critical role of angiopoietin pathway in ischemia

CRITICAL ROLE OF ANGIOPOIETIN PATHWAY IN ISCHEMIA-REPERFUSION INJURY IN CARDIAC TRANSPLANTATION SIMO SYRJÄLÄ 2014

3

CRITICAL ROLE OF ANGIOPOIETIN PATHWAY IN

ISCHEMIA-‐REPERFUSION INJURY IN CARDIAC TRANSPLANTATION

Simo Syrjälä, MD

Cardiopulmonary Research Group, Transplantation Laboratory, Haartman Institute,

University of Helsinki, Helsinki, Finland

Academic dissertation

To be publicly discussed with the permission of the Faculty of Medicine, University of Helsinki, in Lecture Hall 3, Meilahti Hospital,

Haartmaninkatu 4, on 12th December, at 12 o’clock noon

4

Helsinki 2014 Supervised by

Professor Karl Lemström, MD, PhD Cardiopulmonary Research Group,

Transplantation Laboratory, Haartman Institute, University of Helsinki,

Helsinki, Finland

Reviewed by

Professor Timo Paavonen, Department of Pathology, School of Medicine,

University of Tampere, Tampere, Finland

And

Professor Hannu Sariola, Developmental Biology, Institute of Biomedicine

University of Helsinki, Helsinki, Finland

Discussed with

Professor Daniel Goldstein, Yale Cardiovascular Research Center, School of Medicine,

Yale University, New Haven, CT, USA

5

To my family

6

ISBN 978-‐951-‐51-‐0390-‐1 ISBN 978-‐951-‐51-‐0391-‐8 Author contact information: Transplantation Laboratory, Haartman Institute P.O. Box 21 (Haartmaninkatu 3) FI-‐00014 University of Helsinki, Helsinki, Finland Tel: +358-‐9-‐19126582 Fax: +358-‐9-‐2411227 E-‐mail: [email protected]

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TABLE OF CONTENTS

ORIGINAL PUBLICATIONS ............................................................ 9

ABBREVIATIONS ........................................................................ 10

ABSTRACT ................................................................................. 12

INTRODUCTION ......................................................................... 15

REVIEW OF THE LITERATURE ..................................................... 16

1. Clinical heart transplantation ............................................. 16

1.1. Background ...................................................................... 16 1.2. General histology of the heart ........................................ 17 1.3. Indications, patient characteristics, and outcome .......... 18 1.4. Risk factors ...................................................................... 20

2. Ischemia-‐reperfusion injury ............................................... 23

2.1. Ischemia and hypothermia .............................................. 23 2.2. Reperfusion and re-‐oxygenation ..................................... 24 2.3. Microvascular dysfunction .............................................. 26

3. Immunobiology .................................................................. 28

3.1. Innate immune system .................................................... 28 3.2. Alloimmune system ......................................................... 31 3.5. Acute rejection ................................................................ 36 3.6. Immunosuppression ........................................................ 38 3.7. Chronic rejection ............................................................. 40

4. Angiopoietin pathway ........................................................ 44

4.1. General ............................................................................ 44 4.2. Angiopoietin-‐1 ................................................................. 45 4.3. Angiopoietin-‐2 ................................................................. 47

AIMS OF THE STUDY .................................................................. 49

METHODS ................................................................................. 50

1. Experimental rat cardiac transplantation model ................ 50 2. Transmission electron microscopy analysis ....................... 51 3. Immunohistochemistry and immunofluorescence stainings .............................................................................. 52

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4. Histological evaluation ....................................................... 53 5. Enzyme-‐linked immunosorbent assay ................................ 53 6. Cell culture assays .............................................................. 54 7. Microvascular leakage and perfusion assay ....................... 54 8. Gene expression analysis ................................................... 55 9. Heart transplant patients ................................................... 56 10. Statistical analysis ............................................................. 56

RESULTS .................................................................................... 57

1. Prolonged ischemic preservation promoted ischemia-‐ reperfusion injury-‐mediated myocardial injury and inflammation in rat cardiac allografts (I) ............................ 57 2. Prolonged ischemic preservation enhanced chronic rejection (I) ......................................................................... 57 3. Hypoxia induced endothelial cells to release Ang2 ............ 58 4. Cardiac transplantation induces immediate changes in angiogenic growth factor expression in human and in rat (III) ............................................................................. 61 5. Targeting Ang/Tie2 pathway prevented Tie2-‐dependent endothelial destabilization and microvascular dysfunction induced by ischemic preservation (II-‐III) ......... 62 6. Acute rejection can be restrained by endothelial inactivation (III) ................................................................... 64 7. Cardiac allograft vasculopathy development is correlated with preoperative ischemia time and the number of endothelial cells and pericytes (I-‐III) ................. 65

DISCUSSION .............................................................................. 69

YHTEENVETO ............................................................................. 81

ACKNOWLEDGMENTS ............................................................... 84

REFERENCES .............................................................................. 87

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ORIGINAL PUBLICATIONS

The thesis is based on the following original publications referred to

in the text by their Roman numerals:

I Syrjälä SO, Keränen MA, Tuuminen R, Nykänen AI, Krebs R,

Lemström KB: Increased Th17 rather than Th1 alloimmune response

is associated with cardiac allograft vasculopathy after hypothermic

preservation in the rat. J Heart Lung Transplant. 2010 29(9):1047-‐

1057.

II Syrjälä SO, Nykänen AI, Tuuminen R Raissadati A, Keränen MAI,

Arnaudova R, Krebs R, Koh GY, Alitalo K, Lemström KB: Donor Heart

Treatment with COMP-‐Ang1 Limits Ischemia-‐Reperfusion Injury and

Chronic Rejection of Cardiac Allografts. SUBMITTED.

III Syrjälä SO, Tuuminen R, Nykänen AI, Raissadati A, Dashkevich A,

Keränen MAI, Arnaudova R, Krebs R, Leow CC, Saharinen P, Alitalo K,

Lemström KB: Angiopoietin-‐2 Inhibition Prevents Transplant

Ischemia-‐ Reperfusion Injury and Chronic Rejection in Rat Cardiac

Allografts. Am J Transplant. 2014 14(5):1096–108.

10

ABBREVIATIONS AMR antibody-‐mediated rejection Ang angiopoietin AP-‐1 activation protein 1 APC antigen-‐presenting cell CCL21 chemokine (C-‐C motif) ligand 21 CCR7 C-‐C chemokine receptor type 7 CD cluster of differentiation CMC cardiomyocyte CMV cytomegalovirus COMP cartilage oligomeric matrix protein COX cyclooxygenase CTL cytotoxic lymphocyte DA Dark Agouti rat DAMP danger/damage-‐associated molecular pattern DNA deoxyribonucleic acid DC dendritic cell EC endothelial cell ECM extracellular matrix ELISA enzyme-‐linked immunosorbent assay ET-‐1 endothelin-‐1 FITC fluorescein isothiocyanate FOXP3 forkhead box p3 HAS hyaluronic acid synthase HIF-‐1 hypoxia-‐inducible factor-‐1 HLA human leukocyte antigen HMBG1 high-‐mobility box group 1 ICAM-‐1 intracellular adhesion molecule-‐1 IFN-‐g interferon gamma IL interleukin IP-‐10 IFN-‐g-‐inducible protein 10 IRI ischemia-‐reperfusion injury i.v. intravenously KLF-‐2 Krüppel-‐like factor-‐2 LFA1 leukocyte function antigen 1 MHC major histocompatibility complex NF-‐AT nuclear factor of activated T cells NF-‐κB nucleic factor kappa B PAMP pathogen-‐associated molecular pattern

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PBS phosphate-‐buffered saline PC pericyte PMNC polymorphonuclear cell Rbc red blood cell RhoA Ras homolog gene family member A ROR retinoic acid receptor-‐related orphan receptor ROS reactive oxygen species RT-‐PCR reverse-‐transcription polymerase chain reaction s.c. subcutaneously SLO secondary lymphoid organ SMA smooth muscle actin SMC smooth muscle cell STAT signal transducer and activator of transcription TGF transforming growth factor Th T helper cell TLR Toll-‐like receptor TNFa tumor necrosis factor alpha TnT troponin T TOR target of rapamycin Treg regulatory T cell VCAM-‐1 vascular endothelial growth factor-‐1 VE-‐cadherin vascular endothelial cadherin WF Wistar Furth rat

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ABSTRACT

Heart transplant is disconnected from the circulation and preserved

in hypothermia before transplantation. Paradoxically,

revascularization of the heart transplant results in ischemia-‐

reperfusion injury described as myocardial injury, microvascular

dysfunction, and innate and adaptive immune activation. The innate

response consists mainly of neutrophils and macrophages and

innate lymphoid cells and may lead to sustained adaptive immune

response leading to chronic rejection and late graft failure. The

heart is especially susceptible for lack of oxygen; therefore, the

ischemic time in clinical practice is critical. Prolonged ischemic time

– due to long distance between hospitals or technically difficult

operation – is an independent risk factor for primary graft

dysfunction and chronic rejection.

Angiopoietin-‐1 and -‐2 (Ang1 and 2) are vascular growth factors

binding to Tie2 receptor with indispensible role in embryonic

vascular development, but also in endothelial maintenance in

mature vasculature. Vascular supporting cells constantly secrete

Ang1, which maintains the endothelium in quiescent state. In

contrast, Ang2 is produced and released from the endothelial cells in

response to stress stimuli, such as hypoxia and inflammation,

destabilizing and activating the endothelium in order to ease

inflammatory cell accumulation and transmigration.

13

This study utilized experimental animal model to describe the effect

of cardiac allograft ischemia-‐reperfusion injury on innate immune

activation and adaptive immune responses, such as the

development of acute and chronic rejection. This study further

investigated the effects of either activating or inhibiting the

angiopoietin/Tie2-‐signaling pathway in this disease process. The

results show that prolonged hypothermic preservation enhanced

ischemia-‐reperfusion injury-‐related innate immune activation and

adaptive immune and worsened the prognosis of the cardiac

allografts. Analysis of samples from clinical heart transplant

recipients revealed increase in peripheral blood Ang2 levels during

the first day after the operation. Similar findings were evident in the

recipients of rat cardiac allografts.

Ang1 was proven protective when injected into allograft coronaries

prior to the preservation: the treatment stabilized the endothelium,

reduced myocardial injury and inflammation, and hindered the

development of chronic rejection. Donor heart treatment with

Ang2-‐blocking antibody had similar effects on endothelium, but

further inhibited the activation of endothelial cells, acute and

chronic rejection. Furthermore, recipient treatment with multiple

doses of the anti-‐Ang2 antibody immediately after transplantation

significantly prolonged allograft survival and had superior effect

when compared to heart donor treatment.

14

Primary and late graft dysfunction, either due to ischemia-‐

reperfusion injury, or acute and chronic rejection, limit the survival

of patients with solid organ transplant. According to this study,

targeting Ang/Tie2-‐signaling prevents early allograft endothelial

activation and inflammatory cell accumulation. Of studied treatment

protocols, early systemic recipient treatment with anti-‐Ang2

antibody had the most robust effect in preventing allograft

dysfunction. Ang2-‐targeted antibody treatment would have clinical

implications in induction therapy of transplant patients, as the

dosage of other immunosuppressive drugs may be lowered and the

adverse side effects of these drugs avoided. These results encourage

further studies to determine the clinical significance of Ang/Tie2-‐

pathway modification.

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INTRODUCTION

Cardiovascular diseases are the leading cause of death in Western

countries. After advances in surgical techniques and the

introduction of immunosuppressive medication, cardiac

transplantation has become plausible treatment for many end-‐stage

heart diseases. The shortage of organ donors limits the availability

of heart transplants and presents challenges to donor management.

Acute rejection, primary graft dysfunction, infections, malignancies,

and chronic allograft dysfunction limit the survival of cardiac

transplant patients. Of these, acute rejection and infections are

effectively managed with adjustments in immunosuppressive

medication and antibiotics; however, the side effects of

immunosuppressive drugs and the development of chronic rejection

continue to puzzle the clinicians and scientists.

Angiopoietins are vascular growth factors with indispensible role in

embryonic vascular development, but also in the stabilization of

mature vasculature. The angiopoietin signaling has been suggested a

potential immunomodulatory pathway in regulating microvascular

dysfunction and inflammation after cardiac transplantation.

The purpose of this study was to characterize, in experimental rat

cardiac transplantation model, the effects of ischemia-‐reperfusion

injury on transplant inflammation and to elaborate the therapeutic

potential of angiopoietin-‐1 supplementation and angiopoietin-‐2

blocking in this setting.

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REVIEW OF THE LITERATURE

1. Clinical heart transplantation

1.1. Background

The first documentations of tissue replacement are based on the

work of Gasparo Tagliacozzi – an Italian surgeon performing

successful autologous skin transplantations in the 16th century. He

also repeatedly failed with allogeneic transplantations, introducing

the idea of rejection in his publication De Curtorum Chirurgia per

Instionem in 1596. Alexis Carrel and Charles Guthrie developed new

suturing techniques for transplanting arteries and veins,

subsequently enabling vascular anastomosis operations and solid

organ transplantation (Carrel and Guthrie 1905). Joseph Murray and

J. Hartwell Harrison performed the first technically successful kidney

transplantation between identical twins in 1954 (Guild et al. 1955),

raising further interest in clinical solid organ transplantation.

Eventually, the development of heart-‐lung machine enabled

surgeons to perform open-‐heart surgery, and Christiaan Barnard to

perform the first allogeneic heart transplantation in 1967 (Barnard

1967). The first patient died unfortunately due to pneumonia early

after the successful operation. Nevertheless, Barnard inspired

others to establish several heart transplantation programs. Acute

rejection resulting from tissue type mismatching was fatal to most

of the recipients, however, and the hype quickly subsided. The

introduction of immunomodulatory drugs – corticosteroids,

azathioprine, and most importantly cyclosporine in 1976 (Borel et al.

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1976) – enabled long-‐time survival of recipient and finally made

solid organ transplantation a plausible treatment for many end-‐

stage diseases.

1.2. General histology of the heart

Histologically, the heart consists of myocardium, supporting

connective tissue, and vascular structures. Vascular structure can be

divided into large coronary arteries, veins, arterioles, venules,

capillaries, and lymphatic vessels. Lymphatic vessels are responsible

for trafficking inflammatory cells from the heart into secondary

lymphatic organs. The vasculature feeds the myocardium with

oxygen and nutrients, but also enables circulating inflammatory cells

to enter the tissue. Mesenchyme derived pericytes (PC), smooth

muscle cells (SMC), fibroblasts, and extra-‐cellular matrix (ECM) form

the connective tissue surrounding and supporting the vascular

structures and the myocardium. The myocardium is formed by

delicately organized and interconnected cardiomyocytes that are

responsible for the contractile function of the myocardium.

Endothelial cells (EC) line the inner lumen of the blood vessels,

cardiac chambers, and the lymphatics forming a barrier between the

circulation and the tissues. EC play important role in inflammation,

as inflammatory cells must pass through the endothelium in order to

reach target tissue. As a response to inflammation, EC express

adhesion molecules on their luminal surface. These adhesion

18

molecules enable circulating leukocytes to attach to vascular wall,

slow down, and eventually transmigrate through the endothelium.

All cells express self-‐antigens on their cell surface with major

histocompatibility complex (MHC) class I receptors. Antigen-‐

presenting cells (APC; macrophages and dendritic cells) also express

MHC class II receptors and are able to present foreign peptides – i.e.

non-‐self antigens – to T cells with these receptors. In MHC-‐

mismatched organ transplantation, the donor tissue type differs

from the recipient tissue type – the transplant is therefore

allogeneic and called an allograft.

1.3. Indications, patient characteristics, and outcome

The International Society of Heart and Lung Transplantation (ISHLT)

Registry data show that between 2006 and 2012, 22 318 adult

transplantations have been performed around the world with the

most common indications being non-‐ischemic cardiomyopathy (54%

of the patients) and coronary artery disease (37%). Other indications

were valvular (2.8%), congenital heart disease (2.9%), and

retransplantation (2.5%). Over 35% of patients were bridged to the

transplantation with mechanical circulatory assist devices. The

median age of the heart transplant recipients was 54 years, and the

median age of the donors was 35 years. The cause of death for

organ donation was head trauma (46 %), stroke (24%), or other

(30%) (Lund et al. 2013).

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The survival of cardiac transplant recipients has changed little during

the last 30 years; the median survival of cardiac transplant recipients

is 11 years, however, patients surviving the first year after the

transplantation have median survival of 14 years (Stehlik et al.

2011). The latest data shows that 1-‐year survival of all cardiac

transplant patients is 81%, and 5-‐year survival is 69%. Figure 1

demonstrates the median survival of transplant recipients on

different decades. The patient mortality is the highest during the

first year after the transplantation due to graft failure (36% of

deaths), non-‐CMV infections (12%), acute rejection (4%), or other

reasons (46%). After the first year, graft failure, cardiac allograft

vasculopathy, and malignancies are the main survival-‐limiting

factors (Lund et al. 2013).

Figure 1. The median survival of all cardiac transplant patients operated between 1982-‐2011. Modified from Stehlik et al 2013.

Average annual center heart transplant volume (all

20

After the transplantation, the patients usually gain significant

improvement of life and can perform normally in daily activities. The

Registry data show, however, that only 35% of the recipients are

employed 1 year after the transplantation, and 46% at 3 years,

possibly due to regional employer-‐related factors (Lund et al. 2013).

1.4. Risk factors

Immunologic and non-‐immunologic factors pose risks to allograft

and patient survival. The donor-‐derived non-‐immunologic factors

are the cardiovascular status of the donor, the body-‐mass index,

blood glucose levels and direct donor organ trauma. Immunologic

risk factors include donor brain death, tissue-‐type mismatch

between the donor and the recipient, and recipient antibody

production. Donor brain death produces systemic cytokine storm

making the grafts even more susceptible to IRI and rejection (Takada

et al. 1998; Wilhelm et al. 2000).

Due to the acute nature of cardiac diseases leading to

transplantation and shortage of organ donors, cardiac

transplantations are performed between donors and recipients with

mismatching tissue type, or major histocompatibility class (MHC;

human leukocyte antigen, HLA, in humans). Furthermore, due to the

shortage of organ donors, older and sicker patients are accepted as

donors. Episodes of acute rejection are common during the first year

after transplantation, as 25% of patients are diagnosed with mild-‐to-‐

severe acute rejection. However, acute rejection is efficiently

21

treated with immunosuppressive medication, as acute rejection

accounts only 4% of deaths during the first year. The age of the

donor has linear correlation with the risk of mortality during the first

year after the transplantation. Similarly, prolonged allograft

ischemia – especially when exceeding 200 min – is associated with

increased early mortality risk. Other recipient-‐derived risk factors for

early mortality are renal dysfunction, and the need for mechanical

circulatory support before transplantation. In addition to the 1-‐year

risk factors, risk factors for mortality during the first 5 years after

transplantation include episodes of acute rejection, the need for

dialysis or treated infection during hospitalization, and the absence

of certain immunosuppressive drugs 1 year after the

transplantation. Interestingly, donor age and ischemic time continue

to affect the survival of the recipients 15 years after transplantation

(Stehlik et al. 2012).

Immunosuppression fails, however, to protect the allograft and the

recipient from several important risk factors: ischemia-‐reperfusion

injury, chronic rejection, the toxicity of immunosuppressive therapy,

and the exposure to infections. Episodes of acute rejection are

linked to chronic rejection development, and depending on the

severity of rejection, even one acute rejection requiring treatment

decreases the overall survival of the patients.

The use of immunosuppressive medication is necessary to prevent

allograft rejection, but inhibition of the normal function of T cells

22

increases the risk of infections. Viral infections pose the greatest

risks to solid organ transplant recipients, but also to the allograft

itself. Cytomegalovirus (CMV) infection increases the risk of allograft

rejection and cardiac allograft vasculopathy. Immunosuppression

also increases the risk of malignancies. The incidence of non-‐skin

malignancies increases progressively and accounts for 20% beyond 5

years after transplantation (Stehlik et al. 2012).

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2. Ischemia-‐reperfusion injury

2.1. Ischemia and hypothermia

The procurement of heart transplants requires that the blood flow

to the organ be stopped for the transportation and during the

surgery. Lack of circulation renders the transplant into oxygen and

nutrient deprived ischemic state. Current organ preservation

techniques rely on cooling of the allograft (Jacobs et al. 2010).

Ischemia results in accumulation of anaerobic metabolites, changes

in electrolyte balance, and hypoxic tissue injury (McCord 1985).

Ischemic time depends on the distance between the donor hospital

and the transplantation center, and with heart transplantation, is

approximately 3 hours (Stehlik et al. 2012). The kidney, however,

endures ischemia better, and can be transplanted even after 16

hours of ischemia (Southard and Belzer 1995). Transplantation-‐

related ischemia is divided into cold and warm ischemia, of which

cold ischemia is regarded as organ protective and warm ischemia

organ damaging phase. Physiologically, during hypothermia, the cell

metabolism and oxygen consumption are reduced, whereas during

warm ischemia, the tissue remains metabolically active but lacks

oxygen and nutrients driving itself into anaerobic state.

Furthermore, hypothermia protects endothelial cells from apoptosis

(Yang et al. 2009). Ischemia, on the other hand, results in

mitochondrial damage and release of reactive oxygen species,

accumulation of lactate, and downregulation of Krüppel-‐like factor 2

(KLF2) expression, as endothelial shear stress is diminished (Dekker

24

et al. 2002). KLF2 is essentially involved in vascular development and

sustaining physiological quiescence by negatively regulating vascular

inflammation, permeability and angiogenesis (SenBanerjee et al.

2004; Bhattacharya et al. 2005; Dekker et al. 2006; Lin et al. 2006).

Hypoxia-‐inducible factor-‐1 (HIF1) is a transcription factor constantly

produced in various cell types in response to tissue hypoxia and it is

rapidly degraded in normoxia by von Hippel-‐Lindau protein (Wang

and Semenza 1993; Maxwell et al. 1999). HIF1 affects wide range of

downstream proteins, most relevant to microvascular dysfunction

being vascular endothelial growth factor (VEGF), angiopoietin-‐1, and

angiopoietin-‐2 (Semenza 2014). VEGF is pro-‐angiogenic and pro-‐

inflammatory growth factor, partly inducing its effects via increase

in endothelial permeability and recruiting smooth muscle cells and

endothelial cells to form new vessels (Yancopoulos et al. 2000).

VEGF also induces adhesion molecule expression on the luminal

surfaces of the EC, luring inflammatory cells to the site of it’s

secretion (Kim et al. 2001a). Interestingly, KLF2 plays major role in

ischemic allograft as it also regulates HIF1 expression (Kawanami et

al. 2009). Figure 2 describes the factors involved and their interplay.

2.2. Reperfusion and re-‐oxygenation

Revascularization of the transplant is vital for the organ but,

paradoxically, results in ischemia-‐reperfusion injury (IRI).

Reperfusion of ischemic tissue with oxygenated blood results in

release of lactate, reactive oxygen species (ROS), capillary perfusion

and permeability disorder, endothelial activation, and inflammatory

25

cell influx (Eltzschig and Carmeliet 2011; Lampe and Becker 2011). In

allogeneic environment, initial macrophage and neutrophil influx is

followed immediately by sustained NK and T cell infiltration (El-‐Sawy

et al. 2004). Therefore, IRI of allogeneic solid organ transplant differs

from IRI of other origin, such as revascularization in acute coronary

syndrome and in stroke. IRI of transplant is referred as Tx-‐IRI from

now on to emphasize the importance of the difference.

The IRI induces the release of endogenous molecules called

danger/damage-‐associated molecular patterns (DAMP), which are

structural proteins normally bound to, or part of the extracellular

matrix. These molecules are recognized by the TLR-‐receptors of the

innate immune system cells, which in allogeneic environment may

accelerate alloimmune and rejection responses.

26

Figure 2. The effects of hypoxia and ischemia on microvascular wall in the heart. Ang, angiopoietin; COX, cyclooxygenase; ET-‐1, endothelin-‐1; EC, endothelial cell; PC, pericyte; CMC, cardiomyocyte; ICAM-‐1, intracellular adhesion molecule-‐1; IL, interleukin; HIF-‐1, hypoxia-‐inducible factor-‐1; Rbc, red blood cell; RhoA, Ras homolog gene family member A; SMA, smooth muscle actin; TLR, Toll-‐like receptor; TNF-‐a, tumor necrosis factor alpha; TnT, troponin T, VCAM-‐1, vascular endothelial growth factor-‐1; VE-‐cadherin, vascular endothelial cadherin; VEGF, vascular endothelial growth factor. 2.3. Microvascular dysfunction

Hypoxia induces EC instability by formation of cell membrane

protrusion and disintegration (Aono et al. 2000). Ischemia and

reperfusion activates cytoskeletal modulators of endothelial cells,

Extracellular space

EC

HIF1a

VEGFAng2

PC

ECCOX-2

PGEPGI 2

2

Capillary lumen

Macrophage

Granulocyte

VCAM-1

IL-1`TNF-_

VEGF

RhoA

_-SMA

Ang1Ang2ET-1

HYPOXIA

HIF1a

TnT

CMC

VEGF

EC

VEGFR-1

Tie2

Macrophage

ICAM-1

TLR2/4

VE-Cadherin

VE-Cadherin

Rbc

Rbc

27

but more importantly results in PC constriction reducing

microvascular blood flow and tissue perfusion (Yemisci et al. 2009).

Rho GTPases Rac1, and RhoA regulate these membrane morphology

changes, and PC and SMC constriction. In detail, RhoA and Rac1

have opposite function in hypoxia/reoxygenation, as RhoA regulates

endothelial barrier function and stress-‐fiber formation, whereas

Rac1 is required for endothelial recovery. (Wang et al. 2001) Rho-‐

kinase phosphorylates adducin and therefore, phosphorylated

adducin may be regarded as a parameter for Rho activity (Fukata et

al. 1999).

The perfusion defect worsens allograft function and may inflict

fibrosis development. Tx-‐IRI also induces EC-‐EC junction disruption

and microvascular leakage. Leaky vessels are more susceptible to

tissue edema and inflammatory cell influx. IRI affects microvascular

endothelium, PC and underlying tissue resulting in microvascular

dysfunction, endothelial barrier function disruption and

cardiomyocyte damage in cardiac allografts (Tuuminen et al. 2011).

The activation of EC during ischemia and Tx-‐IRI induces expression

of endothelial cell adhesion molecules attracting circulating

macrophages, neutrophils, NK cells, and T cells. Therefore,

microvascular dysfunction results in local inflammation,

accumulation of inflammatory cells and innate and adaptive

immune activation in cardiac allografts (Carden and Granger 2000;

Boros and Bromberg 2006; Dumitrescu et al. 2007).

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3. Immunobiology

3.1. Innate immune system

The innate immune system consists of plasma complement system,

circulating inflammatory cells, such as neutrophil granulocytes,

monocytes, and natural killer (NK) cells, and of circulating and tissue

residing macrophages, and dendritic cells (Janeway and Medzhitov

2002). It is congenital first-‐line defense against invading pathogens

but is also responsible for the cleaning and degradation of injured

tissue (Xu et al. 2006). The complement system may directly destroy

pathogens or help the other inflammatory cells to do so (Müller-‐

Eberhard 1986).

Neutrophils and monocytes/macrophages are phagocytes capable of

internalizing and ingesting pathogens and particles. They originate

from same common myeloid progenitor cells and subsequently from

myeloblasts. Neutrophils are the first-‐responders to inflammation,

and migrate to the site of inflammation within minutes with the help

of IL-‐8-‐ and C5d-‐mediated chemotaxis. They also need to adhere to

the vascular wall and transmigrate through the endothelium by

interacting with selectins, integrins, and adhesion molecules – most

predominantly P-‐selectin, LFA-‐1, ICAM-‐1, and VCAM-‐1. Neutrophils

have characteristic cytoplasmic granules containing substances, such

as myeloperoxidase (MPO), lysozyme, and collagenase that enable

them to degrade phagocytized bacteria and obliterate internalized

particles (Kolaczkowska and Kubes 2013).

29

Macrophages participate in host’s first-‐line defense against invading

pathogens, but also take part in scavenging aging cells and debris.

Furthermore, macrophages are able to boost adaptive immune

response in transplantation by presenting internalized antigens to T

cells, but also directly attacking allogeneic T cells (Xu et al. 2006; Liu

et al. 2012; Canton et al. 2013).

Toll-‐like receptors (TLR) of innate immune cells recognize pathogen-‐

associated molecular patterns (PAMP), such as bacterial

lipopolysaccharide, lipoproteins, peptidoglycan, and flagellin, viral

DNA and RNA (Aderem and Ulevitch 2000). When encountered with

appropriate ligand, TLR activates innate immune cells to induce

adaptive immune responses. Of 10 identified functional human TLR

(13 in mouse and rat), TLR2 and 4 also recognize endogenous

structural molecules exposed during tissue injury (Roach et al. 2005;

Land 2011). These danger/damage-‐associated molecular patterns

(DAMP) include biglycan, fibrinogen, fibronectin, hyaluronic acid

(HA), heat-‐shock proteins and high-‐mobility group box 1 (HMGB1)

(Smiley et al. 2001; Tsan and Gao 2004; Schaefer et al. 2005; Yu et

al. 2006). TLR2 or 4 activation on APC surface results in MyD88-‐

dependent NF-‐kB and mitogen-‐activated protein kinase signaling,

and innate immune activation (Barton and Medzhitov 2003). Innate

immune activation includes DC maturation seen as increased

superficial co-‐stimulatory molecule expression, and release of pro-‐

inflammatory chemokines and cytokines (Figure 3) (Janeway and

Medzhitov 2002; Rossi and Young 2005; Kaczorowski et al. 2007).

30

Figure 3. The activation and maturation of immature dendritic cells upon encountering of danger/damage-‐associated molecular patterns, and allogeneic, foreign peptides. The DC recognize DAMPs with TLR-‐receptors and begin expressing proinflammatory cytokines through NF-‐kB transcription factor activation. CCR7, C-‐C chemokine receptor type 7; CD, cluster of differentiation; DC, dendritic cell; DAMP, danger/damage-‐associated molecular patterns; MHC, major histocompatibility complex; NF-‐kB, nucleic factor kappa B; TLR, Toll-‐like receptor. Maturation increases the superficial expression of costimulatory

molecules CD80, CD83, CD86, CD40. DC migration to secondary

lymphoid organs (SLO), such as lymph nodes and spleen is facilitated

by increased expression of CCR7 – a receptor for constantly secreted

lymphatic chemokine CCL19 and 21 (Banchereau and Steinman

1998; Förster et al. 2008). Activation of DC is important step in

linking innate and alloimmune responses (Figure 4).

TLR4 CD80

MHC-II

CD83

CD86

CD40

CCR7

MHC-II

DAMPs

Foreign

antigen

NFgB

Immature DC Mature DC

31

Figure 4. Cross-‐linkage of innate and adaptive immune responses during ischemia-‐reperfusion injury. CCL21, Chemokine (C-‐C motif) ligand 21; CCR7, C-‐C chemokine receptor type 7; CD, cluster of differentiation; CMC, cardiomyocyte; DAMP, danger/damage-‐associated molecular pattern; DC, dendritic cell; EC, endothelial cell; IFN-‐g, interferon gamma; IL, interleukin; PMNC, polymorphonuclear cell; Th, T helper cell; TLR, Toll-‐like receptor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. 3.2. Alloimmune system

The alloimmune system consist of B and T cells, of which the latter

can be further divided into CD4+ T helper cells (Th), and CD8+

cytotoxic lymphocytes (CTL). The CD4+ T cells are further classified

into subtypes presented in Table 1. CD4+CD25+FoxP3+ subset of T

cells is a crucial cell population for immunological balance and

Capillary lumen

SMC

TLR2TLR4

DC

NF-gB

Macrophage

CCL21

SLO

Th17expansion

DC

IL-6VEGF-A

Th1expansion

IL-2

IFN-a

IL-12

IL-23

Th17

IL-17

IL-6IL-8

CD4+

CD4+

CD4+

CD4+

VEGFR-2

CCR7

VEGFR-3

Cardiac parenchyma

TLR2TLR4

DAMPs

VEGF-C

CD80

CD83

IL-1IL-6TNF-_

EC

CMC

PMNC

32

tolerance. These cells are named regulatory T cells (Treg) for their

suppressive properties (Wood et al. 2012; Lazarevic et al. 2013).

In direct allorecognition, the donor-‐derived passenger DC and

macrophages of the allograft travel to SLO and present donor

peptides with MHC class I receptors directly to cytotoxic CD8+ T

cells, or with MHC class II to naïve CD4+ T-‐cells. T cells may also

recognize foreign peptides directly on allograft endothelial cell MHC

class I receptors (Ali et al. 2013).

In indirect allorecognition, once the recipient APC encounter foreign

protein, they internalize it, activate and increase their expression of

CD80, CD83, CD86, and CCR7 on their surfaces and migrate to SLO to

present the internalized foreign material to naïve CD4+ T cells with

MHC class II receptors (Janeway and Medzhitov 2002; Rossi and

Young 2005; Kaczorowski et al. 2007). The T cells recognize peptides

presented in MHC class II receptors with their T cell receptors (TCR),

and if accompanied with co-‐stimulatory signal between CD28 and

CD80/CD86, the transcription factor of activated T cells (NF-‐AT) is

activated by calcineurin. This leads to interleukin-‐2 (IL-‐2)

transcription and, by paracrine signaling through IL-‐2R, to clonal

proliferation of alloreactive T cells (Figure 5) (Lee et al. 1994).

In a relatively lately discovered phenomenon, semi-‐direct

allorecognition, donor-‐derived peptides are presented unprocessed

33

to T cells by recipient APC. This constitutes a small minority of

allorecognition and probably has little clinical significance.

Table 1. Different T helper cell subsets. IL, interleukin; IFN, interferon; STAT, signal transducer and activator of transcription; TGF, transforming growth factor; ROR, retinoic acid receptor-‐related orphan receptor; FOXP3, forkhead box p3. T cell subtype

Function normally / In transplantation

Transcription factors and hallmark cytokines

Th1 Cellular immune defence; boosts macrophage and CD8+ T cell killing ability / Cell-‐mediated rejection

T-‐bet; IL-‐2, IL-‐12, IFN-‐γ, STAT4

Th2 Humoral immune defence; B cell stimulation / Antibody-‐mediated rejection

GATA3; IL-‐4, IL-‐10, STAT6

Th3 Mucosal immunity in the gut / unknown

IL-‐4, IL-‐10, TGF-‐β

Th17 Anti-‐microbial immunity / Cell-‐mediated rejection

RORγ, STAT3; IL-‐6, IL-‐17, IL-‐23

Treg Immune balance / Tolerance?

FOXP3; IL-‐10, TGF-‐b

34

Figure 5. Antigen presentation and costimulation between dendritic cell (DC) and naïve CD4+ T cell. If accompanied with costimulatory signal, antigen presentation results in clonal expansion of alloreactive T cells. CD, cluster of differentiation; DC, dendritic cell; IL, interleukin; NF-‐AT, nuclear factor of activated T cells; MHC, major histocompatibility complex. Alloreactive cytotoxic CD8+ T cells are the prime effector cells

responsible for allograft injury and may inflict graft rejection in the

absence of CD4+ T cell help. Acute rejection is considered to

originate from direct allorecognition and from MHC class-‐I signaling

between CD8+ T cells and allogeneic cells, whereas chronic rejection

is indirect allorecognition-‐mediated (Liu et al. 1993; Rogers and

Lechler 2001; Schmauss and Weis 2008).

Th17 T cells produce mainly IL-‐17A and participate normally in host

pathogen defense, but also in multiple autoimmune diseases and

chronic inflammation. IL-‐17A has been linked to neutrophil

multiplication and recruitment via granulocyte colony-‐stimulating

MHC-II

CD40

TCR

CD40L

Antigen

CD80/86

CD28

Mature DCNaïve CD4+ T cell

NF-AT

IL-2R

IL-2Calcineurin

35

factor and CXC chemokines (Laan et al. 1999; Ley et al. 2006). IL-‐23

signaling via IL-‐23R is crucial for Th17 T cells effector function and IL-‐

17 mediated inflammation (Korn et al. 2009). The role of Th17

response in allograft rejection was demonstrated with T-‐box21-‐

deficient mice, which lack Th1 alloimmune response. The findings of

Yuan et al. suggest that the absence of Th1-‐transcription factor T-‐

box21 (murine analog for Tbet) results in clonal expansion of IL-‐17

producing T cells and accelerated allograft rejection. (Yuan et al.

2008) Furthermore, in wild-‐type mice, TLR-‐signaling promotes IL-‐6

and IL-‐17-‐dependent acute rejection bridging Th17 response and

innate immune activation (Chen et al. 2009).

Tregs are a subset of T cells naturally originating from thymus with

important role in balancing immune system during everyday life,

especially during microbial infections and pregnancy. Disruption in

Treg population may result in unwanted conditions such as

autoimmune diseases, allergies, and tumor immunity. Furthermore,

generation of alloantigen-‐specific Tregs may induce transplant

tolerance by inhibiting costimulatory signals of T cells and thus

preventing generation of alloreactive effector T cells. (Sakaguchi

2005; Ochando et al. 2006) Recent findings suggest clinical potential

for adoptive transfer of ex vivo-‐expanded antigen-‐specific Tregs

generated from naïve T cells in prevention of allograft rejection

(Takasato et al. 2014).

36

3.5. Acute rejection

Fully allogeneic transplant is foreign tissue to the recipient and,

therefore, is considered a threat. PMC, macrophages, and NK cells

are the first allograft-‐infiltrating inflammatory cells early after the

reperfusion. Leukocyte infiltration is physiological response to IRI

and occurs both in syn-‐ and allografts, and may inflict early graft

injury without antigen processing and allorecognition. In syngrafts,

this acute inflammation subsidizes in hours. In allografts, however,

the direct and indirect allorecognition produces alloreactive effector

T-‐cells, which invade the allograft. The presence of CD8+ T cells

boosts innate immune mediated inflammation and inflicts

prolonged neutrophil-‐mediated response. The CD8+ T-‐cells are also

responsible for direct, MHC class-‐I-‐mediated destruction of

allogeneic tissue (El-‐Sawy et al. 2004).

Hyperacute rejection of a solid organ transplant is a rare

phenomenon seen in sensitized recipients with donor-‐specific

antibodies, resulting from pre-‐existing antibodies against

incompatible ABO blood group or HLA-‐antigens. The allograft is

destroyed within minutes by thrombosis and devascularization.

Hyperacute rejection is the main limitation for xenotransplantation

(Williams et al. 1968; Mengel et al. 2012).

Acute cellular rejection results from alloreactive T cell proliferation

and infiltration after allorecognition. CD8+ T cells and NK cells attack

foreign cells either through foreign peptide encountering with MHC

37

class I receptor or via “non-‐self” recognition. CD4+ Th1-‐type T cells

and macrophages are responsible for delayed hypersensitivity and

inflammation. CD4+ Th2-‐type T cells and B cells are responsible for

antibody-‐mediated rejection. (Rogers and Lechler 2001; Lakkis and

Lechler 2013) Table 2. describes the 2004 revised grading of acute

cellular and antibody-‐mediated rejection in heart transplants

according to ISHLT consensus.

Table 2. International Society of Heart and Lung Transplantation standardized grading of cardiac biopsy for acute cellular rejection (R) and antibody-‐mediated rejection (AMR). Modified from Stewart et al. J Heart Lung Transplant, 2005. Grade 0 No rejection Grade 1 R, mild Interstitial and/or perivascular

infiltrate with up to 1 focus of myocyte damage

Grade 2 R, moderate Two or more foci of infiltrate with associated myocyte damage

Grade 3, R severe Diffuse infiltrate with multifocal myocyte damage ± edema, ± hemorrhage ± vasculitis

AMR 0 Negative for acute antibody-‐

mediated rejection No histologic or immunopathologic features of AMR

AMR 1

Positive for AMR Histologic features of AMR Positive immunofluorescence or immunoperoxidase staining for AMR (CD68+, C4d+)

38

3.6. Immunosuppression

In order to prevent acute allograft rejection and to prolong allograft

survival, the alloimmune response is suppressed with various

immunosuppressive drugs (Table 3). Usual clinical protocol with

triple-‐drug maintenance therapy consists of steroids, calcineurin

inhibitor cyclosporine A or tacrolimus, and of antimetabolite

azathioprine or mycophenolate mofetil, or T cell proliferation

inhibitor sirolimus or everolimus. Induction therapy with anti-‐

thymocyte globulin or with anti-‐IL2R-‐antibodies results in profound

perioperative immunosuppression. With immunosuppressive

medication, acute T-‐cell-‐mediated rejection is preventable. The

immunosuppressive drugs, however, have undesirable side effects,

and require constant monitoring. High level of immunosuppression

also increases the risk of opportunistic infections (Lindenfeld et al.

2004a; 2004b; Baran 2013).

39

Table 3. Immunosuppressive drugs, their mechanism of action, and clinical use. AP-‐1, activation protein-‐1; IL, interleukin; NF-‐kB, nucleic factor kappa B; TOR, target of rapamycin. Drug Mechanism Use Corticosteroids AP-‐1, NF-‐kB

inhibition Induction, maintenance, antirejection therapy

Azathioprine Cell cycle inhibitor of T (and B) cells

Maintenance therapy (to lesser extent)

Cyclosporine A Calcineurin (and subsequently IL-‐2) inhibition

Maintenance therapy

Tacrolimus Calcineurin (and subsequently IL-‐2) inhibition

Maintenance therapy

Mycophenolate mofetil

Inhibitor of T and B cell proliferation

Maintenance therapy

Sirolimus TOR-‐dependent inhibition of lymphocyte proliferation

Maintenance therapy

40

3.7. Chronic rejection

Chronic rejection in cardiac allografts is described histologically as

cardiac allograft vasculopathy (CAV) and clinically as allograft

dysfunction resulting probably from prolonged and sustained

chronic inflammation driven by ischemic injury and acute rejection,

and from subsequent vascular remodeling. The pathogenesis of

chronic rejection, however, is poorly understood but prognostic

factors include preoperative graft ischemia time, episodes of acute

rejection, donor age, and cytomegalovirus infection. In contrast to

common coronary artery disease, allograft vasculopathy is

histologically described as diffuse luminal occlusion of cardiac

arteries and fibrosis development. The incidence of cardiac allograft

dysfunction increases over time and limits the survival of transplant

patients. Current immunosuppressive treatment fails to prevent the

development of vasculopathy and cardiac fibrosis and subsequent

dysfunction, and the only effective treatment for the disease is re-‐

transplantation (Tanaka et al. 2005; Stehlik et al. 2012).

The scientific community generally considers indirect allorecognition

and sustained Th1-‐ and IFN-‐g-‐mediated alloimmune inflammation

the driving force behind the development of vasculopathy. Several

other factors, however, contribute to vascular inflammation and

microvascular dysfunction leading subsequently to graft failure

(Figure 6). Therefore, a combination of chronic inflammation and

response-‐to-‐injury better describes the phenomenon.

41

Figure 6. The interactions between factors affecting the pathogenesis of cardiac allograft dysfunction and vasculopathy (modified from Schmauss and Weis, Circulation 2008). Early innate immune response and following alloimmune activation

described above are the initiating events in pathological vascular

remodeling. Inflammatory cytokines IL-‐1, IL-‐6, and TNF-‐α promote

vascular inflammation and SMC proliferation. Microvascular

dysfunction after cardiac transplantation results in disruption in

endothelial barrier function and microvascular leakage, but also in

Alloimmune-independent factors

Alloimmune response

- Brain death- Preservation injury- Ischemia-reperfusion injury- Innate immune response

Direct allorecognition(donor DC)

Monocyte/DCinfiltration/activation

Indirect allorecognition(recipient APC)

T cell activation/clonal expansion

CD8+ T cell cytotoxicityB cell antibody production

Proinflammatory cytokine release

Vascular inflammationEndothelial cell activation - expression of MHC-II, TNF-a, Ang2 - adhesion molecules - chemokines, cytokines

Endothelial dysfunctionJunctional disruption, apoptosis,SMC proliferation, vascular leakage,no-reflow, endothelial-mesenchymaltransition

Recipient-dependent factors- Metabolic disorders (hyperlipidemia, hyperglycemia)- Hypertension

Transplant coronary artery disease (TxCAD)

Infection (CMV)- Systemic and vascular cytokine release- Vascular oxidative stress- eNOS-dysregulation

Inappropriate repairmechanisms- Dysfunction of hematopoietic/ circulating progenitor and cardiac stem cells- Dysregulated intragraft angiogenesis

42

no-‐reflow phenomenon. No-‐reflow occurs as a response to ischemic

insult when the small arterioles and capillaries remain contracted

and blood flow to the tissue is impaired, possibly due to contraction

of endothelial cells and pericytes. Due to loss of capillaries and

myocardial inflammation, the myocardium is gradually replaced with

fibrotic tissue, accompanied with impaired contractile function.

(Reffelmann et al. 2003; Schmauss and Weis 2008; Tuuminen et al.

2011) Chronic cardiac allograft dysfunction manifests histologically

as cardiac fibrosis and allograft vasculopathy, which in turn is

characterized clinically by diastolic dysfunction and heart failure

(Hollenberg et al. 2001; Haddad et al. 2012).

Pollack et al. (2013) have recently reviewed extensively the

diagnostic methods of evaluating CAV. Diagnosis of CAV is

somewhat complicated, as the innervation of the heart is

interrupted after the transplantation and the recipients do not feel

any heart-‐related chest pain. The progressive intimal stenosis results

ultimately in graft failure and, therefore, the clinical symptoms

implicating problems appear too late. Current clinical protocols

usually embrace routine transvenous biopsies taken regularly after

the transplantation. Angiography is the most usual method of

evaluating coronary arteries, however, the method is highly

susceptible for interpretation error, as the occluded arteries seem to

be in better shape than they actually are. Intravenous ultrasound is

the most reliable method of evaluating the degree of intimal

occlusion. Cardiac magnetic resonance is novel method of

43

evaluating risk of CAV development and CAV-‐associated changes,

but does not directly detect or determine severity of CAV (Hofmann

et al. 2014; Braggion-‐Santos et al 2014).

The incidence of CAV increases over time. According to the ISHLT

Registry data, approximately 10% of patients are diagnosed with

CAV one year after the transplantation. At ten years, approximately

half of the patients have developed CAV-‐associated changes. (Stehlik

et al. 2012) Therapeutic options in the treatment of CAV remain

limited, as coronary dilatation with angioplasty or stenting, and

coronary by-‐pass operations have resulted in high morbidity and are

nowadays considered as palliative procedures. (Halle et al. 1995;

Bader et al. 2006) Therefore, re-‐transplantation remains as the only

definitive treatment for CAV, however, patients that have

undergone re-‐transplantation have reduced survival after the

operation (Srivastava et al. 2000; Johnson et al. 2007).

44

4. Angiopoietin pathway

4.1. General

The angiopoietins are vascular growth factors consisting of

angiopoietin-‐1, -‐2, and -‐4 (Ang1, Ang2, Ang4), of which Ang1 and -‐2

act through their shared receptor tyrosine kinase Tie2 (Jones et al.

2001). The physiological role of Ang4 has not yet been defined. Tie1

and Tie2 are endothelial cell membrane receptors with high

structural homology. Although discovered earlier than Tie2 receptor,

little is known about the physiological role of Tie1 receptor. Tie1

seems crucial for endothelial cell survival, but not vasculogenesis

(Partanen et al. 1996). Tie1 has been suggested to have blood flow-‐

dependent impact on arteriosclerotic plaque formation, as Tie1

deletion reduces arteriosclerotic lesion formation (Woo et al. 2011).

Tie1 and Tie2 both have cytoplasmic tyrosine kinase domains and

are composed of two extracellular IgG-‐like domains, followed by

three EGF-‐like domains, another Ig-‐like domain, and three

fibronectin type III domains (Figure 8).

Angiopoietins regulate physiological microvascular integrity but also

pathophysiological dysfunction. Ang/Tie2-‐signaling is essential for

embryonic development, as Tie2-‐defiency results in early embryonic

lethality (Davis et al. 1996).

45

Figure 8. The angiopoietin/Tie2 signaling system. Ang1 and 2 both ligate to the same Tie2 receptor tyrosine kinase. Ang2 acts as a context-‐dependent agonist or antagonist to the receptor. Tie2 phosphorylation results in activation of PI3K/Akt pathway and downregulation of FOXO1 transcription factor, which in turn downregulates Ang2 expression (modified from Augustin et al. Nat Rev Mol Cell Biol 2009).

4.2. Angiopoietin-‐1

Ang1 has essential role in embryonic vascular development and

vessel maturation; Ang1-‐deficient mice die early during the

embryogenesis (Hanahan 1997). Ang1 stabilizes the immature and

leaky vessels by promoting interactions between the ECs and

perivascular structures (Gamble et al. 2000; Saharinen et al. 2008),

and by regulating the endothelial cytoskeleton (Mammoto et al.

Ang1 Ang2

Ig-like domain

EGF-like domain

Fibronectin type III

Tyrosine kinase

Tie2 Tie2

PI3K/Akt

FOXO1

???

46

2007). Ang1 is constitutively secreted by endothelial supporting

cells, such as pericytes and SMC, but also by fibroblasts, and several

types of tumor cells (Davis et al. 1996; Stratmann et al. 1998;

Sugimachi et al. 2003).

Tie2 phosphorylation by Ang1 induces signaling cascade that leads

to recruitment of p85 subunit of phophoinositide 3-‐kinase (PI3K),

and the activation of transcription factor Akt. Akt signaling promotes

survival-‐associated pathways, and suppresses apoptotic pathways.

Interestingly, Akt also inactivates Ang2-‐targeting forkhead

transcription factor FOXO1 resulting in negative feedback loop on

Ang2 production. On the other hand, inactivation of PI3K/Akt

signaling activates FOXO1 and increases Ang2 production and EC

destabilization (Papapetropoulos et al. 2000; Kontos et al. 2002;

Daly et al. 2004; DeBusk et al. 2004).

Ang1/Tie2-‐signaling regulates EC-‐EC permeability by inhibiting VEGF-‐

dependent adherens junction protein vascular endothelial (VE)-‐

cadherin internalization from junctional complexes (Gavard et al.

2008). In addition, Ang1 and Tie2 form junctional complexes further

stabilizing endothelium, but also its binding to extracellular matrix

(Saharinen et al. 2008).

Ang1 acts as an anti-‐inflammatory cytokine as it inhibits leukocyte

recruitment by preventing VEGF-‐induced endothelial adhesion

molecule expression and after LPS induced endotoxic shock (Kim et

47

al. 2001b; Witzenbichler et al. 2005). In addition, Ang1 induces KLF2

expression on endothelial cells counteracting VEGF-‐mediated

VCAM-‐1 expression and inflammatory cell adhesion (Sako et al.

2009). Furthermore, Ang1/Tie2-‐signaling inhibits NF-‐kB-‐mediated

inflammation via A20 binding inhibitor of NF-‐kB (ABIN) protecting

the endothelial cells from inflammation and apoptosis (Hughes et al.

2003; Tadros et al. 2003).

4.3. Angiopoietin-‐2

Originally discovered 1997 as Ang1 and Tie2 receptor antagonist, the

complex mechanism of Ang2 physiology remains somewhat

unknown (Maisonpierre et al. 1997; Eklund and Saharinen 2013).

Ang2 is not crucial for embryogenesis, as seemingly normal mice are

born with Ang2-‐knockout background. However, the mice lacking

Ang2 soon develop severe chylous ascites and die postnatally. Ang2

is normally stored in the Weibel-‐Palade bodies of EC with

endothelin-‐1 (ET-‐1) and von Willebrand factor and IL-‐8, and is

exocytosed upon stress, i.e., in ischemia, shear stress, and VEGF.

Hypoxia induced RhoA activation and cytoskeletal changes have

essential role in Weibel-‐Palade body exocytosis. (Vischer et al. 2000;

Fiedler et al. 2004; Kuo et al. 2008). Recent studies have further

shed light on Ang2 secretion by showing that Ang2 is released in

exosomes when PI3K and Akt are inhibited, and that the release was

regulated by syndecan/syntenin pathway. (Tsigkos et al. 2006; Ju et

al. 2014) Ang2 selectively competes with Ang1 in binding to the Tie2

receptor and is considered to have antagonistic function on the

48

receptor signaling in resting endothelium (Maisonpierre et al. 1997).

However, exogenous and endogenous Ang2 has been demonstrated

to induce Tie2 phosphorylation under certain conditions (Kim et al.

2000; Daly et al. 2006). Ang2 has, therefore, context dependent

function and can enhance neovascularization, promote EC survival,

or increase vascular permeability (Oliner et al. 2004; Nag et al. 2005;

Gallagher et al. 2007).

Ang2 expression increases in peripheral blood in several types of

malignancies. Its role in tumor environment has been suggested to

be closely linked to neovascularization, tumor growth and

metastasizing. (Etoh et al. 2001; Scholz et al. 2007; Schulz et al.

2011) Furthermore, increased expression of Ang2 has been

associated with primary graft failure in lung transplant patients and

cardiac allograft vasculopathy development (Diamond et al. 2012;

Daly et al. 2013). The inflammatory function of Ang2 partly

manifests as sensitization of endothelial cells to TNF-‐a and by

increase of endothelial adhesion molecules (Fiedler et al. 2006).

49

AIMS OF THE STUDY

The aim of this study was to characterize the effects of hypothermic

preservation on ischemia-‐reperfusion injury and innate and adaptive

immune responses in rat cardiac allografts, and to investigate the

therapeutic effects of targeting the angiopoietin-‐1 and -‐2 signaling

with clinically relevant protein and antibody treatment in this animal

model.

The specific aims of were:

1. to describe the effects of hypothermic preservation on

ischemia-‐reperfusion injury and innate and adaptive immune

responses, and on the development of cardiac fibrosis and

allograft vasculopathy;

2. to investigate the effect of donor heart treatment with

recombinant and stable variant of native angiopoietin-‐1 on

ischemia-‐reperfusion injury and subsequent development of

innate and adaptive immune responses in cardiac allografts;

3. to investigate the kinetics and mechanisms of angiopoeitin-‐2

during ischemia-‐reperfusion injury and adaptive immune

responses in rat cardiac allografts.

50

METHODS

1. Experimental rat cardiac transplantation model

Specific pathogen-‐free, inbred Dark Agouti (DA, RT1av1) rats

weighing 200-‐250 g were used as cardiac allograft donors. The

donor rat heart was perfused with ice-‐cold heparinized phosphate-‐

buffered saline (PBS), and excised. In the treatment studies, the

coronaries were then perfused with 200 µl of anti-‐Ang2 antibody

(MEDI3617; 150 ng/ml, in PBS; MedImmune), or non-‐specific IgG

(150 ng/ml, in PBS; MedImmune), or with recombinant Ang2 (50

µg/ml, in PBS)(Hwang et al. 2005), COMP-‐Ang1 (10 µg/ml, in PBS) or

PBS. The donor heart was preserved in PBS at 4°C (cold ischemia)

for 2 to 4 h, depending on the study model (Tuuminen et al. 2011).

Warm ischemia was standardized to 1 h. After preservation,

heterotopic cardiac transplantations were performed between fully

MHC-‐mismatched, pathogen-‐free, inbred 8-‐ to 12-‐week-‐old male DA

donor and Wistar Furth (WF, RT1u) recipient rats (Harlan

Laboratories, Boxmeer, The Netherlands).

To prevent irreversible episodes of acute allograft rejection, to

achieve long-‐term allograft survival, and to enable the development

of chronic rejection, the recipients in chronic rejection groups

received cyclosporine A (Novartis, Basel, Switzerland) 2 mg/kg/day

s.c. for the first 7 days and 1 mg/kg/day thereafter. For anesthesia

and perioperative analgesia, the recipient rats inhaled isoflurane

51

(Isofluran, Baxter, Deerfield, IL) and received s.c. buprenorphine

(Temgesic, Schering-‐Plough, Kenilworth, NJ).

The effect of cold preservation on the integrity of microvascular

endothelial cells was analyzed by transmission electron microscopy

in the non-‐transplanted DA donor hearts. After reperfusion, the

recipients of cardiac allografts were sacrificed at 30 min to analyze

the effect on microvascular perfusion and permeability with

Lycopersicon esculentum (Tomato) lectin perfusion and modified

Miles assay, at 6 h to analyze endothelial activation and

inflammatory cell influx with immunohistochemistry, myocardial

injury with serum troponin T (TnT) analysis, and innate immune

activation with real-‐time quantitative reverse-‐transcription PCR, and

at 8 weeks to analyze the degree of cardiac fibrosis and allograft

vasculopathy with histological stainings.

The State Provincial Office of Southern Finland approved all animal

experiments. The animals received care in compliance with the

Guide for the Care and Use of Laboratory Animals as outlined by the

National Academy of Sciences (ISBN 0-‐309-‐05377, revised 1996).

2. Transmission electron microscopy analysis

The mid-‐cardiac specimens were fixed in 2.5% glutaraldehyde in

phosphate buffer (pH 7.4). The Advanced Microscopy Unit

(Haartman Institute, University of Helsinki, Helsinki, Finland)

prepared the samples by TEM-‐routine: samples were post-‐fixed in

52

1% osmium tetroxide, then dehydrated in graded ethanol, and

embedded in Epoxy resin LX-‐112. Ultra-‐thin sections were cut at

around 80 nm. Electron staining was performed with uranyl acetate

and lead citrate in Leica EM STAIN automatic stainer. The sections

were then examined with JEM-‐1400 TEM (JEOL, Tokyo, Japan) at 80

KV and analyzed the incidence of endothelial cell (EC)-‐EC junction

gaps and EC blebbing with 25000x magnification.

3. Immunohistochemistry and immunofluorescence stainings

Cryostat sections were stained for subsets of inflammatory cells and

microvascular vessels using the peroxidase ABC method (Vectastain

Elite ABC Kit, Vector Laboratories) and the reaction was developed

with 3-‐amino-‐9-‐ethylcarbazole (AEC, Vectastain).

Immunofluorescent stainings were performed using Alexa 568 red

and Alexa 488 green (Promega, Madison, WI) secondary antibodies.

The following antibodies and dilutions were used: Ang2 (10μg/ml,

AF623, R&D Systems); RECA-‐1 for rat endothelium (50 μg/ml,

MCA97, AbD Serotec, Dusseldorf, Germany); FLAG for COMP-‐Ang1

(10μg/ml, Sigma-‐Aldrich Co, St. Louis, MO); CD4 for T cells (5 µg/ml,

22021D, BD Pharmingen, San Diego, CA), CD8 for T cells (5 µg/ml,

22071D BD Pharmingen), ED1 for macrophages (5 µg/ml, 22451D,

BD Pharmingen); MPO for neutrophils (20 µg/ml, ab9535, Abcam,

Cambridge, UK); OX62 for dendritic cells (10 µg/ml, MCA 1029G,

Serotec, Oxford, UK); VCAM-‐1 (10 μg/ml, MMS-‐141P, Covance,

Princeton, NJ); ICAM-‐1 (10 μg/ml, 1A29, Seikagaku, Tokyo, Japan).

53

The immunoreactivity was quantified in a blinded manner using

400x magnification.

4. Histological evaluation

Paraformaldehyde-‐fixed paraffin mid-‐cardiac cross sections were

used for histological stainings. The degree of cardiac fibrosis was

determined from cross sections subjected to Masson’s trichrome

staining and computer-‐assisted image processing (Zeiss Axiovision

4.4, Munich, Germany) by measuring the average proportional area

stained for fibrosis from photographs captured with 100x

magnification. Cardiac allograft vasculopathy was evaluated from

cross sections stained with hematoxylin-‐eosin, and Resorcin-‐Fuchsin

for internal elastic lamina, by measuring the area between the

internal elastic lamina and vessel lumen. The ratio of neointimal

area to internal elastic lamina determined the arterial occlusion

percentage.

5. Enzyme-‐linked immunosorbent assay

To quantify Ang1 and Ang2 from human and rat serum samples,

ELISA was performed according to the Manufacturer’s instructions

with following kits: human Ang1 (DANG10; R&D Systems Inc.,

Minneapolis, MN); human Ang2 (DANG20; R&D Systems); rat Ang2

(E90009Ra; Uscn Life Science Inc., Wuhan, China).

54

6. Cell culture assays

Human dermal blood microvascular endothelial cells (BECs)

(PromoCell, Heidelberg, Germany) were maintained on fibronectin-‐

coated dishes in Endothelial Cell Basal Medium MV (ECBM,

PromoCell) with growth supplements provided by the manufacturer

and used in passages 2-‐6. Hypoxic treatment (1% O2) was carried out

in Invivo2 400 hypoxia chamber (Ruskinn Technology, Bridgend, UK).

Tie2-‐GFP retroviral transfected cells were produced as

described(Saharinen et al. 2008). Cells were treated with non-‐

specific IgG (2 mg/ml; MedImmune, Gaithesburg, MD) or anti-‐Ang2

antibodies (MEDI3617; 2 mg/ml; MedImmune) or with COMP-‐Ang1

(Cho et al. 2004) (50 ng/ml). The cells were fixed, permeabilized, and

stained for Ang2, Tie2, or COMP-‐Ang1.

7. Microvascular leakage and perfusion assay

A modified Miles assay was used to measure extravasation of

plasma proteins from the microvasculature into the interstitial space

of cardiac allografts: immediately after reperfusion, the recipients

were injected i.v. with Evans Blue dye (Sigma-‐Aldrich; 30 mg/ml

diluted in 0.9 % NaCl), and allowed this to circulate for 30 min. To

detect perfused vessels 30 min after reperfusion, 50 ml FITC-‐labeled

Lycopersicon esculentum (Tomato) lectin (Vector Laboratories,

Burlingame, CA) diluted in 150 ml of 0.9% NaCl was injected to the

aortic root a few minutes before removing the allografts.

Immediately thereafter, the right auricle was incised and the

coronary network was flushed with 5 ml of 1 % PFA in 0.05 M citrate

55

buffer, pH 3.5. For quantification of extravasated Evans Blue, 100 mg

of apical myocardium was incubated into 500 ml of formamide on a

shaker at +60 °C for 24 h. The absorbance of dissolved Evans Blue

dye was then measured from the formamide by spectrophotometry

at 610 nm wavelength. The mean density of FITC positive

microvascular vessels was quantified from mid-‐axial cross sections

from 10 random fields of each cardiac cross section by fluorescence

microscopy.

8. Gene expression analysis

Total RNA was isolated from tissue samples dissected from the

myocardial midpiece of the allografts using RNeasy kit according to

the Manufacturer’s instructions (Qiagen, Hilden, Germany) and

reverse transcribed by using the High-‐RNA-‐to-‐cDNA kit (Applied

Biosystems, Foster City, CA). Quantitative real-‐time PCR was

performed on a RotorGene-‐6000 (Corbett Research, Doncaster,

Australia) using 2X DyNAmo Flash SYBR Green Master mix

(Finnzymes, Espoo, Finland) and the mRNA quantities of the

following factors were measured from each group: innate immune

receptors TLR2 and TLR4, their ligands biglycan, HAS1-‐3, HMGB1,

transcription factor NF-‐kB, dendritic cell (DC) maturation markers

CD80, CD86, and CD83, as well as inflammatory cytokines TNF-‐a, IL-‐

1b, IP-‐10, TGF-‐b, IL-‐6 and IL-‐10. The number of mRNA copies of the

gene of interest was calculated from a corresponding standard curve

using the RotorGene software. Of the tested housekeeping genes

(18SRNA, GAPDH, b-‐actin and TBP), 18SRNA was most stably

56

expressed and therefore all RT-‐PCR data were normalized against

18SRNA.

9. Heart transplant patients

The data consists of 11 cardiac allograft donors (8 men) and 11

recipients (8 men) operated between 2010 and 2011 in Helsinki

University Central Hospital, Helsinki, Finland. Healthy volunteers

were used as a control group (n = 24; 9 men). The mean age of the

donors was 40.9±14.1 years (range 19-‐56 years), of the recipients

47.4±11.4 years (range 29-‐63 years), and of the healthy volunteers

34.9±11.9 years (range 22-‐61). Blood samples were collected

preoperatively from cardiac allograft donors and postoperatively

from the recipients 1, 6, and 24 h after transplantation and ELISA

was used to measure the serum levels of Ang1 and Ang2. The Ethical

Committee of University of Helsinki, Helsinki, Finland approved the

use of the patient samples, and each patient gave written informed

consent.

10. Statistical analysis

All data are two group comparisons were performed by Mann-‐

Whitney U test; multiple group comparisons were performed by

Kruskal-‐Wallis analysis with Dunn correction, or by repeated

measures ANOVA using PASW Statistics 19.0 (SPSS Inc., Chicago, IL).

P<0.05 was considered statistically significant.

57

RESULTS

1. Prolonged ischemic preservation promoted ischemia-

reperfusion injury-mediated myocardial injury and inflammation

in rat cardiac allografts (I)

Myocardium is highly susceptible to hypoxic injury. Cardiomyocyte

injury is therefore inevitable during organ recovery and

preservation. Prolonged ischemic time resulted in profound

myocardial injury, but also increased the influx of inflammatory cells

into the allograft. The acute Tx-‐IRI phase was characterized by

increased expression of innate immune activating endogenous

danger molecules and influx of innate immune macrophages and

neutrophils alongside with induction of inflammatory cytokines IFN-‐

γ, IL-‐6, and TNF-‐α.

2. Prolonged ischemic preservation enhanced chronic rejection

(I)

As regards alloimmune activation, allografts transplanted after

prolonged ischemic preservation exhibited increased influx of CD4+

and CD8+ T cells, as well as macrophages, 10 days after the

transplantation and under subclinical immunosuppression.

Interestingly, during Th1-‐inhibiting CsA immunosuppression,

prolonged preoperative ischemic preservation resulted in shifting of

the cytokine profile of allograft-‐infiltrating T cells towards Th17

alloimmune response. This was seen as increased IL-‐17 and IL-‐23p19

gene expression. Paradoxically, Treg-‐associated FoxP3 transcription

factor expression was also robustly induced during alloimmune

58

response. Increased myocardial injury, acute and sustained

inflammation reflected as accelerated the development of cardiac

fibrosis, loss of functional capillary network, and arterial luminal

occlusion.

These findings emphasize the importance of early allograft injury

and inflammation in the development of late dysfunction, and

suggests early protection of allografts as potential target for

treatment with long-‐lasting effects.

3. Hypoxia induced endothelial cells to release Ang2

(unpublished)

Previous findings indicate that hypoxia regulates Ang2 expression in

human umbilical vein endothelial cells (Pichiule et al. 2004). Human

blood microvascular endothelial cells were incubated in hypoxic

environment for 1, 3, 8, 12, or 24 hours, and the culture medium

was analyzed for Ang2 with ELISA. In congruence with previous

findings, the data showed 2-‐fold increase in Ang2 secretion in

response to hypoxia during 24-‐hour incubation, when compared to

cells incubated in normoxia (Figure 9).

59

Figure 9. Ang2 release by human microvascular cells in normoxia or hypoxia. When Tie2-‐overexpressing ECs were incubated in hypoxia, the

secreted Ang2 localized into EC-‐EC junctions and associated with

Tie2. Application of anti-‐Ang2 antibody blocked this Ang2/Tie2 co-‐

localization (Figure 9A). Similarly, exogenous Ang1 applied to the

culture localized with Tie2 receptor in normoxia, but to lesser extent

during hypoxia indicating competitive binding to Tie2 with Ang2

(Figure 10 B-‐C).

1 3 8 12 24

3000

4000

2000

5000

0

1000

Med

ium

Ang

2 (p

g/m

l)

Incubation time (h)

Normoxia

Hypoxia

60

Figure 10. Hypoxia induces Ang2 deposition in endothelial cell-‐cell junctions and inhibits Ang1-‐Tie2 binding in endothelial cells. A, Human dermal blood microvascular cells (BECs) transfected with Tie2-‐GFP retrovirus were subjected to normoxia or hypoxia (1% O2) for 16 h in the presence of control-‐IgG or anti-‐Ang2 antibody (2 μg/ml). Immunofluorecent staining shows the localization of Tie2 (green) and Ang2 (red); DAPI indicates nuclei (blue). B, Tie2-‐GFP retrovirus transfected BECs were cultured in normoxia or hypoxia (1% O2) for 24 h, and stimulated with COMP-‐Ang1-‐FLAG (50 ng/ml) for the last 30 min. C, fractional area of Tie2/FLAG double-‐positive pixels from total area quantified from samples incubated in normoxia and hypoxia with or without COMP-‐Ang1. Immunofluorecent staining shows the localization of Tie2 (green) and COMP-‐Ang1 (FLAG; red). DAPI indicates nuclei (blue). Scale bars = 20 μm.

COMP-

Ang1CTRL

COMP-

Ang1CTRL

Normoxia Hypoxia

25

50

0

Tie2

+/FL

AG

+ar

ea (%

)

IgG IgG anti-Ang2 abNormoxia 16h Hypoxia 16h

Tie2

DA

PIA

ng2D

API

Tie2

Ang

2DA

PI

PBS

Hypoxia 24h

PBS COMP-Ang1

Normoxia 24h

COMP-Ang1

Tie2

DA

PIFL

AG

DA

PITi

e2FL

AG

DA

PI

A B

C

61

4. Cardiac transplantation induces immediate changes in

angiogenic growth factor expression in human and in rat (III)

Heart transplant donors and recipients were analyzed for serum

levels of Ang1 and Ang2 before organ donation and during the first

24 h after the transplantation. No changes were observed in the

serum levels of Ang1 in the donors (Figure 11A). The expression of

Ang2 was increased in brain dead heart donors compared to healthy

volunteers (Figure 11B). In the recipient patients, heart

transplantation induced progressive decrease in Ang1 levels and

increase Ang2 expression during the first 24 h (Figure 11C,D).

Similarly, in experimental rat cardiac transplantation, allogeneic

heart transplantation resulted in serum release of Ang2, but

interestingly reduced expression of Ang1 in peripheral blood.

Syngeneic transplant recipients, however, showed no changes in

serum levels of Ang2.

62

Figure 11. Peripheral blood analysis for Ang1 and 2 levels in cardiac allograft donors after brain death and in cardiac allograft recipients after transplantation. A and B, Human plasma samples from hemodynamically stable cardiac allograft donors (D; n = 19) before organ procurement and compared to healthy controls (CTRL; n = 24) analyzed by enzyme-‐linked immunosorbent assay (ELISA). C and D, Human plasma samples form cardiac allograft recipients (n=19) before transplantation (0 h) and 6, and 24 h after transplantation analyzed by ELISA. Data are given as scatter plot where the black bar indicates mean. ***P<0.001 by Repeated ANOVA measurements. 5. Targeting Ang/Tie2 pathway prevented Tie2-dependent

endothelial destabilization and microvascular dysfunction

induced by ischemic preservation (II-III)

Rat heart procurement and hypothermic preservation resulted in

endothelial destabilization, morphological changes in EC, and

disruption of EC-‐EC connections observed with transmission

electron microscopy analysis. When the allografts were ex vivo

perfused either with recombinant Ang1 or with anti-‐Ang2 antibody,

these changes were absent.

CTRL

10

0

5

7.5

2.5Hu

ma

n p

las

ma

An

g1

(n

g/m

l)

CTRL D DCTRL

Hu

ma

n p

las

ma

An

g2

(n

g/m

l)

10

0

5

7.5

2.5

CTRL6h 24h

10

0

5

7.5

2.5Hu

ma

n p

las

ma

An

g1

(n

g/m

l)

6h 24h

20

10

30

0

Hu

ma

n p

las

ma

An

g2

(n

g/m

l)

*********A B C D

63

Allograft tissue perfusion was measured from the apex of the heart

with laser Doppler velocimetry immediately after reperfusion. Ex

vivo donor heart treatment either with COMP-‐Ang1 or with anti-‐

Ang2 antibody resulted in significantly improved myocardial tissue

perfusion during the first 10 minutes after reperfusion. This effect

was dependent on functional capillaries, as the histologically

determined numbers of perfused vessels were increased 30 min

after the reperfusion in the treatment groups, whereas the absolute

numbers of vessels were indifferent.

Endothelial stability was further assessed by microvascular leakage

in modified Miles assay, in which the recipient is perfused with

albumin-‐binding Evans Blue dye, and the extravasation of the dye is

assessed 30 min after the reperfusion. Both donor heart-‐targeted ex

vivo COMP-‐Ang1 and anti-‐Ang2 antibody treatment stabilized the

allograft endothelium and preserved functional capillary network,

whereas Tx-‐IRI in control allografts resulted in reduced capillary

density and increased Evans Blue extravasation.

Ex vivo donor heart treatment with COMP-‐Ang1 or anti-‐Ang2

antibody prevented such early inflammatory response by reducing

the influx of inflammatory cells as well as downregulating the gene

expression of TLR-‐activating ligands. Importantly, these findings

were associated with significantly lower immunoreactivity of

endothelial adhesion molecules ICAM-‐1 and VCAM-‐1.

64

6. Acute rejection can be restrained by endothelial inactivation

(III)

Alloimmune response is imminent after allogeneic solid organ

transplantation. Prolonged preoperative ischemia prior to allogeneic

cardiac transplantation, however, worsens the acute innate immune

response in the absence of immunosuppressive medication. In our

experimental model, the fully MHC-‐mismatched cardiac allografts

are rejected in 5 or 6 days, if the recipients are not

immunosuppressed. When daily administration of CsA 1mg/kg i.p.

was applied, the allograft survival extended to 10-‐15 days. Recipient

treatment with single-‐dose of anti-‐Ang2 antibody failed to prolong

allograft survival with this background immunosuppression.

However, when the administration was continued on days 1, 3, and

5 after the transplantation, the survival was highly significantly

prolonged (Figure 12).

65

Figure 12. The effect of systemic recipient anti-‐Ang2 antibody treatment on rat cardiac allograft survival. Red dash line represents the survival of allografts transplanted after 4-‐h hypothermic preservation and without any immunosuppression. Red line represents allograft survival in recipients treated CsA 1 mg/kg s.c. and with non-‐specific IgG i.p. 4h before the transplantation. Green line represents allograft survival in recipients treated CsA 1 mg/kg s.c. and with anti-‐Ang2 antibody i.p. 4h before the transplantation. Blue line represents allograft survival in recipients treated CsA 1 mg/kg s.c. and with anti-‐Ang2 antibody i.p. 4h before, and on days 1, 3, and 5 after the transplantation. Data are presented by Kaplan-‐Meier survival plot. ***P<0.001 by Log Rank analysis. 7. Cardiac allograft vasculopathy development is correlated

with preoperative ischemia time and the number of endothelial

cells and pericytes (I-III)

Cardiac allograft vasculopathy was associated with gradual loss of

rat endothelial cell antigen-‐1 (RECA-‐1) positive capillaries, but also

NG2-‐positive pericytes. Donor heart treatment with COMP-‐Ang1

inhibited early endothelial activation, but also preserved the

numbers of capillaries and the pericytes surrounding them (Figure

13).

Surv

ival

(%) 80

60

40

20

0

Time (weeks)654321 7 80

100

IgG + CyA 1mg/kg/d1x Anti-Ang2 + CyA 1mg/kg/d4x Anti-Ang2 + CyA 1mg/kg/d

No treatment

***

66

Figure 13. The density of RECA-‐1+ EC (A) and NG2+ pericytes (B) in COMP-‐Ang1-‐ and PBS-‐treated allografts at 6 h, 10 d, 56 d. Representative immunofluorescence images from different timepoints (C). Scale bar = 40 µm. Data are given as mean ± SEM. *P<0.05, **P<0.01 by the Mann-‐Whitney U test. Ischemic time in clinical cardiac transplantation is an independent

risk factor for cardiac allograft vasculopathy development. In a

chronic rejection model, rat cardiac allografts were transplanted to

recipient rats receiving subclinical immunosuppression to prevent

episodes of acute rejection. This treatment protocol enabled chronic

rejection associated arterial luminal occlusion and myocardial

fibrosis development in 2-‐month follow-‐up. Cardiac allograft

vasculopathy changes were correlated with allograft preoperative

ischemic time (Figure 14).

500

0

1500

1000

REC

A-1

+ce

lls/m

m2

A

**

500

0

1500

1000

NG

2+ce

lls/m

m2

B

*

6 h 10 d 56 d

PBS

COMP-Ang1 C

CO

MP

-An

g1

PB

S

RECA-1NG2

6 h

RECA-1NG2

10 d 56 d

RECA-1NG2

RECA-1NG2

RECA-1NG2

RECA-1NG2

67

Figure 14. Cardiac allograft vasculopathy analyzed 56 d after rat allogeneic heart transplantation. A, B, and C, the incidence, occlusion, and representative histological images of arterial luminal occlusion in allografts transplanted after 0, 2, or 4 h hypothermic preservation. D and E, myocardial fibrosis analyzed semi-‐quantitatively from Masson-‐Trichrome stainings. Scale bar = 40 µm. Data are given as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 by the Mann-‐Whitney U test.

68

Donor heart ex vivo treatment either with recombinant COMP-‐Ang1

protein, or with anti-‐Ang2 antibody significantly inhibited the

development of aforementioned chronic rejection-‐associated

changes. Systemic recipient treatment with anti-‐Ang2 antibody had

similar effects on allograft vasculopathy development, indicating

that early inflammation is crucial.

69

DISCUSSION

Solid organ transplantation can be regarded as one of the crown

achievements of modern surgery. Replacing failing livers, lungs, or

hearts with functional grafts from brain dead donors gives second

chance to those patients with otherwise virtually no hope for living.

The median survival of heart transplant recipients has exceeded a

decade many years ago, and it keeps prolonging (Stehlik et al. 2012).

Primary graft dysfunction and allograft vasculopathy are important

factors limiting short-‐ and long-‐term survival of cardiac transplant

recipients. This study emphasizes the role of hypothermic

preservation, and moreover, IRI as an inductor of microvascular

dysfunction, and innate and adaptive immune responses leading to

primary and late allograft dysfunction after cardiac transplantation.

The results presented here shed light on the characteristics behind

IRI-‐induced inflammation and allograft dysfunction, and their impact

on the development of cardiac fibrosis and allograft vasculopathy.

Tie2 signaling was proven important, as supplementation of

recombinant Tie2 agonist COMP-‐Ang1 or inhibition of Tie2

antagonist Ang2 with specific antibody resulted in significant

improvement in microvascular function and allograft survival, as

well as inhibition of innate and adaptive immune responses and the

development of cardiac fibrosis and allograft vasculopathy.

Donor aspects and preservation

Incredible success of clinical heart transplantation has with

increasing numbers of heart transplant recipient candidates resulted

70

in chronic shortage of donated organs. This shortage has required

extending the donor pool to marginal, or extended criteria donors,

who are older and sicker than previously accepted. Furthermore,

the brain death of organ donor may induce a release of

catecholamines, a systemic inflammatory response, microvascular

endothelial cell and pericyte dysfunction, and myocardial injury

(Venkateswaran et al. 2009). Both longer ischemic time and

increased donor age are independent risk factors for 1-‐year

mortality after the transplantation (Lund et al. 2013).

Grafts from donors with extended criteria are especially susceptible

to primary graft dysfunction and injury requiring novel treatment

strategies to prevent Tx-‐IRI-‐induced effects (Iyer et al. 2011;

Abecassis et al. 2012). An experimental rat study demonstrated

worse cardiac function in grafts from older donor rats during early

reperfusion phase compared to grafts from younger donors

(Korkmaz et al. 2013). Recipients with grafts from marginal donors

have higher risk for primary graft failure and require temporary

circulatory support with i.e. extracorporeal membrane oxygenation

(Listijono et al. 2011). The use of temporary circulatory support,

however, is again a significant risk factor for 1-‐ and 5-‐year mortality

(Lund et al. 2013).

Current clinical organ preservation protocols abrogate Tx-‐IRI only

partially. With current immunosuppressive regimen, the episodes of

acute rejection are preventable, but chronic rejection still

71

significantly limits the survival of cardiac allograft recipients (Lund et

al. 2013). According to 2012 ISHLT Registry data, median ischemic

time for cardiac transplant is 3.1 hours. At the same time, ischemic

time exceeding 200 minutes is an independent risk factor for cardiac

allograft vasculopathy development. (Stehlik et al. 2012)

The problems with donors with extended criteria and ischemic time

may be manageable with novel preservation techniques, solutions,

or treatment options. Ex vivo heart perfusion machines are long

studied with little progression regarding the heart (Jacobs et al.

2010). However, Organ Care System for ex vivo perfusion of lung

allografts has shown promising potential and is currently further

investigated (Warnecke et al. 2012). Novel preservation solutions

are investigated in order to preserve grafts longer and in warmer

temperatures (Thatte et al. 2009; Lowalekar et al. 2013). Similarly,

solution with small interfering RNA against proinflammatory

cytokines and complement has proven potential in prolonged

allograft preservation (Zheng et al. 2009).

Reperfusion phase

Ischemia is not the sole determining factor for myocardial injury in

Tx-‐IRI, as syngeneic rat cardiac transplants with similar preoperative

ischemic time exhibit significantly milder cardiomyocyte injury than

allogeneic transplants (Keränen et al. 2013). Allorecognition initiates

after allogeneic solid organ transplantation regardless of T cell-‐

targeted immunosuppression. The duration of hypothermic

72

preservation, however, dictates the intensity of innate immune

response after reperfusion of the allograft (I).

Tx-‐IRI is an inevitable factor in solid organ transplantation, but

differs greatly from conventional IRI occurring in the treatment of

stroke or acute coronary syndrome. Rapid neutrophilic infiltration is

resolved in 24 h in syngrafts, whereas infiltrating CD8+ T cells induce

sustained neutrophilic influx to allografts (El-‐Sawy et al. 2004).

Furthermore, hypothermic preservation of cardiac allografts during

organ recovery and transportation induces ischemic injury to

cardiomyocytes, but also inflicts endothelial cell and pericyte

detachment from one another and disruption of endothelial barrier

(Tuuminen et al. 2011). Longer preservation promotes endothelial

and pericyte dysfunction that manifests as myocardial capillary

perfusion defect, microvascular leakage, vasoconstriction, and EC

activation. Activated EC increase adhesion molecule expression and

EC-‐EC junction disruption allows easier transmigration of

inflammatory cells. Together with increased accumulation of

inflammatory cells and secreted proinflammatory cytokines,

perfusion defect induces significantly worse cardiomyocyte injury

after prolonged preservation. (II-‐III)

Immunological issues

Foreign tissue transplanted to allogeneic recipient is, without

immunosuppression, rapidly rejected by innate immune cells such

and NK cells and adaptive immune response involving antigen-‐

73

presenting cells and the generation of allograft-‐specific IFN-‐γ

producing Th1-‐type T cells (Lehmann et al. 1997). Endothelial cells

are extremely important in the course of immunological recognition,

inflammatory cell recruitment, and myocardial perfusion. EC express

both MHC class I and class II molecules on their cytoplasmic

surfaces, and therefore, can communicate with both CD4+ and CD8+

T cells and induce IL-‐2 production. EC have, however, been shown to

stimulate only memory, but not naïve CD8+ T cells to become

cytolytic T cells. (Hancock et al. 1982) Activated EC may drive

sustained inflammation behind allograft rejection by inducing local

IFN-‐γ secretion by T cells (Salomon et al. 1991; Tellides and Pober

2007). Type I activation of EC is gene transcription-‐independent and

results from histamine or thrombin, and manifests as nitric oxide or

prostaglandin secretion, or Weibel Palade body exocytosis, resulting

in vasodilatation, increased perfusion, and permeability disorder.

Type II activation is described as transcription-‐dependent adhesion

molecule upregulation induced by cytokines such as TNF-‐α, and IL-‐1

(Pober and Sessa 2007). Activated EC promote leukocyte activation

and transmigration by secretin cytokines and chemokines, i.e.

PECAM-‐1, IL-‐8, IP-‐10, MCP-‐1, and RANTES (Al-‐Lamki et al. 2008).

Endothelium is the physiological first line barrier and defence

between circulating inflammatory cells and the allograft. Therefore,

injury to EC greatly contributes to cardiac allograft vasculopathy

development. In allogeneic environment, macrophages, cytotoxic

CD8+ T cells and natural killer cells induce EC apoptosis by non-‐self

74

recognition, Bax-‐induction, death receptor-‐signaling, or by TNF-‐

related apoptosis-‐inducing ligand (Zheng et al. 2000; Hsieh et al.

2002; Bleackley 2005; Wyburn et al. 2005). EC are, however,

resistant to injury and apoptosis by upregulating anti-‐apoptotic zinc

finger protein A20, heme oxygenase-‐1, Bcl-‐2, or the PI3k/Akt

pathway. (Soares et al. 1999; Madge and Pober 2000)

Our findings suggest that early EC activation may be a key mediator

of chronic inflammation, and that preventing this activation results

as significant improvement in allograft function and survival. (II-‐III)

Cardiac allograft vasculopathy and late graft dysfunction

The early innate immune response following solid organ

transplantation is described by infiltration of inflammatory cells

consisting mainly of macrophages, neutrophils and NK cells

depending on cellular adhesion molecules (El-‐Sawy et al. 2004;

2005). Dendritic cells and macrophages rapidly recognize foreign,

allogeneic antigens, but also allograft-‐derived TLR-‐activating danger

molecules result in innate and alloimmune activation (Goldstein

2004). Immunosuppressive drugs mainly target IL-‐2-‐mediated T cell

activation and proliferation; corticosteroids also inhibit but fail to

completely prevent innate immune activation. Of inflammatory

cytokines, IL-‐6 is extremely important in early CD4+ T cell mediated

inflammation and allograft rejection. In allogeneic murine cardiac

transplant model, recipient anti-‐IL-‐6 antibody treatment results in

prolonged survival (Lei et al. 2010). In a similar experimental setting,

75

but with recipient mice depleted of CD8+, IL-‐6 inhibition results in

virtually non-‐existent rejection, even when administered 6 d after

transplantation (Booth et al. 2011).

The answer to the pathophysiology of cardiac allograft vasculopathy

remains yet to be fully elucidated. On the cellular level, T cell driven

alloimmune response may be considered the main driving force

behind allograft vasculopathy development. Accelerated Th17

alloimmune response results in allograft rejection in Th1-‐deficient

mice (Yuan et al. 2008), and also seemed to promote chronic

rejection development in our experimental model (I). Different T cell

depletion strategies are used in clinical practice mainly as an

induction therapy in kidney transplantation, but to lesser extent in

other organs. Rapid release of cytokines, opportunistic infections,

lymphoproliferative diseases, and T cell repopulation are most

common problems, and despite of effectively promoting

immunosuppression early after transplantation, T cell depletion fails

to induce tolerance (Page et al. 2013).

As described above, EC have high potential in inducing or regulating

inflammatory responses after allogeneic solid organ transplantation.

Based on results presented in papers II and III, endothelial

stabilization and inactivation are crucial for allograft survival and

evasion from rejection. With inactivated endothelium, the need for

T cell depletion-‐mediated induction therapy may reduce.

76

Angiopoietins in solid organ transplantation

Angiogenic growth factors are intensively studied in the field of

cancer science, as angiogenesis is crucial for tumor growth and

metastasis (Welti et al. 2013). Interestingly, the same factors that

promote tumor growth elicit vascular instability in solid organ

transplantation.

Endothelial detachment is essential for neovascularization and VEGF

is a potent angiogenic growth factor inducing rapid and prominent

vascular leakage (Nagy et al. 2012). VEGF activates EC in

transplantation setting, and neutralization of VEGF has anti-‐

inflammatory effect in cardiac allografts by inhibiting the expression

of endothelial cell adhesion molecules and the production of several

chemokines (Lemström et al. 2002; Reinders et al. 2003).These VEGF

mediated pro-‐inflammatory effects are possibly downstream of

Ang2 signaling, and preventable by Ang2 inhibition (III). Another

study shows that neutralization of Ang2 reduces endothelial

adhesion molecule expression in murine ischemic limb model

(Tressel et al. 2008). Rat cardiac allograft treatment ex vivo with

anti-‐Ang2 antibody reduces VEGF and TGF-‐b mRNA expression after

the transplantation and subsequently inhibits vascular permeability

and perfusion disorder, and fibrosis development (III).

Ang1 elicits vascular stability and homeostasis during normal

physiology. Ang1 signaling through Tie2 receptor promotes anti-‐

apoptotic signals particularly by activating PI3k/Akt pathway and

77

ABIN (Papapetropoulos et al. 2000; Tadros et al. 2003).

Furthermore, type I endothelial activation may be avoided by Ang1,

which prevents VEGF-‐induced plasma leakage and enhances EC-‐EC

connections at cell junctions. Ang1 also reinforces blood vessel

coating by pericytes and surrounding extracellular matrix. (Thurston

et al. 2000; Saharinen et al. 2008) Donor heart-‐targeted Ang1

treatment stabilized microvascular endothelium of the allografts

and inhibited innate and alloimmune responses (II). Maintaining EC

viability and quiescence is therefore of great importance for

allograft survival.

Future prospects

A. Donor perspective

Currently, transplant units suffer from chronic shortage of donated

organs. Recognizing potential organ donors is a key factor in

increasing the numbers of solid organ transplants. In addition, due

to the shortage of organ donors, older and sicker patients are

accepted as organ donors. This makes donor management

important issue. Brain death of heart transplant donors causes

systemic cytokine storm. Circulating inflammatory cytokines may

prime the heart for subsequent inflammation and injury. Current

clinical protocols include donor treatment with systemic steroids to

prevent the adverse effects of this cytokine storm, but more

targeted treatment may be advantageous. Donor preoperative

simvastatin treatment in experimental rat cardiac transplantation

model protects the allograft from post-‐operative problems, such as

78

Tx-‐IRI and chronic rejection development (Tuuminen et al. 2011).

Our studies have pointed Ang2 to be increased in the donor

peripheral blood before organ procurement, and therefore a

potential target for donor treatment.

B. Allograft (preservation) perspective

The allograft is subjected to hypothermia and ischemia during the

preservation. Organ preservation solutions are designed to protect

the allograft from adverse effects of hypoxia, acidosis, low glucose

levels, and edema (Jacobs et al. 2010). Antioxidants are included

also in some. Potential treatment targets are the oxygen

metabolism producing reactive oxygen species during ischemic

preservation, endothelial protection and inactivation, or prevention

of allorecognition. Solution with small interfering RNA against TNF-‐

α, C3, and Fas has shown promising potential in prolonged allograft

preservation (Zheng et al. 2009). Also, novel preservation solutions

are designed to enable the allografts to be preserved in milder

hypothermia, or even normothermia (Thatte et al. 2009; Lowalekar

et al. 2013). Restoration of circulation with perfusion system after

allograft recovery has been studied for decades, however, the costs

and perfusion system management issues have resulted this

technique to be concluded as inferior to static hypothermic

preservation of the grafts (Cobert et al. 2008; Jacobs et al. 2010).

79

C. Recipient perspective

Current immunosuppressive protocols mostly target IL-‐2-‐mediated

adaptive immunity. Novel immunomodulatory strategies that target

anything else may reduce the need for classical

immunosuppressants. Recipient treatment with statins has long

been standard treatment for their anti-‐inflammatory properties

(Kobashigawa et al. 1995; Shimizu et al. 2003). Angiopoietins play

crucial role in maintaining EC quiescence. The endothelium acts as

the definitive barrier between the transplant and the effector cells

of alloimmune system, and therefore it may be hypothesized that

the alloimmune response may be absent if the endothelium is

preserved intact and non-‐adhesive to inflammatory cells. Our results

show that Ang1 substitution or Ang2 neutralization inhibit early

influx of inflammatory cells, and also the early alloimmune

response, as the gene expression of hallmark cytokines IL-‐2, IL-‐6, IL-‐

12 were downregulated. Ang1 has therefore potential in allograft

protection during the preservation. Ang2 competes with Ang1 in

binding to the Tie2 receptor, promotes inflammation and

endothelial activation, and acts as a chemokine to inflammatory

cells. Neutralization of Ang2 may therefore be beneficial during the

preservation, but also during the acute phase of inflammation after

the reperfusion.

80

Limitations

The experimental rat cardiac allograft model used in this study is a

non-‐functional and heterotopic model. Therefore, the model

represents clinical cardiac transplantation mainly immunologically.

The model excludes donor brain death, which produces systemic

cytokine storm and induces early innate immune response in clinical

patients. The treatment agents used in this study are not in clinical

use, but are currently in clinical trials.

Conclusions

This study emphasizes the importance of ischemia and reperfusion

on endothelial integrity in cardiac allografts. With functional and

intact endothelium, the key mediators of inflammation and

alloimmune response are unable to reach the allograft. This results

in delayed allorecognition and subsequently reduced development

of cardiac allograft vasculopathy. Angiopoietin signaling was proven

important in endothelial stabilization, either by supplementation of

Ang1 or inhibition of Ang2. Further studies are warranted to

investigate the clinical potential of Ang1 and inhibition of Ang2 in

human solid organ transplant recipients. In human patients,

recombinant Ang1 may be added to cardioplegia solutions and anti-‐

Ang2 antibody may be beneficial in induction therapy of the

allograft recipient.

81

YHTEENVETO

Elinsiirrettä irrotettaessa verenkierto katkaistaan ja elin

jäähdytetään kudosvaurion minimoimiseksi. Verenkierron

palauttaminen siirteeseen aiheuttaa paradoksaalisesti iskemia-‐

reperfuusiovaurioksi kutsutun ilmiön, johon liittyy verisuoniston

seinämän aktivoituminen ja synnynnäisen immuniteetin aiheuttama

neutrofiili-‐ ja makrofagivaltainen tulehdusreaktio. Tämä alkuvaiheen

tulehdusreaktio altistaa elinsiirteen myöhäisvaiheen krooniselle

tulehdusreaktiolle, jonka vuoksi siirteen toiminta voi pettää. Sydän

on erityisen herkkä hapenpuutteelle, minkä vuoksi sydänsiirteen

iskemia-‐aika pyritään pitämään mahdollisimman lyhyenä.

Pidentynyt iskemia-‐aika – sairaaloidenvälisen pitkän matkan tai

teknisesti vaikean leikkauksen vuoksi – on itsenäinen riskitekijä

myöhään kehittyvälle hyljintäreaktiolle.

Angiopoietiini-‐1 ja -‐2 (Ang1 ja 2) ovat Tie2-‐reseptoriin sitoutuvia

verisuonikasvutekijöitä, joilla on tärkeä merkitys

verenkiertoelimistön sikiöaikaisessa kehityksessä. Verisuonten

tukisolut erittää jatkuvasti Ang1:tä, joka ylläpitää verisuonen

seinämän tasapainoa kehittyneessä verisuonistossa. Ang2

puolestaan vapautuu verisuonen seinämän endoteelisoluista

hapenpuutteen tai tulehduksen vaikutuksesta ja houkuttelee

paikalle tulehdussoluja sekä muuttaa verisuonen seinämän

läpäisevämmäksi.

82

Tässä tutkimuksessa kuvattiin koe-‐eläinmallilla sydänsiirteen

kylmäsäilytyksen vaikutus luonnollisen immuniteetin

aktivoitumiseen ja kroonisen hyljintäreaktion kehittymiseen.

Tutkimuksessa selvitettiin myös Ang1-‐ ja Ang2-‐signalointiteiden

vahvistamisen tai estämisen vaikutusta sydänsiirteen iskemia-‐

reperfuusiovaurioon ja hyljintäreaktion kehittymiseen. Pidentynyt

kylmäsäilytys lisäsi tulehdusvastetta ja huononsi siirteen ennustetta.

Tutkimuksessa havaittiin, että Ang2 vapautuminen verenkiertoon

lisääntyy sydänsiirteen saaneella potilaalla 1. vuorokauden aikana

leikkauksen jälkeen. Sama ilmiö osoitettiin myös rotan

sydänsiirtomallissa ja samalla todettiin tulehdusreaktion olevan

hapenpuutetta merkittävämpi Ang2 vapauttaja.

Tutkimuksessa rotan sydänsiirtomallissa siirteen sepelvaltimoihin

irrotuksen yhteydessä ruiskutettu Ang1-‐proteiini stabiloi siirteen

verisuoniston seinämän ja Ang2-‐vasta-‐aine esti iskemia-‐

reperfuusiovaurioon liittyvän tulehduksen ja sydänlihasvaurion, sekä

hiussuoniston läpäisevyys-‐ ja perfuusiohäiriön. Tämä alkuvaiheen

suojavaikutus hillitsi myös siirteiden kroonisen hyljinnän muutoksien

kehittymistä. Sydänsiirteen vastaanottajalle systeemisesti annettu

vasta-‐aine ei vaikuttanut verisuoniston läpäisevyyteen, mutta esti

siirteen tulehduksen, akuutin hyljintäreaktion ja kroonisen

hyljintäreaktion kehittymisen.

Elinsiirtopotilaan ennustetta rajoittavat merkittävästi siirteen

varhaisvaiheen pettäminen ja kroonisen hyljintäreaktion aiheuttama

83

hitaasti kehittyvä siirteen toiminnan hiipuminen. Tutkimuksen

tulosten perusteella Ang1-‐signaloinnin vahvistaminen ja Ang2-‐

signaloinnin vaimentaminen estää sydänsiirteen verisuoniston

seinämän endoteelisoluja aktivoitumasta, houkuttelemasta ja

läpäisemästä tulehdussoluja. Vasta-‐ainehoidolle olisi kliinistä

käyttöä elinsiirtopotilaiden induktiohoidossa, jolloin

vaihtoehtoisella, elimistön immuunipuolustukseen

vaikuttamattomalla hoidolla voidaan vähentää potilaiden

tarvitsemien immunosuppressiivisten lääkkeiden määrää ja näin

ollen lääkkeistä aiheutuvia haittavaikutuksia.

Tässä tutkimuksessa osoitettiin koe-‐eläinmallissa, että estämällä

angiopoietiini-‐2 (Ang2) vaikutus vasta-‐aineella paikallisesti

sydänsiirteessä tai siirteen vastaanottajassa vähennetään siirteen

varhaisvaiheen sydänlihasvauriota ja tulehdusvastetta.

Varhaisvaiheen hoidolla oli myös pitkäaikainen hyöty, sillä hoidetut

siirteet selvisivät pidempään ilman akuuttia ja kroonista

hyljintäreaktiota.

84

ACKNOWLEDGMENTS

This study was carried out in the Transplantation Laboratory,

Haartman Institute, University of Helsinki, and Helsinki University

Central Hospital. This work was supported by grants from the

Helsinki University Central Hospital Research Funds, the University

of Helsinki, the Sigrid Juselius Foundation, the Academy of Finland,

the Finnish Foundation for Cardiovascular Research, the Päivikki and

Sakari Sohlberg Foundation, the Paulo Foundation, the Aarne

Koskelo Foundation, the Ida Montin Foundation, and the Emil

Aaltonen Foundation.

I wish to express my sincere gratitude to my Supervisor Professor

Karl Lemström for his enthusiastic guidance and passion for science.

You have allowed me to quite freely fulfill myself scientifically, yet

you have kept my feet on the ground and persistently demanded

top performance and hard work. I feel privileged to have been able

to work for and with you.

I also wish to return special thanks to:

The Reviewers of this work appointed by the Faculty of Medicine,

Professor Timo Paavonen and Professor Hannu Sariola for your

advice and insight over the topic.

Professor and Dean of the Faculty of Medicine Risto Renkonen, the

Head of the Transplantation Laboratory, for introducing me to the

academic world and to my Supervisor, and for constant

encouragement and support.

85

Professor Emeritus Pekka Häyry for establishing our laboratory, and

for inspiring stories of science, traveling, and the dawn of

transplantation medicine.

My co-‐authors Kari Alitalo for sharing invaluable ideas and

connections around the world; Gou Young Koh for providing

recombinant Ang1 and 2; Ching Ching Leow from MedImmune for

providing anti-‐Ang2 antibody; Pipsa Saharinen for sharing your

expertise of angiopoietin signaling; Markku Tammi for your help

with hyaluronic acid; Antti Nykänen for your help, friendship, and

excellent discussions; Raimo Tuuminen, Alireza Raissadati, Alexey

Dashkevich for your help and friendship throughout the years; Ralica

Arnaudova for your help in lab work; Rainer Krebs for being the

wizard you are; Mikko Keränen for introducing me to the everyday

life of the Transplantation Laboratory, for your help, friendship, and

shared experiences around the world.

The other members of our research group: Eeva Rouvinen for

teaching me laboratory practice and for your extremely invaluable

technical assistance; Jussi Tikkanen, Jussi Ropponen, and Janne

Jokinen for your friendship, support, and for our shared journeys

over the years.

Leena Saraste for your humor, assistance with everyday problems,

and for reviewing our manuscripts, letters, and presentations.

The staff of Transplantation Laboratory and H3 Animal Unit for your

kind help and support. The heart and lung transplant coordinators

86

Marja-‐Liisa Hellstedt and Catharina Yesil from Helsinki University

Central Hospital for obtaining the clinical samples.

All my friends for support, encouragement, and for the

extracurricular activities: Joonas, Kalle, Sandor, and Tomi; all the

friends in the medical school, especially Tuomas J., Jussi, Juhani, Ora,

Antti, and Lauri, and also Ilkka, Tuomas K., Juhana, Timo, Kapo, Vera,

Julia, and the late Petri for sharing your time during (scientific!)

lunch, tennis, work, skiing, or whatever reason; all the friends at the

Blood Service, especially Aleksi, Heidi, John, Tea K., and Tea S.

My family: Satu and Martti, Ossi and Rina, Mikko and Anna for your

love, support, and encouragement throughout my life; and all my

other relatives for your support. Tintti and Hanski, Hessu and Anna,

Harri and Hannis for your support and good times spent together.

My deepest gratitude I owe to the most important person in my life,

Heli, thank you for your support, love, and patience during this

project.

In Helsinki, October 2014

87

REFERENCES

Abecassis M, Bridges ND, Clancy CJ, Dew MA, Eldadah B, Englesbe MJ, Flessner MF, Frank JC, Friedewald J, Gill J, Gries C, Halter JB, Hartmann EL, Hazzard WR, Horne FM, Hosenpud J, Jacobson P, Kasiske BL, Lake J, Loomba R, Malani PN, Moore TM, Murray A, Nguyen MH, Powe NR, Reese PP, Reynolds H, Samaniego MD, Schmader KE, Segev DL, Shah AS, Singer LG, Sosa JA, Stewart ZA, Tan JC, Williams WW, Zaas DW, and High KP. Solid-‐organ transplantation in older adults: current status and future research. Am J Transplant. 2012. 12(10):2608–22.

Aderem A, Ulevitch RJ. Toll-‐like receptors in the induction of the innate immune response. Nature. 2000 406(6797):782–7.

Al-‐Lamki RS, Bradley JR, Pober JS. Endothelial cells in allograft rejection. Transplantation. 2008 86(10):1340–8.

Ali JM, Bolton EM, Bradley JA, Pettigrew GJ. Allorecognition pathways in transplant rejection and tolerance. Transplantation. 2013 96(8):681–8.

Aono Y, Ariyoshi H, Sakon M, Ueda A, Tsuji Y, Kawasaki T, Monden M. Human umbilical vein endothelial cells (HUVECs) show Ca(2+) mobilization as well as Ca(2+) influx upon hypoxia. J Cell Biochem. 2000 Jun 6;78(3):458–64.

Bader FM, Kfoury AG, Gilbert EM, Barry WH, Humayun N, Hagan ME, Thomas H, Rendlun D. Percutaneous coronary interventions with stents in cardiac transplant recipients. J Heart Lung Transplant. 2006 25(3):298–301.

Banchereau J, Steinman R. Dendritic cells and the control of immunity. Nature. 1998 392(6673):245–52.

Baran DA. New directions in immunosuppression after heart transplantation. Nat Rev Cardiol. 2013 10(7):422–7.

Barnard CN. The operation. A human cardiac transplant: an interim report of a successful operation performed at Groote Schuur Hospital, Cape Town. S Afr Med J. 1967 41(48):1271–4.

88

Barton GM, Medzhitov R. Toll-‐like receptor signaling pathways. Science. 2003 300(5625):1524–5.

Bhattacharya R, SenBanerjee S, Lin Z, Mir S, Hamik A, Wang P, Mukherjee P, Mukhopadhyay D, Jain MK. Inhibition of vascular permeability factor/vascular endothelial growth factor-‐mediated angiogenesis by the Kruppel-‐like factor KLF2. J Biol Chem. 2005 280(32):28848–51.

Bleackley RC. A molecular view of cytotoxic T lymphocyte induced killing. Biochem Cell Biol. 2005 83(6):747–51.

Booth AJ, Grabauskiene S, Wood SC, Lu G, Burrell BE, Bishop DK. IL-‐6 promotes cardiac graft rejection mediated by CD4+ cells. The Journal of Immunology. 2011 187(11):5764–71.

Borel JF, Feurer C, Gubler HU, Stähelin H. Biological effects of cyclosporin A: a new antilymphocytic agent. Agents Actions. 1976 6(4):468–75.

Boros P, Bromberg JS. New cellular and molecular immune pathways in ischemia/reperfusion injury. Am J Transplant. 2006 6(4):652–8.

Braggion-‐Santos MF, Lossnitzer D, Buss S, Lehrke S, Doesch A, Giannitsis E, Korosoglou G, Katus HA, Steen H. Late gadolinium enhancement assessed by cardiac magnetic resonance imaging in heart transplant recipients with different stages of cardiac allograft vasculopathy. Eur Heart J Cardiovasc Imaging. 2014 15(10):1125-‐32

Canton J, Neculai D, Grinstein S. Scavenger receptors in homeostasis

and immunity. Nat Rev Immunol. 2013 13(9):621–34.

Carden D, Granger D. Pathophysiology of ischaemia-‐reperfusion injury. J Pathol. 2000 190(3):255–66.

Carrel A, Guthrie CC. Functions of a transplanted kidney. Science. 1905 22(563):473.

89

Chen L, Ahmed E, Wang T, Wang Y, Ochando J, Chong A, Alegre M. TLR signals promote IL-‐6/IL-‐17-‐dependent transplant rejection. J Immunol. 2009 182(10):6217–25.

Cho C, Kammerer R, Lee H, Steinmetz M, Ryu Y, Lee S, Yasunaga K, Kim K, Kim I, Choi H, Kim W, Kim S, Park S, Lee G, Koh G. COMP-‐Ang1: a designed angiopoietin-‐1 variant with nonleaky angiogenic activity. Proc Natl Acad Sci USA. 2004 101(15):5547–52.

Cobert ML, West LM, Jessen ME. Machine perfusion for cardiac allograft preservation. Curr Opin Organ Transplant. 2008 13(5):526–30.

Daly C, Pasnikowski E, Burova E, Wong V, Aldrich TH, Griffiths J, et al. Angiopoietin-‐2 functions as an autocrine protective factor in stressed endothelial cells. Proc Natl Acad Sci U S A. 2006 103(42):15491–6.

Daly C, Wong V, Burova E, Wei Y, Zabski S, Griffiths J, Lai K-‐M, Lin HC, Ioffe E, Yancopoulos GD, Rudge JS. Angiopoietin-‐1 modulates endothelial cell function and gene expression via the transcription factor FKHR (FOXO1). Genes Dev. 2004 18(9):1060–71.

Daly KP, Seifert ME, Chandraker A, Zurakowski D, Nohria A, Givertz MM, Karumanchi SA, Briscoe DM. VEGF-‐C, VEGF-‐A and related angiogenesis factors as biomarkers of allograft vasculopathy in cardiac transplant recipients. J Heart Lung Transplant. 2013 32(1):120–8.

Davis S, Aldrich TH, Jones PF, Acheson A, Compton DL, Jain V, Ryan TE, Bruno J, Radziejewski C, Maisonpierre PC, Yancopoulos GD. Isolation of angiopoietin-‐1, a ligand for the TIE2 receptor, by secretion-‐trap expression cloning. Cell. 1996 87(7):1161–9.

DeBusk LM, Hallahan DE, Lin PC. Akt is a major angiogenic mediator downstream of the Ang1/Tie2 signaling pathway. Exp Cell Res. 2004 298(1):167–77.

90

Dekker RJ, Boon RA, Rondaij MG, Kragt A, Volger OL, Elderkamp YW, Meijers JCM, Voorberg J, Pannekoek H, Horrevoets AJG. KLF2 provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium. Blood. 2006 107(11):4354–63.

Dekker RJ, van Soest S, Fontijn RD, Salamanca S, de Groot PG, VanBavel E, Pannekoek H, Horrevoets AJG. Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-‐like factor (KLF2). Blood. 2002 100(5):1689–98.

Diamond JM, Porteous MK, Cantu E, Meyer NJ, Shah RJ, Lederer DJ, Kawut SM, Lee J, Bellamy SL, Palmer SM, Lama VN, Bhorade SM, Crespo M, Demissie E, Wille K, Orens J, Shah PD, Weinacker A, Weill D, Arcasoy S, Wilkes DS, Ware LB, Christie JD. Elevated plasma angiopoietin-‐2 levels and primary graft dysfunction after lung transplantation. PLoS ONE. 2012 7(12):e51932.

Dumitrescu C, Biondi R, Xia Y, Cardounel A, Druhan L, Ambrosio G, Zweier J. Myocardial ischemia results in tetrahydrobiopterin (BH4) oxidation with impaired endothelial function ameliorated by BH4. Proc Natl Acad Sci USA. 2007 104(38):15081–6.

Eklund L, Saharinen P. Angiopoietin signaling in the vasculature. Exp Cell Res. 2013 319(9):1271–80.

El-‐Sawy T, Belperio JA, Strieter RM, Remick DG, Fairchild RL. Inhibition of polymorphonuclear leukocyte-‐mediated graft damage synergizes with short-‐term costimulatory blockade to prevent cardiac allograft rejection. Circulation. 2005 112(3):320–31.

El-‐Sawy T, Miura M, Fairchild R. Early T cell response to allografts occurring prior to alloantigen priming up-‐regulates innate-‐mediated inflammation and graft necrosis. Am J Pathol. 2004 165(1):147–57.

91

Eltzschig HK, Carmeliet P. Hypoxia and inflammation. N Engl J Med. 2011 364(7):656–65.

Etoh T, Inoue H, Tanaka S, Barnard GF, Kitano S, Mori M. Angiopoietin-‐2 is related to tumor angiogenesis in gastric carcinoma: possible in vivo regulation via induction of proteases. Cancer Res. 2001 61(5):2145–53.

Fiedler U, Reiss Y, Scharpfenecker M, Grunow V, Koidl S, Thurston G, Gale NW, Witzenrath M, Rosseau S, Suttorp N, Sobke A, Herrmann M, Preissner KT, Vajkoczy P, Augustin HG. Angiopoietin-‐2 sensitizes endothelial cells to TNF-‐alpha and has a crucial role in the induction of inflammation. Nat Med. 2006 12(2):235–9.

Fiedler U, Scharpfenecker M, Koidl S, Hegen A, Grunow V, Schmidt JM, Kriz W, Thurston G, Augustin HG. The Tie-‐2 ligand angiopoietin-‐2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-‐Palade bodies. Blood. 2004 103(11):4150–6.

Förster R, Davalos-‐Misslitz AC, Rot A. CCR7 and its ligands: balancing immunity and tolerance. Nat Rev Immunol. 2008 8(5):362–71.

Fukata Y, Oshiro N, Kinoshita N, Kawano Y, Matsuoka Y, Bennett V, Matsuura Y, Kaibuchi K. Phosphorylation of adducin by Rho-‐kinase plays a crucial role in cell motility. J Cell Biol. 1999 145(2):347–61.

Gallagher DC, Bhatt RS, Parikh SM, Patel P, Seery V, McDermott DF, Atkins MB, Sukhatme VP. Angiopoietin 2 is a potential mediator of high-‐dose interleukin 2-‐induced vascular leak. Clin Cancer Res. 2007 13(7):2115–20.

Gamble J, Drew J, Trezise L, Underwood A, Parsons M, Kasminkas L, Rudge J, Yancopoulos G, Vadas M. Angiopoietin-‐1 is an antipermeability and anti-‐inflammatory agent in vitro and targets cell junctions. Circ Res. 2000 87(7):603–7.

92

Gavard J, Patel V, Gutkind JS. Angiopoietin-‐1 prevents VEGF-‐induced endothelial permeability by sequestering Src through mDia. Dev Cell. 2008 14(1):25–36.

Goldstein DR. Toll-‐like receptors and other links between innate and acquired alloimmunity. Curr Opin Immunol. 2004 16(5):538–44.

Guild WR, Harrison JH, Merrill JP, Murray J. Successful homotransplantation of the kidney in an identical twin. Trans Am Clin Climatol Assoc. 1955 67:167–73.

Haddad F, Khazanie P, Deuse T, Weisshaar D, Zhou J, Wook Nam C, Vu TA, Gomari FA, Skhiri M, Simos A, Schnittger I, Vrotvec B, Hunt SA, Fearon WF. Clinical and Functional Correlates of Early Microvascular Dysfunction Following Heart Transplantation. Circ Heart Fail. 2012 5(6):759-‐68

Halle AA, DiSciascio G, Massin EK, Wilson RF, Johnson MR, Sullivan

HJ, Bourge RC, Kleiman NS, Miller LW, Aversano TR. Coronary angioplasty, atherectomy and bypass surgery in cardiac transplant recipients. J Am Coll Cardiol. 1995 26(1):120–8.

Hanahan D. Signaling vascular morphogenesis and maintenance. Science. 1997 277(5322):48–50.

Hancock WW, Kraft N, Atkins RC. The immunohistochemical demonstration of major histocompatibility antigens in the human kidney using monoclonal antibodies. Pathology. 1982 14(4):409–14.

Hollenberg SM, Klein LW, Parrillo JE, Scherer M, Burns D, Tamburro P, Oberoi M, Johnson MR, Costanzo MR. Coronary endothelial dysfunction after heart transplantation predicts allograft vasculopathy and cardiac death. Circulation. 2001 104(25):3091–6.

93

Hofmann NP, Steuer C, Voss A, Erbel C, Doesch A, Ehlermann P, Giannitsis E, Buss SJ, Katus HA, Korosoglou G. Comprehensive Bio-‐Imaging Using Myocardial Perfusion Reserve Index During Cardiac Magnetic Resonance Imaging and High-‐Sensitive Troponin T for the Prediction of Outcomes in Heart Transplant Recipients. Am J Transplant. 2014, epub ahead of print; Oct 7.

Hsieh CL, Obara H, Ogura Y, Martinez OM, Krams SM. NK cells and

transplantation. Transpl Immunol. 2002 9(2-‐4):111–4.

Hughes DP, Marron MB, Brindle NPJ. The antiinflammatory endothelial tyrosine kinase Tie2 interacts with a novel nuclear factor-‐kappaB inhibitor ABIN-‐2. Circ Res. 2003 92(6):630–6.

Hwang S-‐J, Choi HH, Kim K-‐T, Hong HJ, Koh GY, Lee GM. Expression and purification of recombinant human angiopoietin-‐2 produced in Chinese hamster ovary cells. Protein Expr Purif. 2005 39(2):175–83.

Iyer A, Kumarasinghe G, Hicks M, Watson A, Gao L, Doyle A, Keogh A, Kotlyar E, Hayward C, Dhital K, Granger E, Jansz P, Pye R, Spratt P, Macdonald PS. Primary graft failure after heart transplantation. J Transplant. 2011;2011:175768.

Jacobs S, Rega F, Meyns B. Current preservation technology and future prospects of thoracic organs. Part 2: heart. Curr Opin Organ Transplant. 2010 15(2):156–9.

Janeway C, Medzhitov R. Innate immune recognition. Annu Rev Immunol. 2002 20:197–216.

Johnson MR, Aaronson KD, Canter CE, Kirklin JK, Mancini DM, Mehra MR, Radovancevic B, Taylor DO, Webber SA. Heart retransplantation. Am J Transplant. 2007 7(9):2075–81.

Jones N, Iljin K, Dumont D, Alitalo K. Tie receptors: new modulators of angiogenic and lymphangiogenic responses. Nat Rev Mol Cell Biol. 2001 2(4):257–67.

94

Ju R, Zhuang ZW, Zhang J, Lanahan AA, Kyriakides T, Sessa WC, Simons M. Angiopoietin-‐2 secretion by endothelial cell exosomes: regulation by the phosphatidylinositol 3-‐kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) and syndecan-‐4/syntenin pathways. J Biol Chem. 2014 289(1):510–9.

Kaczorowski D, Nakao A, Mollen K, Vallabhaneni R, Sugimoto R, Kohmoto J, Tobita K, Zuckerbraun B, McCurry K, Murase N, Billiar T. Toll-‐like receptor 4 mediates the early inflammatory response after cold ischemia/reperfusion. Transplantation. 2007 84(10):1279–87.

Kawanami D, Mahabeleshwar GH, Lin Z, Atkins GB, Hamik A, Haldar SM, Maemura K, Lamanna JC, Jain MK. Kruppel-‐like factor 2 inhibits hypoxia-‐inducible factor 1alpha expression and function in the endothelium. J Biol Chem. 2009 284(31):20522–30.

Keränen MAI, Tuuminen R, Syrjala S, Krebs R, Walkinshaw G, Flippin LA, Arend M, Koskinen PK, Nykänen AI, Lemström KB. Differential Effects of Pharmacological HIF Preconditioning of Donors Versus Recipients in Rat Cardiac Allografts. Am J Transplant. 2013 13(3):600-‐10.

Kim I, Kim JH, Moon SO, Kwak HJ, Kim NG, Koh GY. Angiopoietin-‐2 at

high concentration can enhance endothelial cell survival through the phosphatidylinositol 3'-‐kinase/Akt signal transduction pathway. Oncogene. 2000 19(39):4549–52.

Kim I, Moon SO, Kim SH, Kim HJ, Koh YS, Koh GY. Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-‐1), vascular cell adhesion molecule 1 (VCAM-‐1), and E-‐selectin through nuclear factor-‐kappa B activation in endothelial cells. J Biol Chem. 2001a 276(10):7614–20.

Kim I, Moon SO, Park SK, Chae SW, Koh GY. Angiopoietin-‐1 reduces VEGF-‐stimulated leukocyte adhesion to endothelial cells by reducing ICAM-‐1, VCAM-‐1, and E-‐selectin expression. Circ Res. 2001b 89(6):477–9.

95

Kobashigawa JA, Katznelson S, Laks H, Johnson JA, Yeatman L, Wang XM, Chia D, Terasaki PI, Sabad A, Cogert GA. Effect of pravastatin on outcomes after cardiac transplantation. N Engl J Med. 1995 333(10):621–7.

Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol. 2013 13(3):159–75.

Kontos CD, Cha EH, York JD, Peters KG. The endothelial receptor tyrosine kinase Tie1 activates phosphatidylinositol 3-‐kinase and Akt to inhibit apoptosis. Mol Cell Biol. 2002 22(6):1704–13.

Korkmaz S, Bährle-‐Szabó S, Loganathan S, Li S, Karck M, Szabó G. Marginal donors: can older donor hearts tolerate prolonged cold ischemic storage? Aging Clin Exp Res. 2013 25(5):597–600.

Korn T, Bettelli E, Oukka M, Kuchroo V. IL-‐17 and Th17 Cells. Annu Rev Immunol. 2009 27:485–517.

Kuo M-‐C, Patschan D, Patschan S, Cohen-‐Gould L, Park H-‐C, Ni J, Addabbo F, Goligorsky MS. Ischemia-‐induced exocytosis of Weibel-‐Palade bodies mobilizes stem cells. J Am Soc Nephrol. 2008 19(12):2321–30.

Laan M, Cui ZH, Hoshino H, Lötvall J, Sjöstrand M, Gruenert DC, Skoogh BE, Lindén A. Neutrophil recruitment by human IL-‐17 via C-‐X-‐C chemokine release in the airways. J Immunol. 1999 162(4):2347–52.

Lakkis FG, Lechler RI. Origin and biology of the allogeneic response. Cold Spring Harb Perspect Med. 2013 3(8).

Lampe JW, Becker LB. State of the art in therapeutic hypothermia. Annu Rev Med. 2011 62:79–93.

Land WG. Emerging role of innate immunity in organ transplantation Part II: potential of damage-‐associated molecular patterns to generate immunostimulatory dendritic cells. Transplant Rev. 2012 26(2):73-‐87.

96

Lazarevic V, Glimcher LH, Lord GM. T-‐bet: a bridge between innate and adaptive immunity. Nat Rev Immunol. 2013 13(11):777–89.

Lee RS, Grusby MJ, Glimcher LH, Winn HJ, Auchincloss H. Indirect recognition by helper cells can induce donor-‐specific cytotoxic T lymphocytes in vivo. J Exp Med. 1994 179(3):865–72.

Lehmann M, Graser E, Risch K, Hancock WW, Muller A, Kuttler B, Hahn HJ, Kupiec-‐Weglinski JW, Brock J, Volk H-‐D. Anti-‐CD4 monoclonal antibody-‐induced allograft tolerance in rats despite persistence of donor-‐reactive T cells. Transplantation. 1997 64(8):1181–7.

Lei J, He F, Wu M, Zheng X, Chen X, Chen Z. Administration of anti-‐interleukin-‐6 monoclonal antibody prolongs cardiac allograft survival. Transpl Int. 2010 23(12):1271–81.

Lemström KB, Krebs R, Nykänen AI, Tikkanen JM, Sihvola RK, Aaltola EM, Häyry PJ, Wood J, Alitalo K, Ylä-‐Herttuala S, Koskinen PK. Vascular endothelial growth factor enhances cardiac allograft arteriosclerosis. Circulation. 2002 105(21):2524–30.

Ley K, Smith E, Stark MA. IL-‐17A-‐producing neutrophil-‐regulatory Tn lymphocytes. Immunol Res. 2006 34(3):229–42.

Lin Z, Hamik A, Jain R, Kumar A, Jain MK. Kruppel-‐like factor 2 inhibits protease activated receptor-‐1 expression and thrombin-‐mediated endothelial activation. Arterioscler Thromb Vasc Biol. 2006 26(5):1185–9.

Lindenfeld J, Miller GG, Shakar SF, Zolty R, Lowes BD, Wolfel EE, Mestroni L, Page RL, Kobashigawa J. Drug therapy in the heart transplant recipient: part I: cardiac rejection and immunosuppressive drugs. Circulation. 2004a 10(24):3734–40.

Lindenfeld J, Miller GG, Shakar SF, Zolty R, Lowes BD, Wolfel EE, Mestroni L, Page RL, Kobashigawa J. Drug therapy in the heart transplant recipient: part II: immunosuppressive drugs. Circulation. 2004b 110(25):3858–65.

97

Listijono DR, Watson A, Pye R, Keogh AM, Kotlyar E, Spratt P, Granger E, Dhital K, Jansz P, Macdonald PS, Hayward CS. Usefulness of extracorporeal membrane oxygenation for early cardiac allograft dysfunction. J Heart Lung Transplant. 2011 30(7):783–9.

Liu W, Xiao X, Demirci G, Madsen J, Li XC. Innate NK cells and macrophages recognize and reject allogeneic nonself in vivo via different mechanisms. J Immunol. 2012 188(6):2703–11.

Liu Z, Sun YK, Xi YP, Maffei A, Reed E, Harris P, Suciu-‐Foca N. Contribution of direct and indirect recognition pathways to T cell alloreactivity. J Exp Med. 1993 177(6):1643–50.

Lowalekar SK, Lu XG, Thatte HS. Further evaluation of somah: long-‐term preservation, temperature effect, and prevention of ischemia-‐reperfusion injury in rat hearts harvested after cardiocirculatory death. Transplant Proc. 2013 45(9):3192–7.

Lund LH, Edwards LB, Kucheryavaya AY, Dipchand AI, Benden C, Christie JD, Dobbels F, Kirk R, Rahmel AO, Yusen RD, Stehlik J. The Registry of the International Society for Heart and Lung Transplantation: thirtieth official adult heart transplant report-‐-‐2013; focus theme: age. J Heart Lung Transplant. 2013 32(10):951–64.

Madge LA, Pober JS. A phosphatidylinositol 3-‐kinase/Akt pathway, activated by tumor necrosis factor or interleukin-‐1, inhibits apoptosis but does not activate NFkappaB in human endothelial cells. J Biol Chem. 2000 275(20):15458–65.

Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Compton D, McClain J, Aldrich TH, Papadopoulos N, Daly TJ, Davis S, Sato TN, Yancopoulos GD. Angiopoietin-‐2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science. 1997 277(5322):55–60.

Mammoto T, Parikh SM, Mammoto A, Gallagher D, Chan B, Mostoslavsky G, Ingber DE, Sukhatme VP. Angiopoietin-‐1 requires p190 RhoGAP to protect against vascular leakage in vivo. J Biol Chem. 2007 282(33):23910–8.

98

Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, Wykoff CC, Pugh CW, Maher ER, Ratcliffe PJ. The tumour suppressor protein VHL targets hypoxia-‐inducible factors for oxygen-‐dependent proteolysis. Nature. 1999 399(6733):271–5.

McCord JM. Oxygen-‐derived free radicals in postischemic tissue injury. N Engl J Med. 1985 312(3):159–63.

Mengel M, Husain S, Hidalgo L, Sis B. Phenotypes of antibody-‐mediated rejection in organ transplants. Transpl Int. 2012 25(6):611–22.

Müller-‐Eberhard HJ. The membrane attack complex of complement. Annu Rev Immunol. 1986 4:503–28.

Nag S, Papneja T, Venugopalan R, Stewart DJ. Increased angiopoietin2 expression is associated with endothelial apoptosis and blood-‐brain barrier breakdown. Lab Invest. 2005 85(10):1189–98.

Nagy JA, Dvorak AM, Dvorak HF. Vascular hyperpermeability, angiogenesis, and stroma generation. Cold Spring Harb Perspect Med. 2012 2(2):a006544.

Ochando JC, Homma C, Yang Y, Hidalgo A, Garin A, Tacke F, Angeli V, Li Y, Boros P, Ding Y, Jessberger R, Trinchieri G, Lira SA, Randolph GJ, Bromberg JS. Alloantigen-‐presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts. Nat Immunol. 2006 7(6):652–62.

Oliner J, Min H, Leal J, Yu D, Rao S, You E, Tang X, Kim H, Meyer S, Han SJ, Hawkins N, Rosenfeld R, Davy E, Graham K, Jacobsen F, Stevenson S, Ho J, Chen Q, Hartmann T, Michaels M, Kelley M, Li L, Sitney K, Martin F, Sun J-‐R, Zhang N, Lu J, Estrada J, Kumar R, Coxon A, Kaufman S, Pretorius J, Scully S, Cattley R, Payton M, Coats S, Nguyen L, Desilva B, Ndifor A, Hayward I, Radinsky R, Boone T, Kendall R. Suppression of angiogenesis and tumor growth by selective inhibition of angiopoietin-‐2. Cancer Cell. 2004 6(5):507–16.

99

Page E, Kwun J, Oh B, Knechtle S. Lymphodepletional strategies in transplantation. Cold Spring Harb Perspect Med. 2013 3(7); a015511.

Papapetropoulos A, Fulton D, Mahboubi K, Kalb RG, O'Connor DS, Li

F, Altieri DC, Sessa WC. Angiopoietin-‐1 inhibits endothelial cell apoptosis via the Akt/survivin pathway. J Biol Chem. 2000 275(13):9102–5.

Partanen J, Puri MC, Schwartz L, Fischer KD, Bernstein A, Rossant J. Cell autonomous functions of the receptor tyrosine kinase TIE in a late phase of angiogenic capillary growth and endothelial cell survival during murine development. Development. 1996 122(10):3013–21.

Pichiule P, Chavez JC, Lamanna JC. Hypoxic regulation of angiopoietin-‐2 expression in endothelial cells. J Biol Chem. 2004 279(13):12171–80.

Pober JS, Sessa WC. Evolving functions of endothelial cells in inflammation. Nat Rev Immunol. 2007 7(10):803–15.

Reffelmann T, Hale SL, Dow JS, Kloner RA. No-‐reflow phenomenon persists long-‐term after ischemia/reperfusion in the rat and predicts infarct expansion. Circulation. 2003 108(23):2911–7.

Reinders MEJ, Sho M, Izawa A, Wang P, Mukhopadhyay D, Koss KE, Geehan CS, Luster AD, Sayegh MH, Briscoe DM. Proinflammatory functions of vascular endothelial growth factor in alloimmunity. J Clin Invest. 2003 112(11):1655–65.

Roach JC, Glusman G, Rowen L, Kaur A, Purcell MK, Smith KD, Hood LE, Aderem A. The evolution of vertebrate Toll-‐like receptors. Proc Natl Acad Sci U S A. 2005 102(27):9577–82.

Rogers NJ, Lechler RI. Allorecognition. Am J Transplant. 2001 1(2):97–102.

Rossi M, Young J. Human dendritic cells: potent antigen-‐presenting cells at the crossroads of innate and adaptive immunity. J Immunol. 2005 175(3):1373–81.

100

Saharinen P, Eklund L, Miettinen J, Wirkkala R, Anisimov A, Winderlich M, Nottebaum A, Vestweber D, Deutsch U, Koh GY, Olsen BR, Alitalo K. Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-‐cell and cell-‐matrix contacts. Nat Cell Biol. 2008 10(5):527–37.

Sakaguchi S. Naturally arising Foxp3-‐expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-‐self. Nat Immunol. 2005 6(4):345–52.

Sako K, Fukuhara S, Minami T, Hamakubo T, Song H, Kodama T, Fukamizu A, Gutkind JS, Koh GY, Mochizuki N. Angiopoietin-‐1 induces Kruppel-‐like factor 2 expression through a phosphoinositide 3-‐kinase/AKT-‐dependent activation of myocyte enhancer factor 2. J Biol Chem. 2009 284(9):5592–601.

Salomon RN, Hughes CC, Schoen FJ, Payne DD, Pober JS, Libby P. Human coronary transplantation-‐associated arteriosclerosis. Evidence for a chronic immune reaction to activated graft endothelial cells. Am J Pathol. 1991 138(4):791–8.

Schaefer L, Babelova A, Kiss E, Hausser H, Baliova M, Krzyzankova M, Marsche G, Young M, Mihalik D, Gotte M, Malle E, Schaefer R, Grone H. The matrix component biglycan is proinflammatory and signals through Toll-‐like receptors 4 and 2 in macrophages. J Clin Invest. 2005 115(8):2223–33.

Schmauss D, Weis M. Cardiac allograft vasculopathy: recent developments. 2008 117(16):2131–41.

Scholz A, Rehm VA, Rieke S, Derkow K, Schulz P, Neumann K, Koch I, Pascu M, Wiedenmann B, Berg T, Schott E. Angiopoietin-‐2 serum levels are elevated in patients with liver cirrhosis and hepatocellular carcinoma. Am J Gastroenterol. 2007 102(11):2471–81.

Schulz P, Fischer C, Detjen KM, Rieke S, Hilfenhaus G, Marschall von Z, et al. Angiopoietin-‐2 drives lymphatic metastasis of pancreatic cancer. FASEB J. 2011 25(10):3325–35.

101

Semenza GL. Hypoxia-‐inducible factor 1 and cardiovascular disease. Annu Rev Physiol. 2014 76:39–56.

SenBanerjee S, Lin Z, Atkins GB, Greif DM, Rao RM, Kumar A, Feinberg MW, Chen Z, Simon DI, Luscinskas FW, Michel TM, Gimbrone MA, García-‐Cardeña G, Jain MK. KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation. J Exp Med. 2004 199(10):1305–15.

Shimizu K, Aikawa M, Takayama K, Libby P, Mitchell RN. Direct anti-‐inflammatory mechanisms contribute to attenuation of experimental allograft arteriosclerosis by statins. Circulation. 2003 108(17):2113–20.

Smiley S, King J, Hancock W. Fibrinogen stimulates macrophage chemokine secretion through toll-‐like receptor 4. J Immunol. 2001 167(5):2887–94.

Soares MP, Lin Y, Sato K, Stuhlmeier KM, Bach FH. Accommodation. Immunol Today. 1999 20(10):434–7.

Southard JH, Belzer FO. Organ preservation. Annu Rev Med. 1995 46:235–47.

Srivastava R, Keck BM, Bennett LE, Hosenpud JD. The results of cardiac retransplantation: an analysis of the Joint International Society for Heart and Lung Transplantation/United Network for Organ Sharing Thoracic Registry. Transplantation. 2000 70(4):606–12.

Stehlik J, Edwards LB, Kucheryavaya AY, Benden C, Christie JD, Dipchand AI, Dobbels F, Kirk R, Rahmel AO, Hertz MI. The Registry of the International Society for Heart and Lung Transplantation: 29th official adult heart transplant report-‐-‐2012. J Heart Lung Transplant. 2012 31(10):1052–64.

Stehlik J, Edwards LB, Kucheryavaya AY, Benden C, Christie JD, Dobbels F, Kirk R, Rahmel AO, Hertz MI. The Registry of the International Society for Heart and Lung Transplantation: Twenty-‐eighth Adult Heart Transplant Report-‐2011. J Heart Lung Transplant. 2011 30(10):1078–94.

102

Stratmann A, Risau W, Plate KH. Cell type-‐specific expression of angiopoietin-‐1 and angiopoietin-‐2 suggests a role in glioblastoma angiogenesis. Am J Pathol. 1998 153(5):1459–66.

Sugimachi K, Tanaka S, Taguchi K, Aishima S, Shimada M, Tsuneyoshi M. Angiopoietin switching regulates angiogenesis and progression of human hepatocellular carcinoma. J Clin Pathol. 2003 56(11):854–60.

Tadros A, Hughes DP, Dunmore BJ, Brindle NPJ. ABIN-‐2 protects endothelial cells from death and has a role in the antiapoptotic effect of angiopoietin-‐1. Blood. 2003 102(13):4407–9.

Takada M, Nadeau KC, Hancock WW, Mackenzie HS, Shaw GD, Waaga AM, Chandraker A, Sayegh MH, Tilney NL. Effects of explosive brain death on cytokine activation of peripheral organs in the rat. Transplantation. 1998 65(12):1533–42.

Takasato F, Morita R, Schichita T, Sekiya T, Morikawa Y, Kuroda T, Niimi M, Yoshimura A. Prevention of allogeneic cardiac graft rejection by transfer of ex vivo expanded antigen-‐specific regulatory T-‐cells. PLoS ONE. 2014 9(2):e87722.

Tanaka M, Mokhtari G, Terry R, Gunawan F, Balsam L, Hoyt G, Lee K, Tsao P, Robbins R. Prolonged cold ischemia in rat cardiac allografts promotes ischemia-‐reperfusion injury and the development of graft coronary artery disease in a linear fashion. J Heart Lung Transplant. 2005 24(11):1906–14.

Tellides G, Pober JS. Interferon-‐gamma axis in graft arteriosclerosis. Circ Res. 2007 100(5):622–32.

Thatte HS, Rousou L, Hussaini BE, Lu X-‐G, Treanor PR, Khuri SF. Development and evaluation of a novel solution, Somah, for the procurement and preservation of beating and nonbeating donor hearts for transplantation. Circulation. 2009 120(17):1704–13.

Thurston G, Rudge JS, Ioffe E, Zhou H, Ross L, Croll SD, Glazer N, Holash J, McDonald DM, Yancopoulos GD. Angiopoietin-‐1 protects the adult vasculature against plasma leakage. Nat Med. 2000 6(4):460–3.

103

Tressel SL, Kim H, Ni C-‐W, Chang K, Velasquez-‐Castano JC, Taylor WR, Yoon Y-‐S, Jo H. Angiopoietin-‐2 stimulates blood flow recovery after femoral artery occlusion by inducing inflammation and arteriogenesis. Arterioscler Thromb Vasc Biol. 2008 28(11):1989–95.

Tsan M, Gao B. Endogenous ligands of Toll-‐like receptors. J Leukoc Biol. 2004 76(3):514–9.

Tsigkos S, Zhou Z, Kotanidou A, Fulton D, Zakynthinos S, Roussos C, Papapetropoulos A. Regulation of Ang2 release by PTEN/PI3-‐kinase/Akt in lung microvascular endothelial cells. J Cell Physiol. 2006 207(2):506–11.

Tuuminen R, Syrjälä S, Krebs R, Keränen MAI, Koli K, Abo-‐Ramadan U, Neuvonen PJ, Tikkanen JM, Nykänen AI, Lemström KB. Donor simvastatin treatment abolishes rat cardiac allograft ischemia/reperfusion injury and chronic rejection through microvascular protection. Circulation. 2011 124(10):1138–50.

Venkateswaran RV, Dronavalli V, Lambert PA, Steeds RP, Wilson IC, Thompson RD, Mascaro JG, Bonser RS. The proinflammatory environment in potential heart and lung donors: prevalence and impact of donor management and hormonal therapy. Transplantation. 2009 88(4):582–8.

Vischer UM, Barth H, Wollheim CB. Regulated von Willebrand factor secretion is associated with agonist-‐specific patterns of cytoskeletal remodeling in cultured endothelial cells. Arterioscler Thromb Vasc Biol. 2000 20(3):883–91.

Wang GL, Semenza GL. General involvement of hypoxia-‐inducible factor 1 in transcriptional response to hypoxia. Proc Natl Acad Sci U S A. 1993 90(9):4304–8.

Wang Z, Jin N, Ganguli S, Swartz DR, Li L, Rhoades RA. Rho-‐kinase activation is involved in hypoxia-‐induced pulmonary vasoconstriction. Am J Respir Cell Mol Biol. 2001 25(5):628–35.

104

Warnecke G, Moradiellos J, Tudorache I, Kühn C, Avsar M, Wiegmann B, Sommer W, Ius F, Kunze C, Gottlieb J, Varela A, Haverich A. Normothermic perfusion of donor lungs for preservation and assessment with the Organ Care System Lung before bilateral transplantation: a pilot study of 12 patients. Lancet. 2012 380(9856):1851–8.

Welti J, Loges S, Dimmeler S, Carmeliet P. Recent molecular discoveries in angiogenesis and antiangiogenic therapies in cancer. J Clin Invest. 2013 123(8):3190–200.

Wilhelm MJ, Pratschke J, Beato F, Taal M, Kusaka M, Hancock WW, Tilney NL. Activation of the heart by donor brain death accelerates acute rejection after transplantation. Circulation. 2000 102(19):2426–33.

Williams GM, Hume DM, Hudson RP, Morris PJ, Kano K, Milgrom F. “Hyperacute” renal-‐homograft rejection in man. N Engl J Med. 1968 279(12):611–8.

Witzenbichler B, Westermann D, Knueppel S, Schultheiss H-‐P, Tschope C. Protective role of angiopoietin-‐1 in endotoxic shock. Circulation. 2005 111(1):97–105.

Woo KV, Qu X, Babaev VR, Linton MF, Guzman RJ, Fazio S, Baldwin HS. Tie1 attenuation reduces murine atherosclerosis in a dose-‐dependent and shear stress-‐specific manner. J Clin Invest. 2011 121(4):1624–35.

Wood KJ, Bushell A, Hester J. Regulatory immune cells in transplantation. Nat Rev Immunol. 2012 12(6):417–30.

Wyburn KR, Jose MD, Wu H, Atkins RC, Chadban SJ. The role of macrophages in allograft rejection. Transplantation. 2005 80(12):1641–7.

Xu W, Roos A, Daha MR, van Kooten C. Dendritic cell and macrophage subsets in the handling of dying cells. Immunobiology. 2006 211(6-‐8):567–75.

105

Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J. Vascular-‐specific growth factors and blood vessel formation. Nature. 2000 407(6801):242–8.

Yang D, Guo S, Zhang T, Li H. Hypothermia attenuates ischemia/reperfusion-‐induced endothelial cell apoptosis via alterations in apoptotic pathways and JNK signaling. FEBS Lett. 2009 583(15):2500–6.

Yemisci M, Gursoy-‐Ozdemir Y, Vural A, Can A, Topalkara K, Dalkara T. Pericyte contraction induced by oxidative-‐nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery. Nat Med. 2009 15(9):1031–7.

Yu M, Wang H, Ding A, Golenbock D, Latz E, Czura C, Fenton M, Tracey K, Yang H. HMGB1 signals through toll-‐like receptor (TLR) 4 and TLR2. Shock. 2006 26(2):174–9.

Yuan X, Paez-‐Cortez J, Schmitt-‐Knosalla I, D'Addio F, Mfarrej B, Donnarumma M, Habicht A, Clarkson MR, Iacomini J, Glimcher LH, Sayegh MH, Ansari MJ. A novel role of CD4 Th17 cells in mediating cardiac allograft rejection and vasculopathy. The Journal of experimental medicine. 2008 205(13):3133–44.

Zheng L, Dengler TJ, Kluger MS, Madge LA, Schechner JS, Maher SE, Pober JS, Bothwell AL. Cytoprotection of human umbilical vein endothelial cells against apoptosis and CTL-‐mediated lysis provided by caspase-‐resistant Bcl-‐2 without alterations in growth or activation responses. J Immunol. 2000 164(9):4665–71.

Zheng X, Lian D, Wong A, Bygrave M, Ichim T, Khoshniat M, Zhang X, Sun H, De Zordo T, Lacefield J, Garcia B, Jevnikar A, Min W. Novel small interfering RNA-‐containing solution protecting donor organs in heart transplantation. Circulation. 2009 120(12):1099–107.

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