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1 PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. Please be advised that this information was generated on and may be subject to change.

2 Assessment of Ischaemia and Reperfusion Injury Constantijn W. Wouters

3 Assessment of Ischaemia and Reperfusion Injury Constantijn W. Wouters

4 ISBN Cover design and lay-out: Promotie In Zicht, Arnhem Print: Ipskamp Drukkers, Enschede The research described in this thesis was performed at the department of Pharmacology and Toxicology, in collaboration with the departments of Cardiology and Nuclear Medicine at the Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands. Funding for this research was provided by The Netherlands Organization for Health Research and Development ( ) and the Netherlands Heart Foundation (2006 T035). This thesis was financially supported by: Maatschap Cardiologie Canisius Wilhelmina Ziekenhuis, ABN Amro Bank, Society Shop Arnhem, Boehringer Ingelheim B.V., Bayer HealthCare, Merck Sharpe & Dohme B.V., Servier Nederland Farma B.V., Withering Stichting, Roche Diagnostics Nederland B.V., Raadsheren B.V., Pfizer B.V., Daiichi Sankyo Nederland B.V., Eli Lilly Nederland C.W. Wouters 2013 All rights reserved. No parts of this publication may be reported or transmitted, in any form or by any means, without permission of the author. The copyright of the articles that have been published has been transferred to the respective journals.

5 Assessment of Ischaemia and Reperfusion Injury Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. mr. S.C.J.J. Kortmann, volgens besluit van het college van decanen in het openbaar te verdedigen op dinsdag 25 juni 2013 om uur precies door Constantijn Willem Wouters geboren op 2 oktober 1976 te Nijmegen

6 Promotoren Prof. dr. G.A. Rongen Prof. dr. W.J.G. Oyen Prof. dr. P.A.B.M. Smits Manuscriptcommissie Prof. dr. M.J. de Boer, voorzitter Prof. dr. J. de Graaf Prof. dr. W.H. van Gilst (UMC Groningen)

7 Gelukkig is de man wiens kennis afkomstig is van onderzoek Euripides, fragment 910 (vert. H.L. van Dolen)

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9 Contents Chapter 1 Ischaemia and reperfusion in the heart 9 Chapter 2 Atorvastatin does not affect ischaemia-induced phosphatidylserine 31 exposition in humans in vivo Journal of Atherosclerosis and Thrombosis 2012 Chapter 3 Angiotensin II type 1 receptor blockade does not enhance apoptotic 43 cell death during ischaemia and reperfusion in humans in vivo Journal of Cardiovascular Pharmacology 2011 Chapter 4 Dipyridamole provides prolonged protection against ischaemia 55 and reperfusion injury in forearm skeletal muscles in humans in vivo Submitted Chapter 5 Upregulation of ecto-5'-nucleotidase by rosuvastatin increases 65 the vasodilator response to ischaemia Hypertension 2010 Chapter 6 Short-term statin treatment does not prevent ischaemia and 77 reperfusion-induced endothelial dysfunction in humans Journal of Cardiovascular Pharmacology 2012 Chapter 7 Impact of ischaemia and reperfusion injury on endothelial function 91 in the radial and brachial artery in humans in vivo Submitted Chapter 8 General summary and conclusions 103 Nederlandse samenvatting en conclusies 111 References 121 Dankwoord 139 Curriculum Vitae 147 List of publications 151

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11 Chapter 1 Ischaemia and reperfusion in the heart

12 Chapter 1 10

13 Ischaemia and reperfusion in the heart Background Coronary artery disease has become a worldwide burden for society. In The Netherlands every year over persons die due to coronary artery disease, presenting either as an acute myocardial infarction (AMI) or ischaemic heart failure. 275 Moreover annually patients are hospitalised with AMI and due to ischaemic heart failure. 275 When initiated through a combination of lifestyle, genetics, and age, atherosclerosis will slowly progress and in time cause coronary stenotic plaque formation. These laesions can finally limit coronary blood flow resulting in myocardial ischaemia. Ultimately, plaque rupture causes acute obstruction of the coronary artery resulting in an acute myocardial infarction. Ischaemia caused by acute and complete occlusion of a coronary artery induces serious injury to the myocardium. The amount of damage after AMI, or myocardial infarct size, was classically considered to depend on 1) the area at risk of the occluded artery and 2) the duration of the ischaemic insult (or the time to reperfusion). As only the latter could be modulated during the occurrence of AMI, researchers developed therapeutic strategies aimed to accomplish early reperfusion. In 1950 the in-hospital mortality of patients presenting with AMI (diagnosed using ECGrecording) was approximately 15%, as no treatment was yet available. 61 With the introduction of effective pharmacologic (thrombolysis) or endovascular (percutaneous coronary intervention; PCI) reperfusion therapies combined with thrombocyte aggregation inhibitors (aspirin and P2Y12 antagonists) and anticoagulants (e.g. heparin), the mortality rate of AMI has decreased significantly. 275,292 A recent meta-analysis showed that in patients suffering a ST elevated myocardial infarction (STEMI) receiving today s standard care (primary PCI and optimal medicinal treatment) 30-day mortality rate is 5.2%. 30 The detrimental effects of ischaemia are known for a long time. However with the introduction of fast and successful reperfusion therapy, a new phenomenon became apparent: reperfusion injury. The deleterious effects of reperfusion were first noticed in 1975 as reperfusion was associated with sudden and explosive cell death in hypoxic rat hearts. 102 Tissue damage due to both the ischaemic injury and the reperfusion injury is termed ischaemia and reperfusion (IR) injury. This finding sparked the thought that reperfusion is a two-faced process; it is an absolute necessity for maximal tissue salvage, but it also triggers processes that limit the benefit of reperfusion by further damaging the ischaemically injured tissue. 33 Although, the beneficial effects of timely reperfusion always outweigh its detrimental effects, the net benefit of reperfusion could be enhanced by prevention of reperfusion-associated injury. 11

14 Chapter 1 In 1986 Murry et al. provided definitive evidence that infarct size not only depends on area at risk and duration of ischaemia but that cardiac tissue can develop tolerance to the sequellae of ischaemia and reperfusion. They showed in dogs that four cycles of 5 minutes of myocardial ischaemia (achieved by circumflex artery occlusion) and 5 minutes of myocardial reperfusion preceding a prolonged ischaemic insult (circumflex artery occlusion for 40 minutes), limited myocardial infarct size to 25% compared to the control group subjected to only 40 minutes of circumflex occlusion. This protective procedure was named ischaemic preconditioning (IP). 183 Thus, Murry found a third determinant of myocardial infarct size: the intrinsic ability of tissue to develop tolerance to ischaemia and reperfusion and thus defined a new target for therapeutic intervention. The recent recognition of ischaemic post-conditioning (controlled reperfusion with repetitive short episodes of reperfusion and ischaemia, prior to definite reperfusion) as a strategy to successfully reduce infarct size both in animals as well as humans 252 provided definite evidence for the existence of reperfusion-associated injury as an important therapeutic target to reduce (myocardial) infarct size. Physiological and Pathological Changes during Ischaemia and Reperfusion Ischaemia Sudden occlusion of a coronary artery results in multiple metabolic changes, appearing within seconds of cessation of blood flow. ATP reserves in the myocardium are limited, and will be depleted within 4 efficient contractions. 123 After consumption of the oxygen trapped in the ischaemic tissue as oxyhaemoglobin or oxymyoglobin, metabolism shifts from aerobic to anaerobic glycolysis. 123 To optimise metabolic performance, cellular glucose uptake is increased by translocation of GLUT-receptors to the cellular membrane and the rate of glycolysis is enhanced by activation of the enzyme 6-phosphofructokinase. Due to inhibition of the enzyme pyruvatedehydrogenase, pyruvate is metabolised to lactate (instead of acetyl Co-A), together with the conversion of NADPH 2 to NAD. 200 At the onset of ischaemia, hydrolysis of residual ATP leads to mild cellular acidosis, which appears to be beneficial since it reduces cardiac contractility and subsequent metabolic demand. With intracellular hydrolysis of ATP, levels of ADP and subsequent AMP and adenosine rise. AMP activates the enzyme AMP-kinase which in part further stimulates glucose uptake and glycolysis rate. 200 Extracellularly, upregulation of the enzyme ecto-5 - nucleotidase (CD73) results in increased adenosine formation, through augmented phosphohydrolysis of AMP. 68 During prolonged ischaemia the end products of the anaerobic glycolysis (lactate and NAD) start to inhibit glucose uptake and glycolysis, resulting in further ATP depletion and severe acidosis. Gradually serious disturbances in the ion homeostasis develop within the 12

15 Ischaemia and reperfusion in the heart ischaemic cells that have important physiological consequences. Because of dysfunction of the ATP dependant Na-K exchanger and enhanced function of the Na-H + exchanger (due to cellular acidosis) sodium enters the cell, leading to cellular oedema. Furthermore, the increase in intracellular sodium causes the activity of the Na-Ca exchanger to reverse and transport calcium into the cell. 200 As the ATP dependent uptake of calcium by the sarcoplasmatic reticulum is inhibited, intracellular calcium overload develops. These cumulative metabolic changes cause damage to cardiomyocytes, resulting in both necrosis and apoptosis in the area at risk. 269 Experiments in dogs have shown that necrosis starts to appear after approximately minutes of myocardial ischaemia. 124 With prolonged duration of ischaemia more tissue becomes necrotic and after 6 hours the entire area at risk has become irreversibly damaged. 214 Thus, for partial salvage of ischaemically injured myocardial tissue, timely reperfusion is an absolute prerequisite. The coronary blood flow can be restored, either spontaneously or aided by thrombolytic agents or PCI. However, as stated before, reperfusion has been referred to by Braunwald and Kloner as the double edged sword, because reperfusion itself leads to myocardial injury beyond that generated by ischaemia. 33,180 Reperfusion Upon reperfusion the aerobic metabolism is restored instantaneously. Post-ischaemic hyperaemia will occur due to the release of potent vasodilatory substances during ischaemia (e.g. adenosine, bradykinin, nitric oxide), leading to a 4-6 fold increase in flow. 136 Restoration of blood flow will wash out lactate and H +, leading to a quick recovery of tissue ph. 305 Calcium overload There is an acute increase in intracellular and mitochondrial calcium concentration due to ischaemic injury to the sacrolemmal-membrane and dysfunction of the sarcoplasmic reticulum. During reperfusion this calcium overload can cause hypercontracture of the myocytes and contributes to opening of the mitochondrial permeability transition pore (MPTP), both leading to cell death. Reactive oxygen species (ROS) Reintroduction of abundant oxygen at the onset of reperfusion evokes a burst of potent free radicals. 180 The most important sources of ROS in cardiomyocytes are the mitochondrial electron transport chain, and as by-products of the enzymes nitric oxide synthase (NOS) and NADPH oxidase. 178 During reperfusion ROS are produced extracellularly by endothelial cells as well as phagocytes. 178 Their reactive nature causes ROS to interact directly with cellular lipids, proteins and (mitochondrial) DNA, resulting in damage to the cell membrane and the membranes of cellular organelles and subsequent organelle or cellular failure

16 Chapter 1 Endothelial dysfunction At reperfusion the enzyme arginase is activated. Arginase competes with NO-synthase for its substrate L-arginine and results in a subsequent decrease in endothelial NOrelease. 67,105,133 This contributes to IR induced endothelial dysfunction, which can be clinically recognised as the no-reflow phenomenon. For example after a successful PCI, blood flow in the affected coronary artery remains limited due to endothelial vasoconstriction and vascular plugging. Occurrence of the no-reflow phenomenon limits the benefits of timely reperfusion, as the myocardial blood flow remains deprived. Inflammation During myocardial ischaemia, multiple chemokines (e.g. monocyte chemoattractant protein (MCP), macrophage inflammatory protein (MIP), several interleukins and tumour necrosis factor (TNF-alpha)) are expressed, which regulate monocyte, lymphocyte and macrophage recruitment and evoke an inflammatory reaction that will ultimately contribute to myocardial healing and scar formation. In the early phase of IR-injury the massive influx of inflammatory cells causes vascular plugging, which diminishes reperfusion. 200 Mitochondria and MPTP Myocardial mitochondria provide energy in the form of ATP for cellular contraction. However mitochondria are very sensitive to alterations in cellular environment and they can quickly change from being a supporter of life to a promoter of cell death. IR-injury can induce mitochondrial fission, which is linked to increased production of ROS, impaired function and initiation of cell death. 196 Mitochondrial failure is caused by formation of the mitochondrial permeability transition pore (MPTP). Opening of this non-selective, large conductance channel initiates a cellular cascade resulting in apoptotic myocardial cell death. 179 Prevention of MPTP opening (e.g. by cyclosporine treatment) has been shown to reduce impact of IR-injury. 206 Clinical consequences The release of ROS and intracellular calcium overload can both result in mitochondrial damage caused by opening of the MPTP, activation of caspases and subsequent cell damage and eventually cell death (by either necrosis or apoptosis). 120 As a result, IR-injury has several clinical consequences in a patient suffering from AMI. It causes myocardial stunning, arrhythmias, increased impedance of microvascular blood flow (no-reflow), myocardial cell death, and endothelial dysfunction, all of which contribute to increased mortality and morbidity after AMI. 133,305 14

17 Ischaemia and reperfusion in the heart Prevention of Ischaemia and Reperfusion Injury Reperfusion Coronary reperfusion after AMI can be established using various therapies. Today the most favourable is the percutaneous coronary intervention (PCI), in which the occlusion is opened by a wire-guided balloon, introduced via the femoral or radial artery. To increase coronary patency most often a stent is deployed at the location of the stenosis and the subsequent occlusion. Prior to the procedure patients receive anticoagulant treatment and at least double thrombocyte aggregation inhibition to prevent coronary re-occlusion after PCI. If a PCI facility is not timely available, thrombolytic therapy can be administered in order to attempt to dissolve the occluding blood clot. However 30-day mortality is higher in STEMI patients receiving thrombolytic therapy than in those receiving PCI (9% vs. 7% respectively). 292 The Netherlands have a high density of PCI facilities, a well organised and swift ambulance service, and a decent infrastructure, which together allow all patients with a STEMI to be transported to a PCI centre for a primary PCI. Today, a 30-day mortality of 12.5% is observed in STEMI patients, in whom reperfusion could not be established. If reperfusion can be achieved by PCI within the first hour after the onset of symptoms mortality is reduced to 4.7%. 292 As the delay between symptom onset and restoration of coronary blood flow increases, mortality rises. If coronary perfusion is restored within 3-6 hours, mortality is 5.6%. 7 A delay of 6-12 hours to reperfusion results in a mortality of 8.5%, indicating that even in case of prolonged ischaemia, the myocardium still benefits from reperfusion through PCI. 30,292 Over the last 30 years interventions aimed at restoring coronary circulation have been improved. Moreover early diagnosis (at home) of STEMI and early preparation of the catheterization laboratories have further reduced patient-to-balloon-time. De Luca et al. (figure 1) have clearly shown the importance of early reperfusion as in their cohort the 1-year mortality triples if coronary reperfusion is just established after 6 hours compared to one hour after onset of complaints. 51 Conditioning As stated earlier, in 1986 Murry et al. have shown that ischaemic preconditioning (IP), consisting of several brief episodes of non-injuring ischaemia and reperfusion prior to the index ischaemia, could reduce IR-injury. 183 The results of Murry have been replicated by many other scientists in animal experiments. Early mechanistic and descriptive studies revealed that IP was associated with 2 phases of cardioprotection. After the preconditioning event, a protective phenotype occurs immediately and lasts for maximally 6 hours (acute preconditioning). 304 Twenty-four hours after the preconditioning trigger, a delayed preconditioning or second window of protection (SWOP) starts. However, little is known about the duration of delayed preconditioning. A duration of at least 72 hours, though no 15

18 Chapter One-year mortality (%) Ischaemia time (min) Figure 1 One-year mortality in patients with STEMI related to time between onset of complaints and coronary reperfusion by PCI, calculated using a quadratic regression model by De Luca et al. (Circulation 2004;109: ). Dotted lines represent 95% confidence interval of predicted mortality. longer than 96 hours has been reported for both ischaemic and adenosine induced delayed preconditioning. 16,18 Further experiments showed that the protective effect of IP was not limited to the heart, as different organs (e.g. kidney, brain) could become preconditioned after exposure to repetitive episodes of non-lethal ischaemia, resulting in improved tolerance to subsequent prolonged ischaemia. 278 Recent experiments report that repetitive hypoxic preconditioning induces long-term tolerance in nervous tissue, lasting 4 to 8 weeks. 255,309 Further research to investigate the possibilities offered by IP revealed several other interesting protective variations. Przyklenk et al. showed in 1993 that the protective effect of ischaemic preconditioning was not limited to the tissue subjected to IP. In dogs, they applied IP to the circumflex coronary artery, followed by a 1 hour of sustained left anterior descending (LAD) coronary artery occlusion. They found that the IP in one vascular bed could protect remote virgin myocardium from IR-injury. This transfer of protection is termed remote ischaemic preconditioning (RIPC). Further experiments have shown that protection against IR can be transferred between various organs and tissues. 97 In 2003 Zhao et al. published their results comparing the myocardial protection provided by IP and ischaemic postconditioning (or controlled reperfusion). Again in dogs, they showed that a single cycle of 5 minutes LAD ischaemia and 10 minutes of reperfusion 16

19 Ischaemia and reperfusion in the heart reduced myocardial infarct size induced by a subsequent 60 minute LAD occlusion. Interestingly, when the 60 minutes LAD occlusion was followed by 3 cycles of 1 minute of reperfusion and 1 minute of re-occlusion, infarct size was smaller compared to control heart, and similar compared to ischaemically preconditioned hearts. 308 Apparently even after occurrence of the index ischaemia, a protective mechanism can be triggered rendering the myocardium less vulnerable to IR-injury. The most recently published type of conditioning is remote ischaemic perconditioning. 132 Kerendi et al. showed that concomitant renal ischaemia, during prolonged cardiac ischaemia reduces myocardial infarct size in rats. Also concomitant ischaemia and reperfusion of the brain 88 or the leg 151 can induce myocardial tolerance against IR. Andreka et al. found a reduced infarct size in pigs, when they applied the remote ischaemic postconditioning stimulus (4x5min hind limb ischaemia and reperfusion) at the onset of myocardial reperfusion. 11 This intervention is very promising because of its clinical applicability, for example in patients suffering from AMI. 97 Whereas preconditioning is only suited for situations with foreseeable IR, and postconditioning requires control over the circulation of the target organ, remote ischaemic perconditioning can be initiated during the index ischaemic event, and applied to for example the lower limb. 238 After the landmark publication of Murry, many scientists aimed their efforts at further unravelling and exploiting the protective mechanism of IP. Taken together, these experiments have provided the insight that protection after conditioning is induced through a complex mechanism; several triggers (e.g. adenosine, bradykinin, opioids, and NO) act through accompanying G-protein coupled receptors to initiate different intracellular cascades (constituted of a variety of protein kinases e.g. PI3K, Akt, ERK, PKC). 107 Subsequent opening of the mitochondrial K ATP -channels is part of a final common pathway on which the signalling pathways seem to converge. The end-effector of protection is ultimately the prevention of opening of the mitochondrial permeability transition pore (MPTP). 99 This MPTP, situated through both inner and outer mitochondrial membrane, remains closed under physiological circumstances by means of multiple pro-survival signal pathways. IR-induced opening of the MPTP triggers a cellular cascade ultimately leading to apoptosis. 179 Experiments clarifying the mechanism of delayed preconditioning have shown involvement of increased transcription of cardioprotective genes and expression of various proteins including inos, COX-2, heme oxygenase and antioxidant enzymes. 254,304 Elucidation of this mechanism has provided multiple targets for pharmacologic modulation of ischaemia and reperfusion injury. Pharmacological interventions mimicking the effect of ischaemic preconditioning are called pharmacologic preconditioning. Drugs that have been proven to be able to reduce infarct size in animal models include several statins, adenosine, NO, dipyridamole, cyclosporine, diazoxide, angiotensin receptor antagonists, isoflurane, and nicorandil

20 Chapter 1 Methods to Study IR-Injury in Humans In Vivo In animal experiments the primary endpoint most frequently used to study the impact of IR is myocardial infarct size. This is usually assessed histologically and expressed as the fraction of the area at risk, which is determined independently. Obviously, in human research other outcomes (surrogate endpoints) are studied. This paragraph summarises different models used to study simulated IR in healthy volunteers and endpoints used in follow-up of patients exposed to IR-injury. Surrogate endpoints in translational models of IR-injury In contrast to clinical trials, translational experimental models are not designed to study clinical outcomes. Translational models of IR-injury can be used to study in detail different aspects of IR-injury and to study the pathogenesis of IR-injury. Also the mechanism of action of interventions and drugs preventing IR-injury can be studied using translational models. The use of healthy volunteers instead of patients reduces burden for patients and facilitates more easy inclusion of participants. IR-induced phosphatidylserine exposition Introduced by Rongen et al. in 2005, this technique enables visualisation and quantification of phosphatidylserine (PS) exposure that occurs after IR in humans in vivo. Ischaemia induced phosphatidylserine exposure on cardiomyocytes has been associated with reversible cellular damage and apoptosis. 189,269 Rongen developed an experimental model to study PS exposure after IR in healthy male volunteers. This model uses radiolabeled recombinant annexin A5 to visualise PS-exposition occurring after 10 minutes of forearm ischaemia combined with isometric exercise. 226 Interventions that have been shown in preclinical and clinical research to modulate myocardial infarct size (ischaemic preconditioning, and pharmacologic preconditioning with adenosine and dipyridamole) similarly modulate annexin A5 targeting in this forearm model. 220,224,226 IR-induced endothelial dysfunction Flow mediated dilation Flow mediated dilation (FMD) represents a shear stress induced, largely NO-mediated 182 endothelium-dependent vasodilation. Originally this method was introduced to assess endothelial function. Endothelial dysfunction is considered to be the first clinical feature of atherosclerosis, and in a large cohort study reduced FMD was an independent predictor of cardiovascular death. 294 In 2001 Kharbanda et al. 133 introduced repeated FMD measurements in the radial artery as a model to study IR-injury in humans in vivo. In this non-invasive and well-tolerated model, they showed in healthy volunteers that 15 minutes of ischaemia and reperfusion reduced FMD from 7.7% at baseline to 3.5%. This endothelial dysfunction proved to be temporary, 18

21 Ischaemia and reperfusion in the heart as after one hour of reperfusion endothelial function had completely recovered. Moreover they showed in this model that ischaemic preconditioning (3 episodes of 5 minutes upper limb ischaemia and reperfusion) could prevent IR-induced endothelial dysfunction. FMD measurement before and after IR has successfully been used to further explore (pharmacologic) interventions improving tolerance against IR-induced endothelial dysfunction. Therapies that prevent endothelial dysfunction after IR include treatment with sildenafil, bradykinin, NO-donors and rosuvastatin, and interventions such as remote ischaemic preconditioning and ischaemic postconditioning. 80,83,155,156,158,159 Plethysmography Another widely used technique to measure endothelial function is venous occlusion plethysmography. In this model mercury-in-silastic strain gauges are placed around the forearm. A pneumatic cuff is placed at the upper arm and inflated to 40mmHg for 6-10 heartbeats. This pressure exceeds venous pressure but remains below arterial pressure. Thus during the venous occlusion, blood flow into the arm is unhindered, while venous return is closed off. The pooling blood will cause the forearm to swell subtly. The increase in forearm diameter is registered by the mercury strain gauges. The speed of the increase of the diameter, is related to the forearm blood flow. 225 Forearm blood flow can be influenced by conduit artery vasodilation and vasoconstriction. These functions are locally controlled by endothelium in both the arterial and venous vascular bed. 277 Endothelium function can be assessed by measuring changes in forearm blood flow in response to different dosages of vasoactive agents (NO donors like nitroprusside or nitroglycerine, endothelium dependent vasoactive agents like acetylcholine and vasoconstrictive agents like L-NMMA). As stated above, endothelial dysfunction is a distinct feature of IR-injury. Venous occlusion plethysmography has been used to test pharmacological interventions aimed at prevention of endothelial dysfunction after IR. 31,207 Endpoints in intervention studies in patients with coronary artery disease Observational clinical data and functional measurements Observation of the clinical course of a patient for example after STEMI, PCI or CABG surgery can provide interesting information. The clinical need of inotropic support or vasopressor therapy and monitoring heart rhythm provide information on the effect of IR-injury on myocardial stunning and arrhythmias. Clinical relevant endpoints used to measure outcome after IR-injury include survival rate, duration of hospital stay, and major adverse cardiac events (MACE; e.g. myocardial death, myocardial infarction, hospital admission due to acute coronary syndrome (ACS) and coronary revascularisation). The effect of IR on myocardial performance can be analysed using functional outcomes, for example left ventricular function 121 (assessed using cardiac ultrasound or MRI), or left ventricular stroke work index

22 Chapter 1 Myonecrosis markers Myocardial damage can be assessed biochemically with myocardial necrosis biomarkers (e.g. troponin-i, troponin-t, MB fraction of creatine kinase (CK-MB)). The amount of released biomarker is related to the extent of myocardial damage and is a predictor of mortality. 216,244 As a small blood sample is sufficient to determine myocardial injury, measuring myonecrosis markers represents a rather easy and rapid way to gather clinically relevant information on IR-injury. Follow-up of myocardial necrosis biomarkers is performed and validated in the course of myocardial infarction, PCI and cardiac surgery. 14,280 MRI assessment of IR-injury Several aspects of myocardial IR-injury can be visualised using cardiac magnetic resonance imaging (MRI). 2 Infarct-associated myocardial oedema, assessed using T2 imaging, depicts the area at risk. Irreversible myocardial injury can be detected with late gadolinium enhancement and enables accurate and reproducible assessment of infarct size. The no-reflow phenomenon can be visualised using gadolinium enhancement. Finally also left ventricular ejection fraction can be measured with MRI reliably. Clinical Studies on Attenuation of Myocardial IR-injury Studies on prevention of IR-injury in the setting of an acute myocardial infarction In a patient presenting with an acute myocardial infarction, the possibilities to prevent IR-injury (apart from swift reperfusion) are limited to interventions that are timed during and after the index ischaemia, being (remote) ischaemic per and postconditioning and pharmacologic per- and postconditioning. Staat et al. were the first to successfully apply ischaemic postconditioning (or controlled reperfusion) in patients undergoing PCI during a STEMI. After opening of the culprit laesion, they exposed the patient to 4 cycles of 1 minute of reperfusion and 1 minute of coronary occlusion by balloon re-inflation. Postconditioning significantly increased blush grade (a measure of patency of the cardiac microcirculation) and reduced release myonecrosis markers (CK-MB) compared to unhindered reperfusion. 252 These findings were confirmed by other research groups 142,283, though not all. 75,251 Piot et al. studied the effect of cyclosporine (inhibitor of MTPT opening) in patients with STEMI. They assessed infarct size using cardiac MRI, and found that absolute mass of infarcted tissue was significantly reduced in patients receiving cyclosporine compared to placebo (37 grams vs. 46 grams respectively). 206 Several retrospective studies have shown that in patients with a history of angina pectoris, survival after STEMI was significantly better (97%) compared to patient without any prodromal symptoms (92%). Also the occurrence of cardiogenic shock after STEMI was 20

23 Ischaemia and reperfusion in the heart lower in patients with prodromal symptoms (1% vs. 6%). As the angina resulted from transient bouts of myocardial ischaemia, the myocardium became ischaemically preconditioned and thus less vulnerable for a future ischaemic insult and subsequent IRinjury. 116,137 Albeit, these retrospective studies could have been confounded, as patients with prodromal symptoms were probably more likely to search medical support, and thereby facilitate early recognition of e.g. myocardial infarction and possibly reduce time to reperfusion. In a Japanese experiment, the long-term follow-up after a single intravenous bolus of nicorandil (a hybrid compound of mitochondrial K ATP -channel opener and NO-donor) in patients with STEMI, significantly reduced occurrence of MACE (6.5%), compared to placebo (16.4%). 115 The Italian ARMYDA-ACS trial showed that atorvastatin treatment just prior to PCI in statin naïve patients reduces MACE at 30 days from 17% to 5% in patients with acute coronary syndrome. 203 Large clinical trials on pharmacological improvement of outcome after myocardial infarction using adenosine and pexelizumab infusion failed to significantly reduce mortality. 12,166,228 Also administering erythropoietin at myocardial reperfusion in patients with STEMI did not convincingly reduce myonecrosis markers nor did it reduce patient mortality. 181 Studies on prevention of IR-injury in the setting of elective PCI In approximately 30% of the patients undergoing elective PCI IR-injury is present (as assessed by a periprocedural rise of myonecrosis markers). 14,135 MRI studies have shown that this rise in myonecrosis factors after PCI represents actual myocardial injury. 216,244 Furthermore a correlation has been found between post-pci increase of myonecrosis markers and increased risk on restenosis 185 and even on long-term mortality. 208 Explanations on the cause of IR-injury after PCI include transient balloon-induced ischaemia, side branch occlusion (either by atheroma or by a stent), no-reflow phenomenon, transient vessel closure due to dissection or spasm and embolization of platelet fibrin aggregates along with atherosclerotic debris. 14,135,187 Deutsch et al. were the first to study the effect of standardised repeated balloon inflations (2x90 seconds) in patients undergoing elective PCI. They found a significant reduction of ST deviation, angina pain score and myocardial lactate production during the second balloon inflation. 53 Initially these findings were questioned because in animal experiments an ischaemic episode lasting 90 seconds did not induce myocardial protection. 144,169 It was proposed that during the first episode of ischaemia collateral flow was recruited, and that the changes during the second episode could be contributed to the increased coronary flow to the ischaemic area of interest. 169 However additional experiments have shown that repetitive brief episodes of coronary occlusion do not recruit significant collateral flow. 48,171 Tomai et al. confirmed the results of Deutsch, and moreover they found that oral 21

24 Chapter 1 glibenclamide (ATP-dependent potassium channel blocker) treatment prior to ischaemic preconditioning completely abrogated the protective effect of IP. 271 Furthermore IP also reduced periprocedural arrhythmias. 3 Hoole et al. showed that remote ischaemic preconditioning (3 cycles of 5 minutes of upper limb ischaemia) can reduce troponin-i release and reduce incidence of MACE with 70% in patients undergoing elective PCI. 110 Parallel to findings in preclinical research, investigators initiated experiments to study pharmacologic preconditioning in patients undergoing elective PCI. Randomised and blinded intracoronary administration of dipyridamole 256 and adenosine 104 increased myocardial resistance against IR. However, more recently intracoronary adenosine did not protect the heart in a randomised study with MRI-detected injury as endpoint. 52 Intravenous administration of nitroglycerin 24 hours prior to PCI also provided protection, indicating the existence of a pharmacological induced second window of protection in humans. 149 A group of drugs extensively studied concerning their ability to induce protection against IR-injury during PCI are statins (HMG-reductase CoA inhibitors). In the first trial studying the relation between statin pretreatment and IR-injury, statin naïve hypercholesterolaemic patients with stable angina pectoris were randomised to receive three months treatment with pravastatin or placebo. To show involvement of adenosine in the alleged protective effect, participants were randomised to receive aminophylline (a potent adenosine receptor antagonist) just prior to PCI. This experiment showed that statin pretreatment provided protection against (measured using ST shift on the ECG) and that this effect could completely be abrogated by aminophylline. 148 Further experiments have shown a reduction in myonecrosis markers after PCI by both a 7 day treatment with atorvastatin 202 and even a single high loading dose of atorvastatin. 34 A double dose of atorvastatin immediately prior to an acute PCI on top of concurrent statin therapy was able to significantly reduce MACE after PCI. 55 Studies on prevention of IR-injury in the setting of CABG In cardiac surgery temporary cardiac arrest is applied to enable valve repair, valve replacement, or placement of coronary bypass grafts. During cardiac arrest an extracorporal circulation (or heart-lung machine) will provide oxygenated blood to the rest of the body, only the heart becomes ischaemic during this period. Extensive experiments on cardioplegic solutions used during cardiac arrest have greatly improved tolerance of the myocardium to this ischaemic phase of the surgical procedure. 38 Nonetheless 99% of CABG patients show increased values of troponin-i after surgery. Moreover in a post-mortem analysis of patients who died shortly after coronary bypass surgery 25% showed histological evidence of extensive IR-injury. 289 Different features of IR-injury are frequently observed in the aftermath of for example coronary artery bypass surgery, including increased levels of myonecrosis markers, myocardial stunning and subsequent need for inotropic therapy, and occurrence of arrhythmias is common during the first postoperative days

25 Ischaemia and reperfusion in the heart Ample experiments have been carried out to investigate and reduce the burden of perioperative IR-injury. In 1993 preliminary data were published by Yellon and Alkhulaifi on the possible benefit and clinical applicability of ischaemic preconditioning in the setting of cardiac surgery. 7,303 After initiation of the cardiopulmonary bypass, 14 patients were randomised to receive either two 3 minute episodes of generalised cardiac ischaemia (induced by aortic cross clamping) and subsequent reperfusion followed by 10 minutes of ischaemic ventricular fibrillation (VF), or just 10 minutes of ischaemic VF. In the cardiac biopsies taken after ischaemic VF in patients subjected to ischaemic preconditioning levels of ATP were higher and levels of lactate were lower than in controls. The authors concluded that IP could beneficially influence myocardial metabolism and thus protect the myocardium against IR. Shortly thereafter Alkhulaifi showed that this protocol also significantly reduces troponin-t release after CABG, further showing that IP in cardiac surgery could reduce myocardial tissue damage. 6 Inspired by these results, many researchers have repeated or modified the IP protocol in cardiac surgery. Most of them reaching similar conclusions 122,152,162,259,298, though not all. 47,131 More recently the possibility of remote ischaemic preconditioning was successfully introduced in the field of cardiac surgery by Cheung. 41 In children undergoing correction of a congenital cardiac defect postoperative troponin-i release could be reduced by 4 episodes of 5 minutes ischaemia and reperfusion in a lower limb. This effect was confirmed in adult cardiac surgery using CK-MB and cardiac troponins. 96,262,279 Surprisingly, by far the largest study on the effect of remote preconditioning during CABG, could not show a beneficial effect of preconditioning on troponin release, improved haemodynamics or enhanced renal or lung protection. In this experiment over 160 patients were randomised to a double-blind, dummy controlled experiment with three cycles of 5 min upper arm ischaemia prior to CABG. 211 Luo et al. have performed two experiments in both children and adults, testing the effect of controlled reperfusion (ischaemic postconditioning). 164,165 In this protocol patients were randomised to direct removal of the aortic clamp and uninterrupted reperfusion or to 3 episodes of 30 seconds of aortic reclamping, initiated 30 seconds after original clamp removal. In both children and adults postconditioning reduced CK-MB release postoperatively. Schlensak et al., showed that mortality could be reduced from 11% to 5%, in patients undergoing urgency CABG, by controlled reperfusion compared to direct reperfusion. 237 However, as this study was a non-randomized patient-control experiment, several forms of bias could have influenced the outcomes. Moreover numerous papers have been published studying pharmacologic preconditioning in the setting of cardiac surgery. A series of different drugs have been administered to patients prior to cardiac surgery, and postoperatively myonecrosis factors were followed up. The results published were rather variable. Here we would like to summarise the most 23

26 Chapter 1 important and best studied. For example adenosine delivered intracoronary appeared to induce protection 146, whereas if it was administered intravenously 19 no effect on IR-injury could be noted. An A1 adenosine receptor agonist did not reduce injury after CABG. 259 Nitric oxide either delivered in the cardioplegia 281 or through the ventilation 79 reduced postoperative myocardial injury. Volatile anaesthetics have also been shown to be able to reduce infarct size in animal experiments. 36 In two experiments in CABG patients isoflurane reduced troponin-i and CK-MB release after CABG surgery 20,94 and improved haemodynamics. 184 However this protective effect of isoflurane could not be confirmed by all. 167 Continues administration of volatile anaesthetics (desflurane or sevoflurane) reduced post CABG troponin-i release and shortened postoperative hospital stay compared to propofol or midazolam based anaesthesia. 50 Finally, in another observational study Jacobsen studied the influence of volatile anaesthetics on IR-injury as opposed to propofol as sedative. For 10,535 patients undergoing CABG in Denmark, multiple parameters were filed in an obligatory national registry. After stratification for case load by the EuroSCORE (a validated scoring system to assess operative mortality risk), no differences were found in 30 day mortality, postoperative AMI or postoperative arrhythmias. 118 Other drugs that have been described reducing myocardial necrosis markers after CABG are bradykinin 287, levosimendan 272, and nicorandil 100. In the MEND-CABG trial, the effect of pyridoxal 5 -phosphate on inhibiting calcium influx in ischaemic cardiomyocytes via ATP (non-selective P2 receptor antagonist 212 ) and subsequent myocardial tolerance for after CABG was tested in 900 patients. Only a post-hoc analysis with stringent definitions of myocardial infarction showed a significant reduction in the composite of death and AMI. 127 Subsequently 3000 CABG patients were included in the MEND-CABG II study. However in this large clinical trial the researchers did not find any difference in postoperative death or AMI after treatment with pyridoxal 5 -phosphate. 5,258 There are several limitations to studying IR-injury in the setting of CABG. The use of extracorporal circulation induces a systemic inflammatory response, which could contribute to the occurrence of vascular plugging and endothelial dysfunction, leading to no-reflow. Moreover, surgical trauma to the heart may also increase myonecrosis factors independent from IR-injury. Ex vivo model on myocardial IR-injury In 1995 Yellon introduced an ex vivo model that measured contractile force recovery after IR-injury in human atrial tissue harvested during cardiac surgery. 282 In brief, the right atrial appendage is collected during cardiac surgery before the initiation of extracorporal circulation. An atrial trabecle with sufficient length and diameter is dissected, vertically suspended in an organ bath and linked to a force transducer. To recover from preparation the trabecle is superfused with a nutritious and well-oxygenated solution. Next the trabecle is exposed to 90 minutes of simulated ischaemia, by removing oxygen and 24

27 Ischaemia and reperfusion in the heart nutrients from the superfusate. After 90 minutes reperfusion was simulated by restoring oxygen and nutrient delivery to the trabecle by the superfusate. During both simulated ischaemia and reperfusion contractile force delivered by the trabecle is continually monitored. Contractile force recovery after simulated ischaemia and reperfusion is influenced by irreversible damage and stunning of the atrial tissue. A modified setup can be used with two trabecles measured simultaneously, one trabecle can be exposed to experimental interventions (e.g. ischaemic preconditioning, or addition of any pharmacologic agent to the superfusate) whilst the other serves as a control. 224 Studies on optimisation of prevention of IR-injury To further elucidate determinants of susceptibility to prevention of IR-injury several translational and clinical experiments have been performed. Two different experiments have studied the effect of age on susceptibility to IP induced protection against IR. Wu et al. showed in CABG patients, that IP could reduce troponin-i release postoperatively in patients younger than 68 years, whereas in patients older than 68 years no effect of IP on troponin-i was observed. 299 In the experiment of DeVan et al. middle-aged volunteers showed larger effect of IR on endothelial dysfunction compared to young volunteers. Moreover, sedentary volunteers were more susceptible to endothelial dysfunction after IR than endurance-trained participants (in both young and middle aged groups). 54 These methodological different experiments cover different aspects of the relation between age and IP. DeVan shows that age and habitual exercise influence the impact of IR on endothelial dysfunction, whereas Wu shows that with ageing efficacy of ischemic preconditioning to reduce IR-injury may decrease. Riksen et al. have shown that in volunteers with a dysfunctional gene for the enzyme adenosine mono-phosphate deaminase-1 (AMPD-1), post-occlusive reactive hyperaemia was augmented and annexin A5 targeting after voluntary ischemic exercise was reduced. AMPD-1 normally catalyses intracellular conversion of adenosine monophosphate (AMP) to inosine monophosphate (IMP), whereas this action is blocked in the 34 C>T variant. It is suggested that during ischaemia in humans with the variant allele, increased cellular levels of AMP are preferentially degraded to adenosine, which will increase tolerance to IR. 221 These results indicate that humans with the 34 C>T variant of AMPD-1 are endogenously protected to ischaemia and reperfusion injury. This observation provides a mechanism for the survival benefit that has been reported in cardiac patients with the 34 C>T variant of the AMPD-1 gene. 9 As stated above, many drugs have been found to be able to either interfere with or to induce tolerance against IR. Several experiments have used sulfonylurea derivates (K ATP -channel blockers, e.g. glibenclamide), to abrogate protection induced by for example IP. 23,161,271 A randomised trial in patients undergoing CABG, showed protection by isoflurane anaesthetics, an opener of K ATP -channels. In diabetic patients on glibenclamide, this protection was completely prevented. 74 However another retrospective cohort study 25

28 Chapter 1 could not confirm this interaction. 70 Ishihara et al. have shown that in patients with diabetes mellitus the protective effect of prodromal angina prior to STEMI was abrogated, compared to non-diabetic patients. This suggests that diabetes mellitus prevents the protective effect of IP. 114 This conclusion is supported by preclinical evidence. 176 In animal research synergistic effects of for example dipyridamole and atorvastatin on protection against IR have been published. 302 Human data on this subject however are still missing. Aim and Outline of this Thesis In this thesis, we describe a series of experiments focused on measurement of ischaemia reperfusion injury and pharmacologic modulation of IR-injury in humans in vivo. We aim to find a model to study in vivo the efficacy and mechanism of various interventions to reduce IR-injury. Moreover, we explored whether these models can predict clinical efficacy of these strategies in patients who present with cardiac ischaemia. For this purpose we investigated atorvastatin and irbesartan, both drugs with a proven benefit on IR-injury in several clinical trials. 128,202,203 In chapters 2,3 and 4 we focus on the use of annexin A5 to assess phosphatidylserine exposition in the course of IR-injury. We studied the effect of several drugs, all known to be protective against IR-injury (pharmacologic preconditioning) in previous animal and clinical experiments. In chapter 2 we tested if short-term treatment with atorvastatin reduces PS exposition after forearm ischaemia and subsequent reperfusion. Atorvastatin, a widely used drug in patients with cardiovascular disease, has been proven to prevent IR-injury in multiple relevant settings (e.g. patients undergoing PCI, either elective 202 or in the setting of an ACS 203 ). As atorvastatin has previously been shown to prevent myocardial damage, we tested whether prevention of PS exposition is a part of the protective mechanism. This experiment used a crossover design, including an extra control group randomised to placebo treatment twice. This control group allowed us to calculate within-individual correlation, thereby further quantifying the reproducibility of annexin scintigraphy after IR-injury. In chapter 3 we studied the effect of a 7-day treatment with the angiotensin receptor blocker (ARB) irbesartan on PS exposition after IR-injury. Despite the preclinical benefits 39,73 angiotensin II type 1 receptor antagonists seem to enhance rather than reduce morbidity and mortality after myocardial infarction compared to angiotensin converting enzyme inhibitors. 57 This may result from unopposed angiotensin II type 2 receptor stimulation, which is associated with enhanced apoptotic cell death and increased infarct size. We studied whether the clinical efficacy of irbesartan is hampered by enhanced apoptotic activity, detected by exposition of phosphatidylserines, during ischaemia and reperfusion in humans in vivo. 26

29 Ischaemia and reperfusion in the heart Our research group has shown previously that a 7-day treatment with dipyridamole reduces annexin A5 targeting after voluntary forearm IR-injury. However, this study was neither blinded nor randomised, and therefore the conclusions have to be considered carefully. 220 To test our hypothesis more rigorously, we decided to readdress the protective effects of dipyridamole against IR in chapter 4 using a randomised double-blind placebocontrolled design. Moreover we further examined the mechanism of protection. To address a possible role of extended nucleoside transport inhibition in the apparent long-term action of dipyridamole, we investigated the 4-week post-treatment effect of a 7-day oral dipyridamole administration on nucleoside transport ex vivo in human erythrocytes. In chapters 5,6 and 7 we used two techniques for the assessment of endothelial function to study the effects of ischaemia and reperfusion injury. Ischaemia and reperfusion reduces endothelial function as assessed by flow-mediated dilatation (FMD). We have previously shown that one week treatment with rosuvastatin improves formation of adenosine in the forearm and increases forearm post-occlusive reactive hyperaemia (PORH). 172 In chapter 5 we further clarified the mechanism behind this effect of rosuvastatin. We have assessed the activity of the enzyme ecto-5 -nucleotidase, which dephosphorylates extracellular AMP, as a potential source of extracellular adenosine. Moreover we investigated the role of adenosine receptor stimulation in rosuvastatin-induced augmentation of PORH. We hypothesized that an adenosine dependent reaction should be abolished by administering caffeine, a potent adenosine receptor antagonist. In chapter 6 we used FMD to compare the effect of two different statins (the lipophilic atorvastatin vs. the hydrophilic rosuvastatin) on their ability to prevent endothelial dysfunction of the human brachial artery induced by IR. Interestingly, hydrophilic and lipophilic statins appear to differ in their effects on patients. Lipophilic statins have been reported to render endothelial and vascular smooth muscle cells more susceptible to apoptosis than hydrophilic statins. 130 Moreover hydrophilic statins have been suggested to be superior in reducing event rate after a primary acute coronary syndrome in normocholesterolaemic patients. 233 Thus, lipophilicity of statins may influence their ability to reduce IR-injury. FMD to assess IR-induced endothelial dysfunction is either measured in the brachial or radial artery. To date, little is known about methodological aspects of this technique. For example, regional heterogeneity in wall architecture 268 and vascular function 265, may alter the impact of IR-injury on endothelial function between these vascular beds. In chapter 7 we examined the difference in endothelial tolerance to IR-injury between the radial and brachial artery, by measuring the effect of IR simultaneously in both arteries ipsilaterally. 27

30 Chapter 1 Abbreviations ACE Angiotensin II converting enzyme ACS Acute coronary syndrome ADP Adenosine diphosphate AMI Acute myocardial infarction AMP Adenosine monophosphate AMPD Adenosine monophosphate deaminase ARA Angiotensin II receptor antagonist ARB Angiotensin II receptor blocker AT 1 Angiotensin II receptor type 1 AT 2 Angiotensin II receptor type 2 ATP Adenosine triphosphate CABG Coronary arterial bypass grafting CD73 Ecto-5 -nucleotidase CV Coefficient of variance CT Computer tomography ECG Electrocardiogram FBF Forearm blood flow FMD Flow mediated dilation HDL High-density lipoprotein IP Ischaemic preconditioning IR Ischaemia and reperfusion IRI Ischaemia and reperfusion injury LAD Left anterior descending coronary artery LDL Low-density lipoprotein MACE Major adverse cardiac event MPTP Mitochondrial permeability transition pore MRI Magnetic resonance imaging NO Nitric Oxide NonSTEMI Non ST elevated myocardial infarction PCI Percutaneous coronary intervention PORH Postocclusive reactive hyperaemia PS Phosphatidylserine RNA Ribonucleic acid RIPC Remote ischaemic preconditioning ROS Reactive oxygen species STEMI ST Elevated myocardial infarction SWOP Second window of protection UV Ultraviolet VF Ventricular fibrillation 28

31 Ischaemia and reperfusion in the heart 29

32

33 Chapter 2 Atorvastatin does not affect ischaemia-induced phosphatidylserine exposition in humans in vivo Constantijn Wouters Patrick Meijer Carola Janssen Geert Frederix Wim Oyen Otto Boerman Paul Smits Gerard Rongen Journal of Atherosclerosis and Thrombosis 2012; 19:

34 Chapter 2 Abstract Aim: Statins can induce pharmacologic preconditioning and thereby reduce infarct size. Cellular phosphatidylserine (PS) exposition occurs in the course of ischaemia and reperfusion and has been associated with IR-injury. In this experiment we studied the effect of atorvastatin on PS exposition after a standardised ischaemia and reperfusion challenge. Methods: In a double-blind randomised cross-over trial 30 healthy volunteers were allocated to 3 day treatment with atorvastatin (80mg/day) and placebo (n=24), or placebo treatment twice (n=6). At the end of each treatment period, volunteers underwent 10 minutes of forearm ischaemic exercise. At reperfusion radiolabeled annexin A5 was administered intravenously and Gamma camera imaging of both hands was performed 1 and 4 hours after reperfusion. Results: Annexin A5 targeting was not different between atorvastatin treatment (26.1 ±9.8% and 24.0 ±9.5% respectively at 1 and 4 hours after reperfusion) and placebo treatment (25.6 ±11.0% and 24.5 ±10.7%) (p=0.99). Our time control experiment did not reveal a carry-over effect. Conclusions: Our results show that treatment with atorvastatin 80mg does not reduce forearm PS exposition after ischaemic exercise. This suggests that the role of PS exposure in the prevention of ischaemia and reperfusion injury by short term treatment with atorvastatin is limited. 32

35 Atorvastatin and phosphatidylserine exposition Introduction The cholesterol lowering action of statins (HMG-coenzyme A reductase inhibitors) and its associated reduction in cardiovascular events have been well documented in numerous clinical trials. 150,210,230,231 Traditionally, this beneficial effect of statins has been explained by lowering of plasma cholesterol and subsequent reduced progression of atherosclerosis. Apart from its cholesterol lowering properties, statins have other ( pleiotropic ) actions, such as upregulation of ecto-5 -nucleotidase 145,198,234, activation of the phosphatidylinositol 3-kinase-Akt pathway 274 and up-regulation of NO synthase 106,138 which all may increase tolerance against ischaemia and reperfusion (IR). 22,32,229 Several animal studies have shown reduction of IR-injury as a result of statin treatment in both the heart and the kidney. 89,101,126,306 This putative protection may explain the clinical observation that early initiation of statin treatment in patients with an acute cardiovascular event improves long-term prognosis as compared with a delayed start of this treatment. 49,60,242 Furthermore in two randomised clinical trials one day treatment with atorvastatin prior to percutaneous coronary intervention reduced myocardial injury and improved clinical outcomes in patients with acute coronary syndromes. 55,203 Ischaemia induced phosphatidylserine (PS) exposure on cardiomyocytes has been associated with reversible cellular damage and apoptosis. 189,269 Treatment to shield exposed PS with diannexin, a homodimer of human annexin A5 (with high affinity for exposed phosphatidylserines) significantly reduces IR-injury in the liver and kidney. 260,290 Likewise, diannexin prevents no-reflow in a rabbit cardiac ischaemia-reperfusion model. 91 These observations suggest a critical role for exposed PS in the development of IR-injury. We have developed an experimental model to study PS exposure after IR in healthy volunteers. Our model uses radiolabeled recombinant annexin A5 to visualise PS-exposition that occurs after 10 minutes of forearm ischaemia combined with isometric exercise. 226 Interventions that have been shown in preclinical and clinical research to modulate myocardial infarct size (ischaemic preconditioning, and administration of adenosine, dipyridamole, and caffeine) similarly modulate annexin A5 targeting in our forearm model, further supporting a role for PS-exposure in IR-injury. 220,224,226 Using annexin A5 scintigraphy after voluntary ischaemic exercise in healthy males, we have recently shown that annexin A5 targeting after a short pretreatment with rosuvastatin 20 mg once daily was significantly reduced (16 ±1% and 18 ±2% respectively at 1 and 4 hours after reperfusion) compared to placebo treatment (21 ±3% and 25 ±3%) (p<0.05). 172 As previously reported for other statins in preclinical research with infarct size as endpoint, this effect of rosuvastatin was inhibited by caffeine, an adenosine receptor antagonist. 172 There is ample evidence from animal and clinical research that treatment for three days with high dose atorvastatin protects against IR-injury. 13,27,89,202,203 However, we do not know whether PS exposure relates to this clinical benefit of atorvastatin, as differences have been observed in the efficacy of various statins to inhibit HMG-CoA-reductase 188 and to induce pleiotropic effects

36 Chapter 2 Aim We aim to assess the role of PS-modulation in the beneficial effect of atorvastatin on IR-injury. We tested the hypothesis that a short pretreatment with atorvastatin 80 mg reduces annexin A5 targeting in our forearm IR-model. Methods After approval of the protocol (NCT ) by the Institutional Review Board of the Radboud University Nijmegen Medical Centre and in accordance with the declaration of Helsinki (2008), 30 healthy male volunteers (age years) signed informed consent. All participants underwent medical screening to exclude cardiovascular disease, hypercholesterolaemia, hypertension, diabetes mellitus, and impaired renal function. Furthermore serum values of creatine kinase (CK) and alanine aminotransferase (ALT) were measured to exclude participants possibly at risk for adverse events due to atorvastatin use (rhabdomyolysis and toxic hepatitis). In this double blind randomised crossover trial (figure 1) participants were allocated to treatment with either atorvastatin and placebo (n=24), or placebo treatment twice (n=6). The latter group served as a time-control. Furthermore, it allowed us to verify our assumptions on within-subject variability and test-to-test correlation of our annexin A5 scans. Placebo or atorvastatin (80 mg) was administered for three days, and at least 4 weeks were allowed between the two treatment periods as a washout. Atorvastatin 80mg (Pfizer, Capelle a/d IJssel, The Netherlands) was capsulated to match placebo by the department of Clinical Pharmacy (Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands according to GMP standards). In both treatment periods the first tablet was administered under supervision of the investigator, after venous blood sampling to determine fasting lipid profile. The participant was instructed to take the second capsule in the morning and to abstain from caffeine during the last 24 hours prior to ischaemic exercise. On day 3 after an overnight fast the last tablet was administered under supervision, again after assessment of fasting lipid profile and serum caffeine level to verify protocol compliance. Sixty minutes after administration of the study medication in all participants maximal voluntary contraction force of the non-dominant forearm was measured with an isometric handgrip dynamometer. Subsequently, the circulation of the non-dominant forearm was occluded for 10 minutes with an upper arm cuff inflated to 200mmHg. Directly after occlusion volunteers were asked to perform isometric contractions of the finger flexors at 50% of their maximum contraction force. These contractions were performed rhythmically with 5 seconds of contraction followed by 5 seconds of relaxation until exhaustion. The total duration of ischaemia was 10 minutes, independent of the duration of contractions. Immediately upon reperfusion 100 microgram recombinant human annexin A5, radio- 34

37 Atorvastatin and phosphatidylserine exposition Atorvastatin Placebo n=12 n=30 n=12 Placebo Atorvastatin n=6 Placebo Placebo IR-injury and Tc-99m annexin A5 scintigraphy Figure 1 Study protocol From the 30 participants 12 were randomised to 3 days of atorvastatin treatment followed by three days of placebo treatment. 12 Participants were randomised to first 3 days of placebo treatment and next 3 days atorvastatin treatment, and 6 participants were randomised to placebo treatment twice. At day three of treatment participants performed 10 minutes of ischaemic forearm exercise. Upon reperfusion Tc-99m labelled annexin A5 was administered and scintigraphy was performed 1 and 4 hours after reperfusion. Between both treatment periods a 4-week interval was observed. labeled with 450 MBq Technetium-99m was administered intravenously into the dominant arm. One and four hours after this administration both hands were scanned simultaneously by use of a gamma camera (Orbiter camera equipped with low-energy high-resolution collimators; Siemens, Hoffman Estates, Ill, USA) as previously described. 226 Caffeine plasma concentrations were determined using reversed-phase HPLC with UV detection set at 273nm. 240 Serum lipid profile (total-, HDL- and LDL-cholesterol and triglycerides), creatine kinase and alaninine amino transferase were determined with a commercially available kit (Aeroset, Abbott, Santa Clara, Cal., USA). Data analysis All the digitised gamma camera images were analysed offline by the same investigator (W. J.O.) using Hermes software (Nuclear Diagnostics, Stockholm, Sweden). A predefined region of interest was placed over the thenar muscle of both hands. Annexin A5 targeting was expressed as the percentage difference in counts/pixel between the experimental (non-dominant) hand and the control hand as previously described

38 Chapter 2 Statistical analysis Values are expressed as mean ± standard error (SE) unless indicated otherwise. This study is the first to use our forearm IR-model in a paired set-up in healthy volunteers. Based on previous studies, a between subject SD of 10% was predicted. 220,224,226 Conservatively assuming a correlation coefficient of 0.5 between first and second paired observation, we calculated that with an alpha of 0.05 and a power of 90%, using 24 subjects allows us to find a difference in annexin A5 targeting of 7%. This is well in the range of our previous observation after treatment with rosuvastatin. 172 Furthermore 6 additional participants were randomised (in a double-blind fashion) to receive placebo treatment twice to serve as time-control experiment. This time-control allows us to verify our assumptions regarding this power analysis afterwards. The effect of atorvastatin treatment on annexin A5 targeting and lipid profile was tested using ANOVA for repeated measurements. All statistical analyses were performed using SPSS 16.0 for Windows. Results Baseline characteristics of all participants are presented in table 1. Caffeine levels were all under 0.85 mg/l, indicating satisfactory compliance to the 24 hours caffeine abstinence. The annexin A5 targeting was not different between atorvastatin treatment (26.1 ±2.0% and 24.0 ±1.9% respectively at 1 and 4 hours after reperfusion) and placebo treatment (25.6 ±2.4% and 24.5 ±2.2%) (p=0.999) (figure 2). Three-day treatment with atorvastatin significantly reduced total cholesterol (from 4.15 ±0.10mmol/L to 3.44 ±0.10mmol/L) and LDL cholesterol (from 2.31 ±0.10mmol/L to 1.84± 0.10mmol/L as compared to placebo treatment; p<0.001 for both comparisons). HDL cholesterol and triglyceride levels were unaffected by atorvastatin (from 1.22 ±0.04 to 1.25 ±0.05 for HDL-C, and from 0.93 ±0.08 to 0.80 ±0.09 for triglycerides). Workload (50% of the maximal strength * duration of ischaemic exercise) did not significantly differ between both study days (4260 ±284kg*sec and 4253 ±219kg*sec after atorvastatin and placebo respectively (p=0.96), indicating that potential differences in ischaemic challenge did not confound the observed lack of effect on annexin A5 targeting. In the time control group (n=6) the annexin A5 targeting after the first visit was 23.5 ±5.5% and 23.2 ±6.5% (respectively at 1 and 4 hours after reperfusion) and 30.2 ±6.1% and 20.3 ±7.8% after the second visit (figure 3). The between-subject standard deviations in annexin A5 targeting were 13.5%, 15.8%, 15.0% and 9.3% at 1 and 4 hours for visit 1 and 2 respectively. Within subject correlation was 0.93 and 0.79 at 1 and 4 hours respectively. This reproducibility should have allowed us to detect a difference in targeting between placebo and atorvastatin of at least 7%, closely resembling our initial power calculation. 36

39 Atorvastatin and phosphatidylserine exposition Table 1 Baseline characteristics of all participants (n=30) Mean ±S.D. Age (years) 23.3 ±6.6 BMI (kg/m 2 ) 22.4 ±2.4 Systolic blood pressure (mmhg) 126 ±8 Diastolic blood pressure (mmhg) 71 ±10 Heart rate (bpm) 62 ±8 Glucose (mmol/l) 4.6 ±0.7 Total cholesterol (mmol/l) 4.0 ±0.6 Triglycerides (mmol/l) 1.19 ±0.94 HDL-C (mmol/l) 1.33 ±0.26 LDL-C (mmol/l) 2.19 ± Annexin A5 targeting (%) Hours after reperfusion Figure 2 Annexin A5 targeting 1 and 4 hours after reperfusion, after placebo (open bars) and atorvastatin (closed bars) treatment Bars represent mean ± SE. 37

40 Chapter 2 40 Annexin A5 targeting (%) Hours after reperfusion Figure 3 Annexin A5 targeting 1 and 4 hours after reperfusion in time-control group (placebo treatment twice), after visit 1 (open bars) and visit 2 (closed bars). Bars represent mean ± SE. Discussion The novel finding of this study is the lack of effect of atorvastatin on annexin A5 targeting after voluntary ischaemic exercise. This indicates that in our forearm model treatment with atorvastatin, in a dose and duration as previously used in clinical trials with patients who present with coronary syndromes 55,202,203, does not modulate PS exposition after IR, in contrast to treatment with rosuvastatin, as reported recently. 172 As the protective action of atorvastatin in a setting of ischaemia and reperfusion has been convincingly shown in multiple animal experiments 13,27 and even in several clinical trials 42,55,203, we conclude that PS modulation is not likely involved in the protective mechanism of atorvastatin. During reperfusion after an ischaemic event, multiple detrimental processes take place simultaneously, including formation of radical oxygen species (ROS), cellular and mitochondrial calcium overload, and a rapid increase (restoration) in ph. 305 These actions result in mitochondrial damage caused by opening of the mitochondrial permeability transition pore (MPTP), activation of caspases and subsequent cell damage and possible cell death (by either necrosis or apoptosis)

41 Atorvastatin and phosphatidylserine exposition PS exposition is recognised to be part of the signalling cascade in the course of apoptosis. 170 It attracts phagocytes which remove the dying cell before its remnants evoke an inflammatory response. 72 The importance of PS exposition in developing tissue damage after IR-injury was clearly demonstrated in several experiments where IR-injury could be reduced by shielding of PS by diannexin treatment. 246,260,290 The time-control experiment excluded a carry-over effect as a potential reason for the lack of observed effect of atorvastatin on annexin A5 targeting. In addition, inter-individual SD and within-subject correlation resulted in a power to detect differences between placebo and atorvastatin that resembles the results of our initial power calculation. In fact, based on these measurements we could calculate a minimal detectable difference of less than 10%, which is well below the previously observed effect of rosuvastatin on annexin A5 targeting. Thus, our sample size was sufficient to detect the effect size as expected from our hypothesis and previous observation with rosuvastatin. The (cardio)protective mechanism of statins is well investigated, though its mechanism has not been fully elucidated. 163 Adenosine appears to play an important role in initiating the cascade (adenosine dependent phosphorylation of ERK1/2, Akt and enos) leading to a protective phenotype after statin therapy. 175 Upregulation of ecto-5 -nucleotidase has been shown in vitro and in animals in vivo for various statins. 197 In humans in vivo however, this effect has only been explored for rosuvastatin, which increases ecto-5 -nucleotidase activity with 50%. 173 In our rosuvastatin trial participants were treated 7 days 172, whereas in this atorvastatin trial participants were treated for 3 days. However we doubt that treatment duration could account for the difference in annexin A5 targeting in these experiments. We have previously shown in humans that activation of ecto-5 -nucleotidase mediates prevention of PS exposure by rosuvastatin after IR. The effect of statins on ecto-5 -nucleotidase appears to occur rapidly. 145,234 For example Sanada et al. found a significant increase of ecto-5 -nucleotidase activity in dogs for different statins (pravastatin, pitavastatin and cerivastatin) already 10 minutes after treatment. 234 Thus, our discrepant results between rosuvastatin and atorvastatin are not likely explained by differences in treatment duration but rather reflect a difference in pharmacodynamics between these two statins. There are significant differences between several statins in their efficacy to lower LDL cholesterol. 125 Moreover, pleiotropic effects have been shown to differ between lipophilic statins (e.g. atorvastatin) and hydrophilic statins (e.g. rosuvastatin). Lipophilic statins have been reported to render endothelial and vascular smooth muscle cells more susceptible to apoptosis than hydrophilic statins. 130 In humans rosuvastatin inhibits Rho/Rho kinase activity to a greater extent than atorvastatin at a dose that equally reduced LDL cholesterol. 213 Moreover hydrophilic statins have been suggested to be superior in reducing event rate after a primary acute coronary syndrome in normocholesterolaemic patients

42 Chapter 2 Conclusion Our results show that treatment with atorvastatin 80mg does not reduce forearm PS exposition after ischaemic exercise. This suggests that the role of PS exposure in the prevention of ischaemia and reperfusion injury by short-term treatment with atorvastatin is limited. 40

43 Atorvastatin and phosphatidylserine exposition 41

44

45 Chapter 3 Angiotensin II type 1 receptor blockade does not enhance apoptotic cell death during ischaemia and reperfusion in humans in vivo Patrick Meijer Constantijn Wouters Wim Oyen Otto Boerman Gertjan Scheffer Paul Smits Gerard Rongen Journal of Cardiovascular Pharmacology 2011; 57:

46 Chapter 3 Abstract Despite the theoretical benefits angiotensin II type 1 receptor antagonists seem to enhance rather than reduce morbidity and mortality after myocardial infarction compared with angiotensin converting enzyme inhibitors. This may result from unopposed angiotensin II type 2 receptor stimulation, which is associated with enhanced apoptotic cell death and increased infarct size. We studied whether the clinical effectiveness of irbesartan is hampered by enhanced apoptotic activity, detected by exposition of phosphatidylserines, during ischaemia and reperfusion in humans in vivo. Twenty healthy male volunteers were randomized to a 1-week treatment with irbesartan (300 mg/d) or placebo in a double-blind fashion. After treatment, all participants underwent 10 minutes of ischaemic exercise of the non-dominant forearm. Upon reperfusion Tc-99m-labeled Annexin A5 was administered and 1 and 4 hours afterward, both hands were scanned using a gamma camera. Targeting of annexin A5, expressed as the percentage difference in radioactivity in the area of interest (thenar muscle) between experimental and control hand, did not differ between participants treated with irbesartan or placebo. Therefore irbesartan does not enhance phosphatidylserine exposition in humans in vivo. The results of this study do not support enhanced apoptotic activity after treatment with irbesartan in a setting of ischaemia and reperfusion. 44

47 Irbesartan and apoptotic cell death during IRI Introduction Angiotensin II functions through 2 receptor subtypes: angiotensin II type 1 (AT 1 ) and angiotensin II type 2 (AT 2 ). Most of the well-known physiological effects of angiotensin II are mediated via the AT 1 receptor, which is widely expressed by most cell types. 77 After myocardial infarction, the rise of angiotensin II levels results in AT 1 receptor stimulation, subsequent vasoconstriction, increased cardiac contractility and cellular proliferation may all worsen the outcome. Indeed, the inhibition of the formation of angiotensin II by angiotensin-converting enzyme (ACE) inhibitors improves survival after acute myocardial infarction. 1 AT 1 receptor antagonists (ARAs) are suggested to have, at least, similar benefits compared with ACE inhibitors as they provide a more intense and sustained inhibition of AT 1 receptor stimulation. Several preclinical studies demonstrated an infarct size limiting effect of ARAs after coronary artery occlusion in a variety of species. 127,243,247 Unexpectedly the optimal trial in myocardial infarction with the ARA losartan (OPTIMAAL) demonstrated an increase in cardiovascular mortality of 17% when treated with losartan compared with when treated with captopril. 57 This impaired clinical effectiveness after acute ischaemic events may result from a difference in ancillary properties. ACE inhibitors inhibit the breakdown of bradykinin, which has additional benefits during ischaemia and reperfusion. 84,241 In contrast ARAs facilitate unopposed AT 2 receptor stimulation. During normal conditions, the expression of the AT 2 receptor is limited and occurs predominantly in foetal tissue where it influences development. 1 However, in response to pathological conditions, including vascular injury, hypertension, myocardial infarction, and congestive heart failure, AT 2 receptor expression is enhanced and may become functionally relevant. 45,186,190,193,235,310 Although originally believed to form a counter regulatory mechanism against the detrimental effects of AT 1 receptor stimulation, it is now apparent that AT 2 receptor stimulation promotes apoptosis. 284,300 Apoptotic cell death plays an important role in the extent of myocardial infarction as shown by the observations that interference in the apoptotic cell death pathway results in an infarct size reduction of 48 to 64%. 35,40,147 Thus, ARAs may facilitate AT 2 receptor stimulation, which enhances myocardial infarct size and worsen outcome following myocardial infarction. Indeed, the inhibition of AT 1 receptor activity promotes apoptosis and increases infarct size whereas AT 2 receptor antagonists improve functional recovery and reduce infarct size after coronary artery occlusion. 39,73,141,284 In summary, clinical data raise concern about the use of ARAs in acute myocardial infarction as it seems to have less beneficial effects in comparison with ACE inhibition. This may result from a reduced clinical efficacy of ARAs as unopposed AT 2 receptor stimulation may increase apoptotic cell death. Due to the undisputed beneficial properties of ACE inhibitors, clinical trials comparing placebo with ARAs in a setting of ischaemia and reperfusion are missing. In addition, preclinical studies addressing the effects of ARAs on ischaemia reperfusion injury (IRI) are equivocal. This mandates human in vivo experiments 45

48 Chapter 3 for proper translation into clinical practice. We developed a human in vivo ischaemiareperfusion model, which facilitates the comparison between placebo and ARAs in a human in vivo setting. 226 This model combines annexin A5 scintigraphy and ischaemic forearm exercise. Annexin A5 scintigraphy detects the loss of membrane asymmetry, which results in phosphatidylserine (PS) exposure on the outer membrane leaflet and occurs shortly after an ischaemic insult as an early, reversible, sign of apoptosis. 93,139 Radiolabeled recombinant annexin A5 binds with high affinity to PS located on the outer leaflet of the cell membrane and can be visualized by annexin A5 scintigraphy. This technique has been applied in the human heart in which it demonstrated the presence of PS exposition after acute myocardial infarction supporting the use of our model as a surrogate end point. 109 The aim of this study is to test the hypothesis that a 1-week treatment with irbesartan augments PS exposure in human forearm skeletal muscle after ischaemic exercise. To exclude possible confounding coexisting morbidity, including hypertension, this study was performed in healthy normotensive volunteers. Methods Subjects The study was performed in accordance with the declaration of Helsinki (2000) of the world medical association and approved by the Institutional Review Board of the Radboud University Nijmegen Medical Centre. Twenty male volunteers without cardiovascular disease, hypertension [Blood pressure (BP) > 140/90 mmhg], hypercholesterolaemia (fasting total cholesterol > 6 mmol/l), reduced kidney function, or diabetes mellitus (fasting glucose > 7.0 mmol/l) signed informed consent for participation. Study protocol Volunteers were randomly divided in 2 groups (n=10 in each group) to receive double blind either a 7-day treatment with placebo (Department of Clinical Pharmacy, Radboud University Nijmegen Medical Centre) or irbesartan (300 mg/d, Sanofi-Aventis, Gouda, The Netherlands). Before the first and last ingestions of the study drug, blood was drawn to determine plasma renin, creatinine, sodium, potassium, and caffeine (only day 7). On the first and sixth study days, the urine produced in 24 hours was collected for analysis of sodium and creatinine excretion. On the seventh day, volunteers entered our research centre after an overnight fast and 24 hours of caffeine abstinence. Subsequently, the last tablet was ingested followed 1 hour later by an ischaemic exercise protocol of the non-dominant forearm. Immediately, on reperfusion Tc-99m-labeled (0.1 mg, 400 MBq) annexin A5 was injected into an antecubital vein of the dominant arm. Annexin A5 targeting was recorded by imaging with a gamma camera using the dominant hand as an internal control. 46

49 Irbesartan and apoptotic cell death during IRI Ischaemic exercise and annexin A5 imaging All procedures were performed as described before. 226 In brief, maximal voluntary contraction was determined in the non-dominant hand with a handgrip dynamometer. The circulation of the non-dominant forearm was occluded for 10 minutes by inflation of an upper arm cuff to 200mmHg. Immediately after occlusion, the volunteers performed rhythmic isometric hand contractions at 50% of their maximal voluntary force for 5 seconds every 10-second period until exhaustion. After 10 minutes, the upper arm cuff was deflated and reperfusion started. Both hands were imaged at 1 and 4 hours of reperfusion by using a gamma camera (Siemens Orbiter, Hoffman Estates, IL) connected to a Hermes Gold image processing system (Nuclear Diagnostics, Stockholm, Sweden). Recombinant human annexin A5 was obtained from Theseus Imaging Corporation and conjugated with succininydylhydrazinonicotinamide under good manufacturing practice conditions (Department of Clinical Pharmacy, Radboud University Nijmegen Medical Centre). Labelling of this conjugated annexin with Tc-99m was performed in the morning of the administration (Department of Nuclear Medicine, Radboud University Nijmegen Medical Centre) as previously described. 224 Laboratory analysis Plasma caffeine concentrations were determined by use of reversed-phase high-performance liquid chromatography with UV detection set at 273 nm according to Schreiber- Deturmeny and Bruguerolle. 240 Plasma renin concentrations were determined by immunoradiometric assay (Cisbio Bioassays, Bagnols-sur-Cèze Cedex, France). Statistical analysis All gamma camera images were analysed by the same blinded investigator (W.O.). A predefined region of interest was drawn on each hand representing the thenar muscle. Within this region of interest, the annexin A5 targeting was expressed as counts per pixel. The effect of ischaemic exercise is presented as the percentage difference in annexin A5 targeting between the experimental and control hand as previously described. 226 The effect of irbesartan on annexin A5 targeting was analysed with an analysis of covariance for repeated measures with treatment as the between-subject factor and workload as a covariate. Workload was defined as the product of 50% of the maximal voluntary force and duration of ischaemic exercise. Statistical analysis was performed using the SPSS software package (version 16.0). Results are expressed as mean ± SE. 47

50 Chapter 3 Results Our placebo and active treatment group did not significantly differ at baseline with respect to age, BP, concentrations of plasma renin nor total and fractional sodium excretion (Table 1). Plasma caffeine concentrations at the start of the experiment on day 7 were all below 0.8 mg/l indicating adequate compliance to the caffeine-free diet. In the placebo group, no changes in plasma or urine parameters were observed after treatment. In contrast, a 1-week treatment with irbesartan increased circulating renin concentrations from 14 ±3 to 86 ±16 meq/l indicating compliance to the study drug and adequate interruption of AT II subtype 1 receptor-mediated feedback inhibition of circulating renin. No significant effect was observed on total and fractional sodium excretion (Table 2). Annexin A5 Scintigraphy There was no significant difference between the workload performed by the volunteers between the placebo and irbesartan group (4389 ±304 vs ±222, p = 0.4, independent t-test). Treatment with Irbesartan did not result in enhanced annexin A5 targeting after 1 and 4 hours of reperfusion (p = 0.9). This result was unaffected when correcting for delivered workload (p = 0.8, Fig. 1). 48

51 Irbesartan and apoptotic cell death during IRI Table 1 Baseline characteristics Placebo (n=10) Irbesartan (n=10) Age (yr) 29 ±11 29 ±13 Systolic BP (mmhg) 124 ± ±9 Diastolic BP (mmhg) 79 ±8 76 ±9 Total cholesterol (mmol/l) 4.7 ± ±0.8 Glucose (mmol/l) 4.4 ± ±0.6 Plasma creatinine (µmol/l) 80 ±4 84 ±8 Values expressed as mean ±SE Table 2 Effect of treatment on plasma parameters and sodium excretion Placebo (n=10) Irbesartan (n=10) Day 1 Renin (mmol/l) 10 ±2 14 ±3 Creatinine (µmol/l) 77 ±2 83 ±3 Sodium (mmol/l) 140 ± ±0.4 Potassium (mmol/l) 3.8 ± ±0.1 Total sodium excretion (mmol) 186 ± ±28 Fractional sodium excretion (%) 0.65 ± ±0.07 Day 2 Renin (mmol/l) 13 ±3 86 ±16* Creatinine (µmol/l) 75 ±2 80 ±2 Sodium (mmol/l) 141 ± ±0.3 Potassium (mmol/l) 3.8 ± ±0.1 Total sodium excretion (mmol) 181 ± ±18 Fractional sodium excretion (%) 0.64 ± ±0.12 Values expressed as mean ±SE *p=

52 Chapter 3 Annexin A5 targeting (%) Placebo Irbesartan Time after reperfusion (hours) Figure 1 Annexin A5 targeting after ischemic exercise is presented as the percentage difference, in counts per pixel within a predefined region of interest (thenar muscle), between the experimental and control hand. In all subjects, ischemic exercise was performed after a 24-hour abstinence from caffeine-containing beverages. The subjects had been randomly allocated to a 1-week treatment with placebo or irbesartan. p = 0.8, analysis of covariance for repeated measures. Data are expressed as mean ± SE. Discussion In this study, irbesartan, in a clinically relevant dose, did not enhance PS exposition on the outer leaflet of the plasma membrane in response to an ischaemic stimulus. Therefore, our results do not support a reduction in clinical effectiveness of irbesartan as a result of increased apoptotic activity during IRI in humans in vivo. In the last 10 years, it has become apparent that apoptotic cell death plays an important role in the extent of myocardial infarction. In this light, we developed a technique that detects apoptosis by visualization of PS exposure, an early hallmark of apoptosis. Phosphatidylserines on the outer membrane leaflet provide binding sites for annexin A5 thereby enabling the visualization of PS-annexin A5 complexes by detecting Tc-99m-labeled recombinant annexin A5 with a gamma camera. Thus, annexin A5 targeting represents the amount of cells vulnerable to ischaemia and subsequently the extent of IRI. Indeed, annexin A5 targeting depends on the intensity of the ischaemic stimulus and is limited by ischaemic preconditioning, adenosine, dipyridamole, and rosuvastatin confirming the present literature. 172,220,226 In addition, shielding of PS exposition 50

53 Irbesartan and apoptotic cell death during IRI opposes IRI after liver transplantation in an experimental model underlining the clinical relevance of PS exposition. 246,260 These findings support the use of our model as a translational research tool to determine the effects of irbesartan on IRI. The results from OPTIMAAL suggest increased cardiovascular mortality after myocardial infarction when treated with an ARA compared with ACE inhibition. We therefore studied the effect of irbesartan on IRI to determine whether unopposed AT 2 receptor stimulation increases apoptotic activity during ischaemia and reperfusion in a human in vivo setting. Based on our results, we conclude that irbesartan does not enhance PS exposition and thereby apoptosis. Therefore, increased apoptotic activity does not interfere with the clinical effectiveness of ARAs during ischaemia and reperfusion. Several observations support these findings. Two trials comparing the long-term effects between ARAs and calcium channel blockers revealed no inferiority of ARAs on the reduction of cardiovascular mortality and morbidity. 129,192,286 In addition, the valsartan in acute myocardial infarction trial demonstrated comparable effects on cardiovascular mortality in patients treated with valsartan or captopril after myocardial infarction complicated by ventricular dysfunction. 205 Interestingly, approximately 40% of patients treated with the ARA in the valsartan in acute myocardial infarction trial received non-study treatment with ACE inhibitors during an average of 5 days after myocardial infarction compared with none in OPTIMAAL. This supports the presence of an additional beneficial effect of ACE inhibitors over ARAs. Indeed, ACE inhibitors administered during acute myocardial infarction within 0-36 hours from the onset of symptoms reduces 30-day mortality by 7% compared with treatment with placebo. 1 More than 80% of this effect was reached within the first week of treatment supporting an early effect on myocardial salvage. Preclinical studies confirm this direct infarct size limiting effects of ACE inhibitors, which is independent of inhibition of the renin-angiotensin system but rather reflects the associated reduction of bradykinin degradation. 177 Bradykinin increases the cellular tolerance against IRI and thereby reduces infarct size after coronary artery occlusion. 84,241 ARAs do not reduce bradykinin degradation but instead facilitate unopposed AT 2 receptor stimulation, which is associated with increased apoptosis and enhanced IRI. 284 However, preclinical studies addressing the effects of ARAs on IRI have been equivocal as results cover the entire range from beneficial to detrimental effects. 56,73,127,217,243,247,253 This may represent differences in study drug, dosing regimens, ischaemia-reperfusion protocols, and species studied. This study in a human in vivo setting does not support a modulating effect of ARAs on IRI in humans in vivo. The results obtained in OPTIMAAL therefore do not seem to result from a detrimental effect of unopposed AT 2 receptor stimulation but may rather reflect the beneficial ancillary effect of ACE inhibitors. This latter statement will need further confirmation in studies addressing the effect of ACE inhibition on IRI in humans in vivo. Potential limitations of our study design need to be addressed. First, we assessed IRI in forearm skeletal muscle and not in the myocardium. However, we do know that the 51

54 Chapter 3 skeletal muscle of the human forearm reacts in a similar fashion to ischaemia and reperfusion as myocardial tissue. In both organs, the extent of injury depends on the intensity of the ischaemic insult and protective strategies applied in human myocardial tissue including ischaemic preconditioning 122,303, and statin therapy 203,239 provides similar protection in the human forearm. 156,159,172,226 Second, we used healthy volunteers instead of patients with an increased cardiovascular risk profile. This method excludes possible coexisting morbidity but may hamper the translation of our results to the patient at risk for IRI. Indeed, the effects of ARAs on IRI may depend on health status and age as AT 2 receptor up-regulation occurs during pathological conditions and advancing age. 15 In contrast, our study design is in line with those of the vast majority of experiments performed with ARAs in the preclinical setting in which young healthy adult animals were studied and effects on IRI were observed albeit equivocally. Nevertheless, we cannot exclude an effect of irbesartan on IRI in the patient population. Conclusions A 1-week treatment with irbesartan does not enhance PS exposition in response to an ischaemic stimulus in healthy volunteers. Thus, increased apoptotic activity does not reduce the clinical effectiveness of irbesartan in a setting of ischaemia and reperfusion. 52

55 Irbesartan and apoptotic cell death during IRI 53

56

57 Chapter 4 Dipyridamole provides prolonged protection against ischaemia and reperfusion injury in forearm skeletal muscles in humans in vivo Constantijn Wouters Patrick Meijer Geert Frederix Otto Boerman Paul Smits Wim Oyen Gerard Rongen Submitted

58 Chapter 4 Abstract Pharmacological protection against ischaemia and reperfusion could benefit patients with coronary artery disease. We tested whether dipyridamole, an adenosine uptake inhibitor, could provide such protection. Ten healthy men were treated in a double-blind randomised crossover design with 2x 200mg dipyridamole SR and matching placebo for 7 days (minimal wash-out period 3 weeks). After treatment volunteers performed 10 minutes of forearm ischaemic exercise. Subsequently, Tc-99m annexin A5 scintigraphy was used to measure IR-injury 1 and 4 hours after reperfusion. Dipyridamole reduced annexin A5 targeting in those volunteers who received placebo first and dipyridamole next (from 27±14% and 30±16% (1 and 4 hours after reperfusion) to 15±9% and 16±10%), compared to those receiving dipyridamole first and placebo next (from 17±13% and 19±6% to 20±14% and 19±6%; p=0.029). One week and 4 weeks after cessation of therapy inhibition of nucleoside transport was no longer observed (-3.8 ±5.7% and -5.2 ±6.2%; p=0.71 and 0.61 respectively). Dipyridamole treatment protects against IR in forearm skeletal muscle of healthy volunteers. Considering the washout period, this effect appears to persist over 4 weeks. A prolonged effect of dipyridamole on nucleoside transport was ruled out as possible explanation of this protective action of dipyridamole. 56

59 Dipyridamole protects against IRI Introduction Atherosclerosis and its associated organ perfusion disturbances are a main cause of death worldwide. Currently, secondary prevention of cardiovascular disease focuses on prevention of new events. However, emerging preclinical and clinical evidence suggests that improvement of tissue tolerance to ischaemia and reperfusion (IR) injury should be prioritised, in particular in the current era of rapid reperfusion therapy. 305 Clinical evidence in the setting of both coronary bypass surgery and percutaneous coronary interventions indicates that reperfusion injury limits the benefit of early reperfusion and that tolerance to ischaemia and reperfusion can be improved pharmacologically. 146,203 Ideally, drugs that improve tolerance to IR should also prevent events. Dipyridamole has such a beneficial profile, as it reduces cerebrovascular events 92 and improves tolerance against IR in animals. 29,257 Presently dipyridamole treatment is part of the standard care of stroke patients. 76 In patients with coronary artery disease, the FDA still advises to use this drug with caution as a coronary steal phenomenon has been observed when dipyridamole was administered intravenously in a high dose. However in the ESPS-2 and ESPRIT trials the addition of dipyridamole to aspirin did not increase the incidence of cardiac events. 58,92 Further evidence that dipyridamole induces protection against IR in humans in vivo, would support the conductance of a phase-2 clinical trial with dipyridamole to reduce cardiac IR-injury in patients suffering from coronary artery disease. Dipyridamole has several pharmacological properties including inhibition of the equilibrative nucleoside transporter (ENT), cyclic-gmp specific phosphodiesterase inhibition (5, 6), and scavenging of free radicals. 24,44,71 Of these properties, inhibition of the ENT has been characterised in detail in humans in vivo and occurs at clinically applied doses. 25,223 The inhibitory effect of dipyridamole on the ENT decreases uptake of endogenous adenosine by erythrocytes and endothelial cells, thereby increasing adenosine levels at its extracellular formation sites. 14,15 A subsequent increase in adenosine receptor (A 1, A 2A and A 3 ) stimulation can induce several tissue-protective effects, including vasodilation, inhibition of inflammation and thrombocyte aggregation and induction of tolerance against IR-injury, so called preconditioning. 16,17 Using our forearm model with Tc-99m-annexin A5 scintigraphy, we have addressed the protective effect of 7-day oral dipyridamole treatment on IR-injury in humans in vivo. 220 In this model, annexin A5 targeting results from phosphatidylserine (PS) exposure on cell membranes, which has been associated with IR-induced cell stress and apoptosis. 72 However, this study was neither blinded nor randomised, and therefore the conclusions have to be considered carefully. To test our hypothesis more rigorously, we decided to readdress the protective effects of dipyridamole against IR using a randomised double-blind placebo-controlled design. To decrease inter-individual variation and thus increase statistical power, the experiment was carried out in a crossover design. As 57

60 Chapter 4 described previously, we used in vivo annexin A5 targeting after forearm ischaemic exercise as a measure of IR-injury. 172,220,224,226 Since our results showed a surprisingly long-lasting effect of dipyridamole on PS-exposition, we also addressed a possible role of extended nucleoside transport inhibition in this long term action of dipyridamole. For this purpose, we investigated the 4-week post-treatment effect of a 7-day oral dipyridamole administration on nucleoside transport ex vivo in human erythrocytes. Methods Subjects Approval was obtained for both experiments from our institutional medical ethical committee (CMO regio Arnhem-Nijmegen, The Netherlands) and all participants gave written informed consent prior to inclusion. Ten healthy male volunteers were included in the in vivo annexin A5 targeting experiment (NCT ) and 7 healthy volunteers were included in the additional study to explore the long-term effect of dipyridamole on ex vivo nucleoside transport. Medical screening was performed to exclude cardiovascular disease, hypercholesterolemia, hypertension, diabetes mellitus, asthma and use of concomitant medication. In the 24 hours prior to the experiment all volunteers agreed to a caffeine free diet. Experimental protocols In vivo annexin A5 targeting after forearm ischaemic exercise (experiment 1) Volunteers were treated for 7 days with dipyridamole (Persantin slow release) 200 mg (Boehringer-Ingelheim, Alkmaar, The Netherlands) or placebo twice daily. Placebos were prepared according to GMP standards by our department of Clinical Pharmacy (Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands). To match placebo, dipyridamole was recapsulated. Between both experimental periods a washout period of at least three weeks was observed. On day one the first capsule was taken under supervision of the investigator. On day 7, the volunteers returned to the research centre after an overnight fast. A blood sample was drawn to determine serum caffeine and steady-state dipyridamole trough concentrations. Subsequently the final capsule was taken. One hour thereafter a blood sample was drawn to determine serum dipyridamole peak concentrations. Subsequently, volunteers underwent ischaemic exercise of the non-dominant forearm and annexin A5 scintigraphy was performed as previously described. 220,224,226 In brief, the circulation of the non-dominant forearm was occluded for 10 minutes with an upper arm cuff inflated to 200 mmhg. Directly after occlusion, participants performed rhythmic hand contractions at 50% of their maximum contraction force until exhaustion. The total duration of ischaemia was 10 minutes, independent of the duration of contractions. Immediately upon reperfusion 58

61 Dipyridamole protects against IRI 0.1 mg annexin A5 radiolabeled with 450 MBq Technetium-99m was administered intravenously into the dominant arm. One and four hours after annexin A5 administration, scans were made of both hands with a gamma camera (Siemens Orbiter equipped with low-energy high-resolution collimators). 226 At least three weeks later this procedure was repeated for the second treatment period. Post treatment effect of dipyridamole on nucleoside transport (experiment 2) Volunteers were treated for 7 days with dipyridamole (Persantin slow release) 200 mg (Boehringer-Ingelheim, Alkmaar, The Netherlands) twice daily. Prior to the start of treatment, blood was sampled for baseline assessment of ENT-mediated nucleoside transport and dipyridamole concentration. At day 7 of dipyridamole treatment this procedure was repeated, one hour after supervised intake of the last capsule. Furthermore, assessment of ENT activity and dipyridamole concentrations were repeated 1 week and 4 weeks after cessation of treatment. All blood samples were collected after at least 2 hours of fasting. Laboratory analysis Caffeine plasma concentrations were determined using reversed-phase HPLC with UV detection set at 273 nm. 240 Dipyridamole plasma concentrations were determined in deproteinised plasma using HPLC with fluorescence detection set at 286/470 nm. 295 To assess ENT activity, nucleoside transport was measured as reported previously. 220 Statistical analysis The baseline characteristics were compared using paired Student T-test. All digitised gamma camera images were analysed offline by the same investigator (WO) using Hermes software (Nuclear Diagnostics, Stockholm, Sweden). A predefined region of interest was placed over the thenar muscle of both hands. Annexin A5 targeting was expressed as the percentage difference in counts/pixel between the experimental hand and the control hand as previously described. 226 The effect of the intervention was analysed using ANOVA for repeated measurements. In addition, the treatment order was entered as a betweensubjects factor in the ANOVA for repeated measurements. Nucleoside transport inhibition after oral treatment with dipyridamole is calculated as percentage change in nucleoside transport compared to baseline and analysed statistically with a paired Student T-test. Results are expressed as mean ± standard error, except when stated otherwise. All statistical analyses were performed using SPSS for Windows. 59

62 Chapter 4 Results In vivo Annexin A5 targeting after forearm ischaemic exercise Mean age of healthy participants was 23.3 ± 3.8 (SD) years, dipyridamole plasma trough concentration was 0.65 ±0.32 (SD) mg/l and peak concentration was 0.77 ±0.44 (SD) mg/l after 7 days of treatment (baseline characteristics are presented in table 1). After placebo, dipyridamole levels remained below the minimal detection limit. In all participants caffeine levels on day 7, of both dipyridamole and placebo treatment, were under 0.75 mg/l, indicating adequate compliance to the 24 hours caffeine abstinence. Table 1 Baseline characteristics of healthy volunteers from the experiment with Annexin A5 targeting (experiment 1) and the experiment on the post treatment effect of dipyridamole (experiment 2). Experiment 1 Mean ±SD (n=10) Experiment 2 Mean ±SD (n=7) Age (yr) 23.3 ± ±4.4 BMI (kg/m 2 ) 23.1± ±1.5 Syst pressure (mmhg) 129 ±6 127 ±7 Diast pressure (mmhg) 78 ±6 77 ±7 Heart rate (bpm) 63 ±6 68 ±8 Glucose (mmol/l) 4.5 ± ±0.8 When treatment order was not taken into account, our analysis showed no effect of dipyridamole treatment on annexin A5 targeting (17.6 ±3.6% at 1 hour and 17.1 ±2.5% at 4 hours) compared to placebo treatment (22.1 ±4.3% at 1 hour and 22.8 ±4.8% at 4 hours; p=0.236). However a subsequent analysis revealed a statistically significant interaction effect of the randomisation order on the effect of dipyridamole on annexin targeting in this crossover experiment (p=0.029). In participants (n=5) who were randomised to first receive placebo followed by dipyridamole, a strong tendency was demonstrated for a reduction in annexin A5 targeting after dipyridamole treatment (14.9 ±4.0% at 1 hour and 15.5 ±4.4% at 4 hours) compared to placebo treatment (26.7 ±6.2% at 1 hour and 30.3 ±7.1% at 4 hours; p=0.057; see figure 1). In participants (n=5) who received dipyridamole first and placebo next, annexin targeting after dipyridamole (20.3 ±6.2% and 18.7 ±2.8% after 1 and 4 hours respectively) and was similar to that of the participants with opposite randomisation order (see above; p=0.62). However in the participants randomised to dipyridamole first, there was no difference between the annexin targeting after dipyridamole treatment (see above) and placebo treatment (17.4 ±6.0% and 15.3 ±5.0% 60

63 Dipyridamole protects against IRI p= p=0.057 p=0.431 p=0.62 Annexin A5 targeting (%) Time after reperfusion (hours) Placebo first (n=5) Dipyridamole first (n=5) Figure 1 Annexin A5 targeting 1 and 4 hours after ischaemic exercise (N=10). Participants are grouped according to randomisation order (placebo first (n=5) or dipyridamole first (n=5)) after dipyridamole treatment (open bars) and placebo treatment (closed bars). after 1 and 4 hours respectively; p=0.431; see figure 1), as opposed to the observed tendency in participants with the opposite randomisation order. Thus, a within subject effect of dipyridamole was only observed in participants who received placebo in the first treatment period and dipyridamole in the second (p=0.029 for the effect of treatment order). The actual mean wash out period was 28 days (range days). Heart rate did not differ between dipyridamole and placebo treatment, respectively 62 ± 2.4 bpm and 60 ± 2.2 bpm (p=0.23), indicating that the dose of dipyridamole in this experiment was sufficiently low to prevent systemic actions due to ENT inhibition. 227 The workload achieved during ischaemic exercise was comparable between dipyridamole (4023 ± 315 kg*sec) and placebo treatment (3988 ± 269 kg*sec; p=0.893). Post treatment effect of dipyridamole on nucleoside transport Mean age of participants was 23.1 ± 4.4 (SD) years, more baseline characteristics are presented in table 1. On the seventh day of treatment, serum dipyridamole concentration 61

64 Chapter 4 was 1.29 ± 0.44 (SD) mg/l and nucleoside transport was reduced with 86.6 ± 2.1 % compared to baseline (p<0.05). One week and 4 weeks after cessation of therapy, dipyridamole levels were below minimal detection limits and inhibition of nucleoside transport was no longer observed (-3.8 ± 5.7% and -5.2 ± 6.2%; p=0.71 and 0.61 respectively). Discussion In this study we show a protective effect of dipyridamole against IR-injury. In a previous study from our group, we observed protection by dipyridamole in a similar forearm model using annexin A5 targeting. However, this previous study used an historical control group. The current study substantiates this finding in a more rigorous randomised placebo-controlled crossover design. Interestingly, our present data suggest that this protection lasts for at least 4 weeks (considering the minimal 3 week wash-out period and subsequent one week placebo treatment). To explore a prolonged effect of dipyridamole on ENT as a potential explanation for this surprising result, we performed a post treatment effect study, which demonstrated a complete absence of dipyridamole in the serum and a full recovery of nucleoside transport one week after cessation of oral dipyridamole treatment. To our knowledge, we are the first to report a protective effect against IR-injury that lasts for at least 4 weeks after a pharmacologic intervention. Large similarities in the mechanism of preconditioning appear to exist amongst different tissues eligible for protection against IR-injury (e.g. myocardial, brain, skeletal muscle, kidney, and liver). 8,59,98 Adenosine is considered to play a pivotal role in the initiation of ischaemic preconditioning. 17,154 Furthermore, administration of exogenous adenosine can induce pharmacologic preconditioning. 18,270 Adenosine-induced protection (either ischaemic or pharmacologic) against IR-injury consists of two different phases. After the preconditioning event, a protective phenotype occurs immediately and lasts for maximally 6 hours (acute preconditioning). 304 Twenty-four hours after the preconditioning trigger, the second window of protection (or delayed preconditioning) starts. This SWOP is dependent on increased transcription of cardioprotective genes and expression of various proteins including inos, COX-2, heme oxygenase and antioxidant enzymes. 254,304 However, little is known about the time course of delayed preconditioning. Baxter et al. reported the delayed protective effect of adenosine receptor stimulation in rabbit myocardium to last at least 72 hours, though no longer than 96 hours. 18 Harralson et al. found similar results after ischaemic preconditioning skeletal muscles (m. latissimus dorsi) in rats. 95 However in mice, repetitive hypoxic preconditioning is reported to induce long-term tolerance against IR in nervous tissue, lasting four to eight weeks. 255,309 Still, there are very few experiments allowing a delay between the preconditioning stimulus and index ischaemia of more than 48 hours. In the post treatment effect study we did not find a prolonged effect of dipyridamole on ENT-mediated nucleoside transport. Thus, potential accumulation of dipyridamole in the 62

65 Dipyridamole protects against IRI cellular membrane did not influence ENT activity after dipyridamole was completely cleared from plasma. This indicates that the prolonged benefit of dipyridamole cannot be explained by on-going nucleoside transport inhibition. Since the serum dipyridamole concentration was under the detection limit and nucleoside transport was no longer inhibited one week after therapy cessation, our results suggest that a 7-day treatment with dipyridamole initiates a chain of events that results in a protective phenotype that lasts for at least 4 weeks. As reported previously by our group, we observed a significant inhibition of the ENT after 7 days of oral treatment with dipyridamole that should be sufficient to enhance the action of endogenous adenosine. 220,227 Normal physical activity of the forearm results in repetitive bouts of extracellular adenosine formation. 46,140 By inhibiting the ENT, dipyridamole intensifies the subsequent activation of adenosine receptors, which may have initiated a long-lasting increased expression of various cytoprotective proteins such as heme oxygenase, inos, and COX-2. 17,21 This resembles the repetitive preconditioning stimuli as described in nervous tissue, also resulting in prolonged protection. 255,309 Conclusion We conclude that treatment with dipyridamole provides protection against IR-injury in forearm skeletal muscle of healthy volunteers as detected with annexin A5 scintigraphy. Moreover, in our forearm model of ischaemia and reperfusion, this protective effect of dipyridamole appears to persist even when nucleoside transport has returned to baseline. These results support the conduct of a phase 2 clinical trial to study the effect of dipyridamole on cardiac IR-injury in patients with coronary artery disease. Confirmation of the long-term protection and exploration of its mechanism warrants further research. Acknowledgements The authors wish to thank Kim Wever (Department of Pharmacology-Toxicology, RUNMC) for her critical review of the manuscript. 63

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67 Chapter 5 Upregulation of ecto-5 -nucleotidase by rosuvastatin increases the vasodilator response to ischaemia Constantijn Wouters Patrick Meijer Petra van den Broek Maarten de Rooij Gertjan Scheffer Paul Smits Gerard Rongen Hypertension 2010; 56:

68 Chapter 5 Abstract 3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins) are effective in the primary and secondary prevention of cardiovascular events. Although originally developed to improve lipid profile, statins have demonstrated a surplus of beneficial pleiotropic effects, including improved endothelial function, reduced inflammation, and increased tolerance to ischaemia-reperfusion injury. In preclinical studies, increased ecto-5 -nucleotidase activity, the key enzyme in extracellular adenosine formation, plays an important role in these effects. Because human data are absent, we explored the effects of rosuvastatin on ecto-5 -nucleotidase activity and the clinical relevance of increased extracellular adenosine during ischaemia in humans in vivo. The forearm vasodilator responses to 3 increasing periods of forearm ischaemia (2, 5, and 13 minutes) were determined during placebo and caffeine (an adenosine receptor antagonist) infusion into the brachial artery. At the end of an 8-day treatment period with rosuvastatin (20 mg per day), this whole procedure was repeated. During both experiments ecto-5 -nucleotidase activity was determined. Vasodilator responses are expressed as the percentage increase in forearm blood flow ratio from baseline. Rosuvastatin increased ecto-5 -nucleotidase activity by 49±17% and enhanced the vasodilator response after 2, 5, and 13 minutes of ischaemia in the absence (146±19, 330±26, and 987±133 to 312±77, 566±107 and 1533±267) but not in the presence of caffeine (98±25, 264±54, 727±111 vs. 95±19, 205±34 and 530±62). Rosuvastatin increases extracellular formation of adenosine in humans in vivo probably by enhancing ecto-5 -nucleotidase activity. This action results in the improvement of reactive hyperaemia and may further enhance the clinical benefit of statins, in particular in conditions of ischaemia. 66

69 Rosuvastatin, ecto-5'-nucleotidase and adenosine Introduction 3-Hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins) are effective in the primary and secondary prevention of cardiovascular events. 63,204 Although originally developed to improve lipid profile, statins have demonstrated a surplus of beneficial pleiotropic effects, including improved endothelial function, reduced inflammation, and increased tolerance to ischaemia-reperfusion injury. 4,143,172,195,218 These effects are independent of plasma cholesterol lowering and improve outcome after cardiovascular events. 203,242 Increased extracellular adenosine formation has been implicated as one of the underlying mechanisms. This is supported by several preclinical studies demonstrating the activation of ecto-5 -nucleotidase (CD73) activity, increased stimulation of adenosine receptors, and adenosine-mediated protection against ischaemia-reperfusion injury by statins. 145,234,301 The enzyme CD73 dephosphorylates extracellular AMP, which forms the rate-limiting step in the formation of extracellular adenosine. 215 We recently demonstrated increased extracellular adenosine formation in healthy human volunteers after a 1-week treatment with rosuvastatin using a pharmacological approach. In addition, we demonstrated rosuvastatin-induced augmentation of postocclusive reactive hyperaemia (PORH). 172 However, we did not assess CD73 activity as a potential source of extracellular adenosine, nor did we investigate the role of adenosine in rosuvastatin-induced augmentation of PORH. CD73 activity plays a pivotal role in the formation of atherosclerosis and increased cellular resilience against ischaemia-reperfusion injury observed after ischaemic preconditioning. 37,68,307 An increase in CD73 activity may therefore reduce atherosclerosis formation and improve cellular tolerance against ischaemia. The extent of PORH in the human forearm correlates well with the vasodilator impairment in the coronary circulation. 236 Indeed, it reflects vascular health status, because increased forearm PORH is associated with a reduced incidence of stroke and cardiovascular disease in patients with essential hypertension. 261 Compared with normotensive subjects patients with essential hypertension have a reduced vasodilator response to reactive hyperaemia and may therefore benefit from therapies aimed at or associated with an improvement of PORH. 108 Insight into the underlying mechanism of rosuvastatin-induced augmentation of reactive hyperaemia may help us in optimizing the beneficial effects of statins and improve treatment of patients at risk for cardiovascular events. We, therefore, assessed the involvement of adenosine in rosuvastatin-induced augmentation of PORH by testing the following hypotheses: a 1-week treatment with rosuvastatin enhances CD73 activity of circulating mononuclear blood cells, and caffeine, an adenosine receptor antagonist, inhibits the effect of rosuvastatin on forearm PORH. 67

70 Chapter 5 Methods Subjects The study was performed in accordance with the Declaration of Helsinki of the World Medical Association and approved by the Institutional Review Board of the Radboud University Nijmegen Medical Centre. Ten healthy volunteers (5 women; aged years) with a normal medical history, physical examination, blood pressure, body mass index, fasting plasma lipid profile, and glucose concentration gave written informed consent before entering the study. Experiments were performed in a temperature-controlled room (23 ±1 C) in the morning after an overnight fast and 24 hours of caffeine abstinence. All of the experiments were performed according to institutional guidelines. Procedures At the start of each experiment, a 27-gauge needle (B. Braun Medical B.V.) was inserted into the brachial artery of the non-dominant arm for intra-arterial drug administration. In both arms, forearm blood flow (FBF) was measured simultaneously with venous occlusion plethysmography using mercury-in-silastic-strain gauges and occluded hand circulation as described previously. 174 Experimental design Figure 1 shows the design of the study. All volunteers received an 8-day treatment with rosuvastatin (20 mg per day) and entered our research facility twice, the first time before the start of treatment with rosuvastatin and the second time on the final day of treatment with rosuvastatin. On both visits forearm vasodilator responses to 3 increasing periods of forearm ischaemia (2, 5, and 13 minutes) were determined, once during the infusion of placebo (saline 0.9%, 50 µl/min per 100 ml forearm volume) and once during the administration of caffeine (90 µg/min per 100 ml forearm volume) into the brachial artery. Ecto-5 -nucleotidase activity We determined the activity of CD73 exposed on the surface of intact mononuclear cells by quantifying the conversion of 1,N 6 -ethenoadenosine 5 -monophosphate to 1,N 6 -ethenoadenosine 119 in the presence and absence of the CD73 specific inhibitor α,ß-methyleneadenosine 5 -diphosphate. The difference in these 2 activities reflects CD73 activity. To eliminate the influence of non-specific dephosphorylation of 1,N 6 -etheno adenosine 5 - monophosphate to 1,N 6 -ethenoadenosine and 1,N 6 -ethenoadenosine transport, CD73 activity was measured in the presence of ß-glycerophosphate and dipyridamole. ß-Glycerophosphate forms a competitive substrate for non-specific phosphatases eliminating the contribution of nonspecific dephosphorylation of AMP. Dipyridamole is an equilibrative nucleoside transport inhibitor that, in this concentration, prevents adenosine transport across the cell membrane. 68

71 Rosuvastatin, ecto-5'-nucleotidase and adenosine Before treatment (visit1) After 8 days of treatment with rosuvastatin (visit2) FBF (ml/dl/min) NaCl 0.9% Caffeine NaCl 0.9% Caffeine Blood sampling Arterial occlusion NaCl 0.9% FBF exp arm FBF con arm Figure 1 Schematic overview of the experimental protocol, indicating arterial occlusion, placebo and caffeine infusion, and FBF measurements. In addition, the graph above the protocol represents the absolute FBFs, at baseline and reperfusion, in the experimental (white) and control (orange) arms (n=8). At the start of each experiment 8 ml of venous blood was sampled and collected in cell preparation tubes (BD Vacutainer CPT), which were centrifuged at 1600g for 20 minutes to isolate mononuclear cells. After harvesting they were washed twice in PBS (5.39 mmol/l of Na 2 HPO 4, 1.29 mmol/l of KH 2 PO 4, and 0.15 mol/l of NaCl [ph 7.4]), followed by centrifugation for 15 minutes at 319g. The obtained pellets were resuspended in 1 ml of Hanks balanced salt solution (Invitrogen). Pilot experiments were performed to confirm optimal (V max ; maximum CD73 velocity) substrate concentrations (data not shown). For analysis 30 µl of homogenate was buffered in Hanks balanced salt solution containing 10.0 mmol/l of MgCl 2, 10.8 mmol/l of ß-glycerophosphate, 20 µmol/l of dipyridamole, 0.2 mmol/l of 1,N 6 -ethenoadenosine 5 - monophosphate with or without 400 µmol/l α,ß-methyleneadenosine 5 -diphosphate. All of the assays were performed for 5 minutes at 37ºC in a total volume of 100 µl. The reaction was terminated by adding 50 µl of perchloric acid 70% and the obtained lysate centrifuged for 3 minutes. The supernatant was used for measurement of 1,N 6 -ethenoadenosine concentration by ion-paired reverse-phase high-performance liquid chromatography with fluorescence detection set at 280/420 nm. For separation, an Altima HP C18 AQ column was used with a mobile phase containing 50 mmol/l of NH 4 H 2 PO 4, 5 mmol/l of hexane sulfonic acid (ph 3.0), and acetonitrile as the organic modifier. Other analytical procedures In all of the volunteers, blood was collected before and at the end of oral treatment to determine fasting serum lipid profile with a commercially available kit (Aeroset, Abbott). Compliance to caffeine abstinence was monitored by determination of plasma caffeine 69

72 Chapter 5 concentration before each experiment. Plasma caffeine concentrations were determined by use of reversed-phase high-performance liquid chromatography with UV detection set at 273 nm according to Schreiber-Deturmeny and Bruguerolle. 240 Solutions The caffeine (Genfarma) solution was freshly prepared to reach a final syringe concentration of 90 µg per 50 µl. Rosuvastatin tablets contained 20 mg per tablet and were obtained from Astra Zeneca SA. Statistical analysis All of the CD73 activity measurements were performed in duplicate, averaged for each subject and visit, and differences between the 2 visits were analysed by paired t tests. To correct for random changes in FBF unrelated to the intervention, the ratio of simultaneously measured FBF in intervention and control arms was calculated (FBF ratio). The FBF ratios of the last 4 minutes of reference measurements (during intra-arterial saline or caffeine, as appropriate) and first 3 minutes of each reperfusion period were averaged to 1 value. To reduce the influence of intra-individual changes between visits and inter-individual variations in the baseline FBF ratio, results are expressed as the percentage increase in FBF ratio from baseline measurements. An ANOVA for repeated measures was performed on FBF ratio with rosuvastatin, caffeine, and arterial occlusion time as within-subject factors to determine the effect of caffeine on rosuvastatin-enhanced PORH. Results are expressed as mean ± SE. Results The experiments were successful in 8 volunteers. Baseline serum caffeine concentrations were <1.5 mg/l during both experimental days, indicating adequate compliance to the caffeine-free diet. Treatment with rosuvastatin significantly reduced fasting total cholesterol and low-density lipoprotein cholesterol (Table). Ecto-5 -nucleotidase activity Rosuvastatin significantly increased CD73 activity with 49 ± 17% compared with baseline (Figure 2). Forearm circulation The absolute changes in FBF are presented in Figure 1. FBF ratio returned to baseline before the start of the second set of ischaemic challenges on each study day. Rosuvastatin treatment did not influence baseline FBF ratios nor the course of FBF in the control arm between study days. Rosuvastatin treatment significantly increased the vasodilator response 70

73 Rosuvastatin, ecto-5'-nucleotidase and adenosine Table Effect of treatment with rosuvastatin on lipid profile Biochemistry Day 1 Day 8 Fasting total cholesterol (mmol/l) 4.2 ± ±0.1* Fasting low-density lipoprotein cholesterol (mmol/l) 2.3 ± ±0.1* Fasting triglycerides (mmol/l) 1.0 ± ±0.1 Fasting high-density lipoprotein cholesterol (mmol/l) 1.4 ± ±0.1 Data are expressed as mean ±SE *p<0.05 day 1 vs. day 8, paired t test (n=8) * * ecto-5'-nucleotidase activity (nmol/min per mg protein) AMP - CP + AMP - CP CD73 Before treatment Rosuvastatin Figure 2 Ecto-5 -nucleotidase (CD73) activity of intact mononuclear cells. Experiments were performed in the presence and absence of the specific CD73 inhibitor α,ß-methyleneadenosine 5 -diphosphate (AMP-CP). The difference between these two measurements reflects CD73 activity (bars). *p<0.05, data are mean ±SE (n=8). to reactive hyperaemia (Figure 3). In the presence of caffeine, this augmentation of the vasodilator response was abolished. When analysing both experimental days separately, caffeine significantly altered the vasodilator response to reactive hyperaemia while on rosuvastatin (p=0.03) but not during baseline conditions (before rosuvastatin treatment, p=0.2). 71

74 Chapter 5 Without caffeine p< 0.05 With caffeine FBF-ratio (% of baseline) Rosuvastatin Before treatment Rosuvastatin Before treatment p = 0.03 p = Arterial occlusion time (min) Figure 3 Percentage increase in FBF ratio after arterial occlusion compared with baseline. P values indicate the level of significance for the effect of rosuvastatin on FBF ratio after arterial occlusion in the absence (left) and presence of caffeine (right) and the interaction between rosuvastatin and caffeine (left vs. right, ANOVA for repeated measures). Data represent mean ±SE (n=8). Discussion We recently demonstrated by using a pharmacological approach that rosuvastatin increases extracellular adenosine formation. We now demonstrate that this is, at least partly, mediated by increased CD73 activity. In addition, we underline the clinical relevance of the increased adenosine formation as it augments PORH after rosuvastatin treatment. Rosuvastatin increases ecto-5 -nucleotidase Previous studies already reported on the stimulatory effect of statins on CD73 activity. This was demonstrated for lovastatin in rat endothelial cells 145, pitavastatin in canine in vivo 234, and atorvastatin in human vascular endothelial cells. 198 Our results support these previous findings and are the first to demonstrate this effect in a human in vivo setting. The underlying mechanism still has to be unravelled. Inhibition of mevalonate is one of the potential mechanisms, because it not only reduces the synthesis of cholesterol but also isoprenoids, which are implicated in isoprenylation of small GTPases, including members 72

75 Rosuvastatin, ecto-5'-nucleotidase and adenosine of the Rho family. Inhibition of Rho may result in reduced endocytosis of CD73 and subsequently increased expression of CD73 on the cell membrane. 145 It has also been demonstrated that phosphatidylinositol-3-kinase and protein kinase C may influence CD73 activity by influencing its phosphorylation state and may thereby form an alternative route for statin-induced enhancement of CD73 activity. 191,234 Regardless of the underlying mechanism, activation of CD73 by statins is likely to contribute to the clinical efficacy of statins in a setting of cardiac infarction, because inhibition of CD73 attenuates the limiting effect of statins on infarct size in animals. 234 Furthermore, we have shown previously in a human forearm model of ischaemia-reperfusion injury that endogenous adenosine is likely to contribute in rosuvastatin-induced protection against ischaemia-reperfusion injury. 172 Rosuvastatin enhances PORH in the absence but not in the presence of caffeine The additional increase in PORH provided by rosuvastatin was abolished by the adenosine receptor antagonist caffeine indicating the involvement of enhanced adenosine receptor stimulation. Previous observations already showed that rosuvastatin did not affect the vasodilator response to adenosine which excludes an effect of rosuvastatin on adenosine clearance, adenosine receptors, or post receptor signalling. 172 Thus, the increased adenosine receptor stimulation observed during this study results from increased availability of extracellular adenosine. This conclusion is supported by data from the current study indicating activation of CD73 on mononuclear cells as a result of treatment with rosuvastatin. Potential limitations to our study design need to be addressed. First, because this study was neither randomized nor placebo controlled, a nonspecific time effect could have interfered. However, we have shown previously that repeated ischaemia on a single day 26 or with a 1-week interval 264 results in similar reactive hyperaemia. This observation makes it superfluous to explore potential carryover effects in this study. We opted for a limited study design to reduce the experimental load for our volunteers as much as possible. Second, we did not test the effect of caffeine on an adenosine-independent vasodilator, and, therefore, our findings may potentially be the result of adenosine-independent effects. A previous study from our institute demonstrated that intra-arterial caffeine (90 µg/min per 100 ml of forearm volume) did not influence the vasodilator response to the endothelium-independent vasodilator sodium nitroprusside, which rules out a nonspecific effect of caffeine on PORH. 248 Implications of our observations Atherosclerosis is an inflammatory disease of large- and medium-sized arteries and the most important precursor of cardiovascular disease. Studies in atherosclerosis-susceptible mice, including CD73-deficient mice, demonstrate the correlation between reduced CD73 73

76 Chapter 5 activity and enhanced vascular inflammation and subsequent atherosclerosis formation. 37,307 Therefore, increased CD73 activity may aid in the prevention of atherosclerosis. In addition, inhibition of CD73 or targeted gene deletion of CD73 intervenes with the infarct size-limiting effect of ischaemic preconditioning. 68,87 This effect can be restored by administration of soluble CD73. Adenosine forms the likely mediator of CD73-dependent effects, because its activity is functionally associated with increased adenosine receptor stimulation. 69 This is also in line with the observation that administration of an adenosine receptor agonist restored the proinflammatory state of CD73-deficient mice to baseline wild-type values. 307 However, because we did not measure other adenosine forming enzymes, for example, alkaline phosphatase, we cannot rule out a significant contribution of this enzyme in rosuvasatin-induced augmentation of adenosine formation. Reactive hyperaemia in the human forearm depends on postocclusive dilation of arteriolar resistance vessels and correlates well with the degree of vasodilator impairment in the coronary circulation. 236 Indeed, PORH correlates with the incidence of cardiovascular events in patients with essential hypertension and decreases with an increase in cardiovascular risk factors. 113,261 In addition, the extent of PORH after coronary angioplasty reflects the clinical effectiveness of revascularization. 232 These findings support the use of PORH as a marker for cardiovascular disease and effectiveness of treatment. The improvement of reactive hyperaemia by rosuvastatin, therefore, at least in part, represents its therapeutic effect. This effect is attenuated by caffeine, which supports the involvement of adenosine in rosuvastatin-induced effects on cardiovascular disease. Intake of 2 cups of coffee results in a plasma caffeine concentration of ~9 mg/l. 249 In our current design we aimed at optimal adenosine receptor blockade, which is obtained with 90 µg/min per 100 ml of forearm volume. 248 During intra-arterial infusion of caffeine, the plasma concentration in the venous effluent depends on FBF and decreases with higher flows. Unfortunately, we did not collect blood during caffeine infusion and are, therefore, not able to provide caffeine concentrations in the venous effluent in the present study. However, in a previous trial with comparable design, 174 we measured plasma caffeine concentrations after 2 and 13 minutes of arterial occlusion and ~6 minutes of reperfusion which were, respectively 15±2 and 8±1 mg/l (mean ±SE, unpublished data). This indicates that local caffeine concentrations in the present study approach values that occur during regular caffeine consumption. We studied the effects of acute caffeine administration. Previous studies have indicated the development of (partial) tolerance to the effects of caffeine on blood pressure. However, in chronic caffeine consumers the blood pressure response to adenosine continuously increased during a 2-week period of caffeine abstinence, suggesting that the interaction between caffeine and adenosine may be less prone to tolerance development. Therefore, we believe that our observations strongly support the advice that patients who are treated with statins should abstain from caffeine consumption to fully utilize its clinical benefit. Additional evidence for this advice would need a large clinical trial in patients treated with statins randomized to long-term regular 74

77 Rosuvastatin, ecto-5'-nucleotidase and adenosine caffeine consumption or caffeine abstinence with mortality and morbidity as clinical endpoints. Such a trial is very expensive and, in our opinion, hardly feasible. Perspectives In the last 20 years statins have been shown to improve outcome observed during acute coronary syndromes 203,242, cardiac bypass surgery 43,199, and percutaneous coronary interventions. 202 The underlying mechanism is extensively studied but nonetheless not fully understood, which may hamper the full potential of this class of drugs. Our results demonstrate for the first time the effect of rosuvastatin on CD73 in a human in vivo setting and confirm the clinical relevance of increased adenosine receptor stimulation in the beneficial effects exerted by statins. These findings are of particular interest because pharmacological modulation, both beneficial and detrimental, of the transport and actions of adenosine is already in widespread use, for example, dipyridamole and caffeine. Indeed the combination of a statin with an inhibitor of adenosine uptake into the cell, dipyridamole and cilostazol, and subsequent increased extracellular adenosine concentrations potentiate the infarct size-limiting effect of statins in animals. 168,302 Our observations support caffeine abstinence in patients treated with a statin to fully utilize its clinical benefit. Acknowledgements We kindly thank Karin Saini and Anja Rasing-Hoogveld for their assistance during the experiments. 75

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79 Chapter 6 Short-term statin treatment does not prevent ischaemia and reperfusion-induced endothelial dysfunction in humans Constantijn Wouters Kimberley Wever Inge Bronckers Maria Hopman Paul Smits Dick Thijssen Gerard Rongen Journal of Cardiovascular Pharmacology 2012; 59: 22-28

80 Chapter 6 Abstract Statins are known to have cholesterol-independent pleiotropic effects, such as upregulation of the enzyme ecto-5 -nucleotidase. These effects may contribute to the protective effect of statins against ischaemia and reperfusion (IR). Interestingly, pleiotropic effects have been shown to differ between hydrophilic and lipophilic statins. Flow mediated dilation (FMD) represents a largely nitric oxide-mediated, endotheliumdependent dilation and has been shown to decrease after exposure to IR in humans. FMD has been validated to study (pharmacological) interventions in IR-injury. We examined the effect of a short-term (3-7 days) statin pretreatment on brachial artery endothelial function before and after IR, and whether the effect on brachial artery endothelial function differs between rosuvastatin (hydrophilic statin) and atorvastatin (lipophilic statin). Our results show that IR significantly decreases FMD, however statin pretreatment did not alter the effect of IR on FMD (irrespective of treatment duration or type of statin used). This experiment suggests that the cardioprotective effects of statins (both lipophilic and hydrophilic) against IR are not mediated through preservation of endothelial function. 78

81 Statins and endothelial dysfunction after IRI Introduction Cardiovascular disease represents the greatest burden in terms of morbidity and death in the Western world. Previous studies demonstrated that statins (3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitors) reduce serum low-density lipoprotein (LDL) cholesterol, which is associated with a significant reduction in cardiovascular events. 63,150,210,230 Statins also have cholesterol-independent pleiotropic effects, such as upregulation of ecto-5 -nucleotidase 145,173,198,234, activation of the phosphatidylinositol 3-kinase-Akt pathway 274, activation of the ERK 1/2 pathway 175 and upregulation of nitric oxide (NO) synthase. 106,138 These pleiotropic actions may contribute to the cardioprotective effects of statins, such as an improved tolerance against ischaemia and reperfusion injury (IR-injury). 22,32,163,229 Indeed, we have recently demonstrated that rosuvastatin reduces IR induced phosphatidylserine exposition in the thenar muscle in humans in vivo, involving activation of adenosine receptors. 172 In addition, we have reported that rosuvastatin improves formation of adenosine in the forearm and increases forearm postocclusive hyperaemia via an adenosine-dependent mechanism. Finally we have shown in humans a 50% increase in ecto-5 - nucleotidase activity on peripheral blood mononuclear cells after rosuvastatin treatment as compared with placebo. 173 Whether this increase is a result of a change in enzyme production, presence or activity remains unclear. Because the vasodilator response to adenosine involves endothelial release of NO, prevention of IR-induced endothelial dysfunction could be involved in these actions of rosuvastatin. 250 Currently, little is known about the potential of statins to prevent IR-injury of the vasculature. Severe endothelial dysfunction is one of the consequences of IR-injury. 133 Ischaemia activates arginase, which competes with NO synthase for its substrate L-arginine and results in subsequent decrease in endothelial NO-release. 67,105 Flow mediated dilation (FMD) represents a largely NO-mediated 182, endothelium-dependent dilation and has been shown to decrease after exposure to IR-injury in humans. 133,161 Recently, it was demonstrated that a single dose of rosuvastatin could prevent the decrease in FMD after IR-injury, indicating a possible protective effect of the pleiotropic effects of statins on endothelial function. 156 Interestingly, pleiotropic effects have been shown to differ between hydrophilic and lipophilic statins. Lipophilic statins have been reported to render endothelial and vascular smooth muscle cells more susceptible to apoptosis than hydrophilic statins. 130 Moreover hydrophilic statins have been suggested to be superior in reducing event rate after a primary acute coronary syndrome in normocholesterolaemic patients. 233 Thus, lipophilicity of statins may influence their ability to reduce IR-injury. We hypothesize that short-term statin treatment prevents endothelial dysfunction after IR-injury. Therefore, the primary aim of our study is to examine the effect of a short-term 79

82 Chapter 6 statin pretreatment on brachial artery endothelial function before and after IR-injury, and second to examine whether the effect on brachial artery endothelial function differs between a hydrophilic statin (i.e. rosuvastatin) and a lipophilic statin (i.e. atorvastatin). Finally, we assess RNA expression of ecto-5 -nucleotidase before and after statin therapy to elucidate more about the mechanism of increased ecto-5 -nucleotidase activity after statin therapy. Methods After approval of the protocol by our local ethics committee, 48 healthy non-smoking volunteers (age years) were included after signing informed consent. All participants underwent medical screening to exclude cardiovascular disease, hypertension, hypercholesterolemia, impaired renal function, and diabetes mellitus. Serum values of creatine kinase and alanine aminotransferase were measured to exclude participants with an increased risk of adverse events during statin treatment. Subjects with concomitant use of medication or participation to other drug experiments during the past 60 days were excluded. Oral contraceptive use by female participants was permitted, but measurement was planned during the drug-free interval of the oral contraceptive to minimise the influence of the menstrual cycle on the endothelial function measurements. 293 Experimental design In this double-blind randomised controlled trial (NCT ), participants were allocated to daily treatment with either rosuvastatin (20 mg, AstraZeneca, Zoetermeer, The Netherlands), atorvastatin (80 mg, Pfizer, Capelle a/d IJssel, The Netherlands) or placebo (Department of Clinical Pharmacy, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands). Both rosuvastatin and atorvastatin were capsuled by the Department of Clinical Pharmacy (Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands according to good manufacturing practice standards) to match placebo. Participants were equally divided across a 3- or 7-day pretreatment, because the duration of pretreatment may influence the impact of statins on IR-injury. On the first day, drug intake was performed under medical supervision. On subsequent days, capsules were taken individually before breakfast. At the end of the treatment, subjects returned to our laboratory to examine right brachial artery endothelial function before and after IR-injury. The latter was induced using 15 minutes of ischaemia, followed by 15 minutes of reperfusion of the right arm. Measurements In the 24 hours before the test, participants were instructed to refrain from caffeine. Participants were instructed to take the last capsule 3 hours before the test and to fast 4 hours before the test. Before the test, venous blood was taken from the antecubital vein 80

83 Statins and endothelial dysfunction after IRI to assess lipid profile, caffeine level and RNA-expression of ecto-5 -ectonucleotidase in mononuclear cells. Flow mediated dilation Endothelium-dependent, largely NO-mediated, vasodilator function was assessed using FMD. 62,182 Participants rested in the supine position in a temperature-controlled room (22 o C) for at least 15-minutes to facilitate baseline assessment of heart rate and blood flow. To examine brachial artery FMD, the right arm was extended and positioned at an angle of ~80 o from the torso. A rapid inflation and deflation pneumatic cuff (D.E. Hokanson, Bellevue, WA) was positioned on the forearm immediately distal to the olecranon process to enable induction of forearm ischaemia. 263 A 10-MHz multifrequency linear array probe attached to a high-resolution ultrasound machine (T3000; Terason, Burlington, MA) was used to image the brachial arteries in the distal one-third of the upper arm. When an optimal image was obtained, the probe was held stable and the ultrasound parameters were set to optimize the longitudinal, B-mode images of lumen-arterial wall interface. Continuous Doppler velocity assessment was simultaneously obtained using the ultrasound machine, and was collected using the lowest possible insonation angle (always <60 o ), which did not vary during each study. Baseline images were recorded for 1 minute. The forearm cuff was then inflated (>200 mmhg) for 5-minutes. Diameter and blood flow recordings resumed 30 seconds before cuff deflation and continued for 3 minutes thereafter. The baseline FMD measurement was followed by the experimental IR-injury. The pneumatic cuff was placed proximally around the upper arm and inflated to 200mmHg to apply 15 minutes of ischaemia followed by cuff deflation. After 15 minutes of reperfusion, the procedures for FMD assessment as described above were repeated by the same experienced sonographer. FMD analysis Analysis of brachial artery diameters and shear rate were performed using edge-detection and wall-tracking software which is largely independent of investigator bias. 296 Recent articles contain detailed descriptions of our analysis approach. 28 From synchronised diameter and velocity data, blood flow (the product of lumen cross-sectional area and Doppler velocity (ν) was calculated at 30 Hz. Shear rate (an estimate of shear stress without viscosity) was calculated as 4 times mean blood velocity/vessel diameter. 201 Reproducibility of the FMD using this semiautomated software possesses a within subject coefficient of variance of 6.7%-10.5%. 266 Baseline diameter, blood flow, and shear rate were calculated as the mean of data acquired across the 1 minute preceding the cuff inflation period. Peak diameter after cuff deflation was automatically detected using an algorithm that identified the maximum bracket of data subsequent to performance of a moving window smoothing function. FMD was 81

84 Chapter 6 calculated as the percentage rise of this peak diameter from the preceding baseline diameter and was calculated based on standardised algorithms applied to data which had undergone automated edge-detection and wall-tracking, and were therefore observerindependent. 28 In accordance with recent findings 28,209, we calculated the shear rate stimulus responsible for endothelium-dependent FMD after cuff deflation. Blood analysis In all volunteers, blood was collected to determine fasting serum lipid profile (high-density lipoprotein, LDL and total cholesterol and triglycerides) with a commercially available kit (Aeroset, Abbott). Plasma caffeine concentrations were determined by use of reversedphase HPLC with UV detection set at 273nm. 240 Real-time quantitative-polymerase chain reaction procedures All procedures were carried out according to the manufacturers instructions. Whole blood was collected in PAXgene blood RNA tubes and stored at -80 o C., after which RNA was isolated using the PAXgene Blood RNA Kit (both from Qiagen, Venlo, The Netherlands). Total intact RNA was reverse transcribed using pd(n)6 random hexamer primers and Moloney murine leukaemia virus reverse transcriptase (Invitrogen). Real-time quantitativepolymerase chain reaction was performed using the ABI/PRISM 7900HT Gene Expression Micro Fluidic Card (Applied Biosystems). Complementary DNA amplification was performed in Taqman Universal polymerase chain reaction Master Mix, supplemented with 20x solution of primer probe sets for ecto-5 -nucleotidase (Hs _m1), and b-actin (Hs; all from Applied Biosystems). Polymerase chain reactions were analysed using 700 System Sequence Detection Software (version 1.2.3; Applied Biosystems). Sample size and statistical analysis Using the observed difference in FMD after IR between treatment and control, SD and group size reported by Liuni et al., 156 we calculated that the SD of the observed difference in FMD before and after IR was 3.85%. Thus, with 48 volunteers in the present study, and based on the predicted SD of 3.85%, and 80% power and a significance level of 5% we expected to detect a treatment effect of 4% in FMD response after IR. All data are presented as mean ± standard error, unless stated otherwise. To avoid multiple testing, we used analysis of variance (ANOVA) for repeated measures with interaction terms to analyse the effect of different treatments on cholesterol and FMD. Paired t-test was used to test the effect of ischaemia on FMD within the placebo group. Statistical analyses were performed using SPSS For ecto-5'-nucleotidase expression data, 3 measurements were found to have a deviation from the group average of >4 times the SD. This was related to very high b-actin cycle threshold values of these measurements: >5 cycle thresholds above average. Because this suggests that the quality of these RNA samples was compromised, these measurements were excluded from the analysis. 82

85 Statins and endothelial dysfunction after IRI Results Before deblinding, 1 volunteer (placebo) was excluded because of caffeine intake (caffeine level of 1.5mg/L). Baseline characteristics and cholesterol data of all groups are presented in Table 1. Table 1 Baseline characteristics and cholesterol values after treatment with placebo, rosuvastatin or atorvastatin Placebo (n=15) Rosuvastatin (n=16) Atorvastatin (n=16) At baseline Age (years) 21.8 (1.5) 24.4 (7.1) 22.2 (1.3) Female (%) BMI (kg/m 2 ) 22.1 (1.7) 22.7 (2.2) 22.3 (2.2) Systolic blood pressure (mmhg) 119 (9) 122 (10) 122 (7) Diastolic blood pressure (mmhg) 73 (8) 75 (7) 76 (7) Heart rate (bpm) 68 (10) 63 (10) 66 (11) Glucose (mmol/l) 4.7 (0.5) 5.0 (0.8) 5.0 (0.6) Total cholesterol (mmol/l) 4.6 (0.7) 4.3 (0.7) 4.7 (0.8) LDL (mmol/l) 2.8 (0.6) 2.5 (0.6) 2.7 (0.8) After treatment Total cholesterol (mmol/l) 4.6 (0.7) 3.4 (0.7) 3.8 (1.2) LDL (mmol/l) 2.7 (0.6) 1.7 (0.6) 1.9 (0.9) Data represent mean (SD) LDL and total cholesterol were not affected by placebo treatment. Statin treatment significantly reduced LDL and total cholesterol in all groups (ANOVA p<0.05). We found no differences in the effect of treatment on LDL and total cholesterol between rosuvastatin and atorvastatin (ANOVA p=0.98 and 0.86 respectively). Statin pretreatment had no effect on the RNA expression of ecto-5 -nucleotidase in peripheral blood mononuclear cells (figure 1) (ANOVA p=0.38). 83

86 Chapter 6 5'-Ecto mrna (% change from baseline) Before treatment After treatment Placebo Rosuvastatin Atorvastatin Figure 1 Ecto-5 -nucleotidase mrna expression (percentage change from baseline measurement before treatment) before treatment (closed bars) and after treatment (open bars). Error bars represent standard errors. Table 2 Results FMD measurement before and after ischaemia and reperfusion Placebo (n=15) Rosuvastatin (n=16) Atorvastatin (n=16) Before IR-injury Baseline diameter (mm) 0.34 (0.07) 0.36 (0.07) 0.35 (0.06) FMD (mm) 0.37 (0.07) 0.39 (0.07) 0.38 (0.06) FMD (%) 7.7 (3.9) 6.9 (3.4) 8.3 (4.7) Shear rate(s -1 ) (14614) (11349) (8409) Time to peak (s) 63 (24) 56 (23) 50 (16) After IR-injury Baseline diameter (mm) 0.34 (0.07) 0.37 (0.06) 0.34 (0.06) FMD (mm) 0.36 (0.07) 0.39 (0.06) 0.36 (0.05) FMD (%) 5.1 (2.4) 5.8 (3.0) 5.7 (3.4) Shear rate (s -1 ) (11661) (10755) (9676) Time to peak (s) 44 (25) 48 (24) 46 (20) Reduction FMD (%) Data represent mean (SD) 84

87 Statins and endothelial dysfunction after IRI Endothelial function and IR An overview of the FMD measurements is presented in table 2. No differences were observed in baseline diameter or in the shear rate stimulus between the FMD-response before and after IR-injury. In the placebo group, IR significantly decreased FMD (paired t-test p<0.05). Treatment duration (3 vs. 7 days) did not affect the results. Therefore, for each statin, data of 3 and 7-day treatment are pooled. Statin pre-treatment did not alter the effect of IR on FMD, irrespective of the type of statin used (see figure 2). Figure 3 shows the reduction in FMD (FMD before IR minus FMD after IR) for all individual participants in all treatment groups Before IRI After IRI p<0.05 p= FMD (%) Placebo Rosuvastatin Atorvastatin Figure 2 Brachial artery flow mediated dilation (presented as percentage change from baseline) in all pooled treatment groups before IR (open bars) and after IR (closed bars). Error bars represent standard errors. 85

88 Chapter 6 15 FMD reduction (%) Placebo Rosuvastatin Atorvastatin Figure 3 Individual results of reduction in FMD after IR (FMD before IR minus FMD after IR) for all treatment groups. Lines represent mean. Discussion Using a double blind, randomised design we examined the effect of a short-term pretreatment (3 to 7-days) of a lipophilic (i.e. atorvastatin) and a hydrophilic (i.e. rosuvastatin) statin to prevent the IR-induced attenuation in endothelial function in humans. First, we confirmed previous observations of a brachial artery endothelial dysfunction after IR-injury in healthy volunteers. However, in this relatively large sample size, the observed effect of IR on brachial FMD was smaller (34%) than reported previously (40-80%). 54,134, ,194 In contrast to Liuni et al., we observed a persistent reduction in FMD by IR in statin-treated individuals, irrespective of the type of statin used. This suggests that the protective effects of statins against IR-injury are not mediated through preservation of NO-mediated endothelial function. We found that IR resulted in endothelial dysfunction in the control group, which confirms observations in previous studies. 80,83,133,134,156 However, the 34% decrease in endothelial function seen in the current study is less than typically observed in most studies. 80,83,133,156 A potential explanation for this finding is that our ischaemic stimulus was relatively mild compared with previous studies. Indeed, using an ischaemic stimulus of 20 minutes, MacAllister s group repeatedly demonstrated a reduction of the brachial artery endothelial function of 50% to 70%. 158,160,161 Because we adopted a methodological design comparable to MacAllister s group, our findings suggest that the duration of ischaemia to induce IR-injury importantly determines the magnitude of decrease in FMD after IR-injury. In 86

89 Statins and endothelial dysfunction after IRI addition, the site of FMD assessment may play a role in the magnitude of endothelial dysfunction after artificial IR-injury. Liuni et al. found a remarkable reduction of 83% in radial artery FMD after a 15-minute ischaemic stimulus. 156 Recent reports have demonstrated heterogeneity in response to physiological stimuli between the brachial and radial artery, which may contribute to variation in FMD decrease after a 15-minute ischaemic stimulus between studies. For example, a 6% constriction in radial artery was found when exposed to 5-minutes low flow 82, whereas a similar stimulus in the brachial artery has no impact on resting diameter 267,288 or even leads to a slight dilation 267. Also, the degree of dilation to a 5-minute ischaemic stimulus differs between both arteries when examined simultaneously within subjects. 265 Taken together, these observations suggest that methodological aspects (e.g. site of measurement, ischaemia-stimulus for IR-injury) must be considered when comparing studies that examine the impact of IR on endothelial function. The site of measurement of endothelial (dys)function in relation to IR is of special interest to ultimately extrapolate the results to more clinically relevant vascular beds, such as the coronary arteries. In the brachial artery a diminished FMD can predict future cardiovascular events 86,111, whereas such prognostic value is less clear for the radial artery. In addition, a correlation was found between coronary artery endothelial function and brachial and radial artery endothelial function. 10,276 Combining the correlation of the coronary circulation with the strong predictive value of the FMD for clinically relevant end-points (future cardiovascular events), the brachial artery seems a logical measurement site for human in vivo models studying IR-injury. It is important to realise that experiments on endothelial dysfunction after IR-injury are almost exclusively carried out in healthy volunteers. At present it is not known to what extent these findings can be extrapolated to patients with ischaemic heart disease. Furthermore, given the current discrepancies between studies adopting a different methodological design, we strongly recommend harmonization of the methodological approach for future studies of the effects of IR on endothelial dysfunction. To further support this process we are currently performing a study to directly compare brachial and radial artery with respect to the effect of 15 minutes of IR on FMD. An important finding of our study is the persistent reduction in FMD after short statin pretreatment on brachial artery endothelial function after IR-injury in a relatively large study population. The effect of statins was independent of the duration of the pretreatment (3 vs. 7 days), also compound lipophilicity did not alter the ability to protect the endothelium against IR-injury. Our findings suggest that statins do not protect the brachial artery against IR induced endothelial dysfunction, which is in contrast to a previous study that demonstrated that a single dose of 40 mg of rosuvastatin completely prevents IR-induced endothelial dysfunction in the radial artery. 156 As discussed above, methodological differences between studies relating to the site of measurement (brachial vs. radial) may 87

90 Chapter 6 contribute to these conflicting findings. In addition, differences in the dose (40 vs. 20 mg) and duration of pretreatment (3-7 days vs. single dose) between our study and that of Liuni et al. 156 may contribute to the distinct findings of these studies. However, based on the pharmacokinetics of rosuvastatin, the estimated serum levels of rosuvastatin at the time of FMD measurement in the experiment by Liuni et al. should be comparable with those in the present study before IR, because of some accumulation of rosuvastatin after repeated dosing. Therefore, the protective effect of statins against endothelial dysfunction after IR may occur in the radial artery but not in the brachial artery. However, future studies should further examine this potentially clinically important topic. Recently, we found that short-term treatment with rosuvastatin reduces ischaemia-induced phosphatidylserine exposition in the thenar muscle as quantified with annexin A5 targeting. 172 This finding indicates that rosuvastatin pretreatment increases tissue tolerance against ischaemia-induced cellular apoptosis and subsequent injury. The results of our present experiment add the novel knowledge that the attenuating effect of rosuvastatin on ischaemia induced phosphatidylserine exposition is not related to endothelial dysfunction after IR-injury. We have previously observed a 50% increase in ecto-5 -nucleotidase activity on peripheral blood mononuclear cells of volunteers who received an 8 day rosuvastatin pretreatment (20 mg daily). 173 In this study we show that RNA-expression of ecto-5 -nucleotidase on peripheral blood mononuclear cells was not increased after short-term statin treatment. Although we did not measure ecto-5 -nucleotidase activity, combining our findings with previous observations suggests the presence of a nongenomic effect of rosuvastatin on ecto-5 -nucleotidase activity, such as changes in posttranslational modification or localization of the enzyme. In accordance with this interpretation, others have demonstrated in endothelial cell culture that lovastatin increases ecto-5 -nucleotidase activity by reducing enzyme endocytosis. 145 In the dog heart, the phosphatidylinositol 3-kinase/Akt pathway has been involved in the upregulation of ecto-5 -nucleotidase by statins. 234 The exact mechanism of up-regulation in humans needs further exploration. Conclusion In the present study, we show a moderate effect of IR on brachial artery endothelial function, which persisted after adequate short-term hydrophilic or lipophilic statin treatment. Because brachial FMD predicts cardiac events, this suggests that the cardioprotective effects of statins are not mediated through preservation of endothelial function after IR. 88

91 Statins and endothelial dysfunction after IRI Acknowledgements We thank Professor Daniel J. Green and Mr Christopher Reed (University of Western Australia) for the provision and development of the FMD-analysis software. 89

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93 Chapter 7 Impact of ischaemia and reperfusion injury on endothelial function in the radial and brachial artery in humans in vivo Constantijn Wouters Karin van Geel Gurpreet Birk Ellen Dawson Gerard Rongen Timothy Cable Daniel Green Dick Thijssen Submitted

94 Chapter 7 Abstract Aim: Flow mediated dilation as a read-out of endothelial function is often used to explore the efficacy of interventions to reduce endothelial ischaemia-reperfusion injury in humans. These proof-of-concept studies translate preclinical findings in animals to humans and facilitate optimal design of future clinical trials. Differences in methodology, such as the used artery, limits comparison of results between various research groups. Between-study comparisons suggest a difference in susceptibility of IR-injury between the brachial and radial artery. Therefore, we directly compared the effect of IR on endothelial function between the radial and the brachial artery. Methods: We simultaneously examined FMD responses in the brachial artery and ipsilateral radial artery before and after IR of brachial and radial artery. We applied 15 minutes of ischaemia and 15 minutes of reperfusion which represents a frequently used and validated method to induce forearm IR-injury. To determine the FMD, we measured brachial and radial artery diameter and blood flow using duplex ultrasound before and after a 5-minute ischaemic stimulus applied distal from the measurement sites. Results: FMD was significantly attenuated after IR in the radial artery from 7.8±3.8% to 6.3±2.7% and in the brachial artery from 5.1±2.4% to 3.6±1.6% (p=0.03 and p=0.01, respectively). We found no difference in the absolute or relative decrease in FMD between the brachial and radial artery (p=0.68 and p=0.97, respectively). Conclusions: We have shown that the radial artery and the brachial artery demonstrate an equal and significant decrease in FMD in response to IR in healthy volunteers. 92

95 FMD measurement in radial and brachial artery Introduction Ischaemia and reperfusion injury (IR-injury) is an important determinant of mortality and morbidity in patients after an ischaemic insult, such as a myocardial infarction or stroke. 305 Interventions such as ischaemic preconditioning, aimed at improving tolerance to IR-injury can reduce infarct size up to 75%, which emphasises the importance to modulate tolerance to IR-injury in the clinical management of ischaemic insults. 183 A distinct feature of IR-injury relates to the presence of endothelial dysfunction. 103 It is believed that IR-induced endothelial dysfunction is caused by the activation of arginase during ischaemia, which competes with NO-synthase for its substrate L-arginine and subsequently impairs NO-release. 105 In a clinical setting, endothelial dysfunction after IR-injury may contribute to the no-reflow phenomenon during reperfusion and thereby increase infarct size. 117 The assessment of the flow-mediated dilation (FMD) before and after artificial ischaemia and reperfusion provides a popular, non-invasive and well tolerated model to study endothelial tolerance to IR in humans in vivo. 133 This model has successfully been used to translate preclinical findings in animals to humans in vivo. These proof-of-concept studies are important to facilitate effective design of clinical trials aimed to reduce IR-injury and subsequent prognosis. The validity of this model is supported by the ability to prevent the decrease in FMD after IR by well established (non)pharmacological interventions that improve IR tolerance. 156,158,161 Both radial and brachial artery can be used for FMD assessment. Regional heterogeneity in wall architecture 268 and vascular function 265, has been demonstrated to alter the magnitude of FMD. Accordingly, the significant difference in baseline diameter between the brachial and radial artery may have an influence on the impact of IR-injury on endothelial function between both arteries. Indeed, a between-study comparison of non-interventional experiments (placebo or control) reveals an apparent larger decrease in FMD after IR-injury in the radial artery, compared to the brachial artery (Table 1). Moreover, such differences between arteries may also explain apparent conflicting results from recent studies. For example, recent reports showed a preserved endothelial function of the radial artery after single dose rosuvastatin treatment prior to forearm IR 156,157, whilst another study found no protective effect of rosuvastatin when examining the brachial artery. 297 These observations highlight the importance to directly compare the change in FMD in response to IR-injury between different artery sites in humans. Therefore, we simultaneously studied the radial and ipsilateral brachial artery FMD before and after IR-injury in healthy subjects. Based on available literature, we hypothesised a larger IR-induced decrease in radial artery FMD compared to the brachial artery. 93

96 Chapter 7 Table 1 Relative decrease in radial (left panel) and brachial (right panel) artery flow-mediated dilation after ischaemia reperfusion (I/R) injury in noninterventional groups (placebo or controls), presented per individual study (with first author, sample size and duration for the ischaemia and reperfusion (I/R) in minutes) and all studies averaged (last row). Radial artery Brachial artery Reference n I/R %Decrease Reference n I/R %Decrease Kharbanda /15 55% Kilian /30 34% Gori /15 85% Kilian /30 80% Dragoni /15 78% Loukogeorgakis /20 44% Liuba /15 63% Loukogeorgakis /20 60% Gori /15 61% Loukogeorgakis /20 65% Dragoni /15 68% Loukogeorgakis /20 65% Liuni /15 83% Okorie /20 61% Dragoni /15 76% Devan /15 37% Gori /15 84% Wouters /15 34% Liuni /15 74% Webb /20 60% Liuni /15 56% Liuni /15 56% Liuni /15 75% Lisi /15 86% Ha /15 62% Average 71±11% Average 54±16% Methods Subjects A total of 20 healthy young volunteers (8 men, 12 women) without a history of cardiovascular disease, diabetes or family history of cardiovascular death (<50 years) were recruited from the community (Table 2). All subjects were non-smokers and normotensive (<140/90mmHg). None of the subjects used any medication known to interfere with the cardiovascular system. The local Ethics Board of the Liverpool John Moores University approved the protocol and written informed consent from all participants was obtained 94

97 FMD measurement in radial and brachial artery Table 2 Subject characteristics for all participants (n=20) presented as mean ± SD Characteristic Mean ± SD Age (years) 24 ± 3 Height (cm) 172 ± 8 Body weight (kg) 65.5 ± 1.1 Body mass index (kg/m²) 19.0 ± 2.6 Systolic blood pressure (mmhg) 119 ± 19 Diastolic blood pressure (mmhg) 69 ± 7 Heart rate (beats/min) 63 ± 12 prior to participation. All experiments were carried out in accordance with the declaration of Helsinki (2000). Experimental design and measurements Testing was performed between 8.30 AM and 4 PM. Previous studies found that the FMD does not change significantly across this time frame, assuming that testing is performed under well-controlled conditions 263. All measurements were performed under standardised conditions in a temperature-controlled room and after at least 6 h of fast and at least 8 h of abstinence from caffeine and alcohol. No subject performed strenuous physical activity for at least 24 h before testing. Flow mediated dilation. Subjects rested in a supine position with the experimental arm extended and immobilised, supported at an angle of ~80 abduction from the torso. Heart rate and mean arterial pressure were determined from an automated sphygmomanometer (GE Pro 300V2, Dinamap, Tampa, FL). For the assessment of FMD, a rapid inflation/deflation pneumatic cuff was placed distal to the olecranon process to provide an ischaemic stimulus to provoke vasodilation and subsequent shear stress. A 10-MHz (T3000, Terason, Aloka, UK) multi-frequency linear array probe attached to a high-resolution ultrasound machine was used to perform imaging. The brachial artery was imaged in the distal third of the upper arm. The radial artery was imaged in the upper part of the forearm, proximal to the occluding cuff. Ultrasound parameters were set to optimise longitudinal B-mode images of the lumen/arterial wall interface. A continuous Doppler velocity assessment was obtained simultaneously, and data were collected using the lowest possible insonation angle (always <60 ), which did not vary during each study. 296 After a resting period of at least 15 minutes, 1 minute of baseline recording of the arterial 95

98 Chapter 7 diameter and velocity was performed (in brachial and ipsilateral radial artery). Subsequently, the occlusion cuff was inflated to 220 mmhg for 5 minutes. The arterial diameter and velocity recordings were restarted at least 30 seconds before cuff deflation and continued for at least 3 minutes after deflation. Peak arterial diameter and flow, and the time to reach this peak after cuff deflation, were recorded. IR-injury. The rapid inflation/deflation pneumatic cuff was positioned proximally around the upper arm to provide an occlusion for 15 minutes, so that the brachial artery was within the ischaemic zone and was exposed to IR-injury. The cuff was inflated for 15 minutes to 220 mmhg, which was followed by 15 minutes of reperfusion. This method is safe and represents a valid and reproducible method to induce transient endothelial dysfunction in vivo in humans. 83,133 This procedure has no effect on blood pressure, heart rate or basal blood flow. 296 After simulated ischaemia and reperfusion, we repeated simultaneous assessment of the FMD in brachial and ipsilateral radial artery, as described above. Arterial diameter and blood flow analysis Analysis of the brachial and radial artery diameter was performed using custom-designed edge-detection and wall-tracking software, which is independent of investigator bias. 296 Briefly, the echo-doppler signal was real-time encoded and stored as a digital file. Subsequent software analysis of the data was performed at 30 Hz using an icon-bases graphical programming language and toolkit (LabView 6.02, National instruments, Austin, TX). Furthermore, our method of blood flow assessment is closely correlated with actual flow through a phantom arterial flow system. 85 Data analysis Baseline diameter and shear rate were calculated as the mean of data acquired across the 1 minute preceding the cuff inflation period. Following cuff deflation, peak diameter following cuff deflation was automatically detected according to an algorithm which identified the maximum bracket of data subsequent to performance of a moving window smoothing function. This smoothing routine calculates the median value from 100 consecutive samples, before the window shifts to the next bracket of data which shares 20% overlap with the preceding bracket. The maximum value of all the calculated median values is then automatically detected and chosen to represent the peak of the diameter curve. FMD was calculated as the percentage rise of this peak diameter from the preceding baseline diameter. Calculation of FMD was therefore observer-independent and based on standardised algorithms applied to data which had undergone automated edge-detection and wall-tracking. 296 Reproducibility of the FMD using this semi-automated software possesses a CV of %. 266 The post-deflation shear rate data, derived from simultaneously acquired velocity and diameter measures at 30 Hz, was used to calculate 96

99 FMD measurement in radial and brachial artery the area under the shear rate curve for data up to the point of maximal post-deflation diameter (FMD) for each individual. 209 Statistics The statistical analyses were performed using SPSS version 16.0 software. All data are reported as means ± SD (unless stated differently), and statistical significance was assumed at p We used paired T-test to compare baseline ultrasound measurements between radial and brachial artery. A 2-way ANOVA was used to examine the effect of IR-injury on FMD (pre versus post IR) and whether this effect was different between the radial and brachial artery. A Shapiro-Wilk s test was used to control for normality. Results Subject characteristics are presented in Table 2. At baseline, brachial artery FMD was significantly lower compared to the radial artery FMD (Table 3). Brachial artery demonstrated a larger diameter, smaller shear rate and similar time-to-peak diameter compared to the radial artery (Table 3). Table 3 Characteristics of flow mediated dilation of the brachial and radial artery before ( Baseline ) and after ( Post-IR ) ischaemiareperfusion-injury in healthy young subjects (n=20). Data is presented as mean ± SD Radial artery Brachial artery Parameter Baseline Post-IR Baseline Post-IR Baseline diameter (mm) 2.4 ± ± ± 0.6 # 3.7 ± 0.7* Peak diameter (mm) 2.6 ± ± ± 0.6 # 3.8 ± 0.7 Change from baseline (mm) 0.17 ± ± 0.06* 0.18 ± ± 0.07 Flow-mediated dilatation (%) 7.8 ± ± 2.7* 5.1 ± 2.4 # 3.6 ± 1.6* Shear rate (s -1 x10 3 ) 25.4 ± ± ± 9.0 # 14.5 ± 6.0 Time to peak (s) 40.0 ± ± 8.7* 37.6 ± ± 18.3 * Significant p<0.05 different from baseline # Significant p<0.05 from baseline of the radial artery 97

100 Chapter 7 We found that IR resulted in a significantly attenuated FMD in both the radial and brachial artery (2-way ANOVA: p<0.01 for IR-injury, Figure 1). However, we found no difference in 10 A 8 FMD (%) B 15 FMD (%) C Radial artery Brachial artery FMD (%) Two way Anova: IR-injury p=0.002 Artery p=0.003 Interaction p=0.973 Baseline Post-IR Figure 1 Results of FMD (%) measurement before and after IR-injury. Individual results for the brachial (A) and radial artery (B) and group results (C) in healthy young subjects (n=20). Error bars represent SE. 98

101 FMD measurement in radial and brachial artery the magnitude of decrease in FMD between the brachial and radial artery (2-way ANOVA interaction; p=0.97). Also in relative terms, we found no difference between the decrease in radial and brachial artery FMD (19% versus 29%, t-test; p=0.68). A significantly shorter time to peak after IR compared to baseline was found in the radial, but not in the brachial artery (Table 3). We have added gender and baseline diameter as a covariate, but this procedure did not alter our results. Discussion The main purpose of this experiment was to examine the difference in endothelial tolerance to IR injury between the radial and brachial artery; i.e. two frequently used sites to study IR injury in humans. First, we confirmed the presence of an immediate decrease in endothelial function after IR in healthy humans in vivo. More importantly, the absolute and relative decrease in endothelial function caused by IR was comparable between the brachial and radial artery in response to the same IR stimulus. This novel finding suggest that, in contrast to our hypothesis and suggestions from available literature, IR-injury leads to an immediate decrease in endothelial function in healthy subjects that is independent of the artery studied (either brachial or radial artery). In agreement with various previous studies, we found that IR injury in healthy volunteers leads to a decrease in brachial and/or radial artery endothelial function. The effect of IR on brachial and radial artery endothelial function (29% and 19%, respectively) was similar to that observed in a previous study from our laboratory 297, but somewhat smaller compared to others (Table 1). Methodological differences in FMD assessment other than forearm artery (e.g. wrist or forearm cuff placement and (dis)continuous ultrasound recording) could partially account for this difference. 83,133 A potential methodological issue that contributes to the relatively small decrease in FMD in our study relates to the intensity of the ischaemic challenge. Indeed, the decrease in FMD observed in our study was comparable to previous studies that used similar 297 (i.e. 15 minutes) or shorter 134 (i.e. 10 minutes) episode of ischaemia, whilst a larger decrease in FMD was observed when adopting a 20 minutes IR stimulus (Table 1). Also subject characteristics may contribute to differences in change in FMD between studies. Recently, Devan et al. demonstrated that the magnitude of decrease in FMD after IR is strongly and positively related with age. 54 In their experiment, young participants showed a 37% decrease in brachial artery FMD, which was significantly lower than the 68% attenuation in middle-aged subjects. 54 The decrease in FMD as well as the age of our participants is well matched with that observed by Devan et al.. Finally, female sex may alter the impact of IR, as the menstrual cycle alters brachial artery FMD. 293 However, we found no difference in the impact of IR on brachial or radial artery FMD between men and women. 99

102 Chapter 7 Regional differences within and between arterial beds in wall architecture and vascular function have been described before. 268 In accordance with previous observations, we found a larger FMD and smaller baseline diameter in the radial artery compared to the brachial artery. We hypothesised that these differences may contribute to a different susceptibility between arteries for IR-injury. However, we found a similar decrease in endothelial function between the radial and brachial artery when exposed to the same IR challenge. Limitations. A strength of this experiment was the simultaneous measurement of the FMD in the radial artery and brachial artery, which effectively excludes possible bias by local and systemic factors that influence vascular function or the impact of IR-injury. A potential limitation of our study relates to the higher baseline diameter of the brachial artery after IR-injury, which is a finding that contrasts with previous experiments 159, including those from our laboratory. 297 Since we found no difference in baseline diameter in the ipsilateral radial artery, which was measured simultaneously with the brachial artery before and after IR, we believe it unlikely that systemic factors contribute to this finding. Moreover, statistically controlling for these changes in diameter did not alter the outcomes of our study. In clinical experiments, measurement of FMD predicts future cardiovascular events in patients with established cardiovascular disease. 111 The prognostic value of IR-induced endothelial dysfunction, however, remains to be elucidated. Nonetheless, the use of FMD before and after an ischaemic event as model to study IR-injury is valid In conclusion, we have shown that the radial artery and the brachial artery demonstrate a significant and comparable decrease in FMD in response to IR in humans in vivo. Acknowledgements We would like to thank Chris Reed for his assistance with software development. 100

103 FMD measurement in radial and brachial artery 101

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105 Chapter 8 General summary and conclusions

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107 General summary and conclusions As stated previously, ischaemia and reperfusion injury contributes significantly to mortality and morbidity in patients suffering from an ischaemic event (e.g. cerebrovascular event, myocardial infarction). Fortunately, Murry et al. in 1986 reported their novel procedure of ischaemic preconditioning, which could spectacularly reduce myocardial infarct size up to 75%. 183 Comparable results have been produced by other researchers in different organs, in different species and with several variations to the preconditioning protocol (as reviewed in chapter 1). Thereupon, extensive research has been devoted to elucidation of this phenomenon. Today much of the protective mechanism of preconditioning has been unravelled. However, translation of the spectacular effect of preconditioning on infarct size in animal experiments to clinically relevant settings in cardiovascular patients has rather been disappointing. Even though some experiments in patients have found (moderate) clinical benefits (e.g. improved MACE after PCI or reduced myonecrosis markers after CABG) 202,279, these results did not have the impact that could have been expected from a 75% reduction in infarct size. As results in clinical experiments fail to live up to the expectations, it is clear more research is needed to clarify this apparent discrepancy between preclinical findings and clinical effects. Future research has to be performed in humans in vivo to further evaluate the possibilities of preconditioning. This calls for validated and well-tolerated models to study IR-injury in humans. In this thesis we have explored three different methods to study different aspects of IR-injury in humans in vivo. Firstly, we used annexin A5 scintigraphy to quantify IR-injury. This technique, developed at the departments of Pharmacology-Toxicology, General Internal Medicine and Nuclear Medicine of the Radboud University Nijmegen Medical Centre 226, uses binding of radiolabeled annexin A5 to phosphatidylserines which get exposed on the outer leaflet of the cellular membrane upon cell stress. In chapter 2 annexin A5 scintigraphy was used to quantify the effect of a short-term treatment with atorvastatin on voluntary forearm ischaemia and subsequent reperfusion. We found no difference in annexin A5 targeting between atorvastatin and placebo treatment. As atorvastatin has previously been shown to prevent myocardial damage 202,203, we conclude that prevention of PS exposition is not part of atorvastatin s protective mechanism. This experiment included an extra control group that was randomised to receive placebo treatment twice. In this control group we calculated a within-individual correlation of annexin A5 targeting of 0.93 and 0.79 at 1 and 4 hours after reperfusion respectively. These results show an adequate reproducibility of annexin scintigraphy after IR-injury, especially for the measurement at 1 hour after reperfusion. In chapter 3 we studied whether the clinical effectiveness of irbesartan is hampered by increased apoptotic activity, detected by exposition of phosphatidylserines, during 105

108 Chapter 8 ischaemia and reperfusion in humans in vivo. Annexin A5 targeting was not influenced by a 7-day treatment with irbesartan 300mg, compared to placebo. The results of this study do not support enhanced apoptotic activity after treatment with irbesartan in a setting of ischaemia and reperfusion. In chapter 4 we further studied the alleged protective effect of dipyridamole against IRinjury. 92,220 Healthy male volunteers were randomised to dipyridamole and placebo treatment in a crossover design. Using annexin A5 scintigraphy we found a carry-over effect between the experimental and placebo treatment. Dipyridamole reduced annexin A5 targeting in those volunteers who received placebo first and dipyridamole next, compared to those receiving dipyridamole first and placebo next. As we implemented a washout period of 3 weeks, this carry-over effect existed for at least 4 weeks. Next, we further examined the observed prolonged protective effect of dipyridamole. To address a possible role of extended nucleoside transport inhibition in the apparent long-term action of dipyridamole, we investigated the 4-week post-treatment effect of a 7-day oral dipyridamole administration on nucleoside transport ex vivo in human erythrocytes. Nucleoside transport was inhibited directly after dipyridamole treatment, however within 1 week of treatment cessation this inhibitive effect had disappeared completely. A prolonged effect of dipyridamole on nucleoside transport is not involved in the apparent prolonged protective effect of dipyridamole on PS-exposition. Even though annexin A5 scintigraphy appears to be a reproducible and suitable method to quantify PS exposure as one of the sequellae of IR-injury, its validity as a tool to translate preclinical observations of strategies to prevent IR-injury to humans in vivo is hampered by the limited role that PS exposure plays in the mechanism of IR-induced cell death. This conclusion is based on our finding that atorvastatin did not prevent annexin A5 targeting, despite its proven efficacy in patients who present with an acute myocardial infarction (chapter 3) and the limited role of PS exposure in renal IR-injury as reported recently by Wever et al. 290,291 Finally, further development of this method is seriously hampered, as human grade annexin A5 is presently no longer available. Clearly, there is a need to explore alternative approaches to study IR-injury in humans in vivo. Therefore, we studied endothelial dysfunction as feature of IR-injury and used two models to quantify the impact of IR on endothelial function. First, we used post-occlusive reactive hyperaemia (PORH) in the forearm of healthy volunteers as a model of post-ischaemic vascular function. As we have previously shown that rosuvastatin increases vasodilation after ischaemia in the human forearm 173, in chapter 5 we further clarified the mechanism behind this effect of rosuvastatin. We have shown that rosuvastatin increases the activity of the enzyme ecto-5 - nucleotidase (which dephosphorylates extracellular AMP to adenosine) with almost 50%. Moreover we have shown the role of adenosine receptor stimulation in rosuvastatin- 106

109 General summary and conclusions induced augmentation of PORH, as rosuvastatin enhanced the vasodilatory response after 2, 5, and 13 minutes of ischaemia in the absence, but not in the presence of caffeine (a potent adenosine receptor antagonist). Finally, we adapted flow mediated dilation (FMD) measurements performed at the Department of Physiology of the Radboud University Nijmegen Medical Centre to be used for quantification of IR-injury, according to scientific literature. 133 This relatively new and non-invasive technique to study IR-induced endothelial dysfunction, appears to be a promising method to study the mechanism of IR-induced endothelial dysfunction and the effect of (pharmacological) interventions. 161 In chapter 6 we used FMD to compare the effect of two different statins (the lipophilic atorvastatin vs. the hydrophilic rosuvastatin) on their ability to prevent endothelial dysfunction of the human brachial artery induced by IR. Our results show that IR significantly decreases FMD, however statin pre-treatment did not alter the effect of IR on FMD, in contrast to a similar experiment conducted recently by Liuni et al. 156 Our results suggest that the cardioprotective effects of short term treatment with statins as observed in clinical trials with patients presenting with an acute myocardial infarction are not mediated through preservation of endothelial function after ischaemia and reperfusion. The conflicting findings between chapter 6 and those of Liuni et al 156, led to the design of chapter 7. Presently, FMD measurements to assess IR-induced endothelial dysfunction are performed in either the brachial or radial artery. In chapter 7 we showed equal susceptibility of the radial and brachial artery to endothelial dysfunction after IR-injury, when we measured the effect of IR simultaneously in both arteries ipsilaterally. With this result we can compare and extrapolate results from previous experiments using FMD to assess endothelial dysfunction after IR. As stated before, measurement of FMD to assess IR induced endothelial dysfunction appears a promising method to investigate IR-injury in humans in vivo. However there are several methodological issues to this model that have not yet been addressed scientifically. To optimally apply this experimental method, knowledge on the exact impact of the total area of ischaemia, duration of ischaemia, or duration of reperfusion on FMD measurement is needed. Subtle differences in methodology may be responsible for the discrepant results as obtained in our study on the effect of statins on IR-induced endothelial dysfunction (chapter 6) and those obtained by others According to our results differences in site of measurement (brachial or radial artery) do not seem to be of methodological relevance. 107

110 Chapter 8 Future perspectives As reviewed by Yellon et al. myocardial IR-injury has several clinically important aspects: cellular death, arrhythmias, myocardial stunning and endothelial dysfunction. 305 Models used to study IR-injury (as reviewed in chapter 1) rarely cover all these different aspects together. For example all the models used in this thesis, study a single aspect of IR-injury: annexin A5 scintigraphy visualises cellular PS exposure as a marker of apoptosis, and FMD and PORH assess endothelial dysfunction. However the clinical consequences of IR-injury to patients are the result of all four aspects (cellular death, arrhythmias, stunning and endothelial dysfunction) together. If human models on IR-injury should be able to study a combination of aspects of IR, and ideally all four aspects (as stated above) together, this should enhance translation of study results to clinical outcomes. Therefore, the search for appropriate (and more complex) models to study IR-injury in humans in vivo should continue. As stated before, unfortunately, much of the protective effect of ischaemic preconditioning is lost in translation from animal experiments to clinical trials in cardiovascular patients. 245 This loss has partially been explained by previous experiments showing that susceptibility to IP is reduced by e.g. increasing age 54, hypercholesterolaemia 273 and diabetes mellitus 114. Moreover susceptibility to IR-injury is influenced by many drugs that are frequently prescribed to patients with cardiovascular disease (e.g. statins, dipyridamole, ARA s, ACE-I, sulphonylurea derivatives). 78,222 Altogether the ability to prevent IR-injury depends on many different factors. In numerous experiments (including those presented in this thesis) healthy young volunteers are used as study objects. The use of medication was an exclusion criterion in all experiments reported here. Can we really translate the results from these young healthy subjects to the aged population of cardiovascular patients with co-morbidity and co-medication? To study clinical benefits and applicability of strategies to prevent IR-injury, we need to include more relevant study populations. Therefore, future research should focus more on patients with relevant co-morbidity, as opposed to the healthy volunteers. Potentially, the use of elective PCI or CABG as models of controlled ischaemia and reperfusion could prove to be a more adequate translational model to study the effects of IR-injury. Using either troponin release as marker of myocardial tissue damage, or MRI or CT-scan to assess both area at risk and the amount of myocardial damage. In this model duration of ischaemia can easily be recorded and different aspects of IR-injury can contribute to the tissue injury. Moreover this model can (only) be applied in patients suffering from cardiovascular disease, with relevant co-morbidity and medication. Another important reason to continue the search for adequate translational models to study IR-injury is the enormous costs of clinical trials. In the end, doctors are not truly 108

111 General summary and conclusions interested in ECG changes or raised biomarkers, they really care about the wellbeing and survival of their patient. To test hard clinical endpoints (such as survival) in a relevant population (i.e. patients suffering from actual IR-injury), large randomised clinical trials are needed. These trials often include up to several thousand patients and are often organised in an international multicentre setting. Organising such large experiments, compliant to standard of Good Clinical Practice is a rather expensive business as costs can reach over a billion euro. 112 To minimise expenses, study designs have to be optimised prior to initiation of the large phase 3 trials. The use of translational experiments can be very helpful. When issues as optimal drug dosing, duration of treatment, timing of treatment, and identification of possible confounders (e.g. co-medication) can be tested using small and relatively inexpensive translational experiments using intermediate endpoints, this facilitates smart and efficient designing of large clinical trials. In conclusion, ischaemia and reperfusion injury causes significant mortality and morbidity in patients suffering from an ischaemic event. In animal experiments successful prevention of IR-injury has shown spectacular reductions in (myocardial) infarct size. In a clinical setting prevention of IR-injury has been proven, however the effect appears to be moderate. To further examine the possibilities and benefits of prevention of IR-injury more research is needed. Translational models on IR-injury can be very helpful to further unravel the mechanism and optimal setting of successful prevention of IR-injury. The models presently available are well suited to perform mechanistic experiments, though do not predict clinical outcomes in cardiovascular patients. Development of new translational models on IR-injury is warranted as these could further improve research on clinical benefits of IR-injury prevention, and aid in clinical trial design. 109

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113 Nederlandse samenvatting en conclusies

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115 Nederlandse samenvatting en conclusies Tijdens een hartinfarct is er sprake van een acute afsluiting van de bloedvoorziening (ischemie) van de hartspier. Wanneer de doorbloeding niet tijdig hersteld wordt, leidt dit onvermijdelijk tot schade aan de hartspier. Bij een patiënt met een acuut hartinfarct is medische behandeling er in eerste instantie op gericht om de doorbloeding van de hartspier zo snel mogelijk weer te herstellen en zo de schade zoveel mogelijk te beperken. Zo kunnen complicaties van hartspierschade, zoals hartfalen of overlijden worden beperkt of voorkomen. Echter, er is gebleken dat ook tijdens het herstellen van de doorbloeding (reperfusie) de hartspiercellen beschadigd raken. Ischemie en reperfusie (IR) schade is derhalve de som van de schade die een orgaan (bijvoorbeeld het hart, de hersenen of de nieren) oploopt ten gevolge van een acute onderbreking van de doorbloeding en het herstel van de doorbloeding. In 1986 ontdekten Murry et al. dat IR-schade kan worden beperkt. Zij toonden dierexperimenteel (bij honden) aan dat wanneer een hartinfarct werd voorafgegaan door enkele kortdurende episodes van ischemie van het hart, de schade van het hartinfarct met maar liefst 75% kon worden verminderd. 183 Het proces van deze voorbereidende kortdurende onderbreking van de doorbloeding noemden Murry et al. ischemische preconditionering (IP). Aanvullend onderzoek liet zien dat ischemische preconditionering ook in andere organen een beschermend effect heeft (bijvoorbeeld de nieren, de lever en de hersenen). Bovendien is aangetoond dat ischemische preconditionering ook werkt bij andere dieren, zoals konijnen, muizen, ratten en varkens. Na de ontdekking van dit beschermende effect van kortdurende ischemie werden onderzoekers nieuwsgierig naar het precieze werkingsmechanisme. Na veel wetenschappelijk onderzoek is dit fenomeen inmiddels grotendeels ontrafeld. Op basis van dit werkingsmechanisme zijn diverse interventies bedacht die IR-schade zouden moeten kunnen verminderen. Hoewel bij dieren deze interventies spectaculaire resultaten toonden (tot 75% afname van de schade), waren de resultaten bij patiënten vooralsnog teleurstellend. Enkele onderzoeken, waarbij statines of ischemische postconditionering als interventie werden toegepast, laten wel enige gunstige effecten zien, zoals minder complicaties bij patiënten die een dotteringreep ondergingen, of lagere bloedwaarden die de mate van hartspierschade weergeven bij patiënten na een bypassoperatie van het hart. 202,279 Andere interventies, zoals met adenosine, lieten zeer teleurstellende resultaten zien. Om de problemen die ontstaan bij de vertaalslag van dier naar mens op te lossen is dringend aanvullend onderzoek nodig. Hiervoor hebben we behoefte aan toegankelijke en gevalideerde onderzoeksmodellen, waarmee we IR-schade in de mens kunnen bestuderen. In dit proefschrift hebben we drie onderzoeksmodellen gebruikt om verschillende aspecten van ischemie en reperfusie-schade in mensen te bestuderen. 113

116 Nederlandse samenvatting en conclusies Allereerst hebben we met behulp van annexin A5-scintigrafie het effect van IR op cel - schade gemeten. Deze techniek is ontwikkeld door de afdelingen Farmacologie-Toxicologie, Algemene Interne Geneeskunde en Nucleaire Geneeskunde van het Universitair Medisch Centrum Nijmegen. 226 Wanneer cellen door IR beschadigd raken, verschijnen aan de buitenkant van deze cellen zogenaamde fosfatidylserines. Deze moleculen spelen een belangrijke rol in het afsterven en netjes opruimen van dode cellen. Het eiwit annexine A5 bindt specifiek aan deze fosfatidylserines. Door het annexine A5 te labelen met radioactief 99m Technetium kan een gammacamera de ophoping van annexine A5 aan de uitgestoken fosfatidylserines in beeld brengen en op deze wijze de intensiteit van de celschade meten. In hoofdstuk 2 is met behulp van annexine A5-scintigrafie het effect van een korte behandeling met atorvastatine op IR-schade onderzocht bij gezonde vrijwilligers. Er was echter geen verschil in annexine A5 binding tussen de groep die werd behandeld met atorvastatine en de groep behandeld met een placebo. In eerder klinisch onderzoek is meermaals bij de mens een duidelijk gunstig effect van atorvastatine op IR-schade is gevonden. 202,203 Op basis van deze gegevens concluderen we dat bij dit beschermende effect van atorvastatine fosfatidylserines geen rol spelen. Om de reproduceerbaarheid van annexine scintigrafie te bepalen hebben we in dit onderzoek een extra controle groep opgenomen, waarin de deelnemers tweemaal een placebo kregen toegediend. De resultaten van deze groep laten een goede reproduceerbaarheid zien van annexine A5-scintigrafie, met name de meting één uur na reperfusie. In hoofdstuk 3 hebben we onderzocht of de ogenschijnlijk gunstige klinische werking een van angiotensine II-receptor (type 1) blokkade negatief wordt beïnvloed door een toename van celdood (apoptose) tijdens IR-schade, dat zou kunnen ontstaan door stimulatie van de type 2 angiotensine II-receptor. De apoptose-activiteit werd hier gemeten met behulp van annexine A5-binding. Proefpersonen die gedurende zeven dagen werden behandeld met irbesartan 300mg lieten geen toename van annexine A5-binding zien in vergelijking met de groep aan wie het placebo was toegediend. Hieruit concluderen wij dat angiotensine II-receptor (type 1) blokkade met irbesartan niet leidt tot toename van apoptose-activiteit bij IR-schade. In hoofdstuk 4 hebben we aanvullend onderzoek verricht naar het veronderstelde beschermende effect van dipyridamol tegen IR-schade ten gevolge van ischemische inspanning van de onderarm. 92,220 Gezonde proefpersonen werden gerandomiseerd naar een behandeling met dipyridamol en placebo, in een cross-over design. De uitkomsten van annexine A5-scintigrafie lieten een carry-over effect zien tussen de experimentele en placebo behandeling. De annexine A5-binding was verminderd bij de proefpersonen die eerst het placebo kregen toegediend en daarna de dipyridamol, vergeleken met de 114

117 Nederlandse samenvatting en conclusies proefpersonen die in de omgekeerde volgorde werden behandeld. Dit carry-over-effect lijkt op basis van onze bevindingen tenminste vier weken aan te houden. Vervolgens hebben we geprobeerd dit beschermende effect van dipyridamol nader te verklaren. Een mogelijk werkingsmechanisme was een langdurig effect van dipyridamol op het nucleoside transport. Dit hebben we onderzocht door het effect van een behandeling met zeven dagen dipyridamol op nucleoside transport van erythrocyten te meten, tot vier weken na het staken van de therapie. Hierbij namen we waar dat het nucleoside transport direct na de behandeling met dipyridamol sterk was verminderd, maar al een week na het beëindigen van de behandeling was het remmende effect weer geheel verdwenen. Hieruit concluderen we dat het langdurige effect van dipyridamol op fosfatidylserine-expositie niet kan worden verklaard door een verlengd remmend effect op het nucleosidetransport. Er is veel preklinisch onderzoek verricht naar interventies en medicijnen die IR-schade kunnen voorkomen. De vertaling van deze bevindingen bij dieren naar de werkzaamheid op het voorkomen van IR-schade bij mensen behoeft aanvullend onderzoek. Annexine A5-scintigrafie lijkt hiervoor echter geen geschikte methode, daar de rol van fosfatidylserine-expositie in het ontstaan van celschade beperkt lijkt. Uit onderzoek van Wever et al 290,291 blijkt dit bij het ontstaan van IR-schade in de nieren van muizen. Voorts toont ons onderzoek met atorvastatine (hoofdstuk 3), een geneesmiddel dat in meerdere humane studies heeft bewezen IR-schade te kunnen beperken, dat atorvastatine geen effect heeft op annexine A5-binding na ischemie en reperfusie bij gezonde proefpersonen. Bovendien wordt de verdere ontwikkeling van deze onderzoeksmethode gehinderd, doordat annexine A5 dat geschikt is om toe te dienen aan gezonde proefpersonen op dit moment nergens wordt geproduceerd en eigen productie te kostbaar is. Er is veel behoefte aan alternatieve onderzoeksmethoden om de gevolgen van ischemie en reperfusie in mensen te onderzoeken. Behalve directe celschade veroorzaakt IR onder meer hartritmestoornissen en verstoort het de functie van het endotheel in het aangedane weefsel. Wij hebben vervolgens onze aandacht gericht op onderzoeksmethoden die het optreden van endotheeldysfunctie na IR-schade kunnen kwantificeren. Allereerst hebben we in de onderarm van gezonde vrijwilligers post-occlusieve reactieve hyperemie (PORH) gemeten om de impact van ischemie en reperfusie op endotheel afhankelijke vaatfunctie te bepalen. Zoals we in een eerdere studie hebben aangetoond, versterkt rosuvastatine de vasodilatatie die optreedt na kortdurende ischemie in de onderarm. 173 In hoofdstuk 5 hebben we het mechanisme van dit effect van rosuvastatine opgehelderd. De activiteit van het enzym ecto-5 -nucleotidase (dat zorgt voor defosforylering van extracellulair AMP tot adenosine) neemt onder invloed van rosuvastatine met 50% toe. Bovendien hebben we aangetoond dat adenosine-receptoren betrokken zijn bij dit effect, aangezien cafeïne 115

118 Nederlandse samenvatting en conclusies (een adenosinereceptor-antagonist) de toename van vasodilatatie na 2, 5 en 13 minuten ischemie geheel ongedaan maakt. Tot slot hebben we de metingen aangepast van flow gemedieerde vasodilatatie (FMD) zoals die veelvuldig worden verricht op de afdeling Fysiologie van het UMCN, om te gebruiken voor metingen van IR-schade conform de inzichten in de literatuur. 133 Deze niet-invasieve techniek lijkt een veelbelovende methode om het effect van ischemie en reperfusie op endotheelfunctie te bestuderen. Hierbij kan zowel het onderliggende mechanisme verder worden opgehelderd als het effect van (farmacologische) interventies op IR geïnduceerde endotheeldysfunctie. 161 In hoofdstuk 6 hebben we met behulp van de FMD het beschermende effect van twee verschillende statines (het lipofiele atorvastatine en hydrofiele rosuvastatine) tegen het optreden van endotheeldysfunctie na ischemie en reperfusie in de onderarm bij gezonde vrijwilligers bestudeerd. Hierbij zien wij een significante daling van de FMD na ischemie, maar er is geen effect van statine voorbehandeling op het effect van IR op de FMD. Deze resultaten suggereren dat het beschermende effect van statine-voorbehandeling in klinisch onderzoek bij patiënten met een hartinfarct, niet kan worden verklaard door een effect van de statines op de endotheelfunctie na ischemie en reperfusie. De resultaten van hoofdstuk 6 zijn echter in tegenspraak met een recent artikel van Liuni et al. 156 Deze onderzoekers bepaalden echter de FMD op een iets andere plaats in de arm (arteria radialis in plaats van de arteria brachialis). Om te onderzoeken of dit verschil in methode een verklaring is voor het verschil in studieresultaten hebben we een experiment opgezet zoals beschreven in hoofdstuk 7. In dit onderzoek tonen wij aan dat het endotheel van deze beide slagaders even gevoelig is voor IR-schade. Deze bevinding stelt ons in staat om eerdere onderzoeksgegevens met elkaar te vergelijken en te extrapoleren. Zoals reeds gemeld, lijkt het meten van de FMD rondom ischemie en reperfusie een veelbelovende methode om IR-schade bij mensen te bestuderen. Echter, voordat dit model betrouwbaar gebruikt kan gaan worden, moet er nog wel een aantal methodologische kwesties worden onderzocht. Zo moet er nog verder worden uitgezocht wat de invloed is van de omvang van het ischemische weefsel op de FMD. Ook is er nog geen onderzoek gedaan naar de invloed van de timing van de ischemie en de reperfusie. Kleine verschillen in de uitvoering van experimenten kunnen leiden tot onverklaarbare verschillen tussen de resultaten van verschillende experimenten, zoals het geval is bij onze resultaten van het effect van statines op IR geïnduceerde endotheeldysfunctie (hoofdstuk 6) en de resultaten van anderen

119 Nederlandse samenvatting en conclusies Toekomst perspectief Ischemie en reperfusie van het hart kan leiden tot de volgende belangrijke klinische effecten: celschade en celdood, hartritmestoornissen, tijdelijke dysfunctie van hartspiercellen en endotheeldysfunctie. 305 De onderzoeksmodellen die gebruikt worden om IR-schade te bestuderen (zoals beschouwd in hoofdstuk 1) focussen echter op een van de hiervoor genoemde aspecten. Zo wordt met de modellen in dit promotieonderzoek steeds maar een van deze gevolgen bestudeerd: annexine-scintigrafie kwantificeert celschade, terwijl de FMD- en PORHmetingen focussen op endotheeldysfunctie. De gevolgen van IR-schade bij een patiënt zijn echter de resultante van alle effecten van ischemie en reperfusie. Een nieuw humaan onderzoeksmodel dat alle aspecten van IR (celschade en celdood, hartritmestoornissen, tijdelijke dysfunctie van hartspiercellen en endotheeldysfunctie) meeneemt in zijn metingen, zou de translatie van de onderzoeksuitkomsten naar klinische uitkomsten sterk kunnen verbeteren. Daarom moeten we vooral doorgaan met het zoeken naar het ideale (en wellicht meer complexe) model om de effecten van ischemie en reperfusie in de mens te bestuderen. Zoals eerder gemeld lijkt het beschermende effect van preconditionering bij patiënten met hart- en vaatziekten veel minder krachtig dan bij proefdieren. 245 Uit onderzoek is gebleken dat het beschermende effect van preconditionering afneemt bij ouderen 54, patiënten met diabetes mellitus 114 en hypercholesterolemie 273. Bovendien wordt de gevoeligheid voor IR-schade beïnvloed door verschillende geneesmiddelen, die regel - matig worden voorgeschreven aan patiënten met hart- en vaatziekten: zoals statines, dipyridamole, angiotensine-2 antagonisten, ACE-remmers of sulfonylureumderivaten. 78,222 Derhalve is het voorkomen of reduceren van IR-schade afhankelijk van veel verschillende factoren. In veel wetenschappelijk onderzoek (ook in dit proefschrift) worden de experimenten verricht bij jonge gezonde vrijwilligers bij wie medicatiegebruik doorgaans een exclusiecriterium is. Is het daarom wel betrouwbaar om de uitkomsten van dit onderzoek bij deze jonge, gezonde deelnemers te extrapoleren naar patiënten die lijden aan hart- en vaatziekten, met co-morbiditeit en het gebruik van verschillende medicamenten? Om op zinvolle wijze onderzoek te kunnen doen naar het klinische effect van preventie van IR-schade, moet dit onderzoek worden verricht in een relevante onderzoekspopulatie. Daarom moet toekomstig onderzoek zich meer richten op patiënten met hart- en vaatziekten, in plaats van op gezonde proefpersoenen. Mogelijk kunnen electieve PCI of CABG als meer geschikt model kunnen dienen voor translationeel onderzoek naar het beperken van ischemie-reperfusie-schade. Door het meten van vrijgekomen troponines als maat van hartspiercelschade, of door met beeldvormende technieken zoals MRI 206 de ernst van de myocardschade na de procedure (en eventuele 117

120 Nederlandse samenvatting en conclusies interventie) als fractie van het totale gebied waarvan de doorbloeding verminderd is geweest te meten, kan de invloed van ischemie en reperfusie schade worden onderzocht. In een dergelijk model kan de ischemie duur (de totale duur van balloninflatie(s) of de duur van het afklemmen van de aorta) worden geregistreerd als potentiële confounder (verstoorder van onderzoeksresultaat). Bovendien is de mate van hartschade gerelateerd aan prognose van de patiënt waardoor de relevantie van dit eindpunt groter is dan het meten van intermediaire gevolgen van IR-schade, zoals phosphatidylserine-expositie of endotheelfunctie. Tot slot is dit model alleen toegankelijk voor patiënten die lijden aan hart- en vaatziekten, met de bijbehorende co-morbiditeit en medicatie, de relevante patiëntencategorie voor dit onderzoek. Een andere belangrijke reden om de zoektocht naar goede methoden om ischemiereperfusie schade bij de mens te meten zijn de enorme kosten van klinisch geneesmiddelen onderzoek met harde klinische eindpunten (zoals sterfte en ziekenhuisopnames). Uiteindelijk zijn artsen minder geïnteresseerd in ECG-veranderingen of verhoogde schademarkers; hun belangstelling gaat uit naar het overleven en welbevinden van hun patiënten. Om onderzoek naar harde klinische eindpunten (zoals overleven) te doen in een klinische relevante populatie (zoals hartpatiënten), zijn grote gerandomiseerde studies nodig. Voor dergelijke studies zijn doorgaans duizenden patiënten nodig. Om zoveel patiënten bijeen te krijgen moeten deze onderzoeken worden uitgevoerd in meerdere ziekenhuizen, en meestal ook nog in verschillende landen. De kosten van de organisatie van dergelijke onderzoeken, uitgevoerd volgens de kwaliteitseisen van Good Clinical Practice, kunnen oplopen tot meer dan een miljard euro per studie. 112 Het gebruik van translationele experimenten kan een waardevolle bijdrage leveren aan het opzetten van dergelijke grote klinische studies. Wanneer vraagstukken als optimale dosering van het te onderzoeken geneesmiddel, de timing van de behandeling en het opsporen van mogelijke confounders, kunnen worden onderzocht in kleine en relatief goedkope onderzoeken die gebruik maken van een adequaat translationeel onderzoeksmodel, kan deze kennis worden gebruikt om het design van een grote klinische studie te optimaliseren en op deze wijze kosten te reduceren. Zoals hierboven beschreven zal een acute onderbreking van de doorbloeding van het hart leiden tot ischemie en reperfusie-schade, waarbij celdood, hartritmestoornissen, dysfunctie van hartspiercellen en endotheeldysfunctie bij de patiënt kunnen leiden tot beschadiging van het hart of zelfs tot het overlijden. In dierwetenschappelijk onderzoek zijn spectaculaire resultaten geboekt bij het reduceren van de omvang van de schade van een infarct. Bij patiënten met hart- en vaatziekten is inmiddels ook aangetoond dat IR-schade kan worden voorkomen. In tegenstelling tot de resultaten bij dieren lijkt bij mensen het effect echter maar gering. Er is meer onderzoek nodig naar de mogelijkheden en potentiële gezondheidswinst van de preventie van IR-schade. Goede translationele 118

121 Nederlandse samenvatting en conclusies modellen kunnen een belangrijke rol spelen in het verder ophelderen van de beschermende mechanismen en de optimale omstandigheden voor preconditionering. De huidige modellen zijn geschikt voor experimenten naar het mechanisme van preconditionering, maar ze zijn niet in staat om uitkomsten te voorspellen bij cardiovasculaire patiënten. Het is van groot belang om nieuwe modellen te ontwikkelen voor onderzoek naar IR-schade. Deze modellen kunnen dan gebruikt worden voor verder onderzoek naar de preventie van IR-schade, en kunnen helpen bij het efficiënt opzetten van grote klinische studies. 119

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139 References 300. Yamada T, Horiuchi M, Dzau VJ. Angiotensin ii type 2 receptor mediates programmed cell death. Proc.Natl. Acad.Sci.U.S.A 1996;93: Ye Y, bu Said GH, Lin Y, Manickavasagam S, Hughes MG, McAdoo DJ, Perez-Polo RJ, Birnbaum Y. Caffeinated coffee blunts the myocardial protective effects of statins against ischemia-reperfusion injury in the rat. Cardiovasc.Drugs Ther Ye Y, Lin Y, Perez-Polo R, Huang MH, Hughes MG, McAdoo DJ, Manickavasagam S, Uretsky BF, Birnbaum Y. Enhanced cardioprotection against ischemia-reperfusion injury with a dipyridamole and low-dose atorvastatin combination. Am.J.Physiol Heart Circ.Physiol 2007;293:H813-H Yellon DM, Alkhulaifi AM, Pugsley WB. Preconditioning the human myocardium. Lancet 1993;342: Yellon DM, Downey JM. Preconditioning the myocardium: From cellular physiology to clinical cardiology. Physiol Rev. 2003;83: Yellon DM, Hausenloy DJ. Myocardial reperfusion injury. N.Engl.J Med. 2007;357: Yokota N, O Donnell M, Daniels F, Burne-Taney M, Keane W, Kasiske B, Rabb H. Protective effect of hmg-coa reductase inhibitor on experimental renal ischemia-reperfusion injury. Am.J.Nephrol. 2003;23: Zernecke A, Bidzhekov K, Ozuyaman B, Fraemohs L, Liehn EA, Luscher-Firzlaff JM, Luscher B, Schrader J, Weber C. Cd73/ecto-5 -nucleotidase protects against vascular inflammation and neointima formation. Circulation 2006;113: Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, Vinten-Johansen J. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: Comparison with ischemic preconditioning. Am.J.Physiol Heart Circ.Physiol 2003;285:H579-H Zhu Y, Zhang Y, Ojwang BA, Brantley MA, Jr., Gidday JM. Long-term tolerance to retinal ischemia by repetitive hypoxic preconditioning: Role of hif-1alpha and heme oxygenase-1. Invest Ophthalmol.Vis.Sci. 2007;48: Zhu YZ, Zhu YC, Li J, Schafer H, Schmidt W, Yao T, Unger T. Effects of losartan on haemodynamic parameters and angiotensin receptor mrna levels in rat heart after myocardial infarction. J.Renin.Angiotensin.Aldosterone. Syst. 2000;1:

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143 Dankwoord Om te beginnen spreek ik graag al mijn waardering uit voor de proefpersonen die met hun deelname ons onderzoek mogelijk hebben gemaakt. De experimenten waar jullie vrijwillig aan meewerkten waren behoorlijk belastend: ischemie is erg pijnlijk, medicijnen kunnen allerlei bijwerkingen hebben en bij een aantal experimenten werd ook nog radioactiviteit toegediend. Gedreven door nieuwsgierigheid naar medisch onderzoek, de behoefte iets bij te dragen aan de medische ontwikkeling en soms verleid door de onkostenvergoeding, meldden jullie je aan voor deelname. Mijn voornemen om al jullie namen hier af te drukken werd (terecht) verhinderd door good clinical practice: ik bezit alleen nog maar geanonimiseerde onderzoeksgegevens, jullie namen liggen veilig achter slot en grendel. Derhalve in een keer: dank jullie wel! Professor G.A. Rongen, beste Gerard, jouw wetenschappelijke creativiteit en tomeloos optimisme waren onmisbaar bij het laten slagen van dit project. Ongehinderd door zorgen over de praktische uitvoerbaarheid, heb je in mijn aanwezigheid voldoende mooie studies bedacht om wel tien proefschriften mee te vullen. Hartelijk dank voor deze promotieplaats en je jarenlange betrokkenheid en toewijding. Om in jouw stijl af te sluiten: high five! Professor W.J.G. Oyen, beste Wim, met de snelheid van het licht (of meer toepasselijk gammastraling, ook fotonen) was je er wanneer we je hulp nodig hadden. Het instellen van de gammacamera, de analyse van de scans of het beoordelen van de manuscripten, binnen no-time kreeg ik de resultaten van je terug. Hartelijk dank voor de prettige en uiterst voortvarende samenwerking. Professor P. Smits, beste Paul, met heel veel plezier heb ik vier jaar lang op jouw afdeling Farmacologie en Toxicologie gewerkt. Als afdelingshoofd was je voor alles in: borrelen, wekelijks soep scheppen, afdelingsuitjes en afterparties. Jouw integere stijl van leidinggeven is inspirerend. En als we er wetenschappelijk even niet uitkwamen, was een half uurtje discussiëren op jouw kamer voldoende om met nieuwe inzichten en frisse moed weer verder te gaan. De manuscript commissie bestaande uit professor M.J. de Boer, professor J. de Graaf en professor W. van Gilst, wil ik graag danken voor hun kritische beschouwing en enthousiaste goedkeuring van het manuscript. Professor F.W.A. Verheugt, beste Freek, al tijdens mijn introductiecoschap wist je me te strikken voor de cardiologie. Tot mijn grote spijt hebben onze klinische wegen zich nauwelijks gekruist door jouw overstap naar Amsterdam. Ik ben je zeer dankbaar voor je vertrouwen in deze coassistent en de geboden mogelijkheden om me te specialiseren in de cardiologie. 141

144 Dankwoord Dr. L. Noyez, beste Luc, als derdejaarsstudent klopte ik bij je aan voor een korte onderzoeksstage epidemiologie. Dit heeft geleid tot een lange en vruchtbare samenwerking, waarin we een aantal artikelen schreven en samen congressen bezochten. Na mijn artsexamen kon ik bij jou op de afdeling (toen nog) Thorax-Hartchirurgie meteen aan de slag. Ons gezamenlijke werk vormde van een belangrijke basis voor de toegewezen agiko-subsidie die leidde tot dit proefschrift. Met veel plezier denk ik terug aan de vele malen dat we het reilen en zeilen van patiënten voor en na hartchirurgie bespraken op het kleinste kantoortje van het UMCN. Geert, Inge, Carola en Maarten: alle vier hebben jullie een wetenschappelijke stage gelopen op de afdeling Farmacologie en Toxicologie en tijdens deze stages hebben jullie een wezenlijke bijdrage geleverd aan ons onderzoek. Jullie wetenschappelijk talent was toen al duidelijk en inmiddels zijn jullie allemaal bezig zijn met een eigen promotieonderzoek, heel veel succes! Researchverpleegkundigen Mariëlle Verstegen, Karin Saini en Aarnout Jansen van Rosendaal, jullie gezelschap en ondersteuning maakte de vele meetdagen en de bijbehorende administratieve afwikkeling uiterst aangenaam. Dank voor jullie hulp. Researchverpleegkundige Anja Rasing, lieve Anja, het was een feest om met je samen te werken. De opzet en uitvoering van een klinische trial (die tot verdriet van Gerard dit proefschrift niet heeft gehaald) kent vele facetten. Met wellicht als hoogtepunt de dagen die we samen doorbrachten in de diepvries (-20 o C), terwijl buiten de mussen van het dak vielen (35 o C). Ik heb nooit zoveel vreemde blikken getroffen als toen wij in deze hittegolf in onze skikleding (met muts, sjaal en handschoenen) door het ziekenhuis banjerden. Hoofdanalist, maar vooral toegewijd kamergenoot Petra van de Broek, lieve Petra, hoewel ik al die jaren met mijn rug naar je toe heb gezeten, hebben we veel plezier gehad in de Farmacologie-Toxicologielounge. Je verdroeg mijn urenlange Pink Floyd-sessies uitermate goed en je was er altijd om mijn promotiefrustraties geduldig aan te horen en van constructief commentaar te voorzien. Ik mis je! Professor D. Thijssen, beste Dick, in het laatste jaar van mijn onderzoek (waarom niet eerder?) hebben we onze krachten gebundeld en jouw expertise van de FMD metingen gebruikt bij het meten van ischemie en reperfusie-schade. Het tempo waarin jij nieuw onderzoek bedenkt, organiseert, uitvoert, analyseert en publiceert is indrukwekkend. Je recente benoeming tot hoogleraar in Liverpool is een welverdiende positie voor een getalenteerd wetenschapper als jij. 142

145 Dankwoord Michael van Riel, beste Michael, op dagen van annexinemetingen was jij al voor dag en dauw bezig met het voorbereiden van het radiofarmacon. Ongevoelig voor mijn ongeduld en met kwaliteit hoog in het vaandel, bereidde je het 99m Technetium-HYNICannexine A5 en kreeg ik het pas mee als alles tiptop in orde was. Dank voor je toewijding en de altijd vrolijke samenwerking (ook in de hele vroege ochtend). Sylvie Kok, lieve Sylvie, zonder jou had mijn promotieonderzoek aanzienlijk langer geduurd. Farmacologisch onderzoek vereist een goede samenwerking met de apotheek. Onderzoekers willen graag alle gewenste preparaten op stel en sprong geleverd hebben en zonder al teveel administratieve rompslomp. Jij maakt dit, en ook nog binnen de huidige wet- en regelgeving, mogelijk. Dergelijke flexibiliteit en betrokkenheid is zeldzaam, dank je wel voor je hulp. Collega-(arts-)onderzoekers, onder meer: Annemarie, Alexander, Bart, Bas, Douwe, Fleur, Joris, Madeleine, Marc, Marlies, Noortje, Saloua, Suzanne en Thijs, in de hoogtijdagen van de wekelijkse agiko-bespreking was het inspirerend om met elkaar onze onderzoeken te bespreken en uitkomsten te interpreteren en bediscussiëren. Een aantal van jullie heeft reeds de felbegeerde doctorsbul mogen ontvangen en voor de rest komt dit moment steeds dichter bij. Het was mij een waar genoegen om samen met jullie in dit schuitje te zitten. Dr. P. Meijer, beste Patrick, veel werk hebben we samen gedaan. Jouw fraaie proefschrift heeft mij geïnspireerd tot het afmaken van mijn deel. Dank voor deze vruchtbare samen - werking. Waarde collega s van de afdelingen cardiologie van het UMCN en het CWZ. Graag wil ik jullie bedanken voor de betrokkenheid en toewijding bij mijn opleiding tot cardioloog. Bovendien ben ik zeer erkentelijk voor het feit dat ik mijn opleiding grotendeels parttime kan volgen, waardoor ik mijn specialisatie en promotie op prettige wijze kan combineren met de zorg voor mijn gezin. Dr. E. Bollen, beste Ewald, uiteindelijk waren het jouw spannende, mooie en (soms misschien) sterke verhalen over dokters, patiënten en hun ziektes die mij de geneeskunde in geleid hebben. Inmiddels vertel ik zelf ook enthousiast dergelijke verhalen, dank voor je inspiratie. Mijn paranimfen dr. Kim Wever en mr. Reyer Baas. Lieve Kim, de afgelopen jaren hebben we samen gewerkt aan ons promotieonderzoek en in het laatste stadium (veel te laat) hebben we samengewerkt aan ons promotieonderzoek. Maar veel meer nog dan dat koester ik onze vriendschap die ontstond uit de vele werkbesprekingen, lunches, borrels, 143

146 Dankwoord ijsjes (ook in de sneeuw), cursussen en congressen. Door jouw prachtige voorbeeld op 30 november 2012 om uur precies, weet ik een beetje wat me staat te wachten. Waarde Reyer, van bevlogen studenten in de universitaire politiek tot ploeterende promovendi. Ik mis de tijd dat we wekelijks tot diep in de nacht doorhaalden (vooral met vergaderen weliswaar) of het glas uit de squashbaan sloegen. Ik zie je de laatste jaren veel te weinig, daar ga ik verandering in brengen! Lieve Peter en Will, dankzij jullie ben ik wie ik ben. Ik wil jullie dan ook vanuit de grond van mijn hart bedanken voor het door jullie geboden warme nest, vol liefde en vertrouwen, met veel aandacht voor zelfontplooiing. Jullie moedigden mij aan om (ver weg) te studeren, te reizen, te lezen en musea, kerken (en velerlei andere oude gebouwen) en theaters te bezoeken, en leerden mij zo genieten van kunst, cultuur en historie. Zonder jullie had het ook zeker langer geduurd voordat dit proefschrift klaar was. Jullie altijd beschikbare hulp bij het reilen en zeilen van mijn gezin is van onschatbare waarde voor ons allemaal en inmiddels even inspirerend als onmisbaar. De toewijding en liefdevolle aandacht, waarmee jullie voor ons en onze kindjes zorgen, ontroert mij en biedt ons veel geborgenheid. Lieve Klaartje, zoals het broer en zus betaamt, hebben we van jongs af aan in verschillende toonaarden over de meest uiteenlopende onderwerpen gediscussieerd. Inmiddels bespreken onder het genot van thee, wijn of cola vooral de echt belangrijke zaken van het leven. Je bent aandachtig als geen ander en het voelt heel vertrouwd als je bij ons bent. Allerliefste Hugo en Tijl, kleine boefjes van me. Ieder nadeel heeft zijn voordeel, zei een voetballer ooit; in de periode dat ik bezig was met het afronden van mijn proefschrift lag mijn laptop ook binnen jullie handbereik. Dit opende voor jullie een wereld van youtubefilmpjes over Knabbel en Babbel, Sjakie Schildpad, angry birds en werken op pipapanda. nl. Zo hebben jullie meegenoten van de laatste loodjes. Mannen, met jullie erbij is alles een feest! Lieve Malaika, mijn engel! Waar zou ik zonder jou zijn? Eigenlijk sta ik hier nooit bij stil, want ik heb het veel te druk met genieten van waar ik samen met jou gekomen ben. Je bent mijn steun en toeverlaat, altijd. Ik ben dol op jou en op onze prachtige jongetjes. Samen met jullie zijn (en bij voorkeur ergens ver weg) is wat ik het liefste wil. Ik ben apetrots en dolblij dat je in Stellenbosch JA gezegd hebt en nu kan ik me gaan verheugen op ons grote feest. 144

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151 Curriculum Vitae Op 2 oktober 1976 werd Stijn Wouters geboren in het Radboud Ziekenhuis te Nijmegen. Zijn tienerjaren besteedde hij, naast veel hockey, met name aan het behalen van zijn gymnasiumdiploma aan het Stedelijk Gymnasium te Nijmegen. Na uitgeloot te zijn voor de studie Geneeskunde startte Stijn in 1995 met de opleiding Biomedische Gezondheidswetenschappen aan de Radboud Universiteit Nijmegen (toen nog de Katholieke Universiteit Nijmegen). Door de jaarlijkse uitlotingsberichten van IB-Groep (in totaal zeven) werd hij in de gelegenheid gesteld deze studie helemaal af te maken. Met als hoofdvak Epidemiologie en stages aan het Department of Community Health van de University of Auckland te Nieuw Zeeland en het Julius Centrum van het Universitair Medisch Centrum Utrecht, behaalde hij zijn doctoraal in Dankzij de welwillendheid van het College van Bestuur van de Radboud Universiteit Nijmegen, werd hij in 1999 toegelaten tot de studie geneeskunde. Om meteen een heel studiejaar geen enkel college te volgen, maar hij hield zich intensief bezig met facultair, universitair en landelijk (medisch) onderwijs en studentenbeleid. Dit deed hij onder meer als studentassessor in het faculteitsbestuur Medische Wetenschappen en de Raad van Bestuur van het Universitair Medisch Centrum Nijmegen (UMCN), als lid van de Universitaire Studentenraad van de Radboud Universiteit Nijmegen en als student-lid van het Discipline Overleg Orgaan Medische Wetenschappen van de VSNU. In 2000 begon hij met een versneld doctoraal geneeskunde en in 2002 met de klinische coassistentschappen, om eindelijk in 2004 de artsenbelofte af te mogen leggen. Stijns eerste baan was als arts-assistent op de afdeling Thorax-Hartchirurgie van het UMCN en naast zijn werkzaamheden aldaar, diende hij samen met dr. G.A. Rongen en professor dr. F.W.A. Verheugt een aanvraag voor een AGIKO-beurs in bij ZONMW. Met de toekenning van deze onderzoeksubsidie was ook toelating tot de opleiding Cardiologie beklonken (opleider professor dr. F.W.A. Verheugt en later professor dr. J.L.R.M. Smeets). Tussen 2005 en 2009 werkte hij afwisselend aan dit promotieonderzoek en aan zijn vooropleiding Algemene Interne Geneeskunde in het UMCN (opleider professor dr. J.W.M. van der Meer). Eind 2009 zette hij zijn eerste stappen in zijn toekomstige vakgebied op de afdeling Cardiologie van het UMCN. Tussen 2011 en 2012 werkte hij op de afdeling Cardiologie van het Canisius Wilhelmina Ziekenhuis onder de bezielende opleiding van dr. A.J.M Oude Ophuis en dr. E.J.P. Lamfers. Op dit moment is Stijn weer werkzaam op de afdeling Cardiologie van het UMCN, voor het laatste deel van zijn opleiding. Stijn woont samen met zijn verloofde Malaika Fuchs en samen hebben zij twee waanzinnig leuke, lieve en ondeugende zoontjes: Hugo (2008) en Tijl (2009). 149

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153 List of publications