary artery disease. Ann Intern Med 152:69-77 Davignon J (2004) Beneficial cardiovascular pleiotropic effects of statins. Circulation 109:III39-43 Rang HP and Dale MM, Pharmacology. 2nd ed1991, Edinburgh ; New York: Churchill Livingstone. 955 p. Ross R (1995) Cell biology of atherosclerosis. Annu Rev Physiol 57:791-804 Kunsch C and Medford RM (1999) Oxidative stress as a regulator of gene expression in the vasculature. Circ Res 85:753-66 Gibbons GH and Dzau VJ (1996) Molecular therapies for vascular diseases. Science 272:689-93 Ross R (1999) Atherosclerosis--an inflammatory disease. N Engl J Med 340:115-26 Arnaud C, Veillard NR and Mach F (2005) Cholesterol-independent effects of statins in inflammation, immunomodulation and atherosclerosis. Curr Drug Targets Cardiovasc Haematol Disord 5:127-34 Lazar HL, Bao Y, Zhang Y and Bernard SA (2003) Pretreatment with statins enhances myocardial protection during coronary revascularization. J Thorac Cardiovasc Surg 125:1037-42 Wolfrum S, Jensen KS and Liao JK (2003) Endothelium-dependent effects of statins. Arterioscler Thromb Vasc Biol 23:729-36 Ebrahim S and Smith GD (2000) Statins and risk of coronary heart disease. Jama 283:2935-6 Harrison DG (1997) Endothelial function and oxidant stress. Clin Cardiol 20:II11-7 Libby P and Ridker PM (2004) Inflammation and atherosclerosis: role of Creactive protein in risk assessment. Am J Med 116 Suppl 6A:9S-16S 51 C. Skogastierna 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 52 Rosenson RS and Hurt-Camejo E (2012) Phospholipase A2 enzymes and the risk of atherosclerosis. Eur Heart J Albert CM, Ma J, Rifai N, Stampfer MJ and Ridker PM (2002) Prospective study of C-reactive protein, homocysteine, and plasma lipid levels as predictors of sudden cardiac death. Circulation 105:2595-9 Moreno PR, Falk E, Palacios IF, Newell JB, Fuster V and Fallon JT (1994) Macrophage infiltration in acute coronary syndromes. Implications for plaque rupture. Circulation 90:775-8 Verma S, Wang CH, Li SH, Dumont AS, Fedak PW, Badiwala MV, Dhillon B, Weisel RD, Li RK, Mickle DA and Stewart DJ (2002) A self-fulfilling prophecy: C-reactive protein attenuates nitric oxide production and inhibits angiogenesis. Circulation 106:913-9 Khanapure SP, Garvey DS, Janero DR and Letts LG (2007) Eicosanoids in inflammation: biosynthesis, pharmacology, and therapeutic frontiers. Curr Top Med Chem 7:311-40 Kleemann R, Bureeva S, Perlina A, Kaput J, Verschuren L, Wielinga PY, HurtCamejo E, Nikolsky Y, van Ommen B and Kooistra T (2011) A systems biology strategy for predicting similarities and differences of drug effects: evidence for drug-specific modulation of inflammation in atherosclerosis. BMC Syst Biol 5:125 Bustos C, Hernandez-Presa MA, Ortego M, Tunon J, Ortega L, Perez F, Diaz C, Hernandez G and Egido J (1998) HMG-CoA reductase inhibition by atorvastatin reduces neointimal inflammation in a rabbit model of atherosclerosis. J Am Coll Cardiol 32:2057-64 Aikawa M, Rabkin E, Sugiyama S, Voglic SJ, Fukumoto Y, Furukawa Y, Shiomi M, Schoen FJ and Libby P (2001) An HMG-CoA reductase inhibitor, cerivastatin, suppresses growth of macrophages expressing matrix metalloproteinases and tissue factor in vivo and in vitro. Circulation 103:276-83 Bellosta S, Via D, Canavesi M, Pfister P, Fumagalli R, Paoletti R and Bernini F (1998) HMG-CoA reductase inhibitors reduce MMP-9 secretion by macrophages. Arterioscler Thromb Vasc Biol 18:1671-8 Fukumoto Y, Libby P, Rabkin E, Hill CC, Enomoto M, Hirouchi Y, Shiomi M and Aikawa M (2001) Statins alter smooth muscle cell accumulation and collagen content in established atheroma of watanabe heritable hyperlipidemic rabbits. Circulation 103:993-9 Williams JK, Sukhova GK, Herrington DM and Libby P (1998) Pravastatin has cholesterol-lowering independent effects on the artery wall of atherosclerotic monkeys. J Am Coll Cardiol 31:684-91 Niwa S, Totsuka T and Hayashi S (1996) Inhibitory effect of fluvastatin, an HMG-CoA reductase inhibitor, on the expression of adhesion molecules on human monocyte cell line. Int J Immunopharmacol 18:669-75 Mehta JL, Rasouli N, Sinha AK and Molavi B (2006) Oxidative stress in diabetes: a mechanistic overview of its effects on atherogenesis and myocardial dysfunction. Int J Biochem Cell Biol 38:794-803 Dalton TP, Shertzer HG and Puga A (1999) Regulation of gene expression by reactive oxygen. Annu Rev Pharmacol Toxicol 39:67-101 Kamata H and Hirata H (1999) Redox regulation of cellular signalling. Cell Signal 11:1-14 Thomas EL, Lehrer RI and Rest RF (1988) Human neutrophil antimicrobial activity. Rev Infect Dis 10 Suppl 2:S450-6 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. Nordberg J and Arner ES (2001) Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic Biol Med 31:1287-312. Gromer S, Urig S and Becker K (2004) The thioredoxin system--from science to clinic. Med Res Rev 24:40-89 Yamawaki H, Haendeler J and Berk BC (2003) Thioredoxin: a key regulator of cardiovascular homeostasis. Circ Res 93:1029-33 May JM, Mendiratta S, Hill KE and Burk RF (1997) Reduction of dehydroascorbate to ascorbate by the selenoenzyme thioredoxin reductase. J Biol Chem 272:22607-10 Arner ES, Nordberg J and Holmgren A (1996) Efficient reduction of lipoamide and lipoic acid by mammalian thioredoxin reductase. Biochem Biophys Res Commun 225:268-74 Sun X, Dobra K, Bjornstedt M and Hjerpe A (2000) Upregulation of 9 genes, including that for thioredoxin, during epithelial differentiation of mesothelioma cells. Differentiation 66:181-8. Berggren M, Gallegos A, Gasdaska JR, Gasdaska PY, Warneke J and Powis G (1996) Thioredoxin and thioredoxin reductase gene expression in human tumors and cell lines, and the effects of serum stimulation and hypoxia. Anticancer Res 16:3459-66. Kumar S and Holmgren A (1999) Induction of thioredoxin, thioredoxin reductase and glutaredoxin activity in mouse skin by TPA, a calcium ionophore and other tumor promoters. Carcinogenesis 20:1761-7 Holmgren A (1985) Thioredoxin. Annu Rev Biochem 54:237-71 Holmgren A (1989) Thioredoxin and glutaredoxin systems. J Biol Chem 264:13963-6 Hirota K, Murata M, Sachi Y, Nakamura H, Takeuchi J, Mori K and Yodoi J (1999) Distinct roles of thioredoxin in the cytoplasm and in the nucleus. A twostep mechanism of redox regulation of transcription factor NF-kappaB. J Biol Chem 274:27891-7 Nakamura H, De Rosa S, Roederer M, Anderson MT, Dubs JG, Yodoi J, Holmgren A and Herzenberg LA (1996) Elevation of plasma thioredoxin levels in HIV-infected individuals. Int Immunol 8:603-11 Ericson ML, Horling J, Wendel-Hansen V, Holmgren A and Rosen A (1992) Secretion of thioredoxin after in vitro activation of human B cells. Lymphokine Cytokine Res 11:201-7 Rubartelli A, Bajetto A, Allavena G, Wollman E and Sitia R (1992) Secretion of thioredoxin by normal and neoplastic cells through a leaderless secretory pathway. J Biol Chem 267:24161-4 Nakamura H, Masutani H, Tagaya Y, Yamauchi A, Inamoto T, Nanbu Y, Fujii S, Ozawa K and Yodoi J (1992) Expression and growth-promoting effect of adult T-cell leukemia-derived factor. A human thioredoxin homologue in hepatocellular carcinoma. Cancer 69:2091-7 Bertini R, Howard OM, Dong HF, Oppenheim JJ, Bizzarri C, Sergi R, Caselli G, Pagliei S, Romines B, Wilshire JA, Mengozzi M, Nakamura H, Yodoi J, Pekkari K, Gurunath R, Holmgren A, Herzenberg LA and Ghezzi P (1999) Thioredoxin, a redox enzyme released in infection and inflammation, is a unique chemoattractant for neutrophils, monocytes, and T cells. J Exp Med 189:1783-9. Mustacich D and Powis G (2000) Thioredoxin reductase. Biochem J 346 Pt 1:18 53 C. Skogastierna 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 54 Selenius M, Rundlof AK, Olm E, Fernandes AP and Bjornstedt M (2010) Selenium and the selenoprotein thioredoxin reductase in the prevention, treatment and diagnostics of cancer. Antioxid Redox Signal 12:867-80 Williams CHJ, ed. Lipoamide dehydrogenase, glutathione reductase, thioredoxin reductase and mercuric ion reductase - A family of flavoenzyme transhydrogenase. Chemistry and Biochemistry of Flavoenzyme, ed. Müller F. Vol. 3, 121-211. 1992, CRC Press: Boca, Raton. Tamura T and Stadtman TC (1996) A new selenoprotein from human lung adenocarcinoma cells: purification, properties, and thioredoxin reductase activity. Proc Natl Acad Sci U S A 93:1006-11 Sandalova T, Zhong L, Lindqvist Y, Holmgren A and Schneider G (2001) Three-dimensional structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a selenocysteine-dependent enzyme. Proc Natl Acad Sci U S A 98:9533-8 Miranda-Vizuete A, Damdimopoulos AE, Pedrajas JR, Gustafsson JA and Spyrou G (1999) Human mitochondrial thioredoxin reductase cDNA cloning, expression and genomic organization. Eur J Biochem 261:405-12 Miranda-Vizuete A, Damdimopoulos AE and Spyrou G (1999) cDNA cloning, expression and chromosomal localization of the mouse mitochondrial thioredoxin reductase gene(1). Biochim Biophys Acta 1447:113-8 Soderberg A, Sahaf B and Rosen A (2000) Thioredoxin reductase, a redoxactive selenoprotein, is secreted by normal and neoplastic cells: presence in human plasma. Cancer Res 60:2281-9 Farber E (1984) Pre-cancerous steps in carcinogenesis. Their physiological adaptive nature. Biochim Biophys Acta 738:171-80 Farber E and Sarma DS (1987) Hepatocarcinogenesis: a dynamic cellular perspective. Lab Invest 56:4-22 Kinzler KW and Vogelstein B (1996) Lessons from hereditary colorectal cancer. Cell 87:159-70 Van Gijssel HE, Ohlson LC, Torndal UB, Mulder GJ, Eriksson LC, PorschHallstrom I and Meerman JH (2000) Loss of nuclear p53 protein in preneoplastic rat hepatocytes is accompanied by Mdm2 and Bcl-2 overexpression and by defective response to DNA damage in vivo. Hepatology 32:701-10 Zhivotovsky B and Orrenius S (2003) Defects in the apoptotic machinery of cancer cells: role in drug resistance. Semin Cancer Biol 13:125-34 Rotstein J, Sarma DS and Farber E (1986) Sequential alterations in growth control and cell dynamics of rat hepatocytes in early precancerous steps in hepatocarcinogenesis. Cancer Res 46:2377-85 Kawata S, Yamasaki E, Nagase T, Inui Y, Ito N, Matsuda Y, Inada M, Tamura S, Noda S, Imai Y and Matsuzawa Y (2001) Effect of pravastatin on survival in patients with advanced hepatocellular carcinoma. A randomized controlled trial. Br J Cancer 84:886-91 Graf H, Jungst C, Straub G, Dogan S, Hoffmann RT, Jakobs T, Reiser M, Waggershauser T, Helmberger T, Walter A, Walli A, Seidel D, Goke B and Jungst D (2008) Chemoembolization combined with pravastatin improves survival in patients with hepatocellular carcinoma. Digestion 78:34-8 Platz EA, Leitzmann MF, Visvanathan K, Rimm EB, Stampfer MJ, Willett WC and Giovannucci E (2006) Statin drugs and risk of advanced prostate cancer. J Natl Cancer Inst 98:1819-25 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. Khurana V, Sheth A, Caldito G and Barkin JS (2007) Statins reduce the risk of pancreatic cancer in humans: a case-control study of half a million veterans. Pancreas 34:260-5 Khurana V, Bejjanki HR, Caldito G and Owens MW (2007) Statins reduce the risk of lung cancer in humans: a large case-control study of US veterans. Chest 131:1282-8 Poynter JN, Gruber SB, Higgins PD, Almog R, Bonner JD, Rennert HS, Low M, Greenson JK and Rennert G (2005) Statins and the risk of colorectal cancer. N Engl J Med 352:2184-92 Stearman RS, Grady MC, Nana-Sinkam P, Varella-Garcia M and Geraci MW (2007) Genetic and epigenetic regulation of the human prostacyclin synthase promoter in lung cancer cell lines. Mol Cancer Res 5:295-308 Moncada S, Gryglewski R, Bunting S and Vane JR (1976) An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263:663-5 Ota K, Suehiro T, Arii K, Ikeda Y, Kumon Y, Osaki F and Hashimoto K (2005) Effect of pitavastatin on transactivation of human serum paraoxonase 1 gene. Metabolism 54:142-50 Jia X, Wei M, Fu X, Gu X, Fan W, Zhang J and Xue L (2009) Intensive cholesterol-lowering therapy improves large artery elasticity in acute myocardial infarction patients. Heart Vessels 24:340-6 Frigola J, Munoz M, Clark SJ, Moreno V, Capella G and Peinado MA (2005) Hypermethylation of the prostacyclin synthase (PTGIS) promoter is a frequent event in colorectal cancer and associated with aneuploidy. Oncogene 24:7320-6 Nakahata N (2008) Thromboxane A2: physiology/pathophysiology, cellular signal transduction and pharmacology. Pharmacol Ther 118:18-35 Katagiri H, Ito Y, Ishii K, Hayashi I, Suematsu M, Yamashina S, Murata T, Narumiya S, Kakita A and Majima M (2004) Role of thromboxane derived from COX-1 and -2 in hepatic microcirculatory dysfunction during endotoxemia in mice. Hepatology 39:139-50 Yokoyama Y, Nimura Y, Nagino M, Bland KI and Chaudry IH (2005) Role of thromboxane in producing hepatic injury during hepatic stress. Arch Surg 140:801-7 Pidgeon GP, Tamosiuniene R, Chen G, Leonard I, Belton O, Bradford A and Fitzgerald DJ (2004) Intravascular thrombosis after hypoxia-induced pulmonary hypertension: regulation by cyclooxygenase-2. Circulation 110:2701-7 Cathcart MC, Tamosiuniene R, Chen G, Neilan TG, Bradford A, O'Byrne KJ, Fitzgerald DJ and Pidgeon GP (2008) Cyclooxygenase-2-linked attenuation of hypoxia-induced pulmonary hypertension and intravascular thrombosis. J Pharmacol Exp Ther 326:51-8 Helliwell RJ, Adams LF and Mitchell MD (2004) Prostaglandin synthases: recent developments and a novel hypothesis. Prostaglandins Leukot Essent Fatty Acids 70:101-13 Christman BW, McPherson CD, Newman JH, King GA, Bernard GR, Groves BM and Loyd JE (1992) An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension. N Engl J Med 327:705 Weitz-Schmidt G, Welzenbach K, Brinkmann V, Kamata T, Kallen J, Bruns C, Cottens S, Takada Y and Hommel U (2001) Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat Med 7:687-92 55 C. Skogastierna 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 56 Amarenco P (2001) Hypercholesterolemia, lipid-lowering agents, and the risk for brain infarction. Neurology 57:S35-44 Nakayama T (2010) Genetic polymorphisms of prostacyclin synthase gene and cardiovascular disease. Int Angiol 29:33-42 Warner TD and Mitchell JA (2002) Cyclooxygenase-3 (COX-3): filling in the gaps toward a COX continuum? Proc Natl Acad Sci U S A 99:13371-3 Tohgi H, Konno S, Tamura K, Kimura B and Kawano K (1992) Effects of lowto-high doses of aspirin on platelet aggregability and metabolites of thromboxane A2 and prostacyclin. Stroke 23:1400-3 Sakai H, Suzuki T, Takahashi Y, Ukai M, Tauchi K, Fujii T, Horikawa N, Minamimura T, Tabuchi Y, Morii M, Tsukada K and Takeguchi N (2006) Upregulation of thromboxane synthase in human colorectal carcinoma and the cancer cell proliferation by thromboxane A2. FEBS Lett 580:3368-74 Nie D, Che M, Zacharek A, Qiao Y, Li L, Li X, Lamberti M, Tang K, Cai Y, Guo Y, Grignon D and Honn KV (2004) Differential expression of thromboxane synthase in prostate carcinoma: role in tumor cell motility. Am J Pathol 164:429-39 Kajita S, Ruebel KH, Casey MB, Nakamura N and Lloyd RV (2005) Role of COX-2, thromboxane A2 synthase, and prostaglandin I2 synthase in papillary thyroid carcinoma growth. Mod Pathol 18:221-7 Cathcart MC, Reynolds JV, O'Byrne KJ and Pidgeon GP (2010) The role of prostacyclin synthase and thromboxane synthase signaling in the development and progression of cancer. Biochim Biophys Acta 1805:153-66 Honn KV, Cicone B and Skoff A (1981) Prostacyclin: a potent antimetastatic agent. Science 212:1270-2 Keith RL, Miller YE, Hoshikawa Y, Moore MD, Gesell TL, Gao B, Malkinson AM, Golpon HA, Nemenoff RA and Geraci MW (2002) Manipulation of pulmonary prostacyclin synthase expression prevents murine lung cancer. Cancer Res 62:734-40 Keith RL, Miller YE, Hudish TM, Girod CE, Sotto-Santiago S, Franklin WA, Nemenoff RA, March TH, Nana-Sinkam SP and Geraci MW (2004) Pulmonary prostacyclin synthase overexpression chemoprevents tobacco smoke lung carcinogenesis in mice. Cancer Res 64:5897-904 Moncada S (1992) The 1991 Ulf von Euler Lecture. The L-arginine: nitric oxide pathway. Acta Physiol Scand 145:201-27 Nathan C (1992) Nitric oxide as a secretory product of mammalian cells. FASEB J 6:3051-64 MSD, Zocord, 2011. Parini P, Gustafsson U, Davis MA, Larsson L, Einarsson C, Wilson M, Rudling M, Tomoda H, Omura S, Sahlin S, Angelin B, Rudel LL and Eriksson M (2008) Cholesterol synthesis inhibition elicits an integrated molecular response in human livers including decreased ACAT2. Arterioscler Thromb Vasc Biol 28:1200-6 Ward JM, Rehm S and Reynolds CW (1990) Pathology of tumours in laboratory animals. Tumours of the rat. Tumours of the haematopoietic system. IARC Sci Publ 625-57 Solt DB, Medline A and Farber E (1977) Rapid emergence of carcinogeninduced hyperplastic lesions in a new model for the sequential analysis of liver carcinogenesis. Am J Pathol 88:595-618 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169. Inman SR, Stowe NT, Cressman MD, Brouhard BH, Nally JV, Jr., Satoh S, Satodate R and Vidt DG (1999) Lovastatin preserves renal function in experimental diabetes. Am J Med Sci 317:215-21 Tatsuta M, Iishi H, Baba M, Iseki K, Yano H, Uehara H, Yamamoto R and Nakaizumi A (1998) Suppression by pravastatin, an inhibitor of p21ras isoprenylation, of hepatocarcinogenesis induced by N-nitrosomorpholine in Sprague-Dawley rats. Br J Cancer 77:581-7 Livak KJ, Flood SJ, Marmaro J, Giusti W and Deetz K (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 4:357-62 Livak KJ and Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-8 Engvall E and Perlmann P (1971) Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8:871-4 Van Weemen BK and Schuurs AH (1971) Immunoassay using antigen-enzyme conjugates. FEBS Lett 15:232-236 Porstmann T and Kiessig ST (1992) Enzyme immunoassay techniques. An overview. J Immunol Methods 150:5-21 Samuelsson B, Goldyne M, Granstrom E, Hamberg M, Hammarstrom S and Malmsten C (1978) Prostaglandins and thromboxanes. Annu Rev Biochem 47:997-1029 Dusting GJ, Lattimer N, Moncada S and Vane JR (1977) Prostaglandin X, the vascular metabolite of arachidonic acid responsible for relaxation of bovine coronary artery strips [proceedings]. Br J Pharmacol 59:443P Rosenkranz B, Fischer C, Reimann I, Weimer KE, Beck G and JC FR (1980) Identification of the major metabolite of prostacyclin and 6-ketoprostaglandin F1 alpha in man. Biochim Biophys Acta 619:207-13 Catella F, Nowak J and Fitzgerald GA (1986) Measurement of renal and nonrenal eicosanoid synthesis. Am J Med 81:23-9 Frolich JC (1984) Measurement of icosanoids. Report of the Group for Standardization of Methods in Icosanoid Research. Prostaglandins 27:349-68 Lawson JA, Patrono C, Ciabattoni G and Fitzgerald GA (1986) Long-lived enzymatic metabolites of thromboxane B2 in the human circulation. Anal Biochem 155:198-205 Ciabattoni G, Pugliese F, Davi G, Pierucci A, Simonetti BM and Patrono C (1989) Fractional conversion of thromboxane B2 to urinary 11dehydrothromboxane B2 in man. Biochim Biophys Acta 992:66-70 Misko TP, Schilling RJ, Salvemini D, Moore WM and Currie MG (1993) A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem 214:11-6 Arpino P (1992) Combined liquid chromatography mass spectrometry. Part III. Applications of thermospray. Mass Spectrometry Reviews 11:3-40 Bjorkhem-Bergman L, Lindh JD and Bergman P (2011) What is a relevant statin concentration in cell experiments claiming pleiotropic effects? Br J Clin Pharmacol 72:164-5 Tang XL, Jiang ZY, Dong J, Liu XC, Cai SY, Xiao R and Lu YR (2006) [Expression of tissue factor induced by IL-6 in HUVEC]. Sichuan Da Xue Xue Bao Yi Xue Ban 37:234-7 57 C. Skogastierna 170. 171. 172. 173. 174. 175. 176. 177. 58 Majed BH and Khalil RA (2012) Molecular Mechanisms Regulating the Vascular Prostacyclin Pathways and Their Adaptation during Pregnancy and in the Newborn. Pharmacol Rev 64:540-82 Wright DH, Abran D, Bhattacharya M, Hou X, Bernier SG, Bouayad A, Fouron JC, Vazquez-Tello A, Beauchamp MH, Clyman RI, Peri K, Varma DR and Chemtob S (2001) Prostanoid receptors: ontogeny and implications in vascular physiology. Am J Physiol Regul Integr Comp Physiol 281:R1343-60 Liao JK (2002) Beyond lipid lowering: the role of statins in vascular protection. Int J Cardiol 86:5-18 Kugi M, Matsunaga A, Ono J, Arakawa K and Sasaki J (2002) Antioxidative effects of fluvastatin on superoxide anion activated by angiotensin II in human aortic smooth muscle cells. Cardiovasc Drugs Ther 16:203-7 Miwa S, Watada H, Omura C, Takayanagi N, Nishiyama K, Tanaka Y, Onuma T and Kawamori R (2005) Anti-oxidative effect of fluvastatin in hyperlipidemic type 2 diabetic patients. Endocr J 52:259-64 Vandjelovic N, Zhu H, Misra HP, Zimmerman RP, Jia Z and Li Y (2012) EPR studies on hydroxyl radical-scavenging activities of pravastatin and fluvastatin. Mol Cell Biochem 364:71-7 Imaeda A, Tanigawa T, Aoki T, Kondo Y, Nakamura N and Yoshikawa T (2001) Antioxidative effects of fluvastatin and its metabolites against oxidative DNA damage in mammalian cultured cells. Free Radic Res 35:789-801 Nakamura T, Nishi H, Kokusenya Y, Hirota K and Miura Y (2000) Mechanism of antioxidative activity of fluvastatin-determination of the active position. Chem Pharm Bull (Tokyo) 48:235-7