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«Functional Characterization of the Transcription Factor Early growth response 1 (Egr1) in Arteriogenesis Dissertation zum Erwerb des Doktorgrades der ...»

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Aus dem Institut für Chirurgische Forschung

(im Walter-Brendel-Zentrum für Experimentelle Medizin (WBex))

der Ludwig-Maximilians-Universität München

Vorstand: Prof. Dr. med. Ulrich Pohl

Functional Characterization of the Transcription Factor Early

growth response 1 (Egr1) in Arteriogenesis

Dissertation

zum Erwerb des Doktorgrades der Medizin

an der Medizinischen Fakultät der

Ludwig-Maximilians-Universität zu München

vorgelegt von

Judith-Irina Carola Pagel

aus

Giessen

im Jahr 2014 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: PD Dr. rer. nat. Elisabeth Deindl Mitberichterstatter: Prof. Dr. med. Jens Waschke Prof. Dr. med. Martin Dichgans Prof. Dr. med. Christian Schulz Mitbetreuung durch den Promovierten Mitarbeiter Dekan: Prof. Dr. med. Dr. h.c. M. Reiser, FACR, FRCR Tag der mündlichen Prüfung: 11.12.2014 Meinen Eltern Teile dieser Arbeit wurden publiziert in Thrombosis & Hemostasis 2012; 107(3):562-74.

TABLE OF CONTENTS

I INTRODUCTION

1 CARDIOVASCULAR DISEASES–A CONTINOUSLY SPREADING EPIDEMIC.. 1

1.1 Cause of disease

1.2 New therapeutic strategies

1.3 Discovery of a collateral circulation

2 FORMATION OF BLOOD VESSELS

2.1 Vasculogenesis

2.2 Angiogenesis

2.3 Arteriogenesis

3 MECHANISMS PROMOTING ARTERIOGENESIS

3.1 Hemodynamic forces

3.2 Monocyte recruitment

3.2.1 Monocyte chemoattractant protein 1(MCP-1)

3.2.2 Intercellular adhesion molecule 1 (ICAM-1)

3.2.3 Urokinase-type plasminogen activator (uPA)

3.3 Vascular cell proliferation and differentiation

4 THE TRANSCRIPTION FACTOR EARLY GROWTH RESPONSE (EGR1)...........12

4.1 Structural properties

4.1.1 The Egr1 promoter

4.1.2 The EGR1 protein

4.2. Function and associated downstream targets

4.3 The EGR-family

II OBJECT OF THE STUDY

III MATERIALS AND METHODS

1 MATERIALS

1.1 Mice strains

1.1.1 C57Bl/6N

Egr1-/-

1.1.2

1.2 Surgical instruments and expendable material

1.3 Chemicals and special equipment

1.4 Pharmaceuticals

1.5 Primers for qRT-PCR

1.6 Special buffers and solutions

1.7 Antibodies

1.7.1 Primary antibodies

1.7.2 Secondary antibodies

1.8 Devices

1.9 Software

TABLE OF CONTENTS

2 METHODS

2.1 Animal model

2.1.1 Anesthesia protocols

2.1.2 Femoral artery ligation

2.1.3 Isolation of tissue

2.2 Laser Doppler perfusion measurements

2.2.1 Technical background

2.2.2 Measurements

2.2.3 Evaluation of flux images

2.3 qRT-PCR

2.3.1 Total RNA Isolation

2.3.2 Agarose gel electrophoresis

2.3.3 cDNA Synthesis and qRT-PCR

2.4 Western Blot

2.4.1 Protein isolation and determination of concentration

2.4.2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)........ 34 2.4.3 Blotting

2.4.4 Detection of immunoreactive bands

2.5 Immunohistochemistry

2.5.1 EGR1 staining

2.5.2 Leukocyte quantification

2.6. Fluorescent activated cell sorting (FACS)

2.6.1 FACS analysis of whole blood

2.6.2 FACS analysis of whole adductor muscle

2.7. Statistical analyses

IV RESULTS

1 GENERAL OBSERVATIONS

2 PARTICIPATION OF EGR1 IN COLLATERAL ARTERY GROWTH

2.1 Perfusion recovery in wildtype and Egr1 deficient mice

2.2 Egr1 expression in arteriogenesis

3 EGR1 INFLUENCES ON CELL COUNTS AND LEUKOCYTE DISTRIBUTION... 42

3.1 Leukocyte recruitment

3.2 Expression of ICAM-1, MCP-1 and uPA

4 PARTICIPATION OF THE OTHER EGR FAMILY MEMBERS

5 INFLUENCES ON VASCULAR CELL PROLIFERATION

5.1 Cell cycle progression

5.2 vSMC phenotype switch

TABLE OF CONTENTS

V DISCUSSION

1 THE MURINE ARTERIOGENESIS MODEL

1.1 Why using the murine peripheral femoral artery ligation model?

1.2 Comparison to a coronary ligation model

1.3 Peripheral arteriogenesis models in different species

1.4 The influence of gender

2 EGR1 AND ARTERIOGENESIS

3 LEUKOCYTE RECRUITMENT

4 EXPRESSION OF EGR FAMILY MEMBERS

– EVIDENCE FOR COMPENSATION

5 CELL CYCLE PROGRESSION AND PROLIFERATION

5.1 Expression of cyclins in growing collaterals

5.2 vSMC phenotype switch: role of SF-1 and αSM-actin

VI PERSPECTIVE:

THE PROSPECTS OF CLINICAL ARTERIOGENESIS THERAPY

VII SUMMARY

VIII ZUSAMMENFASSUNG

IX REFERENCES

X LIST OF ABBREVIATIONS

XI DANKSAGUNG

–  –  –

I. INTRODUCTION

1 CARDIOVASCULAR DISEASES – A CONTINOUSLY SPREADING EPIDEMIC

Pathologies affecting heart and blood vessels such as coronary heart disease, cerebrovascular disease or peripheral arterial disease, are grouped under the term cardiovascular diseases (CVDs). They remain the major global cause of death representing 30% of the total number of deaths worldwide. Currently, 17.3 million fatal casualties per year are caused by CVDs, among them 7.3 million due to coronary heart disease and 6.2 million due to stroke.1 These numbers show a strong tendency to enlarge.





For 2030, the World Health Organization (WHO) is currently projecting a number of 23.3 million deaths due to CVDs.1 Therefore, an early case specific medical treatment on the one hand, and prevention on the other hand is and will be vital.

1.1 Cause of disease

In most cases, the etiology and the correspondent histopathologic finding in these patients is atherosclerosis. It is characterized by intima damage resulting from cholesterol deposits into the arterial wall and a subsequent incomplete inflammatory response due to failure of adequate phagocytosis of oxidized cholesterol by macrophages.2 Their degradation to foam cells and the accumulation of cell debris does not only further chronic vessel wall damage and inflammation but also the hardening of the vessel. The accumulation of foam cells, called atheromatous plaque, can be instable (i.e. vulnerable), and is prone to rupture thus occluding the arterial vessel. The clinical presentation of temporary arterial occlusions is, for example, recurrent episodes of chest pain, transient ischemic attacks or intermittent claudication in peripheral arterial disease.

Prevention is the best form of medicine comprising the reduction of risk factors (RFs) leading to atherosclerosis to reduce the incidence of disease. Due to the high morbidity, CVDs have become a serious burden for every health care system in the western world and are already escalating in newly industrialized countries. Ironically, the alleged advantages of a modern, comfortable lifestyle promote their prevalence. Risk factors are generally classified in modifiable risk factors such as an unhealthy diet, physical inactivity, tobacco abuse, hyperlipidemia, hypertension, diabetes or the metabolic syndrome and non-modifiable RFs like age, sex or genetic predisposition evidencing that not every risk factor for atherosclerosis is in fact controllable.3 However, 80% of coronary heart disease and cerebrovascular disease are indeed due to the behavioral risk factors physical inactivity, unhealthy diet and tobacco abuse.1 So although promising, preventive medicine has not been successful to efficiently reduce the prevalence of those behavioral risk factors and the incidence of atherosclerosis related

–  –  –

CVDs in the public. As mentioned before, their prevalence is rather increasing and, accordingly, the demand for a fast, accessible and efficient therapy is also continuously growing.

1.2 New therapeutic strategies The survival rate for a life threatening acute vessel occlusion such as myocardial infarction is still low with adequate primary care, short transport time (“door-to-balloon time”) to a cardiac center providing fast accessible primary percutaneous coronary intervention (PCI) or cardiac bypass surgery being the most relevant factors these days for the outcome of patients (“time is muscle”).4 Although an increasing number of hospitals can provide these procedures, still a considerable percentage of patients cannot be treated this way or the attempted PCI/bypass surgery turns out to be unsuccessful and in 3rd world countries, most patients are unable to gain access to these treatments at all. As already stated, the patients’ survival is merely a matter of time-to-treatment in the acute event but distinct structural preconditions in the vasculature can improve the patients’ outcome significantly. Therefore, aside from educating the population to a healthier lifestyle and the ongoing technical advances in emergency medicine and PCI techniques, a large field of research is currently exploring new ideas to prevent CVDs and/or treat them from a different point of view. The cardiovascular system has an immense capacity to dynamically adapt to different conditions and pathologies. The door to new therapeutic options lies in the understanding of the dynamics between the molecular processes that promote the progression of disease and the compensatory mechanisms that take place in the living organism. In a recent study, it was shown that well developed preexistent collateral networks are associated with an improved long-term survival of patients after ST-segment elevation myocardial infarction (STEMI).5 So apart from medical treatment and prevention, distinct anatomical advantages such as an efficient preexistent collateral circulation can apparently cope better with the incidence of a major artery occlusion by maintaining sufficient oxygen supply for the muscle.

1.3 Discovery of a collateral circulation

The recognition of a preexistent collateral network capable of remodeling preceded a 300-years lasting debate. In 1669, the British physician Richard Lower (1631-1691) reported for the first time findings of collateral arteries he had found between the right and the left coronary artery by injecting fluid into one artery and observing its appearance on the other.6 The Swiss anatomist Albrecht von Haller (1708described 1757 functional, interarterially connecting vessels in the apex region and in the sulcus longitudinalis of the posterior ventricle.7 In 1785, John Hunter (1728-1793), a Scottish surgeon appointed to King George III, developed a new and modern technique for the treatment of traumatic popliteal aneurysms in patients on the basis of animal experiments using collateral artery growth (see Discussion 1.1).8 A 100 years later distinguished anatomists like Hyrtl, Henle and Cohnheim denied

2 INTRODUCTION

the collateral arteries’ existence and proposed these vessels to be “simple” end arteries.9 In the beginning of the 20th century, the works of the anatomist Werner Spalteholz (1862-1940) depicted clearly a coronary collateral circulation.10 Up until then, pathologists and anatomists used three methods: direct preparation, cast corrosion or radiographs after injection of opaque media, which in those times was still in its infancy. Spalteholz developed a new technique to make specimens transparent after injection of vessels with opaque media, to allow an excellent view inside the organ in its nearly untouched threedimensional structural entity,11 a method that is still used today with marginal changes. Although Jamin confirmed his findings using X-ray technique,12 the criticism persisted and it was not until the works of Longland13 (1953) and Fulton14 (1965) when the hypothesis of end arteries was finally disapproved. Longland demonstrated that collateral arteries consist of a stem, mid-zone and re-entrant.13 Fulton described collaterals as anastomoses being detectable in health and disease. He also related distinct vascular growth patterns to the level of ischemic impairment of the myocardium.14 The relationship between collateralization and the atherosclerotic burden in coronary vessels moved into the center of attention. What did stimulate the development of these networks, why did they develop in some patients suffering from atherosclerosis and not in all of them? And more importantly, could the vessels’ growth be stimulated?

2 FORMATION OF BLOOD VESSELS

Tissue homeostasis and vitality is strongly dependent on the adequate oxygen supply maintained by blood within a fine branching vessel network. Blood vessels form not only during the embryonic stages, they adapt in adults in response to changing stimuli, in physiologic and pathologic states. Blood vessel formation and remodeling have been investigated by various disciplines, and different forms of vascular growth have been identified (Figure 1).

2.1 Vasculogenesis

During the early stages of embryonic development, pluripotent hemangioblasts, precursor cells for both hematopoetic and endothelial cells, differentiate from mesodermal cells of the splanchnic layer located in the lateral plate mesoderm.15 They condense to large cellular aggregates (blood islands).16 In the inner layer, hemangioblasts differentiate to hematopoetic stem cells whereas in the outer layer, they become angioblasts, which are capable to migrate considerably17 until they differentiate again to CD34+/CD133+ endothelial precursor cells (EPCs) and form the lining of primary capillary networks (Fig 1A). Growth factors (GF) such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) 2 mediate vasculogenesis. FGF-2 is required for the differentiation of hemangioblasts from the splanchnic mesoderm and the formation of the blood islands16 and VEGF and its receptors Flk1 and Flt1 regulate the differentiation of the angioblasts to form endothelial tubes.18, 19 During 3

INTRODUCTION



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