Markers of inflammation and haemostasis: Associations with myocardial injury, adverse remodelling and future clinical events in patients with ST-

elevation myocardial infarction

PhD thesis

Vibeke N. Ritschel, MD

Center for Clinical Heart Research and Cardiac Intensive Care Research Unit, Department of Cardiology

Oslo University Hospital Ullevaal

&

Faculty of Medicine

University of Oslo,

Oslo Norway

© Vibeke N. Ritschel, 2018 Series of dissertations submitted to the Faculty of Medicine, University of Oslo ISBN 978-82-8377-315-6 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo.

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CONTENTS

Acknowledgements...........................................................................................................5

Abbreviations....................................................................................................................7

Listofpapers.....................................................................................................................9

Introduction......................................................................................................................11Cardiovasculardiseaseandacutemyocardialinfarction............................................................11

The atherosclerotic process and plaque rupture.............................................................................12Haemostasis, coronary thrombosis and fibrinolysis......................................................................13

Inflammation.............................................................................................................................15Interleukin-6 signalling.................................................................................................................15Myocardial injury, remodelling and function................................................................................18CCN2/connective tissue growth factor (CTGF)............................................................................19

Aimsofthethesis.............................................................................................................20Specific Aims................................................................................................................................20

Materialandmethods......................................................................................................21Study subjects and design..............................................................................................................21Coronary angiography and PCI.....................................................................................................22Ischemic postconditioning (iPost).................................................................................................22Blood sampling and laboratory analyses.......................................................................................23Echocardiography..........................................................................................................................24Cardiac magnetic resonance (CMR).............................................................................................24Endpoint registration.....................................................................................................................25Statistics........................................................................................................................................25

Summaryofresults...........................................................................................................26Paper I...........................................................................................................................................26Paper II..........................................................................................................................................27Paper III.........................................................................................................................................28Paper IV........................................................................................................................................29

Discussion.........................................................................................................................31Methodologicalconsiderations..................................................................................................31Generaldiscussion.....................................................................................................................33

Members of the IL-6 family in patients with acute myocardial infarction....................................33Members of the IL-6 family and their association with later clinical events.................................35Inflammation as therapeutic target in patients with acute myocardial infarction..........................37Hypercoagulability in patients with acute myocardial infarction..................................................38CCN2 in patients with acute myocardial infarction......................................................................40

Conclusions.......................................................................................................................42

Futureperspectives..........................................................................................................44

References........................................................................................................................45

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5

Acknowledgements

This project has been carried out at Center for Clinical Heart Research and Cardiac Intensive

Care Research Unit, Department of Cardiology, Oslo University Hospital Ullevaal during the

period of 2010 - 2017. I am grateful for the financial support from Stein Erik Hagen

Foundation for Clinical Heart Research.

I want to express my sincere gratitude to everyone who has contributed to this thesis. First of

all I would like to thank my main supervisor Geir Øystein Andersen who offered me the

position as a research fellow and introduced me to the clinical cardiovascular research. He

brought up the idea and formulated the hypothesis. Being a full-time cardiologist and giving

necessary supervision, continuous and constructive feedback has been essential for this work.

Second, I want to thank my co-supervisor, Ingebjørg Seljeflot, for her knowledge, experience,

constructive feedback and advices. She showed great patience and offered invaluable support

throughout all these demanding years. I am grateful for being a part of the research group led

by her and Harald Arnesen. I want to extend my gratitude to my second co-supervisor, Jan

Eritsland, for his excellent feedback, scientific knowledge and constructive responses.

Third, Harald Arnesen, Thomas Weiss, Sigrun Halvorsen, Limalanathan Shanmuganathan and

Pavel Hoffmann, all been co-authors of the present work, are appreciated for their great job in

order for this thesis to take its definite form.

Special thanks to Christian Shetelig for constructive collaboration and shared authorship of

the fourth article.

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I am grateful to study nurse Charlotte Holst Hansen for her support and collaboration with the

third paper of this thesis. I also want to thank her for help with collecting blood samples and

clinical data for the BAMI biobank and database.

Special thanks to Morten Fagerland for giving me insights in statistical methods and for

guiding me through the statistical work in the second paper. Many thanks to Sissel Åkra for

her tremendous work with the laboratory analyses in this thesis, and to the study nurses and

the staff at the Intensive Cardiac Care Unit and Center for Clinical Heart Research for their

excellent assistance for the BAMI biobank and database.

Further, I want to thank my colleagues, fellow PhD students and seniors at the Center for

Clinical Heart Research and Department of Cardiology at OUS Ullevaal for their support.

Last but not least, I want to thank my husband Michael, my children Leonard and Catarina,

my parents Britt and Steinar and my brother Erlend and his wife Carmen for their patience,

continuous support and love. They helped me balance the Ph-D project and my clinical work

at NIMI during an intensive house-building period. They all are keystones in my life.

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Abbreviations

ACS Acute coronary syndrome AKT Protein kinase B (serine/threonine-specific

protein kinase) AMI Acute myocardial infarction BAMI Biobanking in Acute Myocardial Infarction BMI Body mass index CAD Coronary artery disease CAT Calibrated automated thrombogram CCN2/CTGF CCN family protein 2/connective tissue

growth factor CHD Coronary heart disease CMR Cardiac magnetic resonance CRP C-reactive protein CVD Coronary vascular disease ECG Electrocardiogram ELISA Enzyme-linked immunosorbent assay ETP Endogenous thrombin potential ERK-kinase Extracellular signal regulated kinase F1+F2 Prothrombin fragment 1+2 HbA1c Glycosylated haemoglobin HT Hypertension IL-6 Interleukin-6 IR Ischemia-reperfusion JAK Janus kinase LDL Low-density lipoprotein LT Lag time LV Left ventricular LVEDVi Indexed left ventricular end-diastolic volume LVEF Left ventricular ejection fraction LVESVi Indexed left ventricular end-systolic volume MAP kinase Mitogen-activated protein kinase MVO Microvascular obstruction NT-proBNP N-terminal pro B-type natriuretic peptide PCI Percutaneous coronary intervention POSTEMI Postconditioning in ST-elevation myocardial

infarction pTG Peak thrombin generation P13k Phosphoinositide 3-kinase Q Quartile sgp130 Soluble glycoprotein 130 sIL-6R Soluble interleukin-6 receptor STAT Signal transducer and activator of

transcription family STEMI ST- elevation myocardial infarction TF Tissue factor TGFß1 Transforming growth factor beta 1 TnT Troponin T

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List of papers

1. Ritschel VN, Seljeflot I, Arnesen H, Halvorsen S, Weiss T, Eritsland J, Andersen GØ.

Il-6 signalling in patients with acute ST-elevation myocardial infarction. Results in

Immunology 2014;4:8-13.

2. Ritschel VN, Seljeflot I, Arnesen H, Halvorsen S, Eritsland J, Fagerland MW,

Andersen GØ. Circulating Levels of IL-6 Receptor and gp130 and long-term clinical

outcomes in ST-elevation myocardial infarction. Journal of the American Heart

Association 2016;5(6).

3. Hansen CH, Ritschel V, Halvorsen S, Andersen GØ, Bjørnerheim R, Eritsland J,

Arnesen H, Seljeflot I. Markers of thrombin generation are associated with myocardial

necrosis and left ventricular impairment in patients with ST-elevation myocardial

infarction. Thrombosis Journal 2015;13:31.

4. Ritschel V*, Shetelig C*, Seljeflot I, Limalanathan S, Hoffmann P, Halvorsen S,

Eritsland J, Fagerland MW, Andersen GØ. Evaluation of circulating levels of

CCN2/connective tissue growth factor in patients with ST-elevation myocardial

infarction. Scientific Reports 2017;7:11945.

*Shared authorship

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Introduction

Cardiovascular disease and acute myocardial infarction

The cardiovascular mortality rate is reduced by approximately 50 % in the Western world

during the last 30 years1, 2, but cardiovascular disease (CVD) is still the major cause of death

and remains a challenge to public health2. CVD will remain one of the most frequent causes

of death in the future3, and a huge increase is expected in Eastern countries. CVD is mainly

caused by an ongoing atherosclerotic and inflammatory process in the medium-sized and

large arteries 4, 5, and acute myocardial infarction (AMI) is together with sudden cardiac death

and stroke the most serious consequence of the atherosclerotic process6. Despite progress in

treatment, new clinical adverse events, including AMI, stroke, heart failure, repeated

revascularisation and death, are still frequent in survivors of AMI, thus, it is important to

identify novel risk factors associated with long-term prognosis.

AMI is usually initiated by endothelial erosion or plaque rupture which may lead to an

occluding coronary thrombus5. The consequence is ischemia leading to necrosis of the

affected myocardium7-9. AMI is characterised by symptoms of ischemia with rise and fall in

levels of specific cardiac markers, i.e. troponins with minimum one value above the 99th

percentile. This, together with new ST-segment changes, left bundle-branch block or Q wave

appearance in the ECG, loss of viable myocardium or regional hypokinesia/akinesia judged

by echocardiography or cardiac magnetic resonance (CMR) imaging, or signs of an acute

thrombus on the coronary angiogram or by autopsy, make up the definition of AMI10, 11. AMI

is divided into two groups according to ECG changes: ST-elevation myocardial infarction

(STEMI)11 and non-ST-elevation myocardial infarction (non-STEMI). STEMI may appear as

anterior, inferior, lateral, or posterior, depending on the location of the injured artery. Non-

STEMI is more common than STEMI, the frequency being about 70 % of all AMI12.

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The atherosclerotic process and plaque rupture

The atherosclerotic process is associated with known common risk factors like hypertension,

smoking, elevated blood lipids and diabetes mellitus. Progression of the pathophysiological

changes in the vessel-wall13 may lead to abnormal blood flow, disturbed shear stress,

endothelial injury, plaque rupture and thrombus formation. Low-density lipoprotein (LDL) is

a crucial component in the development of atherosclerosis and plaque formation. In this

process LDL particles are modified into oxidized LDL (oxLDL) by free-radical oxygen

species. OxLDL particles accumulate in the vessel wall (Figure 1)4, 14 and induce a low-grade

inflammatory response in the arterial intima13, 15 and foam cell production. OxLDL particles

thus promotes the atherosclerotic plaque formation, which can lead to plaque rupture with

subsequent activation of platelets and the coagulation system with risk of thrombus

development and potential vessel occlusion 4, 13, 16. It is accepted that both the innate and

adaptive immune system are activated, involving neutrophils and monocyte recruitment,

cytokine activation, migration of smooth muscle cells and recruitment of T-lymphocytes

among others, and both systems may be involved in the plaque development16.

From a clinical point of view, the involvement of inflammatory mediators in the

atherosclerotic process raises the possibility of developing new therapeutic strategies.

13

Fig. 1 Simplified visualization of arterial plaque formation, rupture and thrombus formation. Adapted from Choudhury RP et al with permission. Copyright Clearance Center´s RightsLink ®service14

Haemostasis, coronary thrombosis and fibrinolysis

The haemostatic system balances platelet activation, coagulation and fibrinolysis in order to

maintain normal blood flow 13, 17, 18. Coagulation can be initiated through both the intrinsic

and extrinsic pathways19 (Figure 2), and is amplified by the phospholipid surface of activated

platelets. In the context of plaque rupture, platelets are activated and aggregated and in

addition to amplify thrombin generation and blood clotting also play a role in the

inflammatory response4. Tissue factor (TF), the most important activator of the extrinsic

coagulation cascade, when exposed to circulating blood, binds to and activate coagulation

factor FVII, with further activation of FX to Xa, initiating the conversion of pro-thrombin to

thrombin and further clot formation by converting fibrinogen to fibrin (Figure 2) 17, 19.

Increased thrombin generation has been shown in patients with AMI and is crucial in the

thrombus formation20-24.

14

By measuring split products from the conversion of prothrombin to thrombin (prothrombin

fragments F1+2), an ongoing in vivo thrombotic process can be estimated25. The endogenous

thrombin potential (ETP) can be used as an estimate of ex vivo potential to generate thrombin

(ref. page 23). Small amounts of fibrin formed, also initiate the fibrinolytic system, balancing

the haemostatic system. The conversion of plasminogen to plasmin by tissue plasminogen

activator (t-PA) results in cleavage of fibrin into fibrin degradation products, which can be

assessed by measurement of D-dimer25, reflecting an ongoing activation of both the

coagulation and fibrinolytic systems26. Like prothrombin fragments F1+2, D-dimer has been

shown to be elevated for a prolonged time in patients with AMI22 and an early reduction of D-

dimer is supposed to indicate better prognosis27

Figure 2. The coagulation and fibrinolytic systems. Reproduced from Kohler with permission. Copyright Massachusetts Medical Society ®17

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Inflammation

The inflammatory process during and after an AMI is complex and depends on a variety of

cytokines and their receptors, acting together to initiate complex signalling cascades28, 29. The

inflammatory response is crucial for the repairing process in the injured myocardium30.

However, an excessive and prolonged inflammatory process can be harmful and may

contribute to larger infarct size, adverse remodelling and heart failure development.8 Studies

on circulating biomarkers like N-terminal pro-B-type natriuretic peptide (NT-pro-BNP) and

C-reactive protein (CRP) have identified important mechanistic insight about prognosis and

risk stratification31, 32. However, there are still many unknown mechanisms to explore. In the

present thesis members of the interleukin-6 (IL-6) transsignalling pathway, circulating IL-6,

the soluble IL-6 receptor (sIL-6R), soluble glycoprotein 130 (sgp130), connective tissue

growth factor (CCN2) and their importance in the early phase of an AMI as well as in long-

term prognosis, are focused.

Interleukin-6 signalling

The IL-6 family of cytokines signals through the common receptor unit gp130 and includes in

addition to IL-6, IL-11, leukaemia inhibitory factor, oncostatin M, ciliary neurotropic factor

and cardiotrophin-133.

IL-6, a multifunctional cytokine with both pro- and anti-inflammatory effects34,35, is released

from different cell types (monocytes/macrophages, B and T- lymphocytes, skeletal muscle

cells, endothelial cells, adipocytes, fibroblasts, vascular smooth muscle cells36, 37) and has a

variety of biological functions, especially in the acute-phase response38. IL-6 may be involved

both in the acute inflammatory response accompanying an AMI and in the chronic low-grade

systemic inflammation associated with cardiovascular events38-40.

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IL-6 is produced in hypoxic cardiomyocytes29, 41, which may contribute to cardiac dysfunction

and ischemia-reperfusion (IR) injury28.

Elevated circulating levels of IL-6 after an AMI have also been reported 42, 43. It remains

unclear whether the IL-6 response related to myocardial necrosis is beneficial, detrimental or

both. IL-6 stimulates the production of C-reactive (CRP) from the liver36 and may be the link

between high levels of CRP and increased risk of cardiovascular events and worsening of

heart failure42, 44-50.

There are two different signalling pathways of IL-6, the classical and the transsignalling, and

three main intracellular signalling cascades are thought to be involved: the Jak/STAT1/3, the

ERK 1/2/MAP kinase and the P13K/AKT pathways36, 37, 51. In the classical signalling

pathway, IL-6 binds to the membrane-bound IL-6R, leading to activation of the early immune

response and possibly regenerative or anti-inflammatory processes 35, 36, 52, 53 (Figure 3A). The

classical membrane-bound IL-6R stimulated pathway is relatively specific due to the limited

number of cells (e.g. hepatocytes, white blood cells) that express IL-6Rs in the cell

membrane54. In the transsignalling pathway, in contrast to the IL-6R pathway, the cellular

ligand of the IL-6/sIL-6R complex on the cell surface, the receptor subunit glycoprotein 130

(gp130 protein- ß subunit) is widely expressed in most cells in the body. Shedding of the IL-6

receptor to the blood stream creates a soluble form of the IL-6 receptor (sIL-6R). sIL-6R

binds to circulating IL-6 and binds subsequently to gp130 on the cell surface and initiate

intracellular signalling mediated through the Jak/STAT1/3 cascade35, 55 (Figure 3 A). This

transsignalling pathway may be responsible for the pro-inflammatory effects of IL-635. The

soluble form of gp130 is the natural inhibitor of the sIL-6R/IL6 complex by binding to the

membrane bound gp130 (Figure 3B)35, 55, 56 and thereby reduce activation of the

transsignalling pathway.

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Figure 3. The IL-6R complex, classical and transsignalling pathways, and the inhibitory mechanism of sgp130. (A) The two modes of IL-6 activation. (B) Classical signalling is unaffected by sgp130. Reproduced from Rose-John with permission. Copyright Clearance Center’s RightsLink® service55

The importance of IL-6 and CRP has been extensively studied and they are thought to play a

role in all stages of the atherosclerotic development from early lesions to destabilisation of the

plaque57. They are important for reperfusion injury, repair processes and scar tissue formation

after an AMI28 and both biomarkers have been shown to be associated with increased risk in

patients with CAD6, 37, 58 and with large infarct size in AMI patients59. In contrast, the soluble

receptors of the IL-6 family (sIL-6R and sgp130) have been sparsely studied in AMI patients.

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Myocardial injury, remodelling and function

LV remodelling after an AMI alters ventricular size, shape and function and involve

mechanical, neurohormonal and possibly genetic factors.

Loss of cardiomyocytes and interstitial fibrosis following an AMI induce an inflammatory

response, which is important for the repair process of the injured myocardium, scar healing

and post-infarction remodelling38. Also, molecular, cellular and interstitial changes occur as a

consequence of myocardial cell death after an AMI. Abnormal loading conditions may be

followed by left or right ventricular dilatation, changes of the shape of the LV, as well as

hypertrophy60. The ventricles are prone to dilatation (eccentric remodelling), increased

hypertrophy and myocardial dysfunction after an AMI (Figure 4)38, 61, 62.

Figure 4. Remodelling after an acute AMI with scar tissue formation and dilatation of the left ventricle. Reproduced from Konstam with permission. Copyright Clearance Center´s Rights link service®62

Inflammatory cell activation is part of the first response after AMI63, 64 and together with

chemokines like IL-8 is thought to act chemotactic in the early phase of an AMI, by recruiting

neutrophils and monocytes to the infarct area30. This early response is followed by release of

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different growth factors like CCN2/ connective tissue growth factor known to be involved in

cardiac hypertrophy and fibrosis63.

CCN2/connective tissue growth factor (CTGF)

CCN2/CTGF is a growth factor and member of the CCN family of matricellular proteins65.

Increased expression of CCN2 has been found in models of pressure-overload heart failure, 66,

67 and myocardial infarction68, 69 and is thought to be involved in collagen production leading

to myocardial fibrosis, scar formation and ventricular remodelling. Furthermore, CCN2 is

involved in extracellular matrix production and angiogenesis and exerts its effects on many

cell types involved in tissue repair, like neutrophils, fibroblasts, smooth muscle cells and

cardiomyocytes 63, 70, 71. Transforming growth factor beta-1 (TGFß1), endothelin-1 and

angiotensin II have all been shown to up-regulate CCN2 in different cell types after an AMI

71. In experimental studies CCN2 have mainly been seen as a pro-fibrotic marker. However,

results from experimental studies have also indicated that CCN2 may have cardioprotective

properties by attenuating LV hypertrophy72 and by improving tolerance to IR injury73. In a

small clinical study, high circulating levels of CCN2 were reported to be associated with

improved LV function74, however, there is a lack of data from clinical studies with adequate

sample size and long-term follow up, and the exact role of CCN2 in patients with AMI is still

unknown.

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Aims of the thesis

The overall aims of the thesis were to improve our understanding of the complex mechanism

involved in the interaction between inflammation, thrombosis and remodelling in patients

with STEMI and, furthermore, to identify novel biomarkers and their possible association

with risk factors of CVD, myocardial injury, infarct size, adverse remodelling and long-term

clinical outcome.

Specific Aims

• To study the association between circulating levels of IL-6, sIL-6R, sgp130 and

CRP and 1) known risk factors of CVD and 2) myocardial injury and function in a

cohort of 1028 STEMI patients (Paper I)

• To investigate circulating levels of IL-6, sIL-6R, sgp130 and CRP as related to

long-term clinical outcome in the same STEMI population (Paper II)

• To study markers of thrombin generation and their possible association with

myocardial injury and function in the above mentioned STEMI population (Paper

III)

• To study whether circulating levels of CTGF/CCN2 are associated with infarct

size, LV function and remodelling in another cohort of 272 STEMI patients (Paper

IV)

• To study any association between CCN2 and clinical outcome in the two different

STEMI populations mentioned above (Paper IV)

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Material and methods

The papers in the thesis are based on data from two different study cohorts. All patients have

given written informed consent and the Regional Committee for Medical Research Ethics

approved the studies. Both studies are conducted in accordance with the ethical principles of

the Declaration of Helsinki.

Study subjects and design

BAMI: In the Biobanking in Acute Myocardial Infarction (BAMI) cohort (n=1028), patients

meeting the criteria of an acute STEMI were included the following morning after admission

at Oslo University Hospital, Ullevaal, in the period of June 2007 to August 2011. They were

all investigated with coronary angiography and treated according to international guidelines11.

The exclusion criteria were age <18 years, unstable patient or patient unable to give informed

consent. A blood sample was collected at inclusion, after an overnight fast, median 24h after

start of symptoms and 18h after coronary angiography and, if indicated, percutaneous

coronary intervention (PCI). The samples were stored as the “BAMI biobank” at -80° C until

later analyses of circulating biomarkers from the IL-6 family (Papers I and II), the thrombotic

markers (Paper III) and CCN2/CTGF (Paper IV).

POSTEMI: Briefly, in the Postconditioning in ST-elevation Myocardial Infarction

(POSTEMI) cohort, 272 haemodynamically stable patients with first time STEMI and

symptoms duration < 6 hours were randomised to one of two different reperfusion strategies,

ischemic postconditioning (iPost) or control75. Patients with renal failure (serum creatinine >

200 µmol/L), previous MI, contraindications for CMR or inability to sign informed consent

were excluded. The inclusion period was between January 2009 and August 2012. Blood

samples were drawn before and immediately after the PCI procedure, at Day 1 and after 4

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months, as described in detail in Paper IV. The primary endpoint of the study was infarct size

measured after 4 months by CMR imaging. Secondary endpoints included myocardial

salvage, microvascular obstruction and clinical events during follow-up.

Coronary angiography and PCI

Coronary angiography was performed in all patients included in the two cohorts. PCI was

performed and adjunctive medical treatment was given according to the present guidelines 11.

Ischemic postconditioning (iPost)

Preconditioning the heart by inducing short episodes of ischemia before a prolonged period of

ischemia has been shown to reduce myocardial injury76. In the setting of an acute STEMI,

preconditioning is not possible and iPost may be an alternative approach. iPost is performed

by different protocols sharing the principle of applying short periods of occlusion of the

infarct related artery during PCI after initial opening of the occluded artery. iPost has been

shown in experimental models and small clinical studies to reduces infarct size76, 77, improve

coronary blood flow and ST-resolution78 and reduce markers of myocardial necrosis (CK

release, troponin)77. The primary objective of the POSTEMI trial was to test whether iPost

could reduce infarct size in STEMI patients76. Briefly, after initial reperfusion with wire and

balloon inflation, the balloon was reinflated 4 times for 1 minute (occluding the vessel),

separated by 1 minute of reflow, and further continuing the procedure after the operators’

judgement (Figure 5). The control group was treated according to standard PCI procedure79.

iPost had no significant effect on infarct size or on secondary ischaemic endpoints in the

study75. Consequently, the POSTEMI population was analysed as a whole in the present

investigation.

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Figure 5. Flow chart describing the investigational procedure in the POSTEMI trial. Reproduced from Limalanathan with permission. Copyright Clearance Center’s RightsLink® service79.

Blood sampling and laboratory analyses

Detailed descriptions are given in each paper. In the BAMI cohort serum was prepared for

determination of the circulating cytokines and CCN2, and citrated plasma for the haemostatic

variables. Commercially available enzyme-linked immunosorbent assays (ELISA) were used

for all analyses, except for the Calibrated Automated Thrombogram (CAT) assay,

determining the endogenous thrombin potential (ETP), which was used according to the

manufacturer’s instructions (Thrombinoscope BV, Maastricht, The Netherlands) and

measured on the Fluoroscan Ascent fluorometer (Thermo Fisher Scientific OY, Vantaa,

Finland). More detailed description is given in Paper III.

Troponin T, NT-proBNP and other routine blood samples were analysed with conventional

methods, also described in each paper. The coefficients of variation (CVs) for all variables are

described in each paper.

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Echocardiography

Left ventricular ejection fraction (LVEF) was determined by echocardiography in the BAMI

study (Paper I-III), a.m. Simpson or by visual judgment before hospital discharge or until 3

months after the index event. By several measurements, an average value was estimated.

LVEF data obtained in rehospitalised patients between index event and 3 months later were

not used.

Cardiac magnetic resonance (CMR)

CMR imaging was performed in the acute phase at a median time of 2 days after the index

infarction and repeated at the 4-months’ follow-up visit in the POSTEMI study (Paper IV).

Briefly, a 1.5 T scanner (Philips Intera, release 11 or Philips Achieva, release 3.2, Best,

Netherlands) was used for imaging and further analyses performed on an extended MR Work

Space (Philips Medical Systems). For volume analyses including LVEF, short axis images of

LV were acquired. To analyse the area at risk, defined by a signal intensity (SI) of more than

2 standard deviations above the SI in remote non- infarcted myocardium, T2 weighted

imaging was performed in the short axis plan. For analysing the infarct size, late gadolinium

enhancement (LGE) imaging was used 15 min after contrast injection (0.15 mmol/kg,

gadolinium-DTPA 469 mg/ml (Magnevist; Schering AG, Berlin, Germany) and 2- and 4-

chamber long axis views and short axis views were analysed. Furthermore, myocardial

salvage index was calculated and microvascular obstruction (MVO) was defined as the dark

area within the hyperintense area in the infarcted myocardium. The intra-observer reliability

estimated by the intra-class correlation coefficients for myocardium at risk and infarct size has

been reported80 in detail.

25

Endpoint registration

In the BAMI cohort 989 patients were followed from inclusion until December 2013, median

time 4.6 years (Papers II and IV). Recording of endpoints is described in the papers. In the

POSTEMI study, 272 patients were followed and endpoints were recorded at 4 and 12

months` follow-up. Mortality data were registered from clinical records until median 5.8 years

after inclusion (Paper IV). The primary endpoint in both cohorts was defined as a composite

of all-cause mortality, myocardial infarction, stroke and unscheduled revascularisation ≥3

months after the index event in both cohorts. All-cause mortality was a secondary endpoint in

both cohorts.

Statistics

The statistical approaches and methods used have been described in detail in each paper.

Briefly, continuous data were presented as mean (with standard deviation (SD)) or median

(25th, 75th percentiles) values and categorical data as proportions. Non-parametric tests were

used throughout due to skewed distribution of most of the outcome variables. For association

analyses Spearman’s rho and group analyses (dichotomised or split into quartiles) performed

by Mann-Whitney U test and Kruskal-Wallis test were used for continuous variables, as

appropriate. Chi-square test was used for categorical variables. Multivariate logistic

regression models were used for associations between prothrombotic markers and peak TnT

and LV impairment. Friedman test followed by Wilcoxon signed rank test were used for

comparing CCN2 levels at different sampling points. Cox regression analyses were used for

associations between circulating biomarkers and clinical endpoints. All analyses were

performed by IBM SPSS Software, version 18. (Paper I-III) and version 23.0 (Paper IV)

(SPSS Inc., Chicago, IL).

26

Summary of results

Paper I

We investigated possible associations between members of the IL-6 transsignalling system

including circulating levels of IL-6, sIL-6R, sgp130 and CRP assessed in the acute phase of

an MI and: 1) known risk factors of CAD 2) myocardial injury and LV impairment in 1028

STEMI patients undergoing immediate coronary angiography and PCI if indicated (the BAMI

cohort). Extensive myocardial necrosis defined as the upper quartile of peak TnT (>7140

ng/L), was associated with elevated levels of IL-6 and CRP (p<0.001, both), whereas there

were no significant associations with the novel biomarkers sIL-6R and sgp130 (Figure 1,

Paper I). In addition, levels of IL-6, sgp130 and CRP were higher in patients with elevated

levels of NT-proBNP (> 122ng/L) (p<0.001, p=0.007 and p<0.001, respectively) and with

reduced LVEF (< 40 %) (only IL-6 and CRP (<0.001, both)). Furthermore, significant

associations between sgp130 and both treated diabetes mellitus (p=0.002) and abnormal

glucometabolic parameters (HbA1c p<0.001 and admission glucose p=0.02) were observed

Quartiles of peak TnT ng/L

IL-6

pg

/mL

1 2 3 40

10

20

30

40

Quartiles of peak TnT ng/L

sgp

130

ng/

mL

1 2 3 40

100

200

300

Quartiles of peak TnT ng/L

sIL

-6R

ng

/mL

1 2 3 40

20

40

60

Quartiles of peak TnT ng/L

CR

P m

g/L

1 2 3 40

20

40

60

Figure 1 in Paper I. Associations between IL-6, sgp130, IL-6R and CRP and quartiles of peak TnT in 1028 patients with STEMI. Blood was sampled the following morning after admission, median 18 hours after acute coronary angiography

27

Paper II

In 989 STEMI patients from the BAMI–cohort, we examined possible associations between

sIL-6R, sgp130, IL-6 and CRP, and later clinical events in order to obtain new insights into

the importance of the IL-6/gp130 transsignalling pathway in risk stratification of patients with

STEMI.

Altogether there were 200 primary composite endpoints recorded after 4.6 years, 66 deaths,

61 reinfarctions, 6 strokes, 52 urgent PCI, and 15 readmissions for heart failure. All-cause

mortality was a secondary endpoint, and the number was 82.

We found that high levels of sIL-6R defined as the upper quartile (47.7 ng/mL) were

significantly associated with future cardiovascular events, and also with all cause-mortality in

both univariate and multivariate analyses (adjusted HRs 1.54 (95 % CI 1.08, 2.21) (p=0.02)

and 1.81 (1.04, 3.18) (p=0.04), respectively) (Figure 1 and 2 Paper II). Levels of sgp130 were

also higher in patients suffering a new event, however not statistically significant when

adjusted for covariates. CRP was significantly related to all-cause mortality (p=0.01), whereas

no associations between IL-6 levels and cardiovascular events or all-cause mortality were

observed.

28

Figure 1 and 2 in Paper II. Time-to-event curves in 989 STEMI patients for the highest quartile of sIL-6R (Q4) vs. the 3 lowest quartiles (Q1-Q3) according to a composite of cardiovascular events (upper panel) or all-cause mortality (lower panel). The log rank test was used to compare the survival curves.

Paper III

We investigated markers of hypercoagulability in 987 of the STEMI patients from the BAMI

cohort and their associations with myocardial necrosis defined as peak TnT and with LV

function assessed by NT-pro-BNP levels and LVEF. Patients on anticoagulant treatment were

excluded. D-dimer and F1+2, reflecting in vivo thrombin generation and the endogenous

thrombin potential (ETP), reflecting ex vivo potential to thrombin generation, were

investigated.

29

We found both F1+2 and D-dimer to be significantly associated with peak TnT, also after

adjusting for covariates (p<0.001, both) (Figure 1 Paper III, Panel A). Univariate correlations

showed both F1+2 and D-dimer to be significantly associated also with NT-pro-BNP,

however, only D-dimer remained statistically significant after adjustments (p< 0.001) (Figure

1 Paper III, Panel B). Both markers were inversely, but weekly correlated toLVEF (D-dimer:

p<0.001, F1+2; p=0.013), and patients with LVEF <40 % had higher levels of both markers

(p<0.001 and p=0.016, respectively). An inverse pattern for the ex vivo thrombin generation

assessed by ETP was found for peak TnT, NT-proBNP and LVEF.

Figure 1 in Paper III. D-dimer and F1+2 (medians) in quartiles of peak TnT (Panel A) and NT-ProBNP (Panel B). Panel A *=adjusted for age, sex, BMI, hypertension, time from symptoms to blood sampling, CRP and NT-ProBNP; Panel B *=adjusted for age, sex, BMI, hypertension, time from symptoms to blood sampling and CRP

Paper IV

CCN2 has been linked to cardiac hypertrophy and fibrosis. However, some studies have

suggested cardioprotective properties of CCN2 related to LV remodelling after an AMI. Thus,

we aimed to investigate possible associations between circulating CCN2 levels and infarct

size, LV function, adverse remodelling and clinical outcome in STEMI patients.

0

50

100

150

200

250

300

350

Q1 Q2 Q3 Q40

100

200

300

400

500

600

700

Q1 Q2 Q3 Q4

0

50

100

150

200

250

300

Q1 Q2 Q3 Q40

100

200

300

400

500

600

700

Q1 Q2 Q3 Q4

p for trend<0.001* F1+2(pmol/mL)

D-dimer(ng/mL)

p for trend<0.001*

p for trend=0. 324*p for trend=0.001*F1+2(pmol/mL)

D-dimer(ng/mL)

TnT Q1 ≤1.71 Q2 >1.71≤3.85 Q3 >3.85≤7.25 Q4 >7.25 µg/L

NT-ProBNP Q1 ≤10 Q2 >10≤31 Q3 >31≤118 Q4 >118 pmol/L

Panel A

Panel B

30

We could not demonstrate any significant associations between levels of CCN2 and the

cardiac biomarkers peak TnT and NT-proBNP. Neither were there any significant

associations with CMR measures of myocardial injury, remodelling or function (LVEF,

changes in LV end-diastolic or end-systolic volumes, myocardial salvage or MVO).

Furthermore, we could not find any association between CCN2 and future clinical events

including all-cause mortality, during short-or long-term follow-up, neither in the POSTEMI

cohort nor in the larger BAMI cohort (Figure 2 Paper IV).

Figure 2 in Paper IV. Event-free survival and overall survival according to quartiles of CCN2 measured median 18 hours after PCI in two STEMI cohorts. Upper panels (BAMI) Lower panels (POSTEMI)

31

Discussion

Methodological considerations

There is a selection of patients in both cohorts included in this thesis. Unstable patients (i.e.

heart failure, cardiac arrest, need of assisted ventilation) were excluded in both studies,

resulting in a selection towards somewhat low-risk populations, even though they all had

STEMI. In the BAMI cohort, some patients were excluded for feasibility reasons due to rapid

referral to community hospitals. In the POSTEMI cohort, patients with previous MI,

symptoms >6 hours, severe renal failure (serum creatinine >200 µmol) or contraindications to

CMR were excluded. In both cohorts, the patients had few complications, they had only

slightly reduced LVEF and the mortality rates were low, 8.3 % in BAMI and 2.3 % in the

POSTEMI cohort, which are somewhat lower compared to other reports81, altogether a low-

risk population. However, the large amount of patients in the BAMI cohort, with 20.2 %

events and the long-term follow-up strengthen our results. Another strength of the BAMI

study is that we achieved follow-up data from 989 (96 %) of the 1028 included patients.

The difference in mortality rate between our cohorts may be explained by the difference in

inclusion criteria. In the BAMI cohort, patients with previous MI and heart failure were

included and they had higher frequency of previous hypertension, diabetes mellitus and

hypercholesterolemia compared to the POSTEMI cohort.

In the BAMI cohort, blood samples were collected only once. Thus, the lack of repeated

samples precluded time-course studies of the measured variables and we may have missed the

peak values and possible transient changes, as previously suggested for some biomarkers82, 83.

In the BAMI cohort, patients with more than 6 hours from symptoms to admission were also

included, thus there was a large variation in time from start of symptoms to blood sampling

32

(2-264 hours). There was a significant correlation between time and CRP (r=0.45, p<0.001),

but only a weak correlation between time and IL-6 (r=0.08, p=0.01). This is in line with other

reports showing peak IL-6 the first day after admission in PCI-treated STEMI patients84, 85,

whereas peak level of CRP seems to be as late as 72 hours after PCI85. This is expected from

the well-described induction of CRP expression in the liver by IL-6, which also fits the strong

correlation we observed between CRP and time. However, adjusting for the time frame did

not change our results. There were no significant correlations between time and the other

measured parameters, suggesting the variables to be representative of a STEMI cohort. Also,

adjusting for the time frame did not change the results described in Paper III.

In the BAMI cohort (Papers I-III), the echocardiography investigations were performed by

different investigators and at different time points, which may represent a potential bias.

In the POSTEMI cohort (Paper IV), the serial measurements of CCN2 in the acute phase of a

STEMI strengthen the results, but the lack of blood samples between the acute phase and 4

months is a limitation and we may speculate whether we missed the peak value, i.e. that

CCN2 peaked at a later time point.

In the POSTEMI cohort, the repeated CMR measurements in a relative large number of

patients strengthen our results on myocardial injury and function. Finally, the CCN2 results

regarding clinical outcome are strengthened by the fact that CCN2 was measured in two

different cohorts, both with long-term clinical follow-up.

33

General discussion

Members of the IL-6 family in patients with acute myocardial infarction

We showed in our study that IL-6 and CRP were significantly associated with myocardial

necrosis and impaired LV function, which confirms previous studies39, 59, 86 and fits with the

assumption of a connection between inflammation and infarct size. IL-6 is a pleiotropic

multifunctional cytokine, and both pro- and anti-inflammatory effects of IL-6 have been

suggested35, 36. The diversity of the IL-6 signalling is complex and not fully understood. As

described in the Introduction chapter, most cells express gp130 on the cell surface, while few

cells have membrane-bound IL-6 receptors35. Only cells that have IL-6 receptors on their cell

membrane can bind IL-6 and induce the classical signalling, while the complex of IL-6 and

sIL-6R can bind to all cells that express gp130, inducing the transsignalling. The soluble form

of IL-6R will therefore have a much broader spectrum of target cells35. Transsignalling

mediated by increased levels of soluble IL-6R seems to be responsible for activation of the

immune system and recruitment of mononuclear cells seen in chronic inflammatory diseases

like Crohn`s disease and rheumatoid arthritis35, 55 and possibly the atherosclerotic process.

The classical pathway is also thought to play a role in the early immune response by induction

of the acute-phase IL-6, in which levels in the physiological range are necessary for the

inflammatory response36. However, an excessive activation in the acute phase or the on-going

chronic activation may have detrimental effects by its action trough the transsignalling

pathway35, 36, 55. The exact roles of IL-6, sIL-6R and the signalling receptor protein gp130 are,

however, not fully understood and the effects of inhibition of the different members of the IL-

6 signalling pathways remain unclear. IL-6 showed anti-inflammatory effects of selective

blocking of the transsignalling pathway in experimental models, indicating activation through

the classical pathway35, 87. Interestingly, IL-6 inhibition by the IL-6R antagonist tocilizumab88,

89, licenced for rheumatoid arthritis treatment90, was recently shown to reduce inflammation

34

and also TnT release in patients with non-STEMI91. Furthermore, a single nucleotide

polymorphism in the IL6R gene resulting in reduced levels of IL-6 and CRP, was associated

with reduced risk of coronary heart disease events 92, 93. It might, however, be discussed

whether inhibition of IL-6 members have different effects depending on the timing and the

length of the treatment, i.e. that IL-6 inhibition may play different roles during acute and

chronic inflammation.

sIL-6R was not related to the degree of myocardial necrosis in our study, which is a novel

finding in STEMI patients. A possible explanation may be that the peak level of sIL-6R

occurs at a later time point or that the association between sIL-6R and clinical outcome

(discussed in next section) is independent of the degree of necrosis. There is limited

knowledge of the role of this marker in the setting of an AMI. Elevated levels were shown in

patients with AMI compared to patients with stable CAD94, however, another study reported

no difference in the levels between patients with STEMI and stable CAD95.

It has also been demonstrated that the concentration of sIL-6R measured in the coronary sinus

was significantly lower compared to the concentration in the aorta, in both STEMI and stable

CAD patients95. This could possibly be explained by an ischemia-related increased expression

of gp130 and further augmented binding of sIL-6R/IL-6 complex to the cell membrane in the

ischemic myocardium, thus leading to decreased levels of sIL-6R in the coronary sinus95. In

line with this, a small study of patients with AMI showed a decrease in sIL-6R in the acute

phase followed by a later increase96.

We could not find any significant associations between sgp130 and markers of myocardial

necrosis in our study. The role of sgp130 in the setting of an AMI is unclear, however, our

results showing sgp130 to be significantly associated with NT-proBNP levels can be

discussed along with previous reports showing increased levels in post-ischemic heart

35

failure97 and in heart failure in general98. An association with advanced heart failure leading to

cardiovascular death has furthermore been described99, 100. However, no significant

association between sgp130 and impaired LV function could be seen in our patients. This may

partly be explained by the majority of patients being relatively stable, few patients had

previously known heart failure (n=20), very few in-hospital complications after the index

infarction and possibly also by the time point of blood sampling. We could demonstrate an

inverse association between spg130 and CRP in our STEMI population, confirming previous

findings in a general MI population, and suggest that sgp130 could be beneficial in this

setting, due to its anti-inflammatory properties of inhibiting the transsignalling pathway35.

The observed associations between sgp130 and variables of dysglycemia are to our

knowledge novel observations in an AMI population. We found significantly higher levels of

sgp130 in patients with diabetes mellitus and a significant association with HbA1c, and we

may speculate whether MI patients with impaired glucose regulation have higher levels of

sgp130 related to insulin resistance and endothelial dysfunction, as suggested by others101-103.

High levels of sgp130 have been described in older patients with the metabolic syndrome

compared to controls101 and an inverse association between insulin sensitivity and sgp130 in

obese patients with polycystic ovary syndrome has been reported102.

Members of the IL-6 family and their association with later clinical events

In paper II we investigated if there were any associations between IL-6, sIL-6R, sgp130, CRP

and later cardiovascular events. We found significant associations between sIL-6R and both

the composite endpoint and all-cause mortality. We also found a significant association

between CRP and all-cause mortality after adjustment for relevant covariates, as also

described by others104, 105. In line with our results, Moreno Velasquez et al demonstrated that

36

high levels of sIL-6R measured in the stable phase after an acute STEMI were related to new

events106. These results indicate that sIL-6R may play an important role in the excessive

inflammatory response accompanying the IR-injury and remodelling process following an

acute MI, as also suggested by other investigators107. In line with this, an association between

sIL-6R and reduced myocardial reperfusion after PCI has been reported in STEMI-patients108.

We observed that patients with clinical events had higher levels of sgp130 compared to those

without, however, the association between sgp130 and new events was not statistically

significant after adjustment in a multivariate model. It might be suggested that sgp130 merely

reflect other covariates related to adverse prognosis or we could speculate that our study

population without impaired LV function and heart failure was not suited to evaluate the

importance of sgp130 in patients with large MI and symptomatic heart failure. The value of

sgp130 as a prognostic biomarker in AMI patients remains unclear, whereas sgp130 in

patients with heart failure has been clearly associated with fatal outcome in other studies100.

Interestingly, we could not demonstrate any relation between levels of IL-6, degree of

necrosis (peak TnT) and later clinical events. The reason for this is unclear, but similar

findings have been described earlier109. In clinically stable STEMI patients with small to

medium-size infarctions, different mechanism, including inflammation, may be more

important than the degree of necrosis in the risk of developing new clinical events post-MI.

In a very recent study from our research group, IL-8, but not peak TnT, measured during

STEMI was found to be strongly associated with long-term prognosis and high IL-8 levels

measured at 4-months follow-up remained associated with an increased risk of all-cause

mortality, also when adjusting for CRP, NT-proBNP and final infarct size measured at the

same time point110.

37

Inflammation as therapeutic target in patients with acute myocardial infarction

Experimental studies on endothelial cells have shown that inhibition of IL-6 with a

monoclonal antibody reduced proatherosclerotic and proinflammatory effects of CRP,

indicating that these effects, at least partly, are dependent on IL-6111. Treatment with an anti-

toll-like receptor-2 antibody was shown to reduce infarct size and preserve systolic function in

a pig model of I/R injury112 and reduction of infarct size by corticosteroid administration was

demonstrated in an experimental animal model already in 1973113. However, anti-

inflammatory therapeutic strategies in patients with CVD have until recently been

unsuccessful. Intravenous administration of immunoglobulin in patients after STEMI failed to

improve LV remodelling or function114. Furthermore, anti-tumor necrosis factor-α therapy,

which in early studies was promising, had no beneficial effect on heart failure patients in a

randomised, placebo-controlled clinical trial115, 116. Thus, the effect of anti-inflammatory

treatment in experimental models has been difficult to replicate in human studies and the

inflammatory response may be different in different species117. However, recent results from

the Cantos trial, a large randomised trial where treatment with canakinumab, an antibody

against IL-1ß, an upstream pro-inflammatory cytokine activating the IL-6/CRP cascade,

reduced the incidence of cardiovascular events including cardiovascular mortality in patients

with previous MI and elevated CRP118. Additionally, a meta-analysis demonstrated that low-

dose treatment with methotrexate seems to reduce the risk of new cardiovascular events in

patients with rheumatoid arthritis (RA)119 and, as discussed above, the anti-human IL-6R

monoclonal antibody tocilizumab, reduced CRP and TnT levels in a small clinical trial on

patients with non-STEMI91. The latter results are further explored in the ongoing ASSAIL-MI

study on STEMI patients (ClinicalTrials.gov Identifier: NCT03004703), where the main aim

is to address the effect of a single dose of tocilizumab administered before PCI on myocardial

salvage assessed by CMR.

38

Hypercoagulability in patients with acute myocardial infarction

Generation of thrombin with subsequent fibrin formation in addition to platelet activation is

known to be essential in the development of an intracoronary thrombus, which may result in

an acute coronary occlusion 13, 18, 120.

We measured markers of hypercoagulability in the BAMI cohort excluding patients on

warfarin due to its influence on the coagulation cascade. The significant associations found

between D-dimer and F1+2 and myocardial necrosis (assessed by peak TnT) indicate that

patients with large MIs are in a more hypercoagulable state. Whether this reflects an

inflammatory response in the acute phase cannot be ruled out, although the association

remained significant after adjustment for CRP. Similar findings were reported in another

study when the prothrombotic markers were measured three months after an AMI reflecting a

more stable situation121. Thus, the relation between a hypercoagulable state and myocardial

injury seems likely and possibly these patients would benefit from additional therapy with

anticoagulation as previously described122-124. Increased activation of the coagulation system

reflected in enhanced thrombin generation has been reported in populations with acute

coronary syndromes20, 21 and a persistent hypercoagulable state in these patients has been

shown several months after an AMI22.

In our STEMI cohort we also found a significant association between procoagulant activity

and myocardial function, assessed by NT-proBNP and LVEF, which to our knowledge has

not been described earlier. However, elevated levels of D-dimer in patients with heart failure

have been reported125 and an increased risk of subsequent cardiac mortality in patients with

AMI has been shown to be associated with elevated levels of prothrombotic markers

independent of residual LVEF126. D-dimer was also recently shown to be of importance for

the prediction of heart failure when added into a multibiomarker panel in the LIPID-study127.

39

Whether the levels of D-dimer and F1+2 measured in the acute phase in our BAMI population

are important markers of future clinical outcome is under investigation, and preliminary

results indicate that especially D-dimer seems to be associated with mortality128.

In line with these results, in a larger cohort of patients with stable CAD (n=2209), D-dimer

was shown to be associated with disease severity assessed by the Gensini score and also with

clinical outcome after up to 3 years’ follow-up129.

Ex vivo thrombin generation assessed by the CAT assay, in which ETP is the most used

variable, is a measure of the patients’ potential to generate thrombin ex vivo when exposed to

TF and has been claimed to be a reliable method to determine the degree of

hypercoagulability130. We observed, however, an inverse pattern for the ex vivo potential to

generate thrombin as related to both myocardial injury and function. Similar observations of

an inverse correlation between in vivo and ex vivo thrombin generation has previously been

reported131, and as discussed in Paper III, this might be an exhaustion phenomenon due to the

increased in vivo thrombin generation. In addition, it has been reported that several plasma

coagulation factors, not measured in our study, may influence the determination of ETP132.

Hypercoagulability has been shown to be associated with several traditional cardiovascular

risk factors. However, the prothrombotic markers in the BAMI cohort were not significantly

related to glucometabolic variables in contrast to other reports133-136. This might be due to the

measurements being performed in the acute phase, in which small differences might be

masked. High BMI in stable CAD patients has been shown to be associated with a less

hypercoagulable state 131 and this is in accordance with our findings of BMI to be inversely

related to D-dimer and F1+2. In contrast, all CAT variables were related to BMI, indicating

an increased potential of ex vivo thrombin generation in these patients.

40

CCN2 in patients with acute myocardial infarction

High levels of CCN2 have in experimental models been shown to be associated with smaller

infarct size, suggesting cardio-protective properties of CCN2137. This is somewhat in contrast

to findings in clinical studies of increased levels in patients with symptomatic heart failure138,

139. There are several reports on CCN2 and its association to cardiac fibrosis140-143 where it is

thought to act as a pro-fibrotic marker144, but the exact mechanism is unclear and its role in

acute MI and postinfarction remodelling is still unknown. Increased expression of CCN2 in

cardiac myocytes and fibroblasts after an AMI has been demonstrated, and also increased

CCN2 expression in viable post-infarct myocardium after 180 days has been shown,

indicating a role in late remodelling after AMI 69, 143. There are diverging reports suggesting

CCN2 to have both pathological and protective properties related to IR-injury and

remodelling and there is a lack of reports in acute STEMI populations, as most of the studies

are experimental.

The previous clinical studies suggesting a cardio-protective role of CCN2 have shown

elevated levels to be associated with reduced IR-injury73, 74, 137, and in one combined clinical

and experimental study, transgenic mice with overexpression of CCN2 had smaller infarcts,

less dilated ventricles and less hypertrophy, as well as better survival74. In the same study,

patients with elevated levels of CCN2 were shown to have improved LV function and

possibly reduced LV remodelling after an MI74. We therefore postulated an association

between high levels of CCN2 and enhanced ventricular function, reduced post-MI

remodelling and improved clinical outcome. To answer this question, the POSTEMI cohort

was used. The serial blood sampling, i.e. before PCI, immediately after PCI, at Day 1, and

after 4 months, was used and use of CMR for measures of infarct size and left ventricular

function give strength to our results.

41

We could not find any significant associations between CCN2 measured either in the acute

phase (Day 1) or after 4 months, and myocardial injury, infarct size or reduced left ventricular

function. We found no associations between CCN2, measured before and after reperfusion,

and markers of IR-injury and LV remodelling, assessed by myocardial salvage, MVO and

change in LVEDV or LVESV. There was also no effect of the iPost procedure used in

POSTEMI study on CCN2 levels. However, we did find an increase in CCN2 levels from

inclusion to 4 months follow–up, and the change from hospitalisation to 4 months was weakly

associated with changes in LV volumes, but not with infarct size and myocardial salvage.

We could not find any significant associations between CCN2 levels and new clinical events,

either in the cohort with multiple sampling points (POSTEMI) or in the larger cohort with

long-time follow-up (BAMI). Based on our results, we may conclude that CCN2 measured in

the acute phase of STEMI does not add any prognostic value to the risk of developing large

infarct size, adverse LV remodelling, reduced LV function or new clinical adverse events.

Nevertheless, the present results are based on CCN2 measured in circulating blood and it does

not exclude an important role for locally released CCN2 in the myocardium, as indicated in

previous reports69, 73.

42

Conclusions

- STEMI patients with more severe disease, defined as highest levels of TnT, elevated

levels of NT-proBNP and reduced LVEF, had higher levels of IL-6 and CRP,

confirming the role of inflammation in AMI (Paper I).

- The soluble receptors of the transsignalling pathway, sIL-6R and sgp130 were not

related to the same variables in our cohort (Paper I).

- Contrary to the findings of IL-6, sIL-6R was associated with new adverse events and

all-cause mortality, also after adjustment for covariates, and there was a trend to

higher levels of sgp130 in patients with events, although not significant in adjusted

models, pointing to a role of the IL-6 transsignalling pathway in long-term prognosis

after STEMI (Paper II).

- Haemostatic variables F1+2 and D-dimer were significantly associated with

myocardial injury assessed by peak TnT. D-dimer was also associated with LVEF and

NT-proBNP, indicating a hypercoagulable state in patients with more severe disease in

the acute phase of a STEMI (Paper III).

- CCN2 as a marker of LV remodelling related to IR injury, was not associated with

infarct size, left ventricular dysfunction or new adverse clinical events, indicating that

it is not suitable as a prognostic biomarker in the acute setting of STEMI (Paper IV).

Overall summary of the thesis is that in STEMI patients, final infarct size and left ventricular

function were related to IL-6, CRP and haemostatic variables, indicating a pronounced

proinflammatory and hypercoagulable state in these patients. A novel finding was that sIL-6R

in addition to CRP, was related to new adverse clinical events, including mortality, and

measurement of sIL-6R may improve risk assessment in these patients and indicate possible

43

new targets for future therapy. Finally, our data strongly suggest against a role of CCN2 in

risk stratification of STEMI patients.

44

Future perspectives

The concept of residual inflammation in patients with high-risk atherosclerosis or established

coronary heart disease (CHD) has been introduced during the last years145.

There are several recently published clinical trials as well as on-going studies, targeting

residual inflammation in a chronic setting or excessive inflammation in an acute setting like

an AMI. The landmark Cantos trial, demonstrated that inhibition of IL-1ß lowered the rate of

recurrent cardiovascular events in patients with previous MI and high CRP 118. Low-dose

methotrexate seems to reduce the risk of new cardiovascular events in RA patients119 and the

effect of low-dose methotrexate in secondary prevention of new adverse events in CVD

patients is currently investigated in the CIRT (Cardiovascular Inflammation Reduction Trial)

including 7000 patients146. The ongoing ASSAIL-MI trial will explore the possible effect of

IL-6 inhibition on myocardial salvage and final infarct size in STEMI patients. Another

example of a possible target for more specific therapy is findings from our own study group

indicating a role of IL-8 in LV remodelling and function post-MI110, 147. However, the

inflammatory response in the acute setting of an infarction is complex and targeting

inflammation may be both cardio-protective and in some conditions harmful, depending on

the target of the therapy, degree of inhibition, timing of the intervention and the condition of

the patient. Further studies are warranted to explore the safety of this possible future treatment

in AMI patients.

45

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