THE ROLE OF REELIN IN HUMAN NEUTROPHIL PEPTIDE-INDUCED ENDOTHELIAL DYSFUNCTION AND PLATELET

AGGREGATION

by

Ai Li (Alice) Luo

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Physiology

University of Toronto

© Copyright by Ai Li (Alice) Luo 2014

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Master of Science, 2014 Ai Li (Alice) Luo Graduate Department of Physiology University of Toronto The role of reelin in human neutrophil peptide-induced endothelial dysfunction and platelet aggregation Introduction: Human neutrophil peptides (HNP) may play an important role in the

pathogenesis of atherosclerosis. We have shown that HNP-induced platelet

aggregation was abolished in the absence of low density lipoprotein-related protein-8

(LRP8), a receptor for the extracellular matrix protein reelin. Hypothesis: The HNP-induced endothelial dysfunction and platelet aggregation is

mediated by reelin.

Methods: Human coronary artery endothelial cells (HCAEC) were stimulated with HNP

to assess reelin protein expression and release. A reelin neutralizing antibody was

used to assess endothelial function and platelet aggregation after HNP stimulation.

Results: HNP induced reelin protein expression and release in HCAEC. The

stimulation of recombinant mouse reelin resulted in oxidative stress and decreased

endothelial nitric oxide synthase in HCAEC, and platelet aggregation in human

platelets. The HNP-induced endothelial dysfunction and platelet responses were

attenuated by using a reelin neutralizing antibody.

Conclusion: HNP induce endothelial dysfunction and platelet aggregation mediated by

the reelin-LRP8 signal pathway.

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ACKNOWLEDGEMENTS

I am genuinely privileged to have had the incessant support and guidance of Dr. Haibo

Zhang during my Master tenure at the University of Toronto. His remarkable intellect,

enthusiasm, and innovativeness nurtured me to continually perform with high standards

and confidence, to undertake challenges critically, and to develop professionally and

personally. Dr. Zhang is a true inspirational role model for the pursuit of a successful

career, and I am indebted to have him as my supervisor. This dissertation would not

have been possible without him.

I also owe my deepest gratitude to my program advisory committee members. Their

valuable advice, words of encouragement, and unreserved care for my project were

deeply appreciated. Dr. Wolfgang Kuebler relentlessly offered timely suggestions,

stimulating one-on-one discussions to help me explore new potentials, and the extra

drive to achieve my research goals. To Dr. Martin Post, a generous professor whom

offered his respected recommendations, and took the precious time to refine my

project. Finally, to Dr. Minna Woo, a passionate researcher whom kindly clarified my

queries and improved my research technicalities.

I would like to express my most sincere thanks to my friends and colleagues at the Li

Ka Shing Knowledge Institute for their endless support in my project and research

journey. Dr. Bing Han has taught me all my research techniques and principle without

reservation and was never hesitant in responding to my inquiries. Mrs. Julie Khang will

always hold a special place in my heart, for not only did she provide technical support,

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but also precious moral support while facing my most difficult challenges. To Dr. Kieran

Quinn and Mr. Hanpo Yu, it was an honour having both mentors helping me start my

research journey. I would also like to thank the lab members of Dr. Heyu Ni: Dr. Adili

Reheman and Dr. Yiming Wang, without whom I would not be able to learn the

principles of platelet behaviour. To all my friends in the lab that helped create the

positive environment that I was fortunate to be in, thank you Mirna Ghazarian, Dr.

Yasumasa Morita, Dr. Matteo Parotto, Dr. Jennifer Tsang, Dr. Peter Spieth, Dr. Martin

Kneyber, Dr. Fedrico Pozzi, Dr. Tatiana Maron-Gutierrez, Hajera Amatullah, Dr. Yuexin

Sham, Jean Parodo, and Judy Correa. My experience in the lab could not have been

better without your love.

Most importantly, I would like to give a special thanks to my mom and dad for their

never-ending love and care. Their words of wisdom provided me with the support to

keep moving forward and my head held up high, even when all else looked bleak. You

have motivated me to be a better person everyday. Last but not least, to my beloved

Michael, you were there beside me every step of the journey. Your unconditional love

was what kept me inspired and cheerful every day, and I am truly blessed to have you

by my side.

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TABLE OF CONTENTS ABSTRACT………………………………………………………………………………….….ii

ACKNOWLEDGEMENTS…………………………………………………………………….iii

LIST OF FIGURES…………………………………………………………………….………ix

LIST OF TABLES..…………………………………………………………………………… x

LIST OF ABBREVIATIONS………………………………………………………………… xii

CHAPTER 1: INTRODUCTION………………………………………………………………1

1.1 ATHEROSCLEROSIS…………………………………………………………...……… 1 1.1.1 Pathophysiology of atherosclerosis……………………………………………….... 2 1.1.2 Atherosclerosis as an inflammatory disease………………………………….........2 1.1.2.1 Foam cells…………………………………………………………………………. 4 1.1.2.2 Endothelial dysfunction…………………………………………………………... 5 1.1.2.2.1 Endothelial cell in vascular homeostasis……………………………………….. 5 1.1.2.2.2 Nitric Oxide………………………………………………………………………… 6 1.1.2.2.2.1 Nitric oxide synthase………………………………………………………….. 6 1.1.2.2.2.2 Nitric oxide as an anti-inflammatory molecule……………………………... 7 1.1.2.2.3 Endothelial dysfunction…………………………………………………………… 9 1.1.2.2.4 Endothelial dysfunction assessment……………………………………………. 9 1.1.2.2.5 Nitric oxide bioactivity vs. bioavailability………………………………………..10 1.1.2.3 Platelet and platelet aggregation………………………………………………. 11 1.1.2.3.1 Platelets ………………………………………………………………………….. 11 1.1.2.3.2 Platelet activation and aggregation……………………………………………. 12 1.1.2.3.3 Platelet granules ………………………………………………………………… 13 1.1.2.3.4 Platelets in atherosclerosis …………………………………………………….. 14 1.1.3 Neutrophils in atherosclerosis ……………………………………………………... 15

1.2 DEFENSINS ..…………………………………………………………………………….16 1.2.1 Three families of defensins ………………………………………………………… 16 1.2.1.1 α-Defensins ……………………………………………………………………… 17 1.2.2 HNP neutralization ………………………………………………………………….. 18 1.2.3 Properties of HNP …………………………………………………………………... 19 1.2.3.1 HNP and its anti-microbial role ………………………………………………… 19 1.2.3.2 HNP in inflammation ……………………………………………………………. 20 1.2.3.2.1 HNP as chemoattractant ……………………………………………………….. 20 1.2.3.2.2 HNP and P2Y6 …………………………………………………………………... 21 1.2.4 HNP and atherosclerotic events …………………………………………………... 22 1.2.4.1 Plasma HNP levels in inflammatory disease ………………………………… 23 1.2.4.2 HNP in atherosclerosis …………………………………………………………. 23 1.2.4.3 HNP-induced leukocyte adhesion and migration ……………………………. 28 1.2.4.4 HNP-mediated oxidative stress ……………………………………………….. 29

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1.2.4.5 HNP and foam cells …………………………………………………………….. 29 1.2.4.6 HNP and platelet aggregation………………………………………………….. 30

1.3 LOW-DENSITY LIPOPROTEIN RELATED PROTEIN-8 (LRP8) ………………… 30 1.3.1 LDLR and LRP family ………………………………………………………………. 30 1.3.2 LRP8 and atherosclerosis ………………………………………………………….. 33 1.3.2.1 LRP8………………………………………………………………………………. 33 1.3.2.2 LRP8 in platelets ………………………………………………………………... 33 1.3.2.3 LRP8 in endothelial cells ……………………………………………………….. 35 1.3.3 LRP8 ligands ………………………………………………………………………… 35 1.3.3.1 Reelin …………………………………………………………………………….. 36 1.3.3.1.1 Reelin molecule …………………………………………………………………. 36 1.3.3.1.2 Native vs. active reelin …………………………………………………………. 38 1.3.3.1.3 Reelin in inflammation ………………………………………………………….. 40

1.4 SUMMARY ……………………………………………………………………………… 41

CHAPTER 2: RATIONALE AND OBJECTIVES ……………………….……………….. 43

2.1 RATIONALE AND NOVELTY ………………………………………………………… 44 2.2 HYPOTHESIS …………………………………………………………………………... 44 2.3 OBJECTIVES …………………………………………………………………………… 44 2.3.1 Objective #1: To examine the role of HNP-induced reelin release ……………. 44 2.3.2 Objective #2: To determine the specific effect of reelin in endothelial function and platelet aggregation …………………………………………………………………… 44 CHAPTER 3: MATERIALS AND METHODS……………………………………………. 47

3.1 REAGENTS ……………………………………………………………………………... 47 3.2 CELL CULTURE ……………………………………………………………………….. 48 3.2.1 Primary human coronary endothelial cells (HCAEC) …………………………… 48 3.3 WESTERN BLOTTING ………………………………………………………………… 48 3.3.1 Whole cell lysate preparation ……………………………………………………… 48 3.3.2 Protein concentration quantification ………………………………………………. 49 3.3.3 SDS polyacrylamide gel electrophoresis and transfer ………………………….. 49 3.3.4 Immunoblotting ……………………………………………………………………… 49 3.4 PROTEIN-PROTEIN INTERACTION ………………………………………………… 50 3.4.1 GST-pull down assay ………………………………………………………………. 50 3.5 REELIN PROTEIN EXPRESSION …………………………………………………… 51 3.5.1 HCAEC stimulation …………………………………………………………………. 51 3.5.2 P2Y6 blocked-HCAEC ……………………………………………………………… 51 3.5.3 Measurement of reelin from culture medium and western blot ………………… 51 3.5.3.1 Reelin ELISA……………………………………………………………………. 51

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3.5.3.2 Reelin western blot …………………………………………………………….. 52 3.6 ENDOTHELIAL DYSFUNCTION …………………………………………………….. 53 3.6.1 HCAEC stimulation …………………………………………………………………. 53 3.6.2 Measurement of endothelial dysfunction …………………………………………. 53 3.6.3 Measurement of nitric oxide ……………………………………………………….. 54 3.6.3.1 Measurement of reactive nitrogen species …………………………………. 54 3.7 PLATELET AGGREGATION …………………………………………………………. 54 3.7.1 Platelet-rich plasma (PRP) isolation ……………………………………………… 54 3.7.2 Aggregation protocol ……………………………………………………………….. 55 3.8 STATISTICAL ANALYSIS ……………………………………………………………. 56 CHAPTER 4: RESULTS …………………………………………………………………… 57

4.1 OBJECTIVE #1: To examine the role of HNP-induced reelin release and reelin-LRP8-interaction …………………………………………………………………………… 57 4.1.1 Specific aim 1: HNP induced reelin protein expression and release in HCAEC …………………………………………………………………………………………………. 57 4.1.1.1 Reelin expression by western blot…………………………………………….. 57 4.1.1.1.1 Reelin expression from cell lysate ……………………………………………. 57 4.1.1.1.2 Reelin expression from lyophilized culture medium ………………………... 57 4.1.1.2 Reelin expression by ELISA …………………………………………………... 60 4.1.2 Specific Aim 2: HNP-induced reelin protein expression and release was mediated by the P2Y6-signaling pathway ………………………………………………… 60 4.2 OBJECTIVE #2: To determine the specific effect of reelin in endothelial function and platelet aggregation ………………………………………………………. 61 4.2.1 Specific aim 1: Reelin attenuated eNOS and increased iNOS protein expression …………………………………………………………………………………………………. 63 4.2.1.1 HNP-induced endothelial activation ………………………………………….. 63 4.2.1.2 Reelin-induced endothelial activation ………………………………………… 63 4.2.2 Specific aim 2: Reelin induced reactive nitrogen species production …………. 66 4.2.2.1 HNP-induced nitrosative stress ……………………………………………….. 66 4.2.2.2 Reelin-induced nitrosative stress ……………………………………………... 68 4.2.3 Specific aim 3: Reelin enhanced human platelet aggregation in response to ADP …………………………………………………………………………………………………. 68 4.2.4 Specific aim 4: Neutralized reelin (CR50) alleviated endothelial dysfunction and platelet aggregation …………………………………………………………………………. 71 4.2.4.1 CR50 increase eNOSE and decrease iNOS protein expression ………….. 71 4.2.4.2 CR50 attenuates HNP-induced nitrotyrosine concentration ……………….. 72 4.2.4.3 CR50 attenuates HNP-induced platelet aggregation ……………………….. 75 4.3 RESULT SUMMARY …………………………………………………………………... 75 CHAPTER 5: DISCUSSION……………………………………………………………….. 76

5.1 MAIN FINDINGS ……………………………………………………………………… 76

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5.2 POTENTIAL MECHANISM OF HNP-INDUCED REELIN EXPRESSION AND RELEASE ……………………………………………………………………………………. 76 5.2.1 HNP mediates reelin expression ………………………………………………….. 77 5.2.2 HNP mediates reelin procession ………………………………………………….. 78 5.2.3 HNP mediates reelin release ………………………………………………………. 79 5.3 REELIN-INDUCED ENDOTHELIAL ACTIVATION ………………………………… 80 5.3.1 HNP-induced endothelial activation ………………………………………………. 80 5.3.1.1 HNP-mediated eNOS and iNOS protein expression ………………………. 81 5.3.2 Reelin and NOS …………………………………………………………………….. 82 5.3.2.1 Use of mouse recombinant reelin on human cells …………………………. 82 5.3.2.2 Reelin and NOS ……………………………………………………………….. 83 5.3.2.3 Reelin-mediated endothelial activation through its receptors …………….. 85 5.3.2.3.1 Alternative reelin receptors ………………………………………………… 86 5.4 REELIN-MEDIATED PLATELET AGGREGATION ………………………………... 86 5.4.1 Reelin mediates HNP-induced platelet aggregation …………………………….. 86 5.4.1.1 Reelin mediated HNP-induced platelet aggregation through LRP8 signaling pathway ………………………………………………………………………………………. 87 5.4.2 Reelin expression in platelets ……………………………………………………… 88 5.4.3 HNP-induced platelet aggregation ………………………………………………… 88 CHAPTER 6: SUMMARY, AND FUTURE DIRECTIONS………………………………. 90

6.1 SUMMARY ……………………………………………………………………………… 90 6.2 FUTURE DIRECTION …………………………………………………………………. 90 6.2.1 In vivo studies………………………………………………………………………... 90

REFERENCE:..………………………………………………………………………..…….. 93

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LIST OF FIGURES

Figure 1: HNP enhances lipid deposition in aorta of ApoE-/-/HNP(+) mice fed on high fat

diet.

Figure 2: HNP recruits leukocyte to carotid artery in vivo.

Figure 3: HNP increases cytokine and chemokine production in ApoE-/-/HNP(+) mice.

Figure 4: Reelin fragments

Figure 5: HNP does not bind directly to LRP8.

Figure 6: Potential mechanism of HNP in promoting atherogenesis.

Figure 7: HNP induced reelin 300-kDa protein expression.

Figure 8: HNP decrease reelin full-length secretion.

Figure 9: HNP-induced reelin expression is mediated by P2Y6-signaling pathway.

Figure 10: HNP attenuates eNOS, while augmenting iNOS, protein expression.

Figure 11: Reelin reduces eNOS, and enhances iNOS protein expression in HCAEC.

Figure 12: HNP induces production of nitrogenous species.

Figure 13: Reelin induce nitrotyrosine production.

Figure 14: Platelet aggregation is prolonged in platelets stimulated with ADP and reelin

in comparison to stimulation with ADP alone.

Figure 15: Reelin neutralizing antibody alleviates HNP-induced eNOS decrease and

iNOS increase.

Figure 16: Reelin neutralizing antibody reduces HNP-induced nitrotyrosine production.

Figure 17: Mouse reelin is 95% homologous to human reelin.

Figure 18: Experimental design for the potential therapeutic effect of CR50 against

HNP-mediated atherogenesis in vivo.

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LIST OF TABLES

Table 1: Ligands and functions of LDL receptor family members.

Table 2: Ligands and functions of LRP8.

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LIST OF ABBREVIATIONS

α1-PI - α1 Protease Inhibitor

ADMA – Asymmetric Dimethyl-Arginine

ADP – Adenosine Diphosphate

ATP – Adenosine Triphosphate

ANOVA – Analysis of Variance

APC – Anaphase Promoting Complex

Apo – Apolipoprotein

Bcl-2 – B-Cell Lymphoma 2

BH4 - (6R)-5,6,7,8-tetrahydrobiopterin

BLAST – Basic Local Alignment Search Tool

BSA – Bovine Serum Albumin

Ca2+ - Calcium Ion

CAD – Carotid Artery Disease

CaM – Calmodulin

cAMP – Cyclic Adenosine Monophosphate

CD – Cluster of Differentiation

cGMP – Cyclic Guanosine Monophosphate

CRP – C-Reactive Protein

COX-1 – Cyclooxygenase-1

CXC – A chemokine with the first two amino cysteine residues separated by an amino

acid (X)

DTS – Dense Tubular System

EDCF – Endothelial-Derived Constriction Factor

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EDHF – Endothelial-Derived Hyperpolarizing Factor

ELISA – Enzyme-Linked Immunosorbent Assay

eNOS – Endothelial Nitric Oxide Synthase (NOS III)

E-Selectin – Endothelial Selectin

ET-1 – Endothelin-1

FAD – Flavin Adenine Dinucleotide

Fe - Iron

GAPDH - Glyceraldehyde 3-Phosphate Dehydrogenase

GC – Guanylyl Cyclase

GDP – Guanosine Diphosphate

GM-CSF – Granulocyte/Macrophage Colony Stimulating Factor

GP – Glycoprotein

GTP – Guanosine Triphosphate

HCAEC – Human Coronary Artery Endothelial Cells

hBD – Human β Defensin

HBSS – Hank’s Balanced Salt Solution

HDL – High-Density Lipoprotein

HNP – Human Neutrophil Peptides

ICAM-1 – Intercellular Adhesion Molecule-1

IgG – Immunoglobulin G

IκBα - Nuclear Factor of Kappa Light Polypeptide Gene Enhancer in B Cells Inhibitor α

IL- Interleukin

iNOS – Inducible Nitric Oxide Synthase (NOS II)

IVM – Intravital Microscopy

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JAM-3 – Junction Adhesion Molecule-3

JNK - c-Jun N-terminal kinases

K+ - Potassium Ion

LDL – Low-Density Lipoprotein

LDLR – Low-Density Lipoprotein Receptor

Lp(a) – Lipoprotein (a)

LRs – LDL Receptor-Related Protein Receptor family members

LRP8 – LDL Receptor-Related Protein 8 (ApoER2; Apolipoprotein E Receptor 2)

MAC-1 – Macrophage Antigen-1

MCP-1 – Monocyte Chemotactic Protein-1

Mg2+ - Magnesium Ion

MMP – Matrix metalloproteinase

mRNA – Messenger Ribonucleic Acid

mrRLN – Mouse Recombinant Reelin

Na+ - Sodium Ion

NADPH – Nicotinamide Adenine Dinucleotide Phosphate

NF-κB - Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells

NO – Nitric Oxide

NOHA - Nω-hydroxyl-L-arginine

NOS – Nitric Oxide Synthase

nNOS – Neuronal Nitric Oxide Synthase (NOS I)

ONOO- - Peroxynitrite (PN)

oxLDL – Oxidized LDL

p38MAPK – p38 Mitrogen-Activated Protein Kinase

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PAF – Platelet Activating Factor

PBS – Phosphate Buffered Saline

PCR – Polymerase Chain Reaction

PF4 – Platelet Factor 4

PGH2 – Endoperoxide

PGI2 – Prostacyclin

PIP2 – Phosphatidylinositol 4,5 Biphosphate

PKC – Protein Kinase C

PKG – cGMP-Dependent Kinase

PLA2 – Phospholipase A2

PMN – Polymorphonuclear Neutrophils

PPP – Platelet Poor Plasma

PRP – Platelet Rich Plasma

P-Selectin – Platelet-Selectin

PSGL-1 - P-selectin glycoprotein ligand-1

RANTES - Regulated on Activation, Normal T Cell Expressed And Secreted

RAP – Receptor Associated Protein

RLN - Reelin

RNS – Reactive Nitrogen Species

ROS – Reactive Oxygen Species

RT – Room Temperature

SERCA - Sarco/Endoplasmic Reticulum Ca2+- ATP Monophosphatase (ATPase)

SNAP - Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein

SNARE - Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor

SR-A – Scavenger Receptor A

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STEMI - ST-Segment Elevation Myocardial Infarction

TNF-α - Tumour Necrosis Factor - α

tPA – Tissue-Type Plasminogen Activator

TxA2 – Thromboxane A2

Tyr - Tyrosine

VCAM-1 – Vascular Cell Adhesion Molecule-1

VSMC – Vascular Smooth Muscle Cell

vWF – von Willebrand Factor

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Chapter 1: Introduction

1.1 Atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the large elastic and muscular arteries,

that develops over time to become coronary artery diseases and stroke, diseases which

account for 50% of all deaths in westernized societies [1]. The epidemiology of the disease

encompasses a coupling of environmental and genetic risk factors, and a complex cascade of

cellular events. The elements of atherosclerosis is commonly characterized by sub-

endothelial accumulation of cholesterol and lipid-engorged foam cells, leukocyte trafficking

from the lumen into the vessel intima, vascular smooth muscle cell (VSMC) migration from the

tunica media into the tunica intima, platelet activation and aggregation, T-cell activation, and

production of platelet- and leukocyte-derived cytokines and chemokines [1-5]. Unfortunately,

these events only unfold silently with time as the disease develops in the inner lining of the

arteries. Initially, the buildup of cholesterol-rich lipids in arteries, known as a fatty streak, can

be found as early as the first decade of life [1]. These lesions are precursors to the

development of atheromas, a more advanced lesion characterized by the presence of lipid-

rich necrotic debris perturbed with uncontrolled inflammatory events such as leukocyte

recruitment, foam-cell formation, and cytokine and chemokine production. The proliferative

environment subsequently stimulates migrated VSMC to produce extracellular matrix

molecules, such as interstitial collagen and elastin, and form a fibrous cap to enclose the

plaque [1, 6-9]. The growing plaque, that is continuously fuelled by uncontrolled inflammation

and apoptosis, can cause clinical complications when 1) it produces a flow-restricting stenosis

that causes tissues ischemia; and/or 2) its fibrous cap ruptures, due to enzymatic activity by

collagenolytic enzymes and metalloproteinases, to form thrombi that embolize and lodge in

distal arteries that causes thrombotic complications [9]. It is upon plaque rupture and

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thrombosis that the condition finally manifests itself clinically as acute coronary syndrome,

myocardial infarction or stroke.

Despite the medical advances present today, on a global scale, cardiovascular disease

accounts for 1 in 3 deaths, and is projected to be the single leading cause of death by 2030

[10]. Owing to its overwhelming burden medically and economically, extensive clinical and

laboratory research has been conducted to elucidate the pathophysiology of atherosclerosis

for both preventive and precautious purposes.

1.1.1 Pathophysiology of atherosclerosis

Until the 1970s, atherosclerosis was predominantly viewed as a lipid-retention disease [11].

However, research over the last four decades has increasingly elucidated the significant role

of neutrophils, monocytes and leukocytes to the pathogenesis of atherosclerosis. To date,

over 10,000 peer-reviewed articles have been published under the US National Library of

Medicine that documented the inflammatory events that transpire in the disturbed vascular

equilibrium precipitated in atherosclerosis.

1.1.2 Atherosclerosis as an inflammatory disease

The view of atherosclerosis as a chronic inflammatory disease originated from the crucial

observation that different subsets of leukocytes were found in atherosclerotic lesions at

various stages of atherogenesis [12]. The individual leukocytes, such as

monocytes/macrophages, T- cells, mast cells and dendritic cells, and their roles in

contributing to atherosclerosis were well reviewed in 2008 by Weber et al published in Nature

[5].

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Under homeostatic circumstances, the endothelial monolayer resists the firm adhesion and

recruitment of circulating leukocytes. However, at certain arterial sites with decreased shear

stress and increased turbulence [13], specific adhesion molecules from the endothelium are

expressed, and are responsible for the capture and migration of circulating leukocytes such

as monocytes and T-cells [3, 5, 13]. These adhesion molecules acts as receptors for the

integrins found on the surface membrane of monocytes and T-cells. Once adhered to the

endothelial cells, leukocytes enter into the intima by diapedesis between the endothelial

junctions [13, 14]. The chemotactic migration of circulating leukocytes may be accounted for

by the presence of chemokines such as MCP-1, INF-γ and eotaxin [13]. Once resident in the

arterial intima, activated leukocytes amplify receptors that bind to chemoattractant molecules,

which further activates the cells to induce cell proliferation, and inflammation at the site of

lesion. For instance, monocytes proliferate to become macrophages, which encourage the

cell to generate cytokines (such as TNF-α, IL-1 and TGF-β), proteolytic enzymes (such as

MMPs), and growth factors (such as PDGF) that could further fuel the inflammatory

responses at the lesion site [15]. In addition, activated macrophages are known to express

class II histocompatibility antigens that permit them to present antigens to T-cells [13-15].

Since both CD4+ and CD8+ T-cells are present at the lesion site, they too can become

activated, which results in the additional generation and secretion of cytokines such as INF-γ,

TNF-α and -β [15]. A similar T-cell activation can be elicited by dendritic cells (DC) since DCs

are also antigen-presenting cells [15]. In addition to supplying the intima with cytokines and

chemokines, leukocytes such as mast cells are equipped with high contents of proteases

such as tryptase and chymase [1, 3, 5, 15], which can destabilize the atherosclerotic plaque,

subjecting the plaque to increased susceptibility to rupture. Therefore, the cascade of

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inflammatory responses collectively further endorses the recruitment of circulating leukocytes,

which can ultimately destabilize the atherosclerotic plaque.

1.1.2.1 Foam Cells

Monocytes represent an essential cellular component of the host defense system, particularly

in the native and acquired immunity [16]. By recognizing specific molecular patterns and

interacting with specific receptors, monocytes can mediate phagocytosis and various protein

expressions. Although these functions were evolved to protect the host from microbial

infection, however, these mechanisms also inadvertently contribute to the development of

atherosclerosis.

Monocyte recruitment begins with the attraction to sites of injury by the presence of pro-

inflammatory molecules such as monocyte chemotactic protein (MCP-1), macrophage colony

stimulating factor (M-CSF), TNF-α, endothelin-1 (ET-1), granulocyte/macrophage colony

stimulating factor (GM-CSF) and transforming growth factor-β (TGF-β) [16-20]. Using cell

adhesion molecules expressed on activated endothelial cells, monocytes roll, adhere and

migrate across the endothelium barrier under the influence of chemoattractant protein [16].

Once in the subendothelial space, monocytes proliferate and differentiate into macrophages

with high expressions of scavenger receptors such as CD36 and CD68 [21, 22]. This class of

proteins recognizes polyanionic macromolecules such as phosphatidylserine and oxidized

lipoproteins to serve as a mechanism for lipid clearance [16]. Unfortunately, the continual

uptake of oxidized LDL leads to the formation of large lipid-engorged macrophages known as

foam cells [16]. Consequently, foam cells can 1) release cytokines and chemokines to further

fuel the inflammatory responses; and 2) release metalloproteinases that can destabilize the

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plaque and cause a rupture, which leads to the clinical complication of myocardial infarction

or stroke [16].

1.1.2.2 Endothelial dysfunction

1.1.2.2.1 Endothelial cell in vascular homeostasis

The endothelium is a major regulator of vascular homeostasis by exerting vasoprotective

effects such as vasodilation, inhibition of VSMC migration and proliferation, and suppression

of inflammatory events [23-25]. Strategically situated as the interface between the vascular

lumen and vessel body, it is optimally placed to response to both physical and chemical

signals that regulate cellular adhesion, thrombogenesis and fibrinolysis, VSMC proliferation

and vascular inflammation [25]. The importance of the endothelium was first appreciated

through its role in maintaining vascular tone, which is achieved through the production and

release of vasoactive molecules that would relax or constrict the vessel [25, 26]. Several

endothelial-derived vasodilating molecules have been documented including prostacyclin

(PGI2), endothelial-derived hyperpolarizing factor (EDHF), and nitric oxide (NO) [23, 27].

Briefly, prostacyclin is a major product derived from the activities of vascular cyclooxygenase

that is primarily formed in response to shear stress, bradykinin, and hypoxia [24]. This

endothelium-derived arachidonic acid metabolite has previously been shown to cause VSMC

relaxation by activating adenylate cyclase [28], and inhibit platelet aggregation by diminishing

fibrinogen receptor and P-selectin expression [29, 30]. Now, in addition to PGI2, EDHF, a

product of the phospholipase A2-dependent arachidonic acid metabolism [31], also plays an

important role in inducing endothelium-dependent relaxation. In response to extracellular

mediators such as bradykinin and acetylcholine, EDHF mediates VSMC relaxation by

activating potassium (K+) channels to allow K+ efflux and membrane hyperpolarization [31,

32]. Finally, due to NO’s pivotal role in governing endothelial-dependent vasodilation, I will

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further expand on the molecule’s role and production later in the thesis. Interestingly, under

certain conditions, such as the presence of cytokines, thrombin, and serotonin, the

endothelium may begin to produce endothelial-derived constriction factors (EDCF) such as

endothelin-1 (ET-1), angiotensin II, superoxide anions, endoperoxide (PGH2) and

thromboxane A2 [24, 33-36]. One of the common pathways in which EDCF induces VSMC

constriction is by mobilizing Ca2+ influx to increase the intracellular Ca2+ content and mediate

vascular muscle contraction [37].

1.1.2.2.2 Nitric oxide

1.1.2.2.2.1 Nitric oxide synthase

Now, the chief regulator of vasodilation is nitric oxide (NO). Its vasodilating properties were

first demonstrated by experiments conducted by Furchgott and Zawadzki in 1980. In their

study, rabbit aorta with intact endothelium was relaxed upon the treatment of acetylcholine,

but was found to constrict in aortas with denuded endothelium [38]. The culprit responsible for

acetylcholine-mediated relaxation was later found to be nitric oxide [39, 40]. NO is principally

derived from L-arginine by the enzymatic action of a family known as nitric oxide synthase

(NOS). Currently, there are three known isoforms that can synthesize NO: neuronal NOS

(nNOS, also known as NOS I), endothelial NOS (eNOS, also known as NOS III), and

inducible NOS (iNOS, also commonly known as NOS II). Numerous studies have shown a

similarity in three-dimensional crystallographic structure shared by all three isoforms: a flavin-

containing reductase and an iron protoporphyrin IX (heme)-containing oxygenase domain,

bridged by a flexible and calmodulin (CaM)-containing protein strand [41-44]. In addition to

the dimeric structure, there are tightly-bound cofactors required for the proper functioning of

the enzyme: (6R)-5,6,7,8-tetrahydrobiopterin (BH4), flavin adenine dinucleotide (FAD), and

nicotinamide adenine dinucleotide phosphate (NADPH) [23, 41]. The generation of NO begins

! 7!

with the electron donation of NAPDH to the reductase domain of the enzyme and transfer, via

FAD redox carrier, to the oxygenase domain. There, the electrons interact with the heme-

containing BH4 to catalyze the reaction of oxygen with L-arginine to generate an intermediate

species called Nω-hydroxyl-L-arginine (NOHA), prior to the final oxidation to citrulline and NO

[41, 45, 46]. However, even though the three isoforms share a protein structure, the proteins

also contain distinct differences in expression among each other. Firstly, the three isoforms’

expression varies across different tissues. nNOS is predominately found in neuronal tissues;

eNOS is found in vascular endothelial cells; and iNOS is found in a wide range of cells and

tissues such as macrophages and endothelial cells [41]. Secondly, the genes governing the

expression of the proteins are located on different chromosomes. nNOS is found on 12q24.2-

12q24.3 of chromosome 12; eNOS is found on 17cen-q11.2 of chromosome 17; and iNOS is

found on 7q35-7q36 of chromosome 7 [41]. Finally, while eNOS and nNOS are Ca2+-

dependent enzymes, iNOS is an inducible Ca2+-independent enzyme that is often up

regulated by the presence of cytokines to generate 100-1000-fold more NO than its

constitutive counterparts [46-49]. Paradoxically, high content of NO can begin to lose its anti-

proliferative properties, which will be elaborated further later on [50].

1.1.2.2.2.2 Nitrix oxide as an anti-inflammatory molecule

As a gaseous molecule, NO released from the endothelium is able to diffuse across

neighbouring cell’s plasma membrane and bind to its intracellular receptor: soluble guanylyl

cyclase (GC). By binding onto the heme prosthetic group on GC β1 subunit, NO triggers a

conformational change that increases the catalysis of cyclic guanosine monophosphate

(cGMP) synthesis by several hundred-folds [51-54]. Depending on the cell stimulated, NO-

mediated cGMP production has many implications. For instance, in VSMCs, an increase in

! 8!

cGMP has previously been shown to activate cGMP-dependent kinases (PKG), which can

promote SERCA activity and inhibit L-type Ca2+ channels and Na+/Ca2+ exchange, thus

resulting in a decrease in intracellular Ca2+ content and VSMC relaxation [55-58]. Similarly, in

platelets, a rise in cGMP potently inhibited the activation of thromboxane A2 receptor by

phosphorylating the carboxyl terminus of the receptor, rendering it inactive for platelet

activation and aggregation [59]. Additionally, NO has previously been shown to stimulate

other pathways that resulted in an inhibition of inflammatory responses. For instance, NO was

previously shown to inhibit VSMC proliferation by increasing the ubiquitination and

degradation of UbcH10, an enzyme known to interact with the anaphase promoting complex

(APC) [60]. In a study conducted by Spiecker et al, it was demonstrated that NO was able to

inhibit TNF-α-induced NF-κB activation in endothelial cells by increasing the expression and

nuclear translocation of the NF-κB inhibitor, IκBα. Interestingly, this was also correlated with a

reduction in vascular cell adhesion molecule-1 (VCAM-1) expression [61], a crucial molecule

involved in capturing and recruiting leukocytes for migration into the subendothelial

compartment [3, 62]. Congruently, a study by De Caterina et al showed similar results when

IL-1α-induced VCAM-1 expression in endothelial cells was significantly inhibited in the

presence of NO. Not only did this resulted in a reduction in monocyte adhesion to the

endothelial monolayer, but also in a diminished production of cytokine levels as assessed by

IL-6 and IL-8 concentrations [63]. Collectively, it becomes evident that NO plays a pivotal role

in regulating vascular homeostasis, and that the impairment of production or activity of NO

leads to a loss of endothelium-dependent vasodilation, and a gain in pro-inflammatory,

proliferative and pro-coagulatory environment conditioned for the development of

atherosclerosis [64]. Such environment is the hallmark feature of endothelial activation, which

! 9!

eventually leads to endothelial dysfunction [65]. Now, it is here that I would like to address the

difference between endothelial activation and endothelial dysfunction.

1.1.2.2.3 Endothelial dysfunction

The most recent definition of endothelial activation was illustrated in the Journal of Clinical

Investigation, where it defined the phenomenon as “the endothelial expression of cell-surface

adhesion molecules, such as VCAM-1, ICAM-1, and E-selectin…induced by proinflammatory

cytokines… (that) facilitates the recruitment and attachment of circulating leukocytes to the

vessel wall” [66]. Therefore, endothelial activation is the transformation of endothelial cells

from a quiescent phenotype to one that involves in a host defense response [25]. In contrast,

endothelial dysfunction is characterized by the reduction of NO bioavailability [65, 66].

Endothelial activation can lead to endothelial dysfunction, however, it is not uncommon for

some papers use the two terminologies interchangeably. In order to avoid confusion, this

thesis will regard the two events separately.

Interestingly, over the past 30 years, a growing body of evidence has suggested that

atherosclerosis begins with the disruption of vascular endothelium homeostasis, with

endothelial dysfunction as the pioneer event [25]. It has been suggested that it is the

imbalance between vessel dilation and constriction by NO that compels the pathophysiology

in atherosclerosis, including endothelial permeability, leukocyte recruitment, platelet

aggregation and cytokine production [67]. Remarkably, endothelial dysfunction has recently

been used clinically as a diagnostic marker of early atherosclerosis.

1.1.2.2.4 Endothelial dysfunction assessment

! 10!

A common and non-invasive way of assessing endothelial function is by quantifying the

diameter of coronary arteries before and after infusion of acetylcholine by angiography [68,

69]. As healthy coronary arteries are expected to respond by vasodilation, patients with

endothelial dysfunction would exhibit vasoconstriction [23]. Similarly, venous occlusion

plethysmography can also been used to measure vascular responses after infusion of

acetylcholine into the brachial artery [70]. Alternatively, endothelial dysfunction can also be

measured by high-resolution ultrasound to assess the brachial arterial diameter in response

to reactive hyperemia [70]. Lastly, due to its inverse correlation to endothelial function, serum

C-reactive protein (CRP) has also been used to indirectly assess endothelial impairment [71].

1.1.2.2.5 Nitric oxide bioactivity vs. bioavailability.

The primary driving force for endothelial dysfunction is the net reduction of NO bioavailability

[23, 65, 66, 72]. The mechanisms of diminished NO bioavailability is variable: from a

reduction in eNOS protein expression due to oscillatory shear stress [73, 74], or a reduction in

the eNOS cofactor BH4 [75], to an elevation in eNOS inhibitors such as asymmetric dimethyl-

arginine (ADMA) [76]. However, this is not to be confused with NO bioactivity. In a study

conducted by Minor et al, it was found that rabbits fed on a high cholesterol diet for 6 months

exhibited an increase in NO, but an impairment of endothelial-dependent relaxation [77].

This counter-protective model of NO was first proposed by Stuehr et al when the group

discovered two different catalytic pathways of iNOS [78, 79]. Prior to being surrendered from

the oxygenase domain of iNOS, NO is bound to the FeIII-containing heme, a composite known

as the FeIII-NO complex [50]. The FeIII-NO complex could either: a) release the NO, or b) act

as a nucleophile to attack oxygen and generate the reactive nitrogen species (RNS),

peroxynitrite (PN or ONOO-) [50]. Peroxynitrite is a potent cytotoxic and pro-inflammatory

! 11!

molecule works to nitrosylate the aromatic ring of tyrosine residues on proteins to form 3-

nitrotyrosine, which modifies protein functional activities, and drives reactive oxygen species

production [4, 80-84]. Previous studies have observed that nitrosylation of residue 385

(Tyr385) on the catalytic site of cyclooxygenase (COX) in mice and human atherosclerotic

lesions resulted in an interference of the enzymatic reaction of arachidonic acid to

prostaglandins [85-87]. The underlying NOS that governed the NO-derived PN was later

found to be iNOS [85]. This notion was further supported by in vivo studies conducted by

Upmacis et al, showing attenuation of protein nitrosylation of the heart, lungs and liver in diet-

induced atherosclerotic mice lacking iNOS expression [88]. As a consequence of the

accumulation of RNS, the LDL in the environment undergoes accelerated oxidation to form

oxidative LDL (oxLDL), a modified LDL that promotes foam cell formation and plaque

destabilization [89]. Therefore, it is crucial to appreciate the importance of diminished

bioavailability and bioactivity of NO in contributing to endothelial dysfunction, which then

initiates the inflammatory cascade of events observed in atherosclerosis.

1.1.2.3 Platelets and platelet aggregation

1.1.2.3.1 Platelets

As early as 1874, microscopist Dr. William Osler made a pioneering discovery of a disk-

shaped unit, which he coined the term “the third corpuscle” or “blood plates”, that forms

“irregular masses of colorless globules” upon bleeding [90]. Today, we recognize this discoid

unit as the anuclear fragments shed from bone-marrow derived megakaryotypes [91]. During

the terminal differentiation phase of thrombopoiesis, megakaryotypes form proplatelet

extensions that are eventually shed as platelets [92]. Constituting a dimension of 2.0-µm in

length, and 0.5-µm in thickness, platelets are one of the smallest circulating cell-types in the

! 12!

blood [93]. Although lacking a nucleus, platelets are equipped with protein-synthesizing

machineries, including messenger RNA (mRNA) and translational proteins [94]. Their short

lifespan of 8-10 days is governed by an “internal clock” based on the activities of anti- and

pro-apoptotic Bcl-2 family proteins [95]. With a normal count of 150-400 x 109 platelets/L in

humans [96], they play a vital role in the integrity and homeostasis of vascular thrombosis.

Upon an appropriate signal, platelets’ prime directive is to rapidly form platelet-rich thrombi to

stop vascular bleeding [97, 98]. Nonetheless, beyond the apparent role in hemostasis and

thrombosis, platelets are also considered to play a significant role in facilitating inflammatory

responses by recruiting a subset of leukocytes (monocytes, neutrophils, dendritic cells, and T-

cells) to the lesion site, and releasing inflammatory mediators into the lesion milieu [99]. It is

their inherent inflammatory nature that recognizes them as atherosclerosis promoters.

1.1.2.3.2 Platelet activation and aggregation

Under normal conditions, platelets circulate in a quiescent state, characterized by the lack of

platelet-selectin (P-selectin) expression, an integral membrane glycoprotein responsible for

leukocyte recruitment [99]. Upon exposure to a stimulus, platelets undergo three distinct

phases: 1) platelet tethering, activation and adhesion, 2) platelet aggregation and recruitment,

and 3) thrombus stabilization [100]. Platelet tethering is first achieved by the expression and

binding of glycoprotein (GP) Ibα to the collagen-immobilized protein, von Willebrand factor

(vWF) [101]. The security of the adherence is then further supported by the expression of

integrin receptors αIIbβ3 (GP IIb/IIIa) and α2β1 (GP Ib/IIa) upon platelet GPVI interaction with

collagen [102-105]. Once firmly attached, platelets undergo rapid morphology changes from a

disk-like shape to a sphere with pseudopodia extensions [106]. Subsequently, platelets

release their granular contents composed of platelet agonists, such as ADP and TXA2, that

! 13!

act in a paracrine and positive feedback loop that amplifies platelet activation, and assist with

platelet aggregation by promoting the conformational change of αIIbβ3 integrin into its active

form [107]. Active αIIbβ3 is essential for the bridging of activated platelets with fibrinogen, vWF

and fibronectin in order to form platelet aggregates [108]. These platelet aggregates are then

stabilized by the presence of fibrin converted from fibrinogen by thrombin to form a stable

thrombus [109].

1.1.2.3.3 Platelet granules

Platelets are highly secretory cells, with over 300 distinct molecules organized and packaged

into three main organelles: dense granules, α-granules and lysosomes [107, 110]. Firstly,

dense granules are small granules, with a diameter of 150-nm, containing pro-aggregating

factors including nucleotides (ATP, ADP, GTP, GDP), amines (serotonin 5-HT, histamine) and

Ca2+ [107]. Their opaque dense cores are surrounded by a single membrane that is easily

recognizable under an electron microscope [111]. Secondly, α-granules are large spherical or

ovoid organelles, with a diameter of 200-400-nm, that houses a pool of platelet coagulant

factors, fibrinolysis mediators, adhesive glycoproteins, protease inhibitors, and other

immunological proteins [107]. They represent the majority of granule population in both size

and number in platelets [107]. Finally, the third category of granules concerns the acidic

compartment, lysosomes. Measuring in between the dense and α-granules in size, at 175-

250-nm, the lysosome carries a variety of proteases, cathepsins, and glycohydrolases with

bactericidal activity [107]. Degranulation of these compartments occurs once a platelet

agonist, such as thrombin, collagen and thromboxane A2 (TXA2), binds to its corresponding

receptor and initiate a signal transduction for platelet activation [110]. Following activation,

Ca2+ is moved from the platelet dense tubular system (DTS) into the cytosol, which results in

! 14!

an increase in intracellular Ca2+ from an undetectable concentration of 100-nM to 2-10-µM.

This results in: 1) the movement of granules confined within a bundle of microtubules to the

membranes, and 2) dock and fusion of granular membrane with platelet plasma membrane to

unload its contents by a SNARE- and SNAP- dependent exocytosis [112].

1.1.2.3.4 Platelets in atherosclerosis

As early as the 1960s, studies have shown the presence of platelets at sites of inflammation

and endothelial injury [113]. A growing body of evidence now suggests their crucial role in the

development of atherosclerotic lesions. Fundamentally, platelets are able to adhere to intact

endothelial cells both in vitro and in vivo, even in the absence of endothelial lesion [113-115],

thereby resulting in platelet activation and release of platelet-derived mediators.

Consequently, the platelet-derived mediators have the potential of recruiting leukocytes by

eliciting chemokine production and adhesion molecule expression. For instance, among the

platelet-derived mediators released is interleukin-1β, a potent inflammatory cytokine known to

activate endothelial cells to induce intracellular adhesion molecule-1 (ICAM-1) expression and

cytokine production, particularly IL-6 and granulocyte macrophage colony stimulating factor

(GM-CSF) [116]. These cytokines are known to facilitate neutrophil and monocyte recruitment

[116]. In addition, another very potent mediator released by platelets is RANTES, a

chemokine known to attract monocytes and promote monocyte-endothelial interactions [117].

Finally, platelet factor 4 (PF4) is a CXC chemokine found in the α-granules of platelets at high

concentrations of 1500-µg/mL [118]. Upon binding to low-density lipoprotein related protein

(LRP) on endothelial cells, PF4 has previously been shown to induce endothelial NF-κB

activity, which resulted in an increase in expression of endothelial (E)-selectin, an endothelial-

leukocyte adhesion molecule involved in leukocyte recruitment [118]. Moreover, the same

! 15!

PF4 has also been demonstrated to promote the binding of oxidized low-density lipoprotein

(oxLDL) to endothelial cells and macrophages, which further endorses the development of a

fatty streak in atherosclerosis [119]. Similarly, in a study conducted by Daub et al, it was

shown that platelets were able to bind to LDL to form an LDL-platelet complex that could be

phagocytosed by macrophages to promote the formation of lipid-engorged foam cells [120].

Interestingly, secondary capture by platelets have also been observed to facilitate leukocyte

recruitment to the endothelial vessel wall. Activated and endothelium-adhered platelets

express a wide array of leukocyte-binding receptors including P-selectin, GPIbα and JAM-3,

which can bind to their receptor counterparts PSGL-1 and MAC-1 expressed in leukocytes

[113]. By capturing the circulating leukocytes, platelets are inherently contributing to leukocyte

recruitment that can augment the inflammatory responses found in atherosclerosis.

Collectively, these data compels us to appreciate the significant role of platelets in

atherogenesis.

1.1.3 Neutrophils in atherosclerosis

It is evident that atherosclerosis is a complex inflammatory disease of the vessel, with

established and defined roles of monocytes/macrophages, T-cells, and platelets. However,

only recently has the role of polymorphonuclear neutrophils (PMN) in atherosclerogesis been

evolved, since under a classical inflammatory response, they are the first line of defense that

responds to invading microbials and tissue injury [121-123]. In 2002, using coronary artery

segments from patients with acute myocardial infarction, Naurko et al have observed distinct

neutrophil infiltration in patients with ruptured plaques, suggesting that neutrophil infiltration is

actively associated with acute coronary events [122]. This was in congruent with findings from

Haumer et al, showing patients with higher neutrophil counts were at higher risk for major

adverse cardiovascular events [124]. In addition, similar findings were also shown by Grau et

! 16!

al whereby it was found that high neutrophil counts were independent predictors of recurrent

ischemic events, better than monocyte and lymphocyte counts [125]. In a study conducted by

Rotzius et al in 2010, the group utilized neutrophil- or monocyte- depleted ApoE-/- mice to

quantify the population of leukocytes interacting with the endothelium at the lesion site [126].

Interestingly, the results showed that neutrophils accounted for majority of the leukocyte

infiltration [126]. This first wave of neutrophil extravasation into the lesion site would thus

provide the setting to attract monocytes. In 2008, Soehnlein et al demonstrated that by

depleting neutrophils in C57BL/6 mice, extravasation of inflammatory monocytes were

markedly reduced compared to mice with intact neutrophil count [123]. It was later found that

it was the components of neutrophil secretion that attracted and activated the monocytes.

Collectively, these evidences strongly suggest the pioneer and crucial role of neutrophils in

the development atherosclerosis. Moreover, the mechanism by which neutrophils can trigger

atherosclerosis may lie directly through a neutrophil-derived molecule that contributes to the

development of the disease.

1.2 Defensins

1.2.1 Three families of defensins

Antimicrobial peptides are small cationic polypeptides composed of less than 100 amino acids

with significant roles in host defense. The first study of antimicrobial peptides originated 1960,

when Zeya and Spitznagel isolated an arginine-rich cationic peptide from rabbit and guinea-

pig leukocyte lysates that displayed a broad spectrum of antimicrobial activity [127]. Humans,

especially leukocytes and epithelial cells, express two main families of antimicrobial peptides:

defensin and cathelicidin [128]. Although structurally and evolutionarily similar to one another,

attention is directed more to defensins due to their extensive gene pool, various isoforms, and

ubiquitous participation in infection [128].

! 17!

Defensins are a family of antimicrobial peptides with 30-40 amino acids folded into a protein

of molecular weight 3-5kDa [128, 129]. The members of the family share a characteristic triple

stranded β-sheet fold with three cysteine-disulfide bonds [128]. There are tree subfamilies of

defensins: α-, β- and θ- defensins, with α- and β-defensin playing a more prominent role in

human host defense due to the presence of a pre-mature stop codon in θ-defensins [130].

Therefore, due to the deficiency of θ-defensin expression in human, we will not elaborate θ-

defensins in this thesis. Moreover, although β-defensins play a role in human host defense,

however, unlike α-defensins, they are not expressed in immune cells, but in epithelial cells

[128, 131]. Due to the different avenues of α- and β-defensins in mediating host defense, our

project will only focus on the role of α-defensins in atherosclerosis.

1.2.1.1 α-defensin

Human α-defensins were studied as early as 1985 by Dr. Thomas Ganz [132], and has now

been expanded to a breadth of knowledge with research. α-defensins contain 6 members: 1-4

designated as human neutrophil peptides (HNP-1, HNP-2, HNP3- and HNP-4) due to their

high expression in neutrophils, and human α-defensin 5- and 6 (HD-5 and HD-6) which are

chiefly expressed in intestinal paneth cells [129, 130, 133-135]. HD-5 and HD-6, together, are

termed the enteric defensins due to their nature as an innate defense molecule for the

gastrointestinal surface [136]. As such, since HD-5 and -6 are not involved in neutrophil-

mediated immune response, our project will not focus on these two α-defensins.

Differing only in their first amino acid, HNP-1, HNP-2, and HNP-3 constitute more than 50% of

the azurophilic granule content [137], with HNP-1 and HNP-2 collectively accounting for 90%

! 18!

of the defensin composition [138]. In contrast, HNP-4 contains an amino acid sequence

distinct from HNP1-3, and only accounting for less than 2% of the defensin content [139]. Due

to the prominent concentration of HNP1-3 in neutrophils, herein forth, HNP will be referred to

as a collection of HNP1-3.

In addition to neutrophils, HNPs are expressed by a wide range of immune cells including

monocytes, macrophages, natural killer cells, B cells, dendritic cells and γδ T cells [129, 140-

142]. HNP expressions within the various cells are also observed across different species.

HNP has been isolated from rats, guinea pigs, hamsters, rhesus macaques and paneth cells

from mice, suggesting an evolutionary conservation of HNP [128-130, 143-148].

Synthesis of HNP begins as a 90-100 amino acid prepropeptide with three main components:

a 19 amino acid N-terminus, a 45 amino acid anionic propiece, and a 30 amino acid cationic

C-terminus [149, 150]. Extensive processing and folding occurs in the neutrophil precursor

cells known as promyelocyte, with mature HNPs packaged in the azurophillic (or primary)

granules of neutrophils [129, 137, 151-153]. Once mature neutrophils circulate in the blood,

they no longer synthesize the HNP peptides, nor their mRNAs [129]. Degranulation of HNP is

regulated by the presence of microbial, cytokine or neuroendocrine signals [129].

Consequently, plasma concentrations of HNP can range from an undetectable level of 50-

100-ng/mL in healthy humans to a significant range of 900-170,000-ng/mL in septic patients

[154]. Thus, given its high concentration, HNP can play a substantial role in inflammation

outside of its antimicrobial activities, including atherosclerosis.

1.2.2 HNP neutralization

! 19!

α1-protease inhibitor or anti-trypsin (α1-PI or α1-AT) is a member of the serpins superfamily,

expressed by hepatocytes, monocytes, macrophages and neutrophils [155]. Under a steady-

state condition, plasma α1-PI concentration ranges between 15-30-µM, however, low α1-PI

concentrations (at plasma levels below 11-µM) have previously been shown to have important

clinical implications in atherosclerosis [155, 156]. Clinically, patients with low serum levels of

α1-PI during an acute phase of myocardial infarction are associated with a higher risk of

cardiogenic shock and mortality [157]. In addition, in patients undergoing coronary

angiography, the severity of coronary atherosclerosis has been associated with low plasma

levels of α1-PI [156]. Therefore, low levels of α1-PI have become an important indicator of the

development of atherosclerosis. Interestingly, α1-PI is an endogenous neutralizer of HNP

[158-160].

In 1995, a study conducted by Panyutich et al showed that HNP would bind to α1-PI to form a

complex that would inactivate the two molecules. Using human lung carcinoma cell line A549,

it was found that cytotoxicity induced by HNP was inhibited by the presence of α1-PI [161].

Indeed, using A549 and small airway epithelial cells (SAEC) co-cultured with CD4+

lymphocytes, it was previously demonstrated that HNP-induced IL-8 production was

significantly inhibited by the treatment of α1-PI [162]. Taken together, this may potentially

explain the poor prognosis of patients with low α1-PI, as they become incapable of

neutralizing the burden resulted from high concentrations of HNP during inflammation.

1.2.3 Properties of HNP

1.2.3.1 HNP and its anti-microbial role

! 20!

HNP exhibit host defense activities against a wide array of microbes including gram-positive

and –negative bacteria, fungus, and viruses [163-165]. Its activity can be observed at

concentrations as low as 1-10-µg/mL [128]. However, its activity can be impeded by the

presence of metabolic inhibitors, nutrient deprivation, hyperosmotic conditions, divalent

cations and certain plasma proteins [166].

The mechanism of HNP’s antimicrobial and cytotoxic activity involves two crucial steps: 1) the

binding, and 2) permeabilization of target membranes. Firstly, as a positively charged

molecule, HNP is able to bind to negatively charged molecules by electrostatic interactions.

Specifically, HNP binds to the negatively charged components found on microbial membranes

such as lipopolysaccharides (LPS) in gram-negative bacteria, (lipo)teichoic acids in gram-

positive bacteria polysaccharides, phosphatidyl glycerol and cardiolipin [167-169]. The

importance of such electrostatic interaction supports the diminished activity observed when

HNP is cultured with high salt conditions. Due to HNPs’ higher affinity for negatively charged

microbial surface molecules than the native divalent cations (Ca2+ or Mg2+), HNP can

competitively displace these ions and disrupt the normal outer membrane to create a transient

opening [170]. Using their hydrophobic surfaces, HNPs can coalesce into a hexamer of

dimers to form channels with a diameter of 25 Å [128, 165, 170-172]. The electrically inserted

channels subsequently allows for an increase in membrane permeability that ultimately

inhibits RNA, DNA and protein synthesis, thereby decreasing microbial viability [173].

1.2.3.2 HNP in inflammation

1.2.3.2.1 HNP as chemoattractant

HNP contains an intrinsic chemotactic property for T-cells, mast cells, monocytes and

immature dendritic cells [174, 175], a property that labels HNP as an immunological effector.

! 21!

Indeed, in a study by Yang et al, HNP was demonstrated to induce the chemotactic migration

of T- cells and dendritic cells, a response that was dependent on a Giα-protein coupled

receptor [175]. Likewise, using human monocyte- or murine bone marrow-derived

macrophages and macrophage cell line J774A1, Grigat et al, showed that upon treatment of

HNP, the cells’ chemotactic migration was induced [176].

Intriguingly, aside from HNP’s direct role in chemoattraction, HNP can also induce the

production of secondary mediators to further support leukocyte migration. Our group has

previously demonstrated that intratracheal instillation of HNP over 5 h induced an increase in

production of chemoattractants TNF-α, and MCP-1 in the bronchial alveolar lavage fluid [177].

Tumour necrosis factor-α (TNF-α) is a potent chemokine known to be chemotactic for

neutrophil even at nanomolar concentrations [178]. Similarly, monocyte chemoattractant

protein-1 (MCP-1) is a potent chemokine for monocytes, memory T-cells and basophils [179].

Its importance in recruiting monocytes in atherosclerosis was confirmed when mice deficient

in MCP-1 exhibited a decrease in severity of diet-induced atherosclerosis due to the

attenuation of monocyte accumulation at the site of lesion [180, 181].

1.2.3.2.2 HNP and P2Y6

Purinergic receptors are a class of receptors that bind to extracellular nucleotides, and have

been previously implicated to mediate systemic inflammatory responses [182]. Of particular

interest is the P2Y6 receptor, a seven-transmembrane G-protein-coupled receptor that

conventionally binds to UDP [182]. In a study conducted by Riegel et al, upon treatment of

TNF-α for 24 h in human microvascular endothelial cells (HMEC-1), there was a selective

induction of P2Y6 expression, a response that was not observed in a pool of 14 other

! 22!

purinergic receptors [182]. The role of P2Y6 in mediating vascular inflammation was further

supported in P2Y6-/- mice, whereby treatment of lipopolysaccharide (LPS) had an attenuated

production of VCAM and neutrophil-activating protein 3 (KC) [182].

To evaluate the potential role of P2Y6 in acting as an HNP receptor to induce inflammation,

our lab sought to determine the pathway of HNP-mediated IL-8. IL-8 is a pivotal neutrophil

chemoattractant expressed by a variety of cells including monocytes, lymphocytes,

granulocytes, endothelial cells, epithelial cells, and fibroblasts [183]. In additional to

neutrophils, IL-8 can also attract basophils, and eosinophils [184], immune cells that have well

documented roles in atherosclerosis [3]. Therefore, the ability of HNP to induce IL-8

production would have significant impact in recruiting leukocytes and consequently,

atherogenesis.

Indeed, using A549 and SAEC, our lab has previously found that stimulation with HNP would

induce IL-8 production that was mediated through the purinergic P2 receptor, P2Y6 [185].

Interestingly, stimulation of HCAEC and monocytes with HNP has also been shown to

increase IL-8 mRNA and protein expression [162, 174, 185-190]. The resultant interaction

between HNP and P2Y6 receptor instigated activities of the PI3K/Akt and ERK1/2 pathway in

lung epithelial cells [186] that were responsible in the IL-8 production. Finally, the observed

HNP-mediated responses were shown to be significantly masked in the presence of P2Y6

antisense [185]. Now, although it is possible that HNP can promote atherosclerosis through

the P2Y6 signaling pathway, however, our lab has shown in vitro and in vivo that another

receptor may also play a prominent role.

1.2.4 HNP and atherosclerotic events

! 23!

1.2.4.1 Plasma HNP levels in inflammatory disease

Under normal physiological conditions, plasma HNP concentration ranges from

0.2554±0.007- to 42±53-ng/mL [154, 191]. However, in patients with bacterial and non-

bacterial infections, HNP concentration can significantly increase up to 1,075±101.8-ng/mL

and 816.7±164.5-ng/mL, respectively [191]. In addition to plasma, the enhanced HNP

concentrations can be found in other bodily fluids of patients with infection such as pleural,

bronchoalveolar lavage, urine and cerebrospinal [191]. Interestingly, elevated HNP

concentrations were also clinically observed in patients with bacterial septicemia, whereby

their concentrations can reach as high as 170,000-ng/mL [154]. The detection of elevated

HNP suggests a clinical significance of HNP in mediating inflammatory responses, perhaps

even in the inflammatory events found in atherosclerosis.

1.2.4.2 HNP in atherosclerosis

Recently, a study conducted by Zhao et al compared the systemic concentration of HNP in

patients with and without CAD, and acute ST-segment elevation myocardial infarction

(STEMI). Unlike other antimicrobial peptides found in PMN such as LL-37, HNP levels were

found to be significantly higher in patients with CAD and STEMI, in comparison to patients

without CAD [192]. Clinically, this translated to three times the risk of cardiovascular mortality

compared to patients with lower plasma HNP concentration [193]. These results suggest that

circulating HNP, released from activated PMN during vascular inflammation, can complicate

and exacerbate the events observed in atherosclerosis.

In general, HNP can be detected in three areas: 1) leukocyte and epithelial granules; 2)

systemic circulation; 3) localized tissues. Indeed, previous studies have also detected HNP

! 24!

deposition in the intima of atherosclerotic vessels [194]. Most prominently, HNP was found to

be co-localized with the intimal smooth muscle cells and endothelium [194, 195], suggesting

its potential in activating the endothelial and VSMC. In addition, increases in HNP deposition

on the skin overlaying the femoral artery was significantly correlated with the severity of CAD,

as assessed by coronary angiography [196].

To evaluate the role of HNP in the development of atherosclerosis, our lab designed a double

transgenic mice that expresses HNP in the neutrophils and was prone to development of

atherosclerosis called ApoE-/-/HNP(+). Since mice do not express α-defensins in their

neutrophils [128, 131], our double transgenic mice would express HNP regulated by a

neutrophil elastase promoter, thereby making the mouse a more representative model of the

human physiology. After feeding the mice and their littermate controls (ApoE-/-/HNP(-)) on

chow or high fat for 10 weeks, their aorta was extracted to assess for lipid deposition.

Remarkably, mice expressing HNP showed a positive trend, although not significant, of more

lipid deposition in their aorta when fed on high fat diet (Figure 1) [197]. On an inflammation

perspective, mice expressing HNP also showed a significant increase in leukocyte adhesion

in the carotid artery (Figure 2) [197]. This was perhaps due to an increase in cytokine and

chemokine production. Indeed, HNP-expressing mice plasma was found to have a significant

increase in MCP-1, granulocyte macrophage colony stimulating factor (GM-CSF) and

neutrophil-activating protein-3 (KC) in comparison to the littermate controls (Figure 3).

Although not significant, regulated on activation, normal T cell expressed and secreted

(RANTES) showed a positive trend in HNP-expressing mice. Collectively, these results

provide evidence for HNP’s role in amplifying atherosclerotic lesion, leukocyte recruitment,

and chemokine production.

% T

otal

Aor

tic L

esio

n

Cho

wH

F05101520

ApoE

ApoE

HN

P

Die

t

% Aortic Lesion

% D

esce

ndin

g Ao

rtic

Lesi

on

Cho

wH

F051015

ApoE

ApoE

HN

P

Die

t

% Aortic LesionAp

oE%Ch%%

ApoE

%HF%

ApoE

HNP%HF

%Ap

oEHN

P%Ch

%%

Figu

re 1

. HN

P en

hanc

es li

pid

depo

sitio

n in

aor

ta o

f Apo

E-/- /

HN

P(+)

mic

e fe

d on

hig

h fa

t die

t. A

and

B. T

otal

and

de

scen

ding

aor

tic le

sion

was

sta

ined

by

Oil-

Red

-O (O

RO

) in

Apo

E-/-

/HN

P(-

) and

Apo

E-/-

/HN

P(+

) mic

e fe

d on

cho

w a

nd h

igh

fat.

Aor

tic le

sion

was

qua

ntifi

ed a

s pe

rcen

tage

of t

otal

or d

esce

ndin

g ao

rtic

area

. C. I

mag

es a

re re

pres

enta

tive

imag

es o

f at

leas

t 6 e

xper

imen

ts.

A% B%

C%

25

% A

rea

leuk

ocyt

e ad

hesi

on

Cho

wH

F01020304050

ApoE

ApoE

HN

P

Die

t

% Adherence

*"§"

Plas

ma

HN

P

Cho

wH

F020406080

ApoE

ApoE

HN

PHNP (pg/mL)

*"

*"

Leuk

ocyt

e R

ollin

g in

Car

otid

Arte

ry

Cho

wH

F

0102030Ap

oEAp

oEH

NP

Die

t

Rolling cells/min

Figu

re 2

. HN

P re

crui

ts le

ukoc

yte

to c

arot

id a

rter

y in

viv

o. A

. Pla

sma

HN

P w

as m

easu

red

in m

ale

Apo

E-/-

/HN

P(-

) and

A

poE

-/-/H

NP

(+) m

ice

fed

on c

how

or h

igh

fat d

iet f

or 1

0 w

eeks

(n=6

). B

. Mic

e w

ere

inje

cted

with

150

-µL

of rh

odam

ine

6G to

no

n-sp

ecifi

cally

labe

l leu

kocy

tes.

Mea

n va

lues

of l

euko

cyte

rolli

ng w

as m

onito

red

by in

vitr

o m

icro

scop

y (IV

M).

C. A

dher

ent

leuk

ocyt

es w

ere

quan

tifie

d by

Imag

eJ. D

. Rep

rese

ntat

ive

imag

es o

f leu

kocy

te a

dher

ence

und

er fl

uore

scen

t mic

rosc

opy.

*p

<0.0

5 vs

Apo

E-/-

/HN

P(+

) cho

w; §

p<0.

05 v

s A

poE

-/-/H

NP

(+) h

igh

fat.

A%B%

Apo

E-/-/H

NP(-

) A

poE-/-

/HN

P(+)

Ch

HF"

D%C%

26

MC

P-1

Cho

wH

F

0

100

200

300

ApoE

ApoE

HN

PF.I

GM

-CSF

Cho

wH

F

01020304050Ap

oEAp

oE H

NP

pg/mL

RAN

TES

Cho

wH

F

0204060Ap

oEAp

oE H

NP

F.I

KC

Cho

wH

F0

200

400

600

800

1000

ApoE

ApoE

HN

P

pg/mL*"

*"

*§"

*"*"

Figu

re 3

. HN

P in

crea

ses

cyto

kine

and

che

mok

ine

prod

uctio

n in

Apo

E-/- /

HN

P(+)

mic

e. P

lasm

a fro

m A

poE

-/-/H

NP

(-) a

nd

Apo

E-/-

/HN

P(+

) mic

e w

ere

colle

cted

afte

r 10

wee

ks o

f cho

w o

r hig

h fa

t die

t for

ana

lysi

s of

mon

ocyt

e-ch

emoa

ttrac

tant

pr

otei

n-1

(MC

P-1

), gr

anul

ocyt

e-m

acro

phag

e co

lony

-stim

ulat

ing

fact

or (G

M-C

SF)

, Reg

ulat

ed o

n A

ctiv

atio

n N

orm

al T

cel

l E

xpre

ssed

and

Sec

rete

d (R

AN

TES

), an

d ne

utro

phil

activ

atin

g pr

otei

n 3

(NA

P-3

or K

C).

*p<0

.05

vs A

poE

-/-/H

NP

(-) c

how

or

high

fat,

§p<0

.05

vs A

poE

-/-/H

NP

(+) o

n ch

ow a

nd h

igh

fat.

27

! 28!

1.2.4.3 HNP-induced leukocyte adhesion and migration

Leukocyte infiltration in atherosclerotic lesions plays an adverse role in the pathophysiology of

atherosclerosis. Previously, our laboratory has demonstrated surface expression of CD80,

CD86 and ICAM-1 in primary human small airway epithelial cells and A549 alveolar type II

cells, as well as their counterpart ligands CD28, CD152 and CD11a/CD18 in CD4+ T-

lymphocytes were increased in response to HNP stimulation [162]. Correspondingly, an

increase in leukocyte-epithelial interaction was observed [162]. Likewise, HNP treatment was

also shown to be able to enhance monocyte adhesion and migration across primary human

coronary artery endothelial cells by increasing endothelial ICAM-1 and monocytic CD11b

expression [174]. Functionally, this translated into an increase in leukocyte adherence and

rolling found in mice given a bolus of HNP, as captured by intravital microscopy [174]. By

enhancing the expression of proteins involved in leukocyte recruitment, HNP plays a valuable

role in capturing circulating leukocytes and promoting their transmigration and accumulation

to the site of atherosclerotic lesions, therefore endorsing the inflammatory cascade.

In addition, our group has previously demonstrated that HNP was able to induce leukocyte

rolling in the carotid artery of wild-type mice. Moreover, this response was dependent on a

lipoprotein receptor known as LRP8, since leukocyte rolling induced by HNP was significantly

abolished in LRP8-/- mice in comparison to wild-type mice. Interestingly, in mice deficient of all

LDLR and LRP family members (LDLR-/-/LRP-/- mice), there was no significant difference in

leukocyte rolling in comparison to LRP8-/- mice after receiving a bolus of HNP, thereby

suggesting that HNP-induced leukocyte recruitment was chiefly due to the LRP8 signaling

pathway [174], and not only through a P2Y6-dependent signaling pathway.

! 29!

1.2.4.4 HNP-mediated oxidative stress

Oxidative stress is one of the hallmark characteristics of endothelial dysfunction and a

prominent event in atherosclerosis [198-200]. A study conducted by Kougias et al

demonstrated the influential effect of HNP in reducing endothelial-dependent vasorelaxation

that was associated with an increase in superoxide radical production and a decrease in

eNOS mRNA expression in porcine coronary arteries [201]. This was consistent with findings

in our laboratory where HNP was able to stimulate hydrogen peroxide (H2O2) productions in

murine lung explants [202], and nitrotyrosine in human acute monocytic leukemia cell line

(THP-1) derived macrophage [174]. The buildup of these reactive oxygen and nitrogen

species can perturb atherosclerosis by oxidizing lipids into oxLDL, which activates the

endothelium [4, 203-205], promotes foam cell formation [206-209] and attracts circulating

leukocytes [206].

1.2.4.5 HNP and foam cells

On a cellular level, deposited HNP have been shown to have multiple implications. Firstly, our

lab has previously found that treatment of HCAEC with HNP was able to induce MCP-1

secretion [174], a phenomenon that would promote the recruitment of monocytes to the lesion

site and increase the chances of foam cell formation. Secondly, our lab has also

demonstrated that the same HNP was able to accelerate foam cell formation in human acute

monocytic leukemia cell line (THP-1)-derived macrophages treated with oxLDL [174]. It was

later found that this was due to an increase in expression of macrophage scavenger receptor

responsible for lipid uptake: CD36 and CD68 [174]. Consequently, the HNP-stimulated

macrophages was able to elicit nitrotyrosine production, suggestive of HNP’s role in mediating

nitrosative stress that can further perturb atherosclerosis [174].

! 30!

1.2.4.6 HNP and platelet aggregation

Platelet activation and aggregation plays an integral role in atherosclerosis. Our lab has

previously shown that stimulation of human platelets with HNP can induce platelet activation,

a response assessed by measuring P-selectin (CD62P) expression [174], the surface

adhesion marker responsible for platelet-endothelial interaction [210]. Moreover, when human

platelets were primed with HNP, platelets were able to sustain their aggregation response

once stimulated with ADP [174]. The physiological relevance of these results was

demonstrated in vivo. In mice injected with a bolus of HNP intravenously, platelet aggregation

was significantly increased upon application of ferric chloride in comparison to mice that

received vehicle control. Likewise, the amount of time to occlude the vessel was also

significantly reduced in mice that received HNP [174]. Interestingly, the observed platelet

responses were also mediated by the LRP8-signaling pathway [174]. Therefore, additional

research is warranted to provide a better understanding of the role in LRP8 in mediating the

pathogenesis of atherosclerosis.

1.3 Low-density lipoprotein related protein-8 (LRP8)

1.3.1 LDLR and LRP family

The lipoprotein receptor (LR) superfamily is a class of eleven structurally related

transmembrane surface proteins: 1) LRP1, also known as α-2-macroglobulin; 2) LRP1b, also

known as LR32; 3) megalin/LRP2, previously known as gp320; 4) LDL receptor (LDLR); 5)

very low-density lipoprotein receptor (VLDL receptor); 6) MEG7/LRP4; 7) LRP8/apolipoprotein

E receptor 2; 8-11) LR 3, 5, 6 and 11 [211, 212]. The structural domains found common to

majority of the receptors are: 1) ligand binding domain, consisting of seven cysteine-rich type

A repeats enriched with negatively charged clusters of Ser-Asp-Glu residues; 2) epidermal

growth factor (EGF), a 400 amino acid residue that facilitates acid-dependent ligand

! 31!

dissociation; 3) O-linked sugar domain, a 58 amino acid residue enriched with 18 serines and

threonines containing O-linked carbohydrate chains; 4) transmembrane domain, a single 20

amino acid domain that serves to anchor the receptor to the plasma membrane; and 4)

cytoplasmic tail domain, a 50 amino acid residue the receptor internalization sequence Asn-

Pro-Val-Tyr in the coated pit [212]. Members of the superfamily participate in a wide range of

homeostatic responses including lipid metabolism, neurodevelopment, signal transduction,

nutrient and vitamin trafficking, and ligand endocytosis [211]. The expression of the individual

proteins, their biological function, and corresponding ligands vary across different tissues

(Table 1).

In light of LRs’ role in lipid homeostasis and signal transduction, several studies have been

dedicated to elucidate their role in atherogenesis. Firstly, LRP family members are highly

expressed in the vascular wall and is associated with macrophages and vascular smooth

muscle cells [213], suggesting its role in maintaining vascular integrity. Secondly, many of the

LRs’ ligands are mediators of atherosclerosis such as thrombospondin, ApoE, coagulant

factor Xi, thrombin, tPA, uPA, lipoprotein lipase, hepatic lipase, protease nexin-1 and -2, and

protein C [214, 215], which warrants the assumption that LRs may be responsible in the

signaling transduction of atherosclerosis development. Moreover, in a study using

genomewide linkage scan of 428 families, it was found that genetic variants of LRP8

contribute to the development of premature myocardial infarction and familial CAD [216].

Congruently, mutation in the LDLR gene has previously been found to cause familial

hypercholesterolemia [211], due to cellular loss-in-function of LDL uptake. Together, it

suggests that LRs play an important role in shaping atherosclerosis development.

Rec

epto

r E

xpre

ssio

n Li

gand

s B

iolo

gica

l Fun

ctio

n

LD

LR

Hep

atoc

ytes

, mac

roph

age,

ce

ntra

l ner

vous

sys

tem

A

poE

, Apo

B, L

DL

Cho

lest

erol

hom

eost

asis

LR

P1

(α

2-m

acro

glob

ulin

)

Hep

atoc

ytes

, neu

rons

, V

SM

C, m

acro

phag

e,

troph

obla

st, e

mbr

yoni

c tis

sues

Apo

E, c

hylo

mirc

on re

mna

nts,

a2-

mac

rogl

obul

in, a

myl

oid

perc

urso

r pr

otei

n, tP

A, p

rote

ase

inhi

bito

r co

mpl

exes

, lip

opro

tein

lipa

se,

PD

GF,

TG

Fβ

End

ocyt

osis

, chy

lom

icro

n re

mna

nt

rece

ptor

, pha

gocy

tosi

s of

apo

ptot

ic

cells

, em

bryo

nic

deve

lopm

ent,

PD

GF

rece

ptor

regu

lato

r, C

a2+

curr

ent r

egul

ator

V

LDLR

B

rain

, hea

rt ca

rdio

myo

cyte

s,

endo

thel

ial c

ells

, adi

pose

tis

sue

Apo

E, R

eelin

, lip

opro

tein

lipa

se,

tissu

e fa

ctor

pat

hway

inhi

bito

r N

euro

nal m

igra

tion,

syn

aptic

tra

nsm

issi

on

LRP

8

(Apo

ER

2)

Bra

in, t

estis

, pla

tele

t, en

doth

elia

l cel

ls, h

epat

ocyt

es,

VS

MC

, pla

cent

a, o

varie

s

Apo

E, R

eelin

, thr

ombo

spon

din-

1,

coag

ulan

t fac

tor X

I, an

tipho

spho

lipid

s

Neu

rona

l mig

ratio

n, s

ynap

tic

trans

mis

sion

, mal

e fe

rtilit

y, p

late

let

activ

atio

n an

d ag

greg

atio

n

M

egal

in

(LR

P2,

gp3

20)

Ren

al p

roxi

mal

tubu

le, t

hyro

id

and

para

thyr

oid

glan

d, tr

opho

-ec

tode

rm, v

isce

ral y

olk

sac,

ne

uroe

ctod

erm

Apo

B, A

poE

, Apo

J, A

poH

, al

bum

in, c

ubili

n, re

tinol

-bin

ding

pr

otei

n, v

it-D

-bin

ding

pro

tein

, so

nic

hedg

ehog

, BM

P-4

Nut

rient

sup

ply,

tran

scyt

osis

of

thyr

oglo

bulin

, BM

P-4

sig

nalin

g

LR

P1b

(L

R32

)

Cen

tral n

ervo

us s

yste

m

Syn

apto

tagm

in, l

amin

in re

cept

or

prec

urso

rs

Unk

now

n

M

EG

F7

(LR

P4)

Cen

tral n

ervo

us s

yste

m

Unk

now

n U

nkno

wn

Tabl

e 1.

Lig

ands

and

func

tions

of L

DL

rece

ptor

fam

ily m

embe

rs. T

he ta

ble

high

light

s on

ly a

sel

ectio

n of

LD

L re

cept

or

fam

ily m

embe

rs. (

Ada

ptat

ion

from

May

215 w

ith u

pdat

ed in

form

atio

n fro

m re

cent

pub

licat

ions

).

32

! 33!

1.3.2 LRP8 and atherosclerosis

1.3.2.1 LRP8

Until 1996, lipid metabolism in the central nervous system was poorly understood, even

though it was widely known that lipids played a crucial role in myelin formation [217]. Although

most of the lipids found in the brain are synthesized by the neuronal cells such as astrocytes,

however, constitutive cholesterol, particularly ApoE, and fatty acid uptake from the systemic

circulation also contributes significantly to the lipid content in the central nervous system

(CNS) [218, 219]. It wasn’t until the study conducted by Kim et al that the receptor-mediated

ApoE endocytosis was found to be due to the actions of LRP8 [217]. However, despite

LRP8’s ability to bind and internalize apoE in neurons, transgenic mice deficient in LRP8 do

not exhibit hypercholesterolemia [220]. Indeed, in a study conducted by Li et al, they found

LRP8 had the lowest endocytosis rate when compared to LRP1, LRP4, LRP2 and LDLR

[221]. This suggests vascular LRP8 plays a more prominent role in signal transduction, not

lipid internalization.

1.3.2.2 LRP8 in platelet

Although initially characterized as a receptor responsible for lipid metabolism in the central

nervous system, LRP8 has recently gained more attention to its role in atherosclerosis.

Originally believed to be chiefly expressed in the brain, placenta, testes and ovaries,

expression of LRP8 has now been expanded to include heart, endothelial cells, vascular

smooth muscle cells and platelets [222]. Interestingly, of all the LRP family members, only

LRP8 is found in platelets [223], therefore, many studies have first focused on platelet LRP8

to provide insight on the role of LRP8 in atherogenesis.

! 34!

To date, different splice variants of LRP8 on platelets have been discovered. Using a

homology cloning approach and degenerate PCR primers to amplify the binding domain of

LDL-R, Riddell et al found that platelet LRP8 lacked repeating units 4-6 (LRP8Δ4-6) [223].

Later using immune-blotting, Pennings et al have documented three more LRP8 splice

variants: 1) shApoER2Δ5, lacking LDL binding domains 4, 5, and 6; 2) shApoER2Δ4-5,

lacking LDL binding domains 3, 4, 5, and 6; and 3) shApoER2Δ3-4-5, lacking LDL binding

domains 3, 4, 5, 6 and 7 [224]. Although it remains unclear why platelets contain multiple

LRP8 variants, however, it could suggest the diversity of ligands LRP8 can bind to.

The functional role of LRP8 in platelets was first described by Desai et al in 1989 when they

discovered that high density lipoprotein E inhibited ADP-mediated platelet aggregation

through a surface receptor they called HDL2 [225]. It was later confirmed by Riddell that the

receptor was the LDL receptor family member, LRP8 [223]. Moreover, it was elucidated that

ApoE-induced LRP8 activation mediated an intracellular up regulation of eNOS activity, which

generated a large quantity of NO that augmented guanylate cyclase activity and cGMP

content [226]. Later in 2004, Korporaal et al expanded Riddell’s work to include the

downstream pathway of LRP8 activation. By sensitizing isolated human platelets with an LDL

constituent, apolipoprotein B100, the group found that it induced a rapid phosphorylation of

p38 mitogen-activated protein kinase (p38MAPK), a response that was significantly attenuated

by the presence of the LDL receptor family inhibitor: receptor-associated protein (RAP) [227].

The response was later confirmed ex vivo using platelets isolated from LRP8-deficient mice,

whereby LDL was also unable to induce p38MAPK activation [227]. By inhibiting p38MAPK, it can

attenuate cytosolic phospholipase A2 (cPLA2) activity, which results in a decrease in cytosolic

Ca2+ load, and TxA2 production and release [228]. This suggests a critical role of LRP8 in

! 35!

mediating intracellular signaling for platelet aggregation. Indeed, in 2009, Robertson et al

examined the role of LRP8 in platelet aggregation in vivo. The group demonstrated that in

LRP8-deficient (LRP8-/-) and LRP8 heterozygous (LRP8-/+) mice, platelets treated with ADP or

thrombin exhibited a significant attenuation in platelet activation and aggregation, as

quantified by αIIbβ3 activation and aggregometer, respectively [229]. Correspondingly, in

comparison to the littermate controls, LRP8-/- and LRP8-/+ mice exhibited an increase in

carotid artery occlusion time post topical injection of FeCl3 [229]. It thus becomes evident that

LRP8 can play a critical role in mediating platelet aggregation and shape the course of

atherosclerosis.

1.3.2.3 LRP8 in endothelial cells

With a growing body of evidence for the role of LRP8 in platelet aggregation, studies have

shifted to investigating the role of LRP8 in endothelial cell behaviour. Interestingly, in 2011,

Ramesh et al established the role of LRP8 in mediating leukocyte rolling. The group showed

that activation of LRP8’s first LDL-binding domain in vascular endothelial cells, by anti-

phospholipid antibodies (aPL), was able to induce an increase in phosphatase PP2A activity

[230]. The resultant PP2A activation induced a decline in eNOS activity, and hence NO

bioavailability [230]. Using intravital microscopy, the group also found that in LRP8-expressing

mice, stimulation with aPL mediated an increase in leukocyte adhesion and rolling, a

phenomenon that was absence in LRP8-knock out mice [230].

1.3.3 LRP8 Ligands

Outside of ApoE, several other extracellular ligands of LRP8 have recently been identified

that can induce LRP8 signal transduction: 1) reelin, a 400kDa extracellular matrix protein that

binds to the first ligand binding domain of LRP8; 2) thrombospondin, a trimeric or pentameric

! 36!

protein involved in cell-cell communication; 3) selenoprotein P, a transporter for selenium that

is required for spermatogenesis; and 4) coagulation factor XI, a 160-kDa glycoprotein that

participates in the intrinsic blood coagulation pathway [215, 231-233]. Consequently, the

activation of LRP8 can induce binding of multiple scaffold proteins onto the intracellular

domains of LRP8 such as: 1) disabled-1 and -2, a cytoplasmic docking protein that is

essential for Src, Fyn and MAPK kinase activity; 2) JIP-1 and -2, molecular scaffolds involved

in the C-jun N-terminal kinase (JNK)-signaling pathway; and 3) PSD-95, an adaptor protein

responsible for synaptic and dendritic spine formation [234-236] [Table 2]. Interestingly,

previous studies have linked MAPK activity to the increased production and release of platelet

agonist thromboxane A2 (TxA2) [228], and cholesterol ester accumulation in macrophages to

promote foam cell formation [237]. Likewise, numerous studies have established the key role

of JNK in atherogenesis. Studies have shown that atherosclerotic lesions exhibit an elevation

of JNK-activity, and that inhibition of the JNK pathway attenuated CD-36 and SR-A induced

LDL uptake, foam cell formation and atherogenesis in mice [238-240]. Collectively, this

suggests that activation of LRP8 can induce downstream pathways that promote

atherosclerosis.

1.3.3.1 Reelin

1.3.3.1.1 Reelin molecule

Molecularly, reelin is a 3461 amino acid long protein with a molecular mass of 387,497 Da.

The protein contains a 27 amino acid signaling peptide, a 28-190 amino acid F-spondin-like

(SP) segment, a 191-500 residue H region, and a long chain of 8 repetitive segments, each

approximately 350 amino acid long separated by an epidermal growth factor (EGF) [241].

Finally, the protein terminates with a 33 amino acid C-terminus enriched in basic residues

[242]. Although reelin is first expressed as a full-length protein, however, other fragments of

Lig

and

C

ell

Fu

nct

ion

LDL

End

othe

lial c

ells

E

ndot

helia

l act

ivat

ion

Pla

tele

t α

II bβ

3 an

d P

-sel

ectin

exp

ress

ion

Ant

ipho

spho

lipid

s E

ndot

helia

l cel

ls

eNO

S in

activ

ity

Pla

tele

t Tx

B2

expr

essi

on

Apo

E

Pla

tele

t ↓

Pla

tele

t agg

rega

tion

β2G

PI

Pla

tele

t ↑

TxA

2 ex

pres

sion

Ree

lin

Lym

phat

ic

End

othe

lial C

ell

↑ M

CP

-1 e

xpre

ssio

n

Pla

tele

t S

prea

ding

Neu

ron

Neu

rona

l mig

ratio

n

Thro

mbo

spon

din-

1 P

late

let

Pla

tele

t agg

rega

tion

Act

ivat

ed P

rote

in-C

U

937

mon

ocyt

e

cell

line

Sup

pres

sion

of a

popt

osis

Sel

enop

rote

in P

S

perm

S

perm

atog

enes

is

Coa

gula

tion

fact

o X

I P

late

let

Pla

tele

t agg

rega

tion

!

Tabl

e 2.

Lig

ands

and

func

tions

of L

RP

8. T

he ta

ble

high

light

s th

e va

rious

LR

P8

ligan

ds a

nd a

nd th

eir c

orre

spon

ding

fu

nctio

n in

the

cells

. 37

! 38!

reelin have been found due to reelin processing by metalloproteinase [243]. By comparing

partial recombinant reelin construct, reelin has been shown to be cleaved at 2 locations: 1)

between repeats 2 and 3; and 2) between repeats 6 and 7, to yield reelin fragments with

molecular weight 100-, 120-, 180-, 220- and 300-kDa [244]. Reelin fragment 100-kDa

contains the repeating units 7 to C-terminus (R78C); 120-kDa contains the repeating units 3

to 6 (R36); 180-kDa contains the N-terminus to repeating unit 2 (NR2); 220-kDa contains the

repeating units 3 to C-terminus (R38C); and 300-kDa contains the N-terminus to repeating

unit 7 (NR6) [244-246] [Figure 4]. Interestingly, not all reelin fragments bind to their receptor,

LRP8, or at equal affinity.

1.3.3.1.2 Native vs. active reelin

The ligand-receptor relationship between reelin and LRP8 was first observed when LRP8-

deficient mice exhibited reeler-like phenotype [220]. Later, in 1999, D’Arcangelo et al made

the revolutionary discovery that reelin binds directly to LRP8 to elicit their response [247]. By

constructing recombinant reelin proteins of the various fragments, the binding capacity of

other reelin fragments to LRP8 was assessed in vitro [245, 246]. In addition and of equal

importance, by quantifying Dab1 tyrosine phosphorylation on LRP8, the group was also able

to address the ability of various reelin fragments to induce LRP8 activation [245, 246]. It was

found that recombinant reelin proteins containing the N-terminus, the first four repeating units

and part of the fifth repeating unit showed no significant binding to LRP8 [246, 248, 249].

However, recombinant reelin proteins containing repeating units 3-8 or 3-6, which coincidently

was the central segment of reelin, was able to bind to LRP8 at similar affinities to full-length

reelin [246, 249, 250]. Moreover, recombinant reelin containing the central segment was also

able to stimulate Dab1 phosphorylation on LRP8 [245], thereby suggesting the importance of

reelin’s central fragment in binding to and activating LRP8. Interestingly, although the N-

Figu

re 4

. Ree

lin fr

agm

ents

. Pro

teol

ytic

pro

cess

ing

of re

elin

pro

tein

and

the

corr

espo

ndin

g m

olec

ular

wei

ghts

of t

he

fragm

ent.

Figu

re a

dapt

ed fr

om D

’Arc

ange

lo25

7 .

RR

1 R

R8

RR

7 R

R6

RR

5 R

R4

RR

3 R

R2

Sig

nalin

g P

eptid

e F-S

pond

in-li

ke

Seg

men

t

H-r

egio

n C

-Ter

min

us

EG

F

N-T

erm

inus

NR

2 (1

80-k

Da)

R

38C

(2

20-k

Da)

N

R6

(300

-kD

a;

Act

ive)

R

78C

(1

00-k

Da)

R36

(1

20-k

Da)

Full

leng

th (N

ativ

e)

400-

kDa

39

! 40!

terminal moiety of reelin was not responsible in binding to LRP8, it does play a crucial role in

reelin functioning. Studies have shown that reelin interacts electrostatically with each other on

the N-terminus to form a large homopolymer, which is important for reelin’s functional activity

[251, 252]. By using an N-terminus blocking antibody, CR50, reelin was unable to form an

aggregate and induce LRP8 Dab1 phosphorylation [252]. Indeed, a study by Nakajima et al

showed that intraventricular injection of CR-50 at the embryonic stage of mice was able to

disrupt the organized development of the hippocampus, an event that was in conjunction to

the reeler pattern [253]. Collectively, this suggests that reelin fragment 300-kDa and full-

length reelin are the prime activators of LRP8.

1.3.3.1.3 Reelin in inflammation

Reelin was originally believed to be solely expressed by the central nervous system, however,

traces of reelin have been detected in mammalian blood, liver, pituitary pars intermedia,

adrenal chromaffin cells, hepatic stellate cells, lymphatic endothelial cells and platelets [254-

256]. Interestingly, an up regulation of reelin expression was found in patients with liver

cirrhosis [257] and ocular injury [258], suggesting a mediatory role of reelin during

inflammation. Indeed, recently, a study published by Lutter et al found that in the presence of

SMCs, lymphatic endothelial cells release and proteolytically process the endothelium-derived

reelin. Through a paracine mechanism, the released reelin correspondingly acts on

neighbouring endothelial cells to induce an up-regulation of MCP-1 [255]. This suggests that

reelin is capable in mediating leukocyte recruitment. Interestingly, once the monocytes and

macrophages have been recruited, reelin can also act on their LRP8 receptor to regulate

leukocyte cholesterol efflux. In a study conducted by Chen et al, the group observed that

stimulation of mouse macrophages, RAW264.7 cells, with reelin can induce an up-regulation

! 41!

of the ATP binding cassette transporter A1 (ABCA1), a transporter that regulates cholesterol

efflux [259]. Therefore, reelin may play a crucial role in regulating foam cell formation.

To further expand the role of reelin in inflammation, in 2010, Tseng et al sought to determine

the effect of reelin on platelet activation since LRP8 was the only LRP family member

expressed on platelets [223, 254]. Remarkably, stimulation of reelin in platelets induced

platelet spreading, a phenomenon that is characteristic of platelet activation [254]. This

proposes the notion that circulatory reelin is able to induce platelet activation, a primary and

necessary event preceding platelet aggregation. In summary, the observed association

between reelin and inflammation emphasizes the molecule’s potential role in mediating the

events found in atherosclerosis.

1.4 Summary

The role of neutrophils in the development of atherosclerosis as recently gained wide

attention [5, 126]. As the first line of defense, neutrophils have previously been shown to

infiltrate atherosclerotic lesions prior to the recruitment of monocytes and macrophages [126].

However, due to their short life spans, neutrophils do not reside in the lesion over the entire

duration of the disease. Therefore, attention has been shifted to focus on neutrophil proteins

in mediating the neutrophil-provoked inflammation, specifically HNP.

It is evident that HNP released from activated neutrophils play an immune-modulating role,

however, it is also increasingly becoming apparent that HNP may also serve as an

atherosclerotic-modulating peptide. Although our lab has previously shown that the LRP8

signaling pathway plays a significant role in mediating HNP responses, nevertheless, the link

between HNP and LRP8 was still unclear. Here, we present for the first time the relationship

! 42!

between HNP and LRP8, whereby the linkage between the two is mediated through the reelin

molecule.

! 43!

!Chapter 2: Rationale and Objectives

2.1 Rationale and Novelty

Elevation in neutrophil counts and infiltration to the lesions has been previously observed to

correlate with the severity and progression of atherosclerotic lesions [122, 124]. Human

neutrophil peptides (HNP) are cationic proteins that account for more than 50% of the

azurophilic granule contents in neutrophils [137] and are released into the extracellular milieu

upon PMN activation. This leaves a “foot-print” of the neutrophil’s existence at the site of

lesion [260]. In epithelial cells, HNP is known to bind to P2Y6 and elicit IL-8 production [185],

however, the biological consequence of HNP on P2Y6 in endothelial cells is not known.

Moreover, although primarily known for its antimicrobial properties, recently, HNP has been

found to also mediate inflammatory events found in atherosclerosis including the induction of

leukocyte chemoattraction, adhesion, and migration across the endothelium, and platelet

aggregation [174]. Interestingly, these responses involved the communication between HNP

and the LRP8-signaling pathway. However, HNP does not directly bind to LRP8 in human

microvascular endothelial cells (HMEC-1) [Figure 5]. In platelets, LRP8 is the only known

LRP family member expressed, suggesting that a traditional LRP8 ligand participates in the

crosstalk between HNP and LRP8 to induce platelet aggregation. We believe that HNP is able

to stimulate the production and release of an LRP8-ligand, which acts in a paracrine fashion

to instigate the activation of the LRP8-signaling pathway. Due to its prominent expression

during inflammation and its ability induce inflammatory mediators, reelin may be a candidate

ligand that facilitates the HNP-LRP8 crosstalk.

! 44!

Here, we present for the first time the molecular mechanism of HNP activating the LRP8-

signaling pathway through the expression and release of reelin [Figure 6] in endothelial cells

and platelet.

2.2 Hypothesis

Human neutrophil peptides induce endothelial activation and platelet aggregation mediated by

reelin.

2.3 Objectives

2.3.1 Objective #1: To examine the role of HNP-induced reelin release.

Specific Aim I: To examine the protein expression of reelin in HNP-treated endothelial cells.

Specific Aim II: To evaluate the effect of P2Y6 blocker on HNP-mediated reelin expression.

2.3.2 Objective #2:!To determine the specific effect of reelin in endothelial function and

platelet aggregation.

Specific Aim I: To examine the direct effects of reelin on nitric oxide synthase (eNOS and

iNOS) in human coronary endothelial cells (HCAEC).

Specific Aim II: To determine the direct effects of reelin on measure oxidative stress markers

in HCAEC.

Specific Aim III: To evaluate the direct effect of reelin on platelet aggregation.

Specific Aim IV: To assess the effect of reelin neutralizing antibody on endothelial dysfunction

and platelet aggregation.

GST

-Pul

l Dow

n A

ssay

:

WC

L G

ST

+

+

+

+

- 

GST

GST

- B

eads

H

NP

13

0 kD

a LR

P8

30 k

Da

HN

P +

GST

Figu

re 5

. HN

P do

es n

ot b

ind

dire

ctly

to L

RP8

. HC

AE

C p

rote

in ly

sate

s w

ere

incu

bate

d w

ith G

ST

prot

ein,

GS

T-fu

sed

HN

P,

or b

eads

alo

ne o

vern

ight

at 4

o C o

n a

rota

tor.

Dire

ct p

rote

in-p

rote

in b

indi

ng w

as d

eter

min

ed b

y w

este

rn b

lot a

fter p

robi

ng fo

r LR

P8

and

HN

P.

45

PM

N

HN

P

P2Y

6

Ree

lin

LRP

8

Pla

tele

ts

Thro

mbo

sis

Per

oxyn

itrite

Figu

re 6

. Pot

entia

l mec

hani

sm o

f HN

P in

pro

mot

ing

athe

roge

nesi

s. A

ctiv

ated

PM

N re

leas

e H

NP

to a

ct o

n en

doth

elia

l P

2Y6 s

igna

ling

path

way

and

indu

ce th

e ex

pres

sion

and

rele

ase

of re

elin

. Ree

lin a

cts

on p

late

let L

RP

8, th

e on

ly L

DL

rece

ptor

foun

d on

pla

tele

ts, t

o m

edia

te p

late

let a

ggre

gatio

n an

d th

rom

bosi

s. F

inal

ly, re

elin

act

s on

nei

ghbo

urin

g en

doth

elia

l ce

lls to

indu

ce p

erox

ynitr

ite p

rodu

ctio

n an

d en

doth

elia

l dys

func

tion.

46

! 47!

Chapter 3: Methods and Materials

3.1 Reagents

Rabbit α-human LRP8 mAb was purchased from Epitomics (Burlingame, CA, USA). Mouse

α-human reelin mAb was from EMD Millipore (Billerica, MA, USA). Mouse α- GAPDH and -

His mAb were purchased from Biolegend (San Diego, CA, USA). Rabbit α- human eNOS

mAb was from Cell Signaling (Beverly, MA, USA). Rabbit α-human iNOS and lamin pAb were

purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), goat α-rabbit and

mouse IgG-HRP secondary antibody were from Jackson ImmunoResearch Laboratories Inc.

(WestGrove, PA, USA) and enhanced-chemiluminescence solution was from

ThermoScientific (Waltham, MA, USA). Dithiothritol (DTT) was purchased from BioShip

(Burlington, ON, Canada). Mouse recombinant reelin was purchased from R&D Systems

(Minneapolis, MN, USA), and reelin neutralizing antibody (CR-50) was from MBL (Nagoya,

Japan). Adenosine diphosphate (ADP) and 1,4-Di[3-(-isothiocyanatophenyl)thioureido]butane

(MRS 2578) were from Sigma (St. Louis, MO, USA).

Purified HNP was a mixture of HNP-1, -2, and -3 isolated from the sputum of cystic fibrosis

patients, as previously described [202]. Briefly, 20 cystic fibrosis patient sputums were pooled

prior to purification to minimize variability from one patient to another. Sputum was mixed with

5% acetic acid in a 5-mL volume, homogenized and stirred for 24 h at 4oC in a polypropylene

conical tube. The solution was centrifuged for 30 min at 27,000 G and supernatant was

collected. Ten percent dithiothritol (DTT) was added to the sputum supernatant and loaded

onto a polyacrylamide el permeation colum of Bio-Gel P-10 (Bio-Rad Laboratories, Hercules,

CA, USA) and eluted with 5% acetic acid. Fractions were collected, and HNP-contained

eluents were pooled, lyophilized and reconstituted in PBS. Endotoxin detection assay was

! 48!

performed by using Limulus amebocyte lysate (Associates of Cape Cod Inc, Falmouth, MA,

USA), and bacterial killing was performed to test HNP activity. Protein concentrations were

measured by spectrophotometry at an absorbance of 280-nm.

3.2 Cell Culture

3.2.1 Primary human coronary endothelial cells (HCAEC)

Primary human coronary artery endothelial cells (HCAEC, Cell Applications, San Diego, CA)

were grown in T75 flasks (Sarstedt Inc, Newton, NC) and kept at a density of 0.1-1x106

cells/mL in MesoEndo Cell Growth Medium (Cell Applications, San Diego, CA). Cells within

the first fifteen doublings of growth were used for experiments. Upon 80% confluency, cells

were washed twice with HBSS, trypsinized and seeded at a density of 1x105 cells/mL in 12-

well plates for 16 h at 37oC in MesoEndo Cell Growth Medium to ensure adherence. Prior to

stimulation, cells were washed with HBSS and medium was replaced with Endothelial Serum-

Free defined medium with 1% fetal bovine serum (Sigma, St. Louis, MO) to prevent HNP

inactivation [261].

3.3 Western blotting

3.3.1 Whole cell lysate preparation

Cells were stimulated according to specific experimental protocols. Culture medium was

collected, aliquoted and stored in -80oC freezer. Cells were washed with HBSS and

subsequently lysed with 100-µL of cold lysis buffer (50mM Tris-HCl, 2mM EDTA, 2mM EGTA,

150mM NaCl, 1% Triton X-100, pH 7.54). Cells were scraped on ice, centrifuged at 10,000 G

for 5 min at 4oC. Supernatants containing the proteins were transferred into a fresh eppendorf

tube and store at -80oC.

! 49!

3.3.2 Protein concentration quantification

Protein concentration was quantified using the Bradford Assay in a 96-well plate. Bovine

serum albumin (BSA, Sigma, St. Louis, MO) standard (0- 1.0 µg/µL) was diluted in 1xPBS

and 5-µL of each standard was added into duplicate wells. Similarly, 5-µL of samples were

added to duplicate wells, followed by the addition of 195-µL of 1:5 diluted protein assay dye

(BioRad, San Diego, CA). The samples were incubated for 10 min at room temperature to

allow for colour development. Absorbance was measured at 595-nm with a

spectrophotometer (SpectraMax 340, Molecular Devices) and protein concentration was

determined with the constructed standard curve.

3.3.3 SDS polyacrylamide gel electrophoresis and Transfer

Samples were ran on a BioRad Mini-PROTEAN gel electrophoresis system. Five-µg of

protein were prepared to ensure equal loading of protein. Into each sample, 6X loading buffer

(375mM Tris-HCl, 6% SDS, 48% glyercol, 9% MeSH, 0.03% β-mercaptoethanol, pH 6.8) was

added to yield a final concentration of 1X loading buffer in the sample, and boiled for 5 min.

Samples and protein standard ladder were loaded onto their corresponding SDS

polyacrylamide gels and electrophoresed at 100 V for 1.5 h in 1x running buffer (25mM Tris,

0.1% SDS (w/v), 192 mM glycine). Following electrophoresis, proteins were transferred onto a

0.4-µm pore nitrocellulose membrane using a wet transfer system (BioRad, San Diego, CA).

Electroblotting was conducted for 2.5 h or 16 h at constant 400mA or 15V, respectively,

depending on the specific experimental protocols.

3.3.4 Immunoblotting

Following electroblotting, nitrocellulose membranes were blocked with 5% (w/v) skim milk

! 50!

powder in 0.1% TBS-T (0.5M Tris, 1.5M NaCl, pH 7.4, 0.1% Tween-20) for 1 h at room

temperature on a rotating shaker. Membranes were then rinsed once with 0.1% TBS-T and

incubated with specific primary antibodies in 5% BSA-0.01% TBS-T overnight at 4oC on

rotating shaker. Protein membranes were washed 3 times, 10 min/wash, in 0.01% TBS-T,

followed by the incubation with the respective goat α-rabbit or α-mouse HRP-conjugated IgG

antibody for 1 h at room temperature with gentle shaking. Nitrocellulose membranes were

washed 3 times, 20 min/wash, in 0.01% TBS-T. After the removal of 0.01% TBS-T, 1-mL of

enhanced-chemiluminescence solution (ThermoScientific, Waltham, MA) was added to the

membrane for 1 min. Protein expression were exposed onto a film and developed. Protein

density was determined by GS-800 Calibrated Densitometer (BioRad, San Diego, CA). All

protein band intensities were normalized by the house keeping protein, GAPDH or lamin,

developed from the same membrane.

3.4 Protein-protein interaction

3.4.1 GST-pull down assay

Four hundred-µg of HCAEC protein were incubated with 2-µg mouse recombinant reelin

(mrRLN, R&D Systems, Minneapolis, MN) and 2-µg α-His antibody (Biolegend, San Diego,

CA) for 16 hours at 4oC on a rotator. Subsequently, 30-µL of glutathione-agarose beads

(ThermoScientific, Waltham, MA) were added to the lysates and incubated for 2 hours at

room temperature on a rotator. Sample-bead solution was centrifuged at 8000 rpm

(Centrifuge 5415D) for 1 min at room temperature, and washed twice with high salt solution

(500mM NaCl, 200mM HEPES, 10& glycerol, 0.1% Triton-X 100), followed by 3 times wash

with low salt solution (150nM NaCl, 20mM HEPES, 10% glycerol, 0.1% Triton-X 100). After

the final wash and centrifuge, the supernatant was decanted, and the pellet was prepared for

! 51!

western blot. Briefly, the sample-bead solution was incubated with 50-µL of 1X loading buffer

and boiled for 5 min. The samples were centrifuged at 8000 rpm for 1 min at room

temperature, and the supernatant was collected to run on a 6% polyacrylamide gel. Following

the electrophoresis, proteins were transferred onto a nitrotrocellulose membrane for 2.5 h at

constant 400mA. The membrane was blocked and incubated with LRP8 antibody overnight at

4oC. The membrane was developed the following day with protocols as previously stated.

3.5 Reelin protein expression

3.5.1 HCAEC stimulation

Seeded HCAEC were stimulated with PBS, HNP 1- or 10-µg/mL for 6 h. Culture medium and

protein were collected as previously mentioned. Experiment was repeated to reach a sample

size n = 5.

3.5.2 P2Y6 blocked-HCAEC

To examine the specificity of HNP-induced reelin protein expression, in a separate

experiment, HCAEC were pre-treated with 0.5- or 1-µM MRS2578 (Sigma, St. Louis, MO), or

DMSO control (Sigma, St. Louis, MO) for 30 min in 37oC, followed by the 6 h stimulation of

HNP-10 µg/mL or vehicle control. Culture medium and protein were collected as previously

mentioned. Experiment was repeated to reach a sample size n = 3.

3.5.3 Measurement of reelin from culture medium and western blot

3.5.3.1 Reelin ELISA

In order to explain the mechanism by which HNP induce endothelial dysfunction, it was

important to determine the amount of reelin released from HNP-induced HCAEC. To address

! 52!

this, the presence of full-length reelin in the culture medium was determined according to a

commercially available reelin ELISA kit (USCN, China).

3.5.3.2 Reelin western blot

Reelin is a 400kDa glycoprotein that can undergo protein modification and cleavage, thereby

yielding smaller fragments of reelin [244, 246, 262]. Previous studies have determined the

LRP8 binding site of reelin to be localized in central fragment, R3-6 [262], which are found in

the full-length and 300kDa reelin fragment. Therefore, it is also crucial to determine the

expression of reelin fragments expressed in the cells and released into the culture medium

from HNP-stimulated HCAEC.

To our knowledge, there is currently no commercially available ELISA kit that can detect

specifically other fragments of reelin, therefore, to determine the specific fragments of reelin

released in response to HNP, we lyophilized the HNP-induced HCAEC culture medium and

used western blot technique to address this matter.

Reelin detected from lyophilized culture medium was prepared as follows. Two hundred and

fifty-µL of HNP-stimulated HCAEC culture medium were lyophilized (LabConco, Freeze-Dry

System 4.5) continuously at -40oC into powder form for 4 h. The powder was then

reconstituted in 100-µL of 1XPBS and prepared for western blotting. Finally, 10-µL of 6X

loading buffer was added to 50-µL of lyophilized culture medium. In experiments using cell

lysates to detect reelin, 5-µg of HNP-stimulated HCAEC protein were prepared for western

blot as previously mentioned. All samples were boiled for 3 min prior to electrophoreses.

! 53!

Once prepared, samples and protein standard ladder were loaded onto a 5% SDS

polyacrylamide gel and electrophoresed at 100V for 1.5 h. Proteins were transferred onto a

nitrocellulose membrane at 15V, 4oC for 16 h. Subsequently, the membrane was blocked for

1 h and incubated with reelin antibody overnight at 4oC on a shaker. The following day, the

membrane was washed and incubated with goat α-mouse IgG1 HRP for 1 h at room

temperature, and the protein expressions were developed onto a film as previously stated.

3.6 Endothelial dysfunction

3.6.1 HCAEC stimulation

Seeded HCAEC were stimulated with PBS, mrRLN 10- or 20-µg/mL, HNP 1- or 10-µg/mL

with/without reelin neutralizing antibody, CR-50 (MBL, Nagoya, Japan), or mouse IgG at 10-

or 20-µg/mL for 6 h. Culture medium and protein were collected as previously mentioned.

Experiment was repeated to reach a sample size n = 5. Experiment with CR-50 treatment was

repeated to reach a sample size n = 3.

3.6.2 Measurement of endothelial dysfunction

Endothelial and inducible nitric oxide synthase (eNOS and iNOS, respectively) are nitric-oxide

generating enzymes, and imbalances of the two enzymes are classical markers of endothelial

activation [23, 263-267]. To determine endothelial activation, eNOS and iNOS protein

expression were examined by western blot. Briefly, 5-µg of mrRLN-stimulated HCAEC protein

and protein standard ladder were loaded onto 8% SDS polyacrylamide gel and

electrophoresed at 100V for 1.5 h. Proteins were transferred onto a nitrocellulose membrane

at 400mA for 2.5 h. Subsequent stages of western blotting were conducted as outlined in

section 3.2.4.

! 54!

3.6.3 Measurement of nitric oxide

Nitric oxide is a potent vasodilator and marker of endothelial function. Due to its gaseous

properties, nitric oxide is commonly determined indirectly by measuring the nitrate/nitrite

concentration by the Griess Assay (Cayman Chemicals, Ann Arbor, MI). Culture medium from

HNP-stimulated HCAEC were used to determine the nitrate/nitrite concentration according to

the instructions from the commercially available colorimeter assay kit.

3.6.3.1 Measurement of reactive nitrogen species

In order to assess the consequence of nitric oxide synthase expressions, it was important to

demonstrate how NOS can influence endothelial function. To address this, the presence of

RNS was determined from HNP- and RLN-treated HCAEC protein lysates by a nitrotyrosine

ELISA (Oxis International, Foster City, CA). Nitrotyrosine concentration was then normalized

by the samples corresponding protein concentrations.

3.7 Platelet aggregation

3.7.1 Platelet-rich plasma (PRP) isolation

The following protocol was approved by the Institutional Research Ethics Research Board.

Healthy, non-smoking, non-medicated 20- to 40-year-old male volunteers were recruited for

this study. Venous blood was collected with a 21¾-gauge butterfly needle into a 3.8% sodium

citrate (1/9 v/v) vacutainer. Blood was centrifuged at 300 G for 7 min at room temperature to

obtain the PRP, followed by a centrifugation of 1000 G for 15 min at room temperature to

obtain the platelet-poor plasma (PPP). PRP was counted and diluted with PPP to a final

concentration of 2x108platelets/mL.

! 55!

While previous studies have used syringes to draw blood, we used sodium-citrate coated

vacutainers to collect blood. However, due to the shear stress from the vacutainers, platelets

may have been slightly activated during the collection. Therefore, a lower dosage of ADP was

used to induce the ‘first-wave’ of platelet aggregation.

3.7.2 Aggregation protocol

Adenosine diphosphate (ADP) is a physiological agonist of platelet aggregation, known to

induce minimal granule release at low concentrations [268], thereby inducing the ‘first-wave’

of platelet aggregation. Remarkably, HNP was has previously been shown to sustain ADP-

stimulated platelet aggregation by inducing a ‘second-wave’ of platelet aggregation mediated

by the LRP8-signaling pathway [174]. In order to differentiate and observe the first and

second wave of aggregation, we continued to utilize ADP as our platelet agonist.

Human PRP (250µL, 2x108platelets/mL) were pre-treated with 0.5µM ADP (Sigma, St. Louis,

MO) for 1 min, followed by a stimulation of PBS, 10-µg/mL mouse recombinant reelin (R&D

Systems, Minneapolis, MN), or 10-µg/mL HNP with or without 10-µg/mL CR-50. Aggregation

was evaluated by a computerized Chrono-log Aggregometer (Chrono-Log Corportation,

Havertown, PA; generously lent by Dr. Heyu Ni, St. Michael’s Hospital). Experiment was

repeated to reach a sample size n = 5. Aggregation was determined by the amount of light

passing through the cuvette containing the platelets. As platelets aggregate, more area is

created for light to pass through, which directly correlates to the percentage of aggregation.

The results are plotted as time elapsed vs. % light transmission on a computerized system. A

small magnetic stir bar, adjusted to 1000 rpm, was present to allow continued mixing of

platelets and agonist while aggregation was recorded.

! 56!

3.8 Statistical Analysis

The collected data are presented as mean±standard error of n experiments unless noted

otherwise. Statistical significance was determined using one-way ANOVA, followed by a

Tukey’s Multiple Comparison Test. A P-value less than 0.05 was considered statistically

significant. Analyses were conducted using GraphPad Prism version 5.00c (GraphPad

Software Inc., San Diego, CA).

! 57!

Chapter 4: Results

4.1 Objective #1: To examine the role of HNP-induced reelin release and reelin-LRP8

interaction.

4.1.1 Specific Aim #1: HNP induced reelin protein expression and release in HCAEC

4.1.1.1 Reelin expression by western blot

4.1.1.1.1 Reelin expression from cell lysate

A significant increase in reelin 300-kDa protein expression was observed in HCAEC

stimulated with HNP at 1- and 10-µg/mL, as determined by the band intensities from western

blots, and normalized by the corresponding loading control: lamin. HNP increased reelin 300-

kDa expression from 0.244±0.046 in vehicle control to 0.708±0.24 at 1-µg/mL and 0.846±0.24

at 10-µg/mL HNP (Figure 7A). No significant difference was observed between HNP 1- and

10-µg/mL treatment. Interestingly, endothelial stimulation with 10-µg/mL HNP for 6 h yielded a

significant decrease in full-length reelin protein expression (0.428±0.027 fold-decrease;

p<0.05 vs control). While stimulation with 1-µg/mL HNP had no significant effect, however, a

downward trend was observed (0.504±0.163 fold-decrease vs control) (Figure 7B).

4.1.1.1.2 Reelin expression from lyophilized culture medium

When quantifying the amount of reelin from culture medium of HNP 1- and 10-µg/mL

stimulated HCAEC, we observed a negative trend, but not significant, in full-length reelin

release (0.22±0.146 and 0.062±0.044, respectively, compared to control at 0.882±0.516,

p=0.22) (Figure 8A). Conversely, HNP treatment at 1- and 10-µg/mL only showed a

moderate increase in 300-kDa reelin compared to control (1.598±0.076 and 1.840±0.23,

respectively, compared to control at 1.33±0.19, p=0.266) (Figure 8A). Although not

Veh

HN

P 1

HN

P 10

Ree

lin

300

kDa

Lam

in B

67

kDa

µg/m

L

Reel

in 4

00kD

a

Veh

HN

P 1

HN

P 10

0.0

0.5

1.0

1.5

Band Intensity Fold Change

*"

Veh

HN

P 1

HN

P 10

Ree

lin

400k

Da

Lam

in B

67

kDa

µg/m

L

Figu

re 7

. HN

P in

duce

ree

lin 3

00kD

a pr

otei

n ex

pres

sion

. HC

AE

C w

ere

stim

ulat

ed fo

r 6 h

with

1- o

r 10-

µg/m

L H

NP,

or

vehi

cle

cont

rol i

n 1%

ser

um m

ediu

m. P

rote

in ly

sate

s w

ere

colle

cted

to d

eter

min

e re

elin

pro

tein

exp

ress

ion

by w

este

rn

blot

ting.

*p<

0.05

vs

cont

rol.

A"B"

58

Veh

HNP

1HN

P 10

0.0

0.5

1.0

1.5

Band Intensity(Active reelin/lamin)

* *

µg/m

L

300k

Da

Veh

HN

P 1

HN

P 10

0.0

0.5

1.0

1.5

2.0

2.5

Band Intensity

Veh

HN

P 1

HN

P 10

Ree

lin

300k

Da

µg/m

L

Cul

ture

med

ium

reel

in

Veh

HN

P 1

HN

P 10

0.0

0.1

0.2

0.3

Concentration (ng/mL)

*"

Figu

re 8

. HN

P de

crea

se r

eelin

full-

leng

th s

ecre

tion.

HC

AE

C w

ere

treat

ed w

ith H

NP

1- o

r 10-

µg/m

L, o

r veh

icle

con

trol f

or

6 h.

A. S

peci

fic fr

agm

ents

of r

eelin

rele

ased

wer

e de

term

ined

by

wes

tern

blo

tting

of l

yoph

ilize

d cu

lture

med

ium

. B. C

ultu

re

med

ium

was

col

lect

ed fo

r ana

lysi

s of

reel

in re

leas

e by

ELI

SA

. *p<

0.05

vs

cont

rol.

400k

Da

Veh

HN

P 1

HN

P 10

0.0

0.5

1.0

1.5

Band Intensity

Veh

HN

P 1

HN

P 10

µg/m

L

Ree

lin

400k

Da

B"A"

µg/m

L

59

! 60!

statistically significant, the pattern of the reelin fragments released treads in parallel with the

expression found inside the cell. It is possible that the insignificance may be due to the

degradation of reelin protein during the lyophilization procedure, which is a technical limitation

to our study. Future studies may look into the availability of specific reelin fragment antibodies

to construct a homemade ELISA kit. The specific reelin fragment antibodies could be used to

coat the ELISA plate, and non-lyophilized culture medium would be used in the experiment.

This would avoid the potential degradation of the reelin fragments during the sample

preparation phase.

4.1.1.2 Reelin expression by ELISA

To confirm the potential paracrine property of reelin, we first sought to investigate the effect of

HNP on reelin release. When HCAEC were treated with HNP, a decrease in full-length reelin

concentration in the culture medium was observed (Figure 8B). Stimulation of HNP 1- and

10-µg/mL yielded a reelin full-length concentration of 0.189±0.029- and 0.074±0.025-ng/mL

respectively. Only stimulation of HNP-10-µg/mL yielded a significant decrease in reelin

concentration when compared to vehicle control at 0.219±0.055-ng/mL (p<0.05).

4.1.2 Specific Aim #2: HNP-induced reelin protein expression and release was

mediated by the P2Y6-signaling pathway

To confirm that reelin was indeed induced by HNP stimulation, we examined reelin’s

expression in HCAEC blocked of P2Y6 but stimulated with HNP. P2Y6-blockade was

performed by pre-treating HCAEC with an irreversible P2Y6 antagonist, MRS2578, for 30 min

prior to the stimulation of HNP for 6 h. Like previously, the presence of 10-µg/mL HNP in

HCAEC significantly increased the expression of reelin 300-kDa compared to PBS conditions

! 61!

(0.641±0.45 and 0.135±0.112, respectively; p<0.05). The observed response was significantly

attenuated when HCAEC were pre-treated with MRS 2578 at 0.5-µM and 1-µM (0.038±0.035

and 0.174±0.098, respectively; p<0.05), even in the presence of HNP 10-µg/mL (Figure 9A).

MRS 0.5- and 1-µM stimulation alone had no significant effect in inducing reelin 300kDa

protein expression (0.307±0.134 and 0.330±0.097, respectively, vs control at 0.098±0.057).

We then evaluated if pre-treatment of MRS 1-µM was able to recover the diminished full-

length reelin release upon HNP stimulation. Indeed, the pre-treatment of MRS 1-µM

demonstrated a significant increase in full-length reelin compared to stimulation of HNP alone

(1.66±1.39 vs 0.311±0.11, respectively; p<0.05) (Figure 9B). These results suggest that P2Y6

mediated HNP-induced reelin expression in HCAEC. Future studies would need to confirm

the role of P2Y6 in mediating the release of reelin by determining the reelin content in the

culture medium of HNP-treated HCAEC.

4.2 Objective #2: To determine the specific effect of reelin in endothelial function and

platelet aggregation.

Endothelial dysfunction and platelet aggregation are paradigm events in atherogenesis. Given

the notion that HNP was able to induce reelin protein expression and release, we sought to

explain the functional role of reelin in mediating endothelial dysfunction and platelet

aggregation. Endothelial activation and dysfunction was assessed by measuring the protein

expression of nitric-oxide (NO)-producing enzymes, endothelial and inducible nitric oxide

synthase (eNOS and iNOS, respectively), and NO content [23-25, 65, 264, 269, 270]. PRP

was used in platelet aggregation experiment in order to characterize a more physiological

condition, whereby plasma proteins are present to also participate in the platelet aggregation.

Figu

re 9

. HN

P-in

duce

d re

elin

exp

ress

ion

is m

edia

ted

by P

2Y6-

sign

alin

g pa

thw

ay. H

CA

EC

see

ded

at 1

x105

cells

/mL

wer

e pr

e-tre

ated

in 1

% F

BS

med

ium

with

MR

S25

78 a

t 0.5

- or 1

-µM

, or D

MS

O c

ontro

l 30

min

prio

r to

the

addi

tion

of H

NP

10-µ

g/m

L or

PB

S c

ontro

l for

6 h

. A. R

eelin

300

kDa

was

qua

ntifi

ed b

y w

este

rn b

lot f

rom

pro

tein

lysa

te. B

. Cul

ture

med

ium

w

ere

colle

cted

for a

naly

sis

of fu

ll-le

ngth

reel

in b

y E

LIS

A. *

p< 0

.05 vs

con

trol;

§<0.

05 vs

HN

P 10

-µg/

mL.

A"B"

Ree

lin

300

kDa

Lam

in B

67

kDa

62

PBS DMSO PBS +

DMSOMRS 0.

5µM MRS 1µM

HNP 10µg

/mL

MRS 0.5µ

M + HNP 10

µg/m

L

MRS1µM +

HNP 10µg

/mL

0.0

0.5

1.0

1.5

Band Intensity(Active reelin/lamin)

*

§ §

PBS DMSO PBS + DMSO

MRS 1µM HNP 10

µg/m

L

MRS 1µM +

HNP 10µg

/mL

01234

Native reelinFold Change

§

*

! 63!

4.2.1 Specific Aim #1: Reelin attenuated eNOS and increased iNOS protein expression

4.2.1.1 HNP-induced endothelial activation (proof-of-concept)

Before we could measure the impact of reelin in modulating NOS expression, we had to

validate the role of HNP in modulating NOS protein expression. Application of 10-µg/mL HNP

was found to significantly decrease eNOS protein expression in HCAEC following a 6 h

stimulation (0.045±0.01 vs 0.172±0.055 in control; p<0.05). HNP 1-µg/mL showed a

downward trend in eNOS protein expression, however, it was not significant compared to

control (0.062±0.0173 vs 0.172±0.055; p=0.16) (Figure 10A). Conversely, endothelial

stimulation with 10-µg/mL HNP demonstrated a significant increase in iNOS expression

compared to control cells (0.169±0.034 vs 0.066±0.016; p<0.05) (Figure 10B). Similar to the

eNOS response, HCAEC stimulated with HNP 1-µg/mL for 6 h did not yield a significant

increase in iNOS expression (0.078±0.041 vs 0.066±0.016; p=0.75).

4.2.1.2 Reelin-induced endothelial activation

We evaluated the effect of reelin on NOS protein expressions. Stimulation of HCAEC with

mouse recombinant reelin (mrRLN) at 1- and 5-µg/mL for 6 h resulted in a significant

decrease in eNOS expression (0.098±0.031 and 0.13±0.037, respectively, vs control at

0.376±0.096; p<0.05) (Figure 11A). Such response due to reelin was comparable to HNP 10-

µg/mL stimulation at 0.099±0.036. Correspondingly, parallel to HNP 10-µg/mL stimulation,

treatment of mrRLN 5-µg/mL in HCAEC revealed a significant increase in iNOS expression

from 0.041±0.008 in control cells to 0.097±0.024 (p<0.05) (Figure 11B). Stimulation with

mrRLN at 1-µg/mL had no effect on iNOS expression (0.04±0.003 vs control). The

Veh

HNP

1HN

P 10

0.0

0.1

0.2

0.3

Band Intensity(eNOS/GAPDH)

*

Veh

HN

P 1

HN

P 10

eNO

S 13

0 kD

a

GA

PDH

37

kD

a

µg/m

L Ve

h H

NP

1 H

NP

10

iNO

S 13

0 kD

a

GA

PDH

37

kD

a

µg/m

L

Figu

re 1

0. H

NP

atte

nuat

es e

NO

S, w

hile

aug

men

ting

iNO

S, p

rote

in e

xpre

ssio

n. H

CA

EC

see

ded

at 1

x105

cells

/mL

wer

e st

imul

ated

in 1

% s

erum

med

ium

with

veh

icle

con

trol,

or H

NP

at 1

- or 1

0-µg

/mL

for 6

h p

rior t

o th

e co

llect

ion

of p

rote

in

lysa

tes.

Pro

tein

exp

ress

ion

of e

NO

S (A

) and

iNO

S (B

) wer

e m

easu

red

by w

este

rn b

lots

and

ana

lyze

d by

Bio

Rad

GS

-800

D

ensi

tom

eter

. *p<

0.05

vs

cont

rol.

A"B"

64

Veh

HN

P 1

HN

P 10

0.00

0.05

0.10

0.15

0.20

0.25

Band Intensity(iNOS/GAPDH)

*

µg/m

L µg

/mL

Veh

RLN

1R

LN 5

HN

P 10

0.0

0.1

0.2

0.3

0.4

0.5

Band Intensity(eNOS/GAPDH)

*

Veh

RLN

1

HN

P 10

eNO

S 13

0kD

a iN

OS

130k

Da

RLN

5

Veh

RLN

1

HN

P 10

R

LN 5

µg

/mL

µg/m

L

µg/m

L

GA

PDH

37

kDa

GA

PDH

37

kDa

Figu

re 1

1. R

eelin

redu

ces

eNO

S, a

nd e

nhan

ces

iNO

S pr

otei

n ex

pres

sion

in H

CA

EC. H

CA

EC

wer

e st

imul

ated

in 1

%

seru

m m

ediu

m w

ith v

ehic

le c

ontro

l, or

mou

se re

com

bina

nt re

elin

at 5

- or 1

0-µg

/mL

for 6

h p

rior t

o th

e co

llect

ion

of p

rote

in

lysa

tes.

Pro

tein

exp

ress

ion

of e

NO

S (A

) and

iNO

S (B

) wer

e qu

antif

ied

by w

este

rn b

lots

, and

ban

d in

tens

ities

wer

e an

alyz

ed

by th

e B

ioR

ad G

S-8

00 D

ensi

tom

eter

. *p<

0.05

vs

cont

rol.

A"B"

65

Veh

RLN

1R

LN 5

HN

P 10

0.00

0.05

0.10

0.15

0.20

0.25

Band Intensity(iNOS/GAPDH)

*

*

µg/m

L

! 66!

comparable responses of both HNP and mrRLN serve to suggest that RLN mediates HNP-

induced NOS protein expression. However, the specificity of reelin must still be proven.

The variance observed in eNOS response after stimulation of HNP 10-µg/mL in the proof-of-

concept experiment vs reelin experiment may be accountable by technicalities of the

experiment including different lot numbers of eNOS antibody used in the western blot and

passages of endothelial cells used.

4.2.2 Specific Aim #2: Reelin induced reactive nitrogen species production

In order to address the consequence of RLN-induced eNOS decrease and iNOS increase, we

measured the effects of RLN on peroxynitrite formation by measuring nitrotyrosine

concentration.

4.2.2.1 HNP-induced nitrosative stress (proof-of-concept)

Like previously, we first validated the nitrogen species concentration in HNP-induced HCAEC.

A significant increase in nitrate/nitrite concentration was observed in HCAEC stimulated with

10-µg/mL HNP vs control (14.2±1.68-µM vs 2.05±0.17-µM; p<0.05), but not with 1-µg/mL

HNP (3.18±0.17-µM) (Figure 12A). Compatibly, nitrotyrosine formation also revealed a

similar trend, with a significant increase from 71.33±8.83-nmol/g protein in control to

174.9±23.8- and 183±34.2-nmol/g protein in HNP 1- and 10-µg/mL, respectively (Figure

12B).

Nitr

ate/

Nitr

ite L

evel

Veh

HN

P 1

HN

P 10

05101520

Concentration (uM)

*"

Nitr

otyr

osin

e

Veh

HN

P 1

HN

P 10

050100

150

200

250

Nitrotyrosine (nmol/g protein)

*"*"

µg/m

L

Figu

re 1

2. H

NP

indu

ces

prod

uctio

n of

nitr

ogen

ous

spec

ies.

1x1

05ce

ll/m

L H

CA

EC

wer

e in

cuba

ted

with

1- o

r 10-

µg/m

L H

NP,

or v

ehic

le c

ontro

l for

6 h

. A. C

ultu

re m

ediu

m w

ere

anal

yzed

for p

rodu

ctio

n of

nitr

ate/

nitri

te, r

efle

ctin

g th

e pr

esen

ce o

f ni

tric

oxid

e. B

. Cel

l lys

ates

wer

e co

llect

ed fo

r ana

lysi

s of

nitr

otyr

osin

e, in

dica

ting

the

pres

ence

of O

NO

O- .

*p<0

.05 vs

co

ntro

l.

µg/m

L

A"B"

67

! 68!

4.2.2.2 Reelin-induced nitrosative stress

We quantified the amount of nitrotyrosine concentration in RLN-stimulated HCAEC. RLN

stimulation revealed a significant increase in nitrotyrosine production from 63.5±3.64-nmol/g

protein in vehicle to 158.5±8.7- and 191.2±21.1-nmol/g protein with RLN at 1- and 5-µg/mL,

respectively (Figure 13).

4.2.3 Specific Aim #3: Reelin enhanced human platelet aggregation in response to ADP

To preserve the physiological condition of platelet aggregation, platelet-rich plasma (PRP)

was used to perform the aggregation experiments. After incubating human PRP with 0.5-µM

ADP for 1 min, PRP elicited the ‘first-wave’ of platelet aggregation that was not sustained over

the 12 min of recording time (Figure 14). In addition, no difference in final platelet aggregation

was observed between incubation of PRP with ADP alone and ADP with vehicle control

(4.207±2.43% vs 4.817±1.69%; p=0.804) (Figure 14). Interestingly, after 1 min of ADP

priming, HNP 10-µg/mL generated a second wave of platelet aggregation and significantly

prolonged the aggregation response of ADP, with an end aggregation of 50.871±14.38%

(Figure 14). Subsequently, we evaluated the effect of reelin on platelet aggregation.

Comparable to HNP, reelin at 5-µg/mL also prolonged the transient aggregation response of

human platelets to ADP with an end aggregation of 37.683±15.52% (Figure 14).

Finally, the ADP concentration chosen was based on our dose-dependent ADP curve for

human PRP (data not shown). We have found that 0.5-µM ADP to be the minimal possible

concentration to provoke the ‘first-wave’, but not ‘second-wave’, of platelet aggregation in

human PRP. Although previous studies have used higher doses of ADP to generate the ‘first-

Nitr

otyr

osin

e

Veh

RLN

1R

LN 5

HN

P 10

050100

150

200

250

Nitrotyrosine (nmol/g protein)

*"

µg/m

L

Figu

re 1

3. R

eelin

indu

ce n

itrot

yros

ine

prod

uctio

n. F

ollo

win

g th

e tre

atm

ent o

f HC

AE

C w

ith H

NP

10-µ

g/m

L, re

elin

1- o

r 5-

µg/m

L, o

r veh

icle

con

trol f

or 6

h, c

ell l

ysat

es w

ere

anal

yzed

for t

he p

rodu

ctio

n of

nitr

otyr

osin

e by

ELI

SA

. *p<

0.05

vs

cont

rol

69

Plat

elet

Agg

rega

tion

ADPADP +

Veh

ADP + RLN

5µg/m

L

ADP + HNP 10

µg/m

L

ADP+HNP 10

µg/m

L+CR50

10 µg

/mL

020406080

% Light Transmission

ADP$

+$Ve

h$+$RLN$(5

$μg/mL)$

+$HN

P$$

(10$μg

/mL)$

+$HN

P$(10$μg

/mL)$+$

CR50$(1

0$μg

/mL)$

Figu

re 1

4. P

late

let a

ggre

gatio

n is

pro

long

ed in

pla

tele

ts s

timul

ated

with

AD

P a

nd r

eelin

in c

ompa

riso

n to

st

imul

atio

n w

ith A

DP

alo

ne. A

. Hum

an P

RP

wer

e pr

imed

with

0.5

-µM

AD

P fo

r 1 m

in, f

ollo

wed

by

the

addi

tion

of H

NP

10-

µg/m

L w

ith o

r with

out r

eelin

neu

traliz

ing

antib

ody

(CR

50)

10-

µg/m

L, o

r rec

ombi

nant

reel

in a

t 5-µ

g/m

L. P

late

let a

ggre

gatio

n w

as re

cord

ed fo

r 12

min

with

the

Chr

onol

og A

ggre

gom

eter

. B. A

fter 1

2 m

in o

f rec

ordi

ng, t

he fi

nal a

ggre

gatio

n of

pla

tele

ts

wer

e co

mpa

red

acro

ss g

roup

s. *

p<0.

05 vs

cont

rol;

§p<0

.05 vs

HN

P 10

-µg/

mL.

A"

B"

70

*$*$

§$

! 71!

wave’ of platelet aggregation [174], however, this may be explained by the differences in PRP

isolation protocols.

In summary, like HNP, reelin was able to prolong the platelet aggregation response ADP, that

is not present in ADP stimulation alone.

4.2.4 Specific Aim #4: Neutralized reelin (CR50) alleviated endothelial dysfunction and

platelet aggregation.

To confirm the specificity of reelin in HNP-induced endothelial dysfunction and platelet

aggregation, an inhibition experiment was performed whereby reelin neutralizing antibody

(CR50) was co-treated with HNP 10-µg/mL. CR50 antibody inhibits reelin function by

interrupting the crucial formation of the reelin-reelin homopolymer complex that is required in

activating disabled-1 in LRP8 [252].

4.2.4.1 CR50 increase eNOS and decrease iNOS protein expression

HCAEC treated with HNP 10-µg/mL in the presence of 10- or 20-µg/mL CR50 for 6 h

exhibited a significant recovery of eNOS compared to HNP alone. As reported previously,

HNP 10-µg/mL was able to induce a significant decrease in eNOS protein expression

(0.068±0.01 vs 0.183±0.02, respectively; p<0.05). Remarkably, the presence of reelin

neutralizing antibody (CR50) at 10- and 20-µg/mL was able to block the HNP-induced eNOS

attenuation (0.201±0.06 and 0.298±0.03, respectively; p<0.05). The neutralizing effort of the

antibody was confirmed when mouse IgG controls at the same dosage revealed a response

comparable to stimulation of HNP 10-µg/mL alone (0.094±0.029 at 10-µg/mL and 0.064±0.03

at 20-µg/mL of mouse IgG) (Figure 15A).

! 72!

Congruently, HCAEC treated with CR50 at 10- and 20-µg/mL for 6 h also rescued the HNP-

mediated iNOS elevation. Consistently, endothelial cells treated with HNP 10-µg/mL

enhanced iNOS protein expression compared to control (0.258±0.06 vs 0.111±0.03,

respectively; p<0.05) (Figure 15B). Moreover, a significant reduction in iNOS protein

expression was observed in HCAEC treated with HNP 10-µg/mL in the presence of CR50

(0.12±0.01 at 10-µg/mL and 0.042±0.005 at 20-µg/mL; p<0.05). Once again, the neutralizing

effort of CR50 was supported by the increase in iNOS expression in cells treated with HNP

10-µg/mL in the presence of mouse IgG 10- and 20-µg/mL for 6 h. Although only incubation of

10-µg/mL (0.378±0.1), not 20-µg/mL (0.31±0.2), mouse IgG control and HNP 10-µg/mL

resulted in a significant increase in iNOS compared to vehicle, however, this may be justified

by the low sample size (n=3). Additional repeats of the experiment may off set the presence

of outliers.

4.2.4.2 CR50 attenuates HNP-induced nitrotyrosine concentration

The effect of CR50 in HNP-stimulated HCAEC was further supported by the nitrotyrosine

concentration. As expected, HNP induced a significant increase in nitrotyrosine concentration

from 60.5±5.6-nmol/g protein in control to 116.3±0.3-nmol/g protein (p<0.05). Interestingly,

HCAEC stimulated at HNP 10-µg/mL with CR50 10- or 20-µg/mL resulted in a significant

decrease in nitrotyrosine concentration to 61.67±9.66- and 51.9±18.6-nmol/g protein,

respectively. Compatibly, co-stimulation of HNP 10-µg/mL with 10- or 20-µg/mL mouse IgG

control resulted in nitrotyrosine concentrations comparable to HNP alone (117.9±10.9- and

128.77±6.8-nmol/g protein, respectively) (Figure 16).

Veh HNP 10µg

/mL

HNP10µg

/mL +

CR50

10µg

/mL

HNP10µg

/mL +

CR50

20µg

/mL

HNP10µg

/mL +

IgG 10

µg/m

L

HNP10µg

/mL +

IgG 20

µg/m

L

0.0

0.2

0.4

0.6

Band Intensity(iNOS/GAPDH)

*

*

§ §

Veh HNP 10µg

/mL

HNP10µg

/mL +

CR50

10µg

/mL

HNP10µg

/mL +

CR50

20µg

/mL

HNP10µg

/mL +

IgG 10

µg/m

L

HNP10µg

/mL +

IgG 20

µg/m

L

0.0

0.1

0.2

0.3

0.4

Band Intensity(eNOS/GAPDH)

* *

§

§

eNO

S 13

0kD

a

GA

PDH

37

kDa

Figu

re 1

5. R

eelin

neu

tral

izin

g an

tibod

y al

levi

ates

HN

P-in

duce

d eN

OS

decr

ease

and

iNO

S in

crea

se. H

CA

EC

wer

e st

imul

atio

n w

ith v

ehic

le c

ontro

l, or

HN

P 10

-µg/

mL

in th

e pr

esen

ce o

f CR

50 o

r mou

se Ig

G c

ontro

l at 1

0- o

r 20-

µg/m

L fo

r 6 h

. P

rote

in ly

sate

s w

ere

colle

cted

for t

he a

naly

sis

of A

. eN

OS

and

B. i

NO

S p

rote

in e

xpre

ssio

n by

wes

tern

blo

t. *p

<0.0

5 vs

co

ntro

l, §p

<0.0

5 vs

HN

P 10

-µg/

mL.

iNO

S 13

0kD

a

GA

PDH

37

kDa

73

Figu

re 1

6. R

eelin

neu

tral

izin

g an

tibod

y re

duce

s H

NP-

indu

ced

nitr

otyr

osin

e pr

oduc

tion.

HC

AE

C w

ere

stim

ulat

ed w

ith

vehi

cle,

or H

NP

10-µ

g/m

L w

ith C

R50

or m

ouse

IgG

con

trol a

t 10-

or 2

0-µg

/mL

for 6

h. P

rote

in ly

sate

s w

ere

colle

cted

for t

he

quan

tific

atio

n of

nitr

otyr

osin

e. *

p<0.

05 v

s co

ntro

l; §p

<0.0

5 vs

HN

P 10

-µg/

mL.

Nitr

otyr

osin

e

Veh HNP10µg

/mL

HNP10µg

/mL +

CR50

10µg

/mL

HNP10µg

/mL +

CR50

20 µg

/mL

HNP10µg

/mL +

IgG 10

µg/m

L

HNP10µg

/mL +

IgG 20

µ

050100

150

Nitrotyrosine (nmol/g protein)

*"

§"

*"*"

§"

74

! 75!

4.2.4.3 CR50 attenuates HNP-induced platelet aggregation

The inhibitory effect of CR50 was evaluated in platelet aggregation. Human PRP was primed

with 0.5µM ADP for 1 min prior to the co-stimulation of HNP 10-µg/mL and CR50 10-µg/mL.

Compared to HNP alone, the presence of CR50 significantly reduced the end aggregation

from 50.87±14.4% to 29.16±12.2% (p<0.05) (Figure 14). Although the decrease in

aggregation elicited by CR50 at 10-µg/mL was not significantly different from the response

elicited by reelin at 5-µg/mL, however, a dose-dependent curve with increasing CR50

concentration may address this concern.

4.3 Result summary

In summary, we have shown that HNP induces reelin protein expression and release in

HCAEC. The effect of reelin was specific to HNP since the blocking of P2Y6-receptor

attenuated the HNP-induced reelin expression. Reelin was also shown to attenuate eNOS

and elevate iNOS protein expression in HCAEC. This was consequently found to increase

nitrotyrosine concentrations. Moreover, in platelets, reelin was shown to induce a second-

wave of platelet aggregation following ADP priming. The effect of reelin in inducing

endothelial activation and platelet aggregation was significantly attenuated when cells were

treated with a reelin-neutralizing antibody (CR50).

! 76!

Chapter 5: Discussion

5.1 Main findings

In this study, we have demonstrated a novel mechanism of HNP inducing endothelial

activation and platelet aggregation mediated through an LRP8 ligand, reelin. HNP was shown

to activate the purinergic receptor, P2Y6, on human coronary artery endothelial cells

(HCAEC), and induce endothelial-derived reelin expression and release. Consequently, reelin

down-regulated eNOS and up-regulated iNOS protein expression in HCAEC. This was

paralleled with an observed increase in nitrotyrosine content, suggesting the occurrence of

nitrosative stress in the endothelial cells. In isolated human platelets, reelin was able to

sustain ADP-mediated platelet aggregation by inducing a second wave of platelet

aggregation. Lastly, to confirm the specificity of reelin, the functional activity of reelin was

blocked using a reelin neutralizing antibody: CR50. Indeed, in the presence of CR50, HNP-

induced endothelial dysfunction and platelet aggregation was significantly attenuated.

5.2 Potential mechanism of HNP-induced reelin expression and release

We have demonstrated the ability of HNP to induce a significant increase in reelin 300-kDa,

and a decrease in reelin 400-kDa protein expression in HCAEC. Reelin is a large extracellular

matrix protein that exists in a multitude of fragments after proteolytic cleavage by tPA or

matrix metalloproteinase [244-248, 250, 262]. Correspondingly, studies have determined that

it is the central-containing segment that binds and activates the receptor LRP8 [244, 245,

262], with the N-terminus responsible for forming the compulsory functional reelin homodimer

[245-248, 250, 271]. Therefore, to exert the reelin signaling pathway, much attention is

directed to focus on full-length reelin and reelin fragment 300-kDa. Previous studies have

already documented the overexpression of reelin 300-kDa in patients with liver cirrhosis [257]

and ocular injury [258], suggesting the involvement of reelin 300-kDa in mediating

! 77!

inflammatory events. However, the mechanism by which HNP induces reelin expression

requires additional analysis.

5.2.1 HNP mediates reelin expression

In our study, we have shown that upon HNP stimulation, HCAEC up-regulates the reelin

protein expression, specifically the reelin 300-kDa fragment. However, the mechanism of

HNP-induced reelin expression remained to be further examined. Over the last 10 years,

research has been conducted to determine the various different extracellular signal molecules

that could induce reelin expression. Among them are TGF-β, thyroid hormone, retinoic acid,

β-amyloid, corticosterone, and 17β-estradiol [272-277]. Interestingly, even injury to the adult

mice brain would elicit an increase in reelin expression [278]. The common ground of this

induction is the epigenetic regulation of the reelin promoter. In a study conducted by Chen et

al, it was shown that reelin expression was dependent on the methylation status of its

promoter. Using the methylation inhibitor, aza-2’-deoxycytidine, reelin mRNA was shown to

exhibit a 60-fold increase [279]. Similarly, the use of histone deacetylase inhibitors

(trichostatin A) and valproic acid also induced the expression of the endogenous reelinsw

promoter [279]. Moreover, the start site of the human reelin RNA is very GC-rich (75%) and

forms large CpG islands, a genomic region with high frequencies of cytosine and guanine

linked by a phosphodiester bond [279]. This provides the notion that the reelin promoter is

epigenetically regulated, specifically through changes in the DNA cytosine methylation [280-

283]. Unfortunately, currently there is a deficiency of knowledge of the relationship between

HNP or P2Y6 in regulating histone deacetylase. In addition to histone deacetylases, future

studies may focus on the relationship between HNP or P2Y6 and transcriptional regulators

such as Sp1 and Pax6. In a study conducted by Chen et al in 2007, the group found that Sp1

! 78!

and Pax6 was able to bind to the enhancer site of reelin to induce an increase expression of

reelin mRNA [274].

Alternatively, the reelin promoter has four potential sites for transcriptional regulation, one of

which is NF-κB [284]. Interestingly, P2Y6 has previously been implicated to activate the NF-

κB signaling pathway [182, 285, 286]. This may provide an alternative mechanism through

which HNP-activated P2Y6 may govern the expression of reelin.

5.2.2 HNP mediates reelin processing

As mentioned previously, reelin can undergo proteolytic cleavage by tPA or matrix

metalloproteinase [244-248, 250, 262, 287]. Therefore, a potential mechanism of elevating

reelin 300-kDa is by increasing the cleavage of the full-length molecule. This can be

potentially accomplished by: 1) HNP directly cleaving reelin; or 2) HNP increases the

expression and/or activity of tPA or matrix metalloproteinase. Of the two possibilities, the

former mechanism may be of least likelihood. Unlike their sister family, the serine protease

serprocidins, HNP lacks enzymatic activity [288, 289], which renders them unable to conduct

proteolytic cleavage on other molecules. Furthermore, reelin’s proteolytic processing occurs

at the N- and C-terminus, which recent studies have shown to have specific requirements.

Proteases involved in the N-terminus cleavage of reelin require a zinc ion for its catalytic

activity, contains a furin-like proprotein convertase activity, and has a high binding affinity for

heparin [246]. HNP, once released into the extracellular milieu upon neutrophil activation, is

an active molecule that does not require a co-factor for its proper function. Also, due to the

high basicity of reelin’s C-terminus [279], it is likely that HNP’s cationic charge would be

electrostatically repelled from the cleavage site. Collectively, this would strongly suggest that

! 79!

HNP may mediate reelin processing indirectly, perhaps by increasing the expression or

activity of tPA or matrix metalloproteinase.

Indeed, in a study conducted by Higazi et al in 1996, the group showed that HNP was able to

modulate tPA binding to fibrin and endothelial cells [290], suggesting HNP could alter the

plasmin activity through a direct interaction [260]. Moreover, in a study that was recently

published by Ahn et al, the group should that HNP-1 regulated the production of MMPs, a

process that was dependent on the regulation of the JNK, ERK and NF-κB pathway [291].

Therefore, HNP-stimulated HCAEC could have triggered a signaling cascade that resulted in

an increase in protease expression that was responsible for the increase in reelin processing.

Although it is important to recognize that HNP stimulation of HCAEC induced an increase in

reelin 300-kDa, however, our experiments conducted could not differentiate if the observed

increase was to due: a) an increase in proteolytic activity on basal level of full-length reelin; or

b) an increase in full-length reelin expression that was subsequently subjected to processing

to generate the large pool of reelin 300-kDa fragment; or c) a concurrent experience of both a

and b. Further research should be performed to elucidate the mechanism governing the

downstream pathways affecting reelin expression and processing. Such studies may include

analyzing the mRNA content of reelin using qRT-PCR after HNP stimulation.

5.2.3 HNP mediates reelin release

The role of reelin acting as a secretory molecule was chiefly established by their localization

in the golgi apparatus and secretory organelles [292, 293]. In addition to neuronal cells,

secretion of reelin has also been found in lymphatic endothelial cells and liver [255, 256].

However, the mechanism underlying the release is unknown.

! 80!

In our study, we have shown that HNP was able to induce HCAEC reelin release, since the

presence of a reelin neutralizing antibody (CR50) in the culture medium was able to inhibit

HNP-mediated eNOS attenuation and iNOS elevation. This phenomenon then raises the

question of what is the relationship between HNP and secretory organelles? In a study by

Befus et al, the group showed that HNP was able to induce histamine release in mast cells

that was dependent on a G-protein coupled receptor [294]. Likewise, our lab demonstrated

that HNP binds to the purinergic G-protein coupled receptor, P2Y6, on human lung epithelial

cells to induce IL-8 expression and release [185]. Correspondingly, by knocking down P2Y6,

HNP-mediated IL-8 expression was significantly attenuated [185]. These provide an insight in

the role of purinergic receptors in shuttling secretory organelles to the plasma membrane and

facilitate exocytosis of granular content.

Certainly, P2Y6-signaling has previously been shown to activate PLC to increase intracellular

concentrations of DAG and IP3 in pancreatic β-cells [295]. The resultant increase in Ca2+

content enabled the shuttling of insulin into the extracellular milieu [295]. The importance of

elevating Ca2+ ions to mediate P2Y6-dependent exocytosis was further supported in a study

conducted by Kottgen et al. The group shown that P2Y6 receptor in colonic epithelial cells

stimulated NaCl secretion by a synergistic increase of intracellular Ca2+ and cAMP [296]. It is

therefore possible that secretion of reelin into the extracellular milieu is reliant on the P2Y6

activated Ca2+-dependent exocytosis.

5.3 Reelin-induced endothelial activation

5.3.1 HNP-induced endothelial activation

! 81!

Disruption of vascular integrity is a primitive event observed in early atherosclerosis [1-3, 67,

297]. Under a quiescent state, endothelial cells seldom attract leukocytes, however, upon

endothelial activation, expression of surface adhesion molecules provide a stepping stone for

leukocyte recruitment [23, 298-300]. These surface adhesion molecules include ICAM-1 and

VCAM-1. To elucidate the role of HNP in inducing endothelial activation, our lab has

previously demonstrated that stimulation of HCAEC with HNP elicited an increase in VCAM-1

expression [174]. In addition to endothelial activation, our group has also shown that HNP

was able to induce epithelial activation. Using SAEC and A549 epithelial cells, stimulation of

HNP induced a 2- and 3-fold increase in ICAM-1 expression in SAEC and A549, respectively

[162]. These provide support for the role of HNP in inducing cell activation.

5.3.1.1 HNP-mediated eNOS and iNOS protein expression

Whilst the expression of surface adhesion molecules, another commonly observed activity

during endothelial activation is the attenuation of endothelial nitric oxide synthase (eNOS)

protein expression, the hallmark enzyme that governs basal regulatory function in the

vasculature [301]. Consequentially, the cell begins to encounter a diminished pool of nitric

oxide, but an elevation of reactive oxygen or nitrogen species. This activation of the

endothelium eventually leads to the condition endothelial dysfunction. Endothelial dysfunction

is frequently shown to exhibit an attenuation of eNOS and an elevation of iNOS protein

expression [302-304].

Our study has shown that in the presence of HNP, HCAEC responds with a decrease in

eNOS and an increase in iNOS protein expression. These results are in congruency with

results published by Kougias et al in 2006, whereby it was shown that HNP stimulation on

! 82!

porcine coronary arteries elicited a decrease in eNOS mRNA [201]. Consequently, the

endothelium-dependent vasorelaxation was attenuated [201].

Concurrently, we also observed that HNP-stimulated HCAEC had a profound increase in

nitrotyrosine content, a resultant product from the nitrosylation of proteins by the reactive

nitrogen species, peroxynitrite. The source of the NO that drove the synthesis of peroxynitrite

is believed to be from the increased expression of iNOS. When we quantified the NO content

indirectly by measuring the nitrate/nitrite concentration through the Griess assay, we found

that HNP induced a significant increase in nitrate/nitrite concentration. Since eNOS protein

expression was significantly attenuated upon HNP stimulation, it would suggest that iNOS

would be the major contributor of NO, and eventually, peroxynitrite. iNOS has previously been

shown to induce peroxynitrite production which consequently induced myocardial contractile

failure [305]. Moreover, the rise in peroxynitrite could provide a positive feedback loop to

further increase iNOS protein expression through the activation of NF-κB [306], therefore,

supplementing endothelial activation. Akin to our findings, Kougias et al also found that

stimulation of porcine coronary arteries with HNP also induced an increase in superoxide

anion concentration [201], a trait indicative of oxidative stress and endothelial activation. Now,

the molecular mechanism of HNP-induced endothelial activation has been weakly explored.

However, our study may provide insights on the mechanism, through the signaling pathway of

reelin.

5.3.2 Reelin and NOS

5.3.2.1 Use of mouse recombinant reelin on human cells

To address the molecular mechanism of HNP-induced endothelial activation, we employed a

recombinant reelin to stimulate HCAEC. Prior to discussing the results of the reelin-stimulated

! 83!

HCAEC, I would like to address the matter regarding the use of a mouse recombinant protein

on human cells: 1) although we conducted an interspecies stimulation, however, when we ran

a BLAST search to compare the protein sequence of reelin between human and mouse, it

revealed a 98% homology shared between the two species [Figure 17]; 2) studies have

shown that the human and mouse cDNAs of reelin share an 88% nucleotide identity as well

as a high degree of sequence similarity on the transcription start site [249, 279], and 3) in

consistent with a recently published article by Lutter et al, the group utilized the same

recombinant protein to induce MCP-1 production in primary human lymphatic endothelial cells

[255]. This suggests the functional activity of the recombinant protein to elicit the activation of

appropriate signaling pathways in endothelial cells to mediate an inflammatory response.

Nevertheless, the responses provoked by the mouse recombinant reelin protein in our

experiments should be confirmed with a human recombinant reelin protein.

5.3.2.2 Reelin and NOS

We have shown that stimulation of HCAEC with mouse recombinant reelin exhibited a

comparable response to HNP. Like HNP, mouse recombinant reelin down-regulated eNOS

and up-regulated iNOS protein expression. Moreover, the nitrotyrosine concentration was

also significantly elevated by treatment of reelin in comparison to vehicle control. The

specificity of reelin was confirmed with the use of the reelin neutralizing antibody (CR50). To

our amazement, the presence of CR50 significantly attenuated HNP-induced endothelial

activation.

Due to the prominent expression of reelin in the brain, attention has been more focused on

the relationship between reelin and neuronal NOS (nNOS). nNOS is a constitutively

expressed nitric oxide synthase, with structural and functional similarities to eNOS [41].

Figu

re 1

7. M

ouse

ree

lin is

95%

hom

olog

ous

to h

uman

ree

lin. A

pro

tein

seq

uenc

e bl

ast s

earc

h w

as c

ondu

cted

to

dete

rmin

e th

e pe

rcen

tage

hom

olog

y be

twee

n m

ouse

reel

in a

nd h

uman

reel

in.

84

! 85!

Nevertheless, the relationship between reelin and nNOS may provide us with insight to the

relationship between reelin and eNOS, and iNOS. In a study conducted by Romay-Tallon et

al, reelin was found to be co-expressed with nNOS in the dentate gyrus neuron of mice [307].

Moreover, in a study by Herrman et al, it was shown that reeler mice have an attenuated

nNOS protein expression in the olfactory bulb [308]. This suggests the potential role of reelin

in modulating NOS protein expression. Unfortunately, further study is necessary to elaborate

on the mechanism in which reelin can modulate NOS protein expression.

5.3.2.3 Reelin-mediated endothelial activation through its receptors

Currently, there is a deficiency of knowledge on how reelin may directly modulate the

expression of eNOS and iNOS. However, this does not negate the possibility of reelin

indirectly modulating the NOS expression through the activation of reelin receptors. Recently,

much attention has been focused on the role of LRP8 in mediating the development of

atherosclerosis. Interestingly, LRP8 is a conventional reelin receptor.

In a crucial study conducted by Ramesh et al, the group showed that activation of the LRP8

receptor in vivo yielded a significant increase in leukocyte recruitment to the vascular

endothelium [230]. It was later found that LRP8 increased the activity of the PP2A

phosphatase, which reduced the eNOS protein activity and endorsed endothelial activation

[230]. This was in parallel with our study published in 2011, whereby LRP8-/- mice showed a

significant attenuation in leukocyte rolling when given a bolus of HNP in comparison to wild-

type mice [174]. Interestingly, LRP8 is a surface protein that is commonly found in the

caveolae of cell membrane, a location in which eNOS is also localized in [309, 310]. This

could provide the grounds for upon LRP8 activation, possibly through reelin, in endothelial

! 86!

cells to inactivate eNOS and induce endothelial activation. Nevertheless, future studies may

want to study the role of LRP8 in inhibiting eNOS protein expression, on a genomic level.

5.3.2.3.1 Alternative reelin receptors

As a conventional LRP8 ligand, it is postulated that reelin could act on the LRP8 receptor to

attenuate eNOS and increase iNOS protein expression, however, we have yet to confirm that

LRP8-signaling pathway was indeed activated. Future studies may consider quantifying the

tyrosine phosphorylation of Dab-1 on LRP8 to confirm the activation of the signaling pathway.

Moreover, although reelin is widely known to bind to LRP8, however, reelin is also known to

bind to LRP8’s sister receptor: VLDL receptor. We did not specify if the signaling pathway

induced by reelin was due to LRP8 or VLDL receptor in our experimental model. However,

our lab has previously shown that in mice deficient in all LDL receptor and LRP (LDLR-/-/LRP-/-

), which also includes the deficiency in VLDL receptor, there was no significant difference in

leukocyte recruitment after stimulation with HNP in comparison to LRP8-/- mice [174]. This

suggests that although other LDL receptor family members may play a role in leukocyte

recruitment to the endothelium, it is primarily LRP8 that mediates this response. This

response may also be similar in the reelin-induced endothelial activation. However, to provide

a more evidentiary support, future studies should repeat the experiment with HCAEC

transfected with VLDL receptor siRNA such that the only reelin receptor expressed on the cell

is LRP8.

5.4 Reelin-mediated platelet aggregation

5.4.1 Reelin mediates HNP-induced platelet aggregation

We performed a platelet aggregation experiment to determine if HNP-induced platelet

aggregation was mediated through the reelin molecule. After priming the platelet rich plasma

! 87!

with ADP, HNP was shown to sustain the platelet aggregation responses, confirming the role

of HNP as a novel platelet agonist [174]. Interestingly, when recombinant reelin was used

instead of HNP, the sustenance of platelet aggregation was also observed. Although not as

significant as the HNP stimulation, however that could be due to the discrepancy in molar-

molar stimulation. While 10-µg/mL of HNP was used to stimulate the platelets, recombinant

reelin was only stimulated at 5-µg/mL. Moreover, since HNP is a smaller molecule (~3-4-kDa)

than reelin (300-400-kDa), the molar concentration of HNP used at 10-µg/mL was

substantially higher than reelin at 5-µg/mL. Therefore, the discrepancy in molar ratio could

account for the variance observed in the platelet aggregation effect between HNP and reelin.

Nevertheless, the role of reelin in facilitating HNP-induced platelet aggregation was supported

by the observation that platelet aggregation was attenuated when platelets were co-

stimulated with HNP and CR50. This suggests that HNP induced a release of reelin from

platelets, and it was the reelin that induced the platelet aggregation.

5.4.1.1 Reelin mediates HNP-induced platelet aggregation through LRP8 signaling

It is possible that the platelet aggregation induced by reelin were mediated by the LRP8

signaling pathway since LRP8 is the only LRP family member expressed in platelets [223].

Previously, it has been suggested that activation of LRP8 by LDL could elicit an activation of

the p38 MAPK pathway, which would mediate a rise in cPLA2 activity and arachidonic acid

content. This rise in membrane phospholipids would then facilitate the production of

thromboxane A2, a potent platelet agonist known to induce a conformational change of αIIbβ3-

integrin from inactive to active form [228]. Once active, αIIbβ3-integrin can bind to soluble

fibrinogen and form platelet aggregates. Therefore, activation of LRP8 signaling pathway

mediated by reelin could induce platelet aggregation by increasing the content of

! 88!

thromboxane A2. Alternatively, activation of LRP8 by reelin may have also down-regulate

platelet eNOS protein expression, like the response found in reelin-treated HCAEC.

Consequently, there would have been a diminished content of NO, which coincidentally, also

plays a role in inhibiting platelet aggregation [226].

5.4.2 Reelin expression in platelets

Although platelets have been documented to express reelin [254], however, it is to our

knowledge that it is not known how the expression of reelin is regulated in platelets. Equally to

be elucidated is that it is not known which granules would reelin be stored in to mediate its

release since glycoproteins can be found in both the α- and dense granules [311]. Moreover,

it is also unknown how HNP could induce platelet granular release.

5.4.3 HNP-induced platelet aggregation

Recently, a paper published by Horn et al found that HNP induced platelet aggregation

through the release of thrombospondin-1 [312]. Thrombospondin-1 is a platelet agonist found

primarily in the α-granules of platelets [313], which can stimulate platelet aggregation by

inhibiting the anti-thrombotic NO/cGMP signaling pathway [314]. The study itself was a

ground breaking study that supported HNP’s role in inducing platelet aggregation, however, it

left many questions that perhaps our study could provide additional insight. Firstly, although

HNP was shown to bind to gel-filtered platelets, however, the study did not address the

receptor to which HNP bounded to. This perhaps could have been P2Y6 since our lab has

previously elucidated the ligand-receptor relationship between HNP and P2Y6. Secondly, prior

to the release of granular content, platelets must be activated by an agonist. Although HNP

itself could act as an agonist, it appears that it is reelin that plays the predominant role as the

! 89!

platelet agonist. Purinergic receptors, P2Y1 and P2Y12, are commonly expressed on platelets

to elicit platelet activation in response to ADP [315-318]. Although expressed on platelet

surface in small quantities, P2Y6 itself does not induce platelet activation [315]. Therefore,

perhaps through the same mechanism observed in endothelial cells, HNP binds to platelet

surface P2Y6 to induce reelin expression and release. Subsequently, reelin may again act in

an autocrine manner to bind to platelet LRP8 and induce platelet aggregation. Moreover, in a

previous study conducted by Urbanus et al, it was shown that platelet activation required the

downstream signaling pathway induced by LRP8 activation, including Dab-1 phosphorylation

[319]. It was perhaps through the activation by reelin in which thrombospondin-1 was

released, as observed by the study conducted by Horn et al. To further provide support for

reelin’s role as the mediator of HNP-induced platelet aggregation, future studies may examine

platelet aggregation in the presence of MRS compound, such that HNP would no longer be

able to induce reelin secretion by P2Y6.

! 90!

Chapter 6: Summary and future studies

6.1 Summary

Since the discovery of defensins, HNP has recently gained much attention to its role in

promoting atherosclerosis. While the physiological consequences of HNP in endothelial cells,

platelets and leukocytes have been widely studied, only a handful of published research has

dedicated in finding the mechanism in which HNP induces its pro-atherogenic events. In this

study, we have revealed the molecular mechanism in which HNP induced endothelial

activation and platelet aggregation. By binding to endothelial or platelet P2Y6, HNP can

induce reelin protein expression and release into the extracellular milieu. The released reelin

then acts on endothelial or platelet LRP8 in an paracrine manner to induce endothelial

activation or platelet aggregation, respectively. The induction of the LRP8 signaling pathway

then induces an endothelial decrease in eNOS and increase in iNOS. Although nitric oxide is

generated by iNOS, however, NO may quickly be converted into peroxynitrite and mediate

nitrosative stress. Likewise, the release reelin is able to sustain platelet aggregation by

sustaining the “first-wave” and inducing the “second-wave” of platelet aggregation. The

observed responses mediated by reelin were significantly attenuated in the presence of reelin

neutralizing antibody. Thus, with this knowledge by which reelin is the perpetrator of HNP-

mediated atherogenesis, future therapeutic strategies targeting patient cardiovascular disease

should include the possibility of inhibiting reelin.

6.2 Future Direction

6.2.1 In vivo studies

Lastly, to translate our in vitro model into an in vivo model, which would provide a more

physiological depiction of reelin, we would use our previously established atherosclerotic

mouse model, ApoE-/-/HNP(+), and continuously inject the reelin neutralizing antibody by an

! 91!

osmotic Alzet pump. Following five weeks of high fat diet, the animal will be sacrificed to

analyze for atherosclerotic lesion formation, leukocyte recruitment and platelet aggregation

(Figure 18). These mice will be compared with mice that received a vehicle control, a placebo

that would replace the reelin neutralizing antibody. This would provide a more humanized

representation of atherosclerosis in mice, and allow us to examine the potential therapeutic

effect of CR50 as an intervention for HNP-mediated atherogenesis.

Figu

re 1

8. E

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tal d

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n fo

r the

pot

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rape

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ct o

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References:

1.! Lusis,!A.J.,!Atherosclerosis.!Nature,!2000.!407(6801):!p.!233?41.!2.! Glass,!C.K.!and!J.L.!Witztum,!Atherosclerosis.,the,road,ahead.!Cell,!2001.!104(4):!p.!503?16.!3.! Libby,!P.,!Inflammation,in,atherosclerosis.!Nature,!2002.!420(6917):!p.!868?74.!4.! Stocker,!R.!and!J.F.!Keaney,!Jr.,!Role,of,oxidative,modifications,in,atherosclerosis.!Physiol!

Rev,!2004.!84(4):!p.!1381?478.!5.! Weber,!C.!and!H.!Noels,!Atherosclerosis:,current,pathogenesis,and,therapeutic,options.!Nat!

Med,!2011.!17(11):!p.!1410?22.!6.! Insull,!W.,!Jr.,!The,pathology,of,atherosclerosis:,plaque,development,and,plaque,responses,to,

medical,treatment.!Am!J!Med,!2009.!122(1!Suppl):!p.!S3?S14.!7.! Napoli,!C.,!et!al.,!Fatty,streak,formation,occurs,in,human,fetal,aortas,and,is,greatly,enhanced,

by,maternal,hypercholesterolemia.,Intimal,accumulation,of,low,density,lipoprotein,and,its,oxidation,precede,monocyte,recruitment,into,early,atherosclerotic,lesions.!J!Clin!Invest,!1997.!100(11):!p.!2680?90.!

8.! Ross,!R.!and!J.A.!Glomset,!The,pathogenesis,of,atherosclerosis,(first,of,two,parts).!N!Engl!J!Med,!1976.!295(7):!p.!369?77.!

9.! Libby,!P.,!P.M.!Ridker,!and!G.K.!Hansson,!Progress,and,challenges,in,translating,the,biology,of,atherosclerosis.!Nature,!2011.!473(7347):!p.!317?25.!

10.! Mathers,!C.D.!and!D.!Loncar,!Projections,of,global,mortality,and,burden,of,disease,from,2002,to,2030.!PLoS!Med,!2006.!3(11):!p.!e442.!

11.! Jawien,!J.,!New,insights,into,immunological,aspects,of,atherosclerosis.!Pol!Arch!Med!Wewn,!2008.!118(3):!p.!127?31.!

12.! Weber,!C.,!A.!Zernecke,!and!P.!Libby,!The,multifaceted,contributions,of,leukocyte,subsets,to,atherosclerosis:,lessons,from,mouse,models.!Nat!Rev!Immunol,!2008.!8(10):!p.!802?15.!

13.! Ross,!R.,!AtherosclerosisIIan,inflammatory,disease.!N!Engl!J!Med,!1999.!340(2):!p.!115?26.!14.! Carman,!C.V.!and!T.A.!Springer,!A,transmigratory,cup,in,leukocyte,diapedesis,both,through,

individual,vascular,endothelial,cells,and,between,them.!J!Cell!Biol,!2004.!167(2):!p.!377?88.!15.! Galkina,!E.!and!K.!Ley,!Immune,and,inflammatory,mechanisms,of,atherosclerosis,(*).!Annu!

Rev!Immunol,!2009.!27:!p.!165?97.!16.! Li,!A.C.!and!C.K.!Glass,!The,macrophage,foam,cell,as,a,target,for,therapeutic,intervention.!

Nat!Med,!2002.!8(11):!p.!1235?42.!17.! Terkeltaub,!R.,!W.A.!Boisvert,!and!L.K.!Curtiss,!Chemokines,and,atherosclerosis.!Curr!Opin!

Lipidol,!1998.!9(5):!p.!397?405.!18.! Sheikine,!Y.!and!G.K.!Hansson,!Chemokines,and,atherosclerosis.!Ann!Med,!2004.!36(2):!p.!

98?118.!19.! Steffens,!S.!and!F.!Mach,!Inflammation,and,atherosclerosis.!Herz,!2004.!29(8):!p.!741?8.!20.! Takahashi,!K.,!M.!Takeya,!and!N.!Sakashita,!Multifunctional,roles,of,macrophages,in,the,

development,and,progression,of,atherosclerosis,in,humans,and,experimental,animals.!Med!Electron!Microsc,!2002.!35(4):!p.!179?203.!

21.! Febbraio,!M.,!D.P.!Hajjar,!and!R.L.!Silverstein,!CD36:,a,class,B,scavenger,receptor,involved,in,angiogenesis,,atherosclerosis,,inflammation,,and,lipid,metabolism.!J!Clin!Invest,!2001.!108(6):!p.!785?91.!

22.! Suzuki,!H.,!et!al.,!A,role,for,macrophage,scavenger,receptors,in,atherosclerosis,and,susceptibility,to,infection.!Nature,!1997.!386(6622):!p.!292?6.!

23.! Davignon,!J.!and!P.!Ganz,!Role,of,endothelial,dysfunction,in,atherosclerosis.!Circulation,!2004.!109(23!Suppl!1):!p.!III27?32.!

! 94!

24.! Shimokawa,!H.,!Primary,endothelial,dysfunction:,atherosclerosis.!J!Mol!Cell!Cardiol,!1999.!31(1):!p.!23?37.!

25.! Deanfield,!J.E.,!J.P.!Halcox,!and!T.J.!Rabelink,!Endothelial,function,and,dysfunction:,testing,and,clinical,relevance.!Circulation,!2007.!115(10):!p.!1285?95.!

26.! Newby,!A.C.,!An,overview,of,the,vascular,response,to,injury:,a,tribute,to,the,late,Russell,Ross.!Toxicol!Lett,!2000.!112+113:!p.!519?29.!

27.! Drexler,!H.,!Factors,involved,in,the,maintenance,of,endothelial,function.!Am!J!Cardiol,!1998.!82(10A):!p.!3S?4S.!

28.! Fetalvero,!K.M.,!K.A.!Martin,!and!J.!Hwa,!Cardioprotective,prostacyclin,signaling,in,vascular,smooth,muscle.!Prostaglandins!Other!Lipid!Mediat,!2007.!82(1?4):!p.!109?18.!

29.! Kozek?Langenecker,!S.A.,!et!al.,!Effect,of,prostacyclin,on,platelets,,polymorphonuclear,cells,,and,heterotypic,cell,aggregation,during,hemofiltration.!Crit!Care!Med,!2003.!31(3):!p.!864?8.!

30.! Moncada,!S.,!E.A.!Higgs,!and!J.R.!Vane,!Human,arterial,and,venous,tissues,generate,prostacyclin,(prostaglandin,x),,a,potent,inhibitor,of,platelet,aggregation.!Lancet,!1977.!1(8001):!p.!18?20.!

31.! Coats,!P.,!et!al.,!EndotheliumIderived,hyperpolarizing,factor,:,identification,and,mechanisms,of,action,in,human,subcutaneous,resistance,arteries.!Circulation,!2001.!103(12):!p.!1702?8.!

32.! Ozkor,!M.A.!and!A.A.!Quyyumi,!EndotheliumIderived,hyperpolarizing,factor,and,vascular,function.!Cardiol!Res!Pract,!2011.!2011:!p.!156146.!

33.! Vanhoutte,!P.M.,!Endothelial,dysfunction,and,atherosclerosis.!Eur!Heart!J,!1997.!18,Suppl,E:!p.!E19?29.!

34.! Kanaide,!H.,!et!al.,!Cellular,mechanism,of,vasoconstriction,induced,by,angiotensin,II:,it,remains,to,be,determined.!Circ!Res,!2003.!93(11):!p.!1015?7.!

35.! Kinlay,!S.,!et!al.,!Role,of,endothelinI1,in,the,active,constriction,of,human,atherosclerotic,coronary,arteries.!Circulation,!2001.!104(10):!p.!1114?8.!

36.! Saye,!J.A.,!H.A.!Singer,!and!M.J.!Peach,!Role,of,endothelium,in,conversion,of,angiotensin,I,to,angiotensin,II,in,rabbit,aorta.!Hypertension,!1984.!6(2!Pt!1):!p.!216?21.!

37.! Yoshida,!M.,!A.!Suzuki,!and!T.!Itoh,!Mechanisms,of,vasoconstriction,induced,by,endothelinI1,in,smooth,muscle,of,rabbit,mesenteric,artery.!J!Physiol,!1994.!477,(,Pt,2):!p.!253?65.!

38.! Furchgott,!R.F.!and!J.V.!Zawadzki,!The,obligatory,role,of,endothelial,cells,in,the,relaxation,of,arterial,smooth,muscle,by,acetylcholine.!Nature,!1980.!288(5789):!p.!373?6.!

39.! Palmer,!R.M.,!A.G.!Ferrige,!and!S.!Moncada,!Nitric,oxide,release,accounts,for,the,biological,activity,of,endotheliumIderived,relaxing,factor.!Nature,!1987.!327(6122):!p.!524?6.!

40.! Ignarro,!L.J.,!et!al.,!EndotheliumIderived,relaxing,factor,from,pulmonary,artery,and,vein,possesses,pharmacologic,and,chemical,properties,identical,to,those,of,nitric,oxide,radical.!Circ!Res,!1987.!61(6):!p.!866?79.!

41.! Alderton,!W.K.,!C.E.!Cooper,!and!R.G.!Knowles,!Nitric,oxide,synthases:,structure,,function,and,inhibition.!Biochem!J,!2001.!357(Pt!3):!p.!593?615.!

42.! Fischmann,!T.O.,!et!al.,!Structural,characterization,of,nitric,oxide,synthase,isoforms,reveals,striking,activeIsite,conservation.!Nat!Struct!Biol,!1999.!6(3):!p.!233?42.!

43.! Crane,!B.R.,!et!al.,!Structure,of,nitric,oxide,synthase,oxygenase,dimer,with,pterin,and,substrate.!Science,!1998.!279(5359):!p.!2121?6.!

44.! Raman,!C.S.,!et!al.,!Crystal,structure,of,constitutive,endothelial,nitric,oxide,synthase:,a,paradigm,for,pterin,function,involving,a,novel,metal,center.!Cell,!1998.!95(7):!p.!939?50.!

45.! Behrendt,!D.!and!P.!Ganz,!Endothelial,function.,From,vascular,biology,to,clinical,applications.!Am!J!Cardiol,!2002.!90(10C):!p.!40L?48L.!

! 95!

46.! Kibbe,!M.,!T.!Billiar,!and!E.!Tzeng,!Inducible,nitric,oxide,synthase,and,vascular,injury.!Cardiovasc!Res,!1999.!43(3):!p.!650?7.!

47.! Feng,!Q.,!et!al.,!Increased,inducible,nitric,oxide,synthase,expression,contributes,to,myocardial,dysfunction,and,higher,mortality,after,myocardial,infarction,in,mice.!Circulation,!2001.!104(6):!p.!700?4.!

48.! Morris,!S.M.,!Jr.!and!T.R.!Billiar,!New,insights,into,the,regulation,of,inducible,nitric,oxide,synthesis.!Am!J!Physiol,!1994.!266(6!Pt!1):!p.!E829?39.!

49.! Nathan,!C.!and!Q.W.!Xie,!Nitric,oxide,synthases:,roles,,tolls,,and,controls.!Cell,!1994.!78(6):!p.!915?8.!

50.! Marechal,!A.,!et!al.,!Activation,of,peroxynitrite,by,inducible,nitricIoxide,synthase:,a,direct,source,of,nitrative,stress.!J!Biol!Chem,!2007.!282(19):!p.!14101?12.!

51.! Bellamy,!T.C.,!J.!Wood,!and!J.!Garthwaite,!On,the,activation,of,soluble,guanylyl,cyclase,by,nitric,oxide.!Proc!Natl!Acad!Sci!U!S!A,!2002.!99(1):!p.!507?10.!

52.! Martin,!E.,!et!al.,!Soluble,guanylyl,cyclase:,the,nitric,oxide,receptor.!Methods!Enzymol,!2005.!396:!p.!478?92.!

53.! Friebe,!A.!and!D.!Koesling,!Regulation,of,nitric,oxideIsensitive,guanylyl,cyclase.!Circ!Res,!2003.!93(2):!p.!96?105.!

54.! Cannon,!R.O.,!3rd,!Role,of,nitric,oxide,in,cardiovascular,disease:,focus,on,the,endothelium.!Clin!Chem,!1998.!44(8!Pt!2):!p.!1809?19.!

55.! Moncada,!S.,!R.M.!Palmer,!and!E.A.!Higgs,!Nitric,oxide:,physiology,,pathophysiology,,and,pharmacology.!Pharmacol!Rev,!1991.!43(2):!p.!109?42.!

56.! Lincoln,!T.M.,!T.L.!Cornwell,!and!A.E.!Taylor,!cGMPIdependent,protein,kinase,mediates,the,reduction,of,Ca2+,by,cAMP,in,vascular,smooth,muscle,cells.!Am!J!Physiol,!1990.!258(3!Pt!1):!p.!C399?407.!

57.! Lincoln,!T.M.,!N.!Dey,!and!H.!Sellak,!Invited,review:,cGMPIdependent,protein,kinase,signaling,mechanisms,in,smooth,muscle:,from,the,regulation,of,tone,to,gene,expression.!J!Appl!Physiol!(1985),!2001.!91(3):!p.!1421?30.!

58.! Furukawa,!K.,!et!al.,!Cyclic,GMP,stimulates,Na+/Ca2+,exchange,in,vascular,smooth,muscle,cells,in,primary,culture.!J!Biol!Chem,!1991.!266(19):!p.!12337?41.!

59.! Wang,!G.R.,!et!al.,!Mechanism,of,platelet,inhibition,by,nitric,oxide:,in,vivo,phosphorylation,of,thromboxane,receptor,by,cyclic,GMPIdependent,protein,kinase.!Proc!Natl!Acad!Sci!U!S!A,!1998.!95(9):!p.!4888?93.!

60.! Tsihlis,!N.D.,!et!al.,!Nitric,oxide,inhibits,vascular,smooth,muscle,cell,proliferation,and,neointimal,hyperplasia,by,increasing,the,ubiquitination,and,degradation,of,UbcH10.!Cell!Biochem!Biophys,!2011.!60(1?2):!p.!89?97.!

61.! Spiecker,!M.,!H.B.!Peng,!and!J.K.!Liao,!Inhibition,of,endothelial,vascular,cell,adhesion,moleculeI1,expression,by,nitric,oxide,involves,the,induction,and,nuclear,translocation,of,IkappaBalpha.!J!Biol!Chem,!1997.!272(49):!p.!30969?74.!

62.! Libby,!P.,!P.M.!Ridker,!and!A.!Maseri,!Inflammation,and,atherosclerosis.!Circulation,!2002.!105(9):!p.!1135?43.!

63.! De!Caterina,!R.,!et!al.,!Nitric,oxide,decreases,cytokineIinduced,endothelial,activation.,Nitric,oxide,selectively,reduces,endothelial,expression,of,adhesion,molecules,and,proinflammatory,cytokines.!J!Clin!Invest,!1995.!96(1):!p.!60?8.!

64.! Anderson,!T.J.,!Assessment,and,treatment,of,endothelial,dysfunction,in,humans.!J!Am!Coll!Cardiol,!1999.!34(3):!p.!631?8.!

65.! Bonetti,!P.O.,!L.O.!Lerman,!and!A.!Lerman,!Endothelial,dysfunction:,a,marker,of,atherosclerotic,risk.!Arterioscler!Thromb!Vasc!Biol,!2003.!23(2):!p.!168?75.!

! 96!

66.! Liao,!J.K.,!Linking,endothelial,dysfunction,with,endothelial,cell,activation.!J!Clin!Invest,!2013.!123(2):!p.!540?1.!

67.! Ross,!R.,!Atherosclerosis,is,an,inflammatory,disease.!Am!Heart!J,!1999.!138(5!Pt!2):!p.!S419?20.!

68.! Celermajer,!D.S.,!Endothelial,dysfunction:,does,it,matter?,Is,it,reversible?!J!Am!Coll!Cardiol,!1997.!30(2):!p.!325?33.!

69.! Ludmer,!P.L.,!et!al.,!Paradoxical,vasoconstriction,induced,by,acetylcholine,in,atherosclerotic,coronary,arteries.!N!Engl!J!Med,!1986.!315(17):!p.!1046?51.!

70.! Anderson,!T.J.,!et!al.,!Systemic,nature,of,endothelial,dysfunction,in,atherosclerosis.!Am!J!Cardiol,!1995.!75(6):!p.!71B?74B.!

71.! Fichtlscherer,!S.,!et!al.,!Elevated,CIreactive,protein,levels,and,impaired,endothelial,vasoreactivity,in,patients,with,coronary,artery,disease.!Circulation,!2000.!102(9):!p.!1000?6.!

72.! Schachinger,!V.!and!A.M.!Zeiher,!AtherogenesisIIrecent,insights,into,basic,mechanisms,and,their,clinical,impact.!Nephrol!Dial!Transplant,!2002.!17(12):!p.!2055?64.!

73.! Won,!D.,!et!al.,!Relative,reduction,of,endothelial,nitricIoxide,synthase,expression,and,transcription,in,atherosclerosisIprone,regions,of,the,mouse,aorta,and,in,an,in,vitro,model,of,disturbed,flow.!Am!J!Pathol,!2007.!171(5):!p.!1691?704.!

74.! Gambillara,!V.,!et!al.,!PlaqueIprone,hemodynamics,impair,endothelial,function,in,pig,carotid,arteries.!Am!J!Physiol!Heart!Circ!Physiol,!2006.!290(6):!p.!H2320?8.!

75.! Harrison,!D.G.,!Cellular,and,molecular,mechanisms,of,endothelial,cell,dysfunction.!J!Clin!Invest,!1997.!100(9):!p.!2153?7.!

76.! Cooke,!J.P.,!Does,ADMA,cause,endothelial,dysfunction?!Arterioscler!Thromb!Vasc!Biol,!2000.!20(9):!p.!2032?7.!

77.! Minor,!R.L.,!Jr.,!et!al.,!DietIinduced,atherosclerosis,increases,the,release,of,nitrogen,oxides,from,rabbit,aorta.!J!Clin!Invest,!1990.!86(6):!p.!2109?16.!

78.! Santolini,!J.,!et!al.,!A,kinetic,simulation,model,that,describes,catalysis,and,regulation,in,nitricIoxide,synthase.!J!Biol!Chem,!2001.!276(2):!p.!1233?43.!

79.! Stuehr,!D.J.,!et!al.,!Radical,reactions,of,nitric,oxide,synthases.!Biochem!Soc!Symp,!2004(71):!p.!39?49.!

80.! Misko,!T.P.,!et!al.,!Characterization,of,the,cytoprotective,action,of,peroxynitrite,decomposition,catalysts.!J!Biol!Chem,!1998.!273(25):!p.!15646?53.!

81.! Salvemini,!D.,!T.M.!Doyle,!and!S.!Cuzzocrea,!Superoxide,,peroxynitrite,and,oxidative/nitrative,stress,in,inflammation.!Biochem!Soc!Trans,!2006.!34(Pt!5):!p.!965?70.!

82.! Salvemini,!D.,!et!al.,!Peroxynitrite,decomposition,catalysts:,therapeutics,for,peroxynitriteImediated,pathology.!Proc!Natl!Acad!Sci!U!S!A,!1998.!95(5):!p.!2659?63.!

83.! Squadrito,!G.L.!and!W.A.!Pryor,!The,formation,of,peroxynitrite,in,vivo,from,nitric,oxide,and,superoxide.!Chem!Biol!Interact,!1995.!96(2):!p.!203?6.!

84.! Virag,!L.,!et!al.,!Nitric,oxideIperoxynitriteIpoly(ADPIribose),polymerase,pathway,in,the,skin.!Exp!Dermatol,!2002.!11(3):!p.!189?202.!

85.! Deeb,!R.S.,!et!al.,!Heme,catalyzes,tyrosine,385,nitration,and,inactivation,of,prostaglandin,H2,synthaseI1,by,peroxynitrite.!J!Lipid!Res,!2006.!47(5):!p.!898?911.!

86.! Deeb,!R.S.,!et!al.,!Tyrosine,nitration,in,prostaglandin,H(2),synthase.!J!Lipid!Res,!2002.!43(10):!p.!1718?26.!

87.! Upmacis,!R.K.,!R.S.!Deeb,!and!D.P.!Hajjar,!Role,of,nitrogen,oxides,on,eicosanoid,production,during,atherosclerosis:,understanding,the,controversies.!Curr!Atheroscler!Rep,!2001.!3(3):!p.!181?2.!

! 97!

88.! Upmacis,!R.K.,!et!al.,!Profound,biopterin,oxidation,and,protein,tyrosine,nitration,in,tissues,of,ApoEInull,mice,on,an,atherogenic,diet:,contribution,of,inducible,nitric,oxide,synthase.!Am!J!Physiol!Heart!Circ!Physiol,!2007.!293(5):!p.!H2878?87.!

89.! Leeuwenburgh,!C.,!et!al.,!Reactive,nitrogen,intermediates,promote,low,density,lipoprotein,oxidation,in,human,atherosclerotic,intima.!J!Biol!Chem,!1997.!272(3):!p.!1433?6.!

90.! Cooper,!B.,!Osler's,role,in,defining,the,third,corpuscle,,or,"blood,plates".!Proc!(Bayl!Univ!Med!Cent),!2005.!18(4):!p.!376?8.!

91.! Behnke,!O.!and!A.!Forer,!From,megakaryocytes,to,platelets:,platelet,morphogenesis,takes,place,in,the,bloodstream.!Eur!J!Haematol!Suppl,!1998.!61:!p.!3?23.!

92.! Choi,!E.S.,!et!al.,!Platelets,generated,in,vitro,from,proplateletIdisplaying,human,megakaryocytes,are,functional.!Blood,!1995.!85(2):!p.!402?13.!

93.! Tocantins,!L.M.!and!R.!Rodriguez,!Hemorrhagic,disease,in,children;,notes,on,its,pathological,physiology,and,clinical,management.!Med!Clin!North!Am,!1952.!36(6):!p.!1693?1710.!

94.! Weyrich,!A.S.,!et!al.,!Protein,synthesis,by,platelets:,historical,and,new,perspectives.!J!Thromb!Haemost,!2009.!7(2):!p.!241?6.!

95.! Nayak,!M.K.,!P.P.!Kulkarni,!and!D.!Dash,!Regulatory,role,of,proteasome,in,determination,of,platelet,life,span.!J!Biol!Chem,!2013.!288(10):!p.!6826?34.!

96.! Daly,!M.E.,!Determinants,of,platelet,count,in,humans.!Haematologica,!2011.!96(1):!p.!10?3.!97.! Steinhubl,!S.R.!and!D.J.!Moliterno,!The,role,of,the,platelet,in,the,pathogenesis,of,

atherothrombosis.!Am!J!Cardiovasc!Drugs,!2005.!5(6):!p.!399?408.!98.! Davi,!G.!and!C.!Patrono,!Platelet,activation,and,atherothrombosis.!N!Engl!J!Med,!2007.!

357(24):!p.!2482?94.!99.! Lievens,!D.!and!P.!von!Hundelshausen,!Platelets,in,atherosclerosis.!Thromb!Haemost,!2011.!

106(5):!p.!827?38.!100.! Denis,!C.V.!and!D.D.!Wagner,!Platelet,adhesion,receptors,and,their,ligands,in,mouse,models,

of,thrombosis.!Arterioscler!Thromb!Vasc!Biol,!2007.!27(4):!p.!728?39.!101.! Bergmeier,!W.,!et!al.,!The,role,of,platelet,adhesion,receptor,GPIbalpha,far,exceeds,that,of,its,

main,ligand,,von,Willebrand,factor,,in,arterial,thrombosis.!Proc!Natl!Acad!Sci!U!S!A,!2006.!103(45):!p.!16900?5.!

102.! Munnix,!I.C.,!et!al.,!The,glycoprotein,VIIphospholipase,Cgamma2,signaling,pathway,controls,thrombus,formation,induced,by,collagen,and,tissue,factor,in,vitro,and,in,vivo.!Arterioscler!Thromb!Vasc!Biol,!2005.!25(12):!p.!2673?8.!

103.! Varga?Szabo,!D.,!I.!Pleines,!and!B.!Nieswandt,!Cell,adhesion,mechanisms,in,platelets.!Arterioscler!Thromb!Vasc!Biol,!2008.!28(3):!p.!403?12.!

104.! Furie,!B.!and!B.C.!Furie,!Mechanisms,of,thrombus,formation.!N!Engl!J!Med,!2008.!359(9):!p.!938?49.!

105.! Massberg,!S.,!et!al.,!A,crucial,role,of,glycoprotein,VI,for,platelet,recruitment,to,the,injured,arterial,wall,in,vivo.!J!Exp!Med,!2003.!197(1):!p.!41?9.!

106.! Semple,!J.W.,!J.E.!Italiano,!Jr.,!and!J.!Freedman,!Platelets,and,the,immune,continuum.!Nat!Rev!Immunol,!2011.!11(4):!p.!264?74.!

107.! Rendu,!F.!and!B.!Brohard?Bohn,!The,platelet,release,reaction:,granules',constituents,,secretion,and,functions.!Platelets,!2001.!12(5):!p.!261?73.!

108.! Shattil,!S.J.,!Signaling,through,platelet,integrin,alpha,IIb,beta,3:,insideIout,,outsideIin,,and,sideways.!Thromb!Haemost,!1999.!82(2):!p.!318?25.!

109.! Vorchheimer,!D.A.!and!R.!Becker,!Platelets,in,atherothrombosis.!Mayo!Clin!Proc,!2006.!81(1):!p.!59?68.!

110.! Whiteheart,!S.W.,!Platelet,granules:,surprise,packages.!Blood,!2011.!118(5):!p.!1190?1.!

! 98!

111.! White,!J.G.,!Use,of,the,electron,microscope,for,diagnosis,of,platelet,disorders.!Semin!Thromb!Hemost,!1998.!24(2):!p.!163?8.!

112.! Lemons,!P.P.,!et!al.,!Regulated,secretion,in,platelets:,identification,of,elements,of,the,platelet,exocytosis,machinery.!Blood,!1997.!90(4):!p.!1490?500.!

113.! Lindemann,!S.,!et!al.,!Platelets,,inflammation,and,atherosclerosis.!J!Thromb!Haemost,!2007.!5,Suppl,1:!p.!203?11.!

114.! Massberg,!S.,!et!al.,!A,critical,role,of,platelet,adhesion,in,the,initiation,of,atherosclerotic,lesion,formation.!J!Exp!Med,!2002.!196(7):!p.!887?96.!

115.! Gawaz,!M.,!et!al.,!Vitronectin,receptor,(alpha(v)beta3),mediates,platelet,adhesion,to,the,luminal,aspect,of,endothelial,cells:,implications,for,reperfusion,in,acute,myocardial,infarction.!Circulation,!1997.!96(6):!p.!1809?18.!

116.! Hawrylowicz,!C.M.,!G.L.!Howells,!and!M.!Feldmann,!PlateletIderived,interleukin,1,induces,human,endothelial,adhesion,molecule,expression,and,cytokine,production.!J!Exp!Med,!1991.!174(4):!p.!785?90.!

117.! von!Hundelshausen,!P.,!et!al.,!RANTES,deposition,by,platelets,triggers,monocyte,arrest,on,inflamed,and,atherosclerotic,endothelium.!Circulation,!2001.!103(13):!p.!1772?7.!

118.! Yu,!G.,!et!al.,!Endothelial,expression,of,EIselectin,is,induced,by,the,plateletIspecific,chemokine,platelet,factor,4,through,LRP,in,an,NFIkappaBIdependent,manner.!Blood,!2005.!105(9):!p.!3545?51.!

119.! Nassar,!T.,!et!al.,!Platelet,factor,4,enhances,the,binding,of,oxidized,lowIdensity,lipoprotein,to,vascular,wall,cells.!J!Biol!Chem,!2003.!278(8):!p.!6187?93.!

120.! Daub,!K.,!et!al.,!Platelets,induce,differentiation,of,human,CD34+,progenitor,cells,into,foam,cells,and,endothelial,cells.!FASEB!J,!2006.!20(14):!p.!2559?61.!

121.! Li,!Y.,!et!al.,!A,critical,concentration,of,neutrophils,is,required,for,effective,bacterial,killing,in,suspension.!Proc!Natl!Acad!Sci!U!S!A,!2002.!99(12):!p.!8289?94.!

122.! Naruko,!T.,!et!al.,!Neutrophil,infiltration,of,culprit,lesions,in,acute,coronary,syndromes.!Circulation,!2002.!106(23):!p.!2894?900.!

123.! Soehnlein,!O.,!L.!Lindbom,!and!C.!Weber,!Mechanisms,underlying,neutrophilImediated,monocyte,recruitment.!Blood,!2009.!114(21):!p.!4613?23.!

124.! Haumer,!M.,!et!al.,!Association,of,neutrophils,and,future,cardiovascular,events,in,patients,with,peripheral,artery,disease.!J!Vasc!Surg,!2005.!41(4):!p.!610?7.!

125.! Grau,!A.J.,!et!al.,!Leukocyte,count,as,an,independent,predictor,of,recurrent,ischemic,events.!Stroke,!2004.!35(5):!p.!1147?52.!

126.! Rotzius,!P.,!et!al.,!Distinct,infiltration,of,neutrophils,in,lesion,shoulders,in,ApoEI/I,mice.!Am!J!Pathol,!2010.!177(1):!p.!493?500.!

127.! Zeya,!H.I.!and!J.K.!Spitznagel,!Antibacterial,and,Enzymic,Basic,Proteins,from,Leukocyte,Lysosomes:,Separation,and,Identification.!Science,!1963.!142(3595):!p.!1085?7.!

128.! Ganz,!T.,!Defensins:,antimicrobial,peptides,of,innate,immunity.!Nat!Rev!Immunol,!2003.!3(9):!p.!710?20.!

129.! Klotman,!M.E.!and!T.L.!Chang,!Defensins,in,innate,antiviral,immunity.!Nat!Rev!Immunol,!2006.!6(6):!p.!447?56.!

130.! Tang,!Y.Q.,!et!al.,!A,cyclic,antimicrobial,peptide,produced,in,primate,leukocytes,by,the,ligation,of,two,truncated,alphaIdefensins.!Science,!1999.!286(5439):!p.!498?502.!

131.! Ganz,!T.,!The,role,of,antimicrobial,peptides,in,innate,immunity.!Integr!Comp!Biol,!2003.!43(2):!p.!300?4.!

132.! Ganz,!T.,!et!al.,!Defensins.,Natural,peptide,antibiotics,of,human,neutrophils.!J!Clin!Invest,!1985.!76(4):!p.!1427?35.!

! 99!

133.! Jones,!D.E.!and!C.L.!Bevins,!Paneth,cells,of,the,human,small,intestine,express,an,antimicrobial,peptide,gene.!J!Biol!Chem,!1992.!267(32):!p.!23216?25.!

134.! Jones,!D.E.!and!C.L.!Bevins,!DefensinI6,mRNA,in,human,Paneth,cells:,implications,for,antimicrobial,peptides,in,host,defense,of,the,human,bowel.!FEBS!Lett,!1993.!315(2):!p.!187?92.!

135.! Ouellette,!A.J.!and!C.L.!Bevins,!Paneth,cell,defensins,and,innate,immunity,of,the,small,bowel.!Inflamm!Bowel!Dis,!2001.!7(1):!p.!43?50.!

136.! Cunliffe,!R.N.!and!Y.R.!Mahida,!Expression,and,regulation,of,antimicrobial,peptides,in,the,gastrointestinal,tract.!J!Leukoc!Biol,!2004.!75(1):!p.!49?58.!

137.! Selsted,!M.E.,!et!al.,!Primary,structures,of,three,human,neutrophil,defensins.!J!Clin!Invest,!1985.!76(4):!p.!1436?9.!

138.! Voglis,!S.,!et!al.,!Human,neutrophil,peptides,and,phagocytic,deficiency,in,bronchiectatic,lungs.!Am!J!Respir!Crit!Care!Med,!2009.!180(2):!p.!159?66.!

139.! Wilde,!C.G.,!et!al.,!Purification,and,characterization,of,human,neutrophil,peptide,4,,a,novel,member,of,the,defensin,family.!J!Biol!Chem,!1989.!264(19):!p.!11200?3.!

140.! Kokryakov,!V.N.,!et!al.,!Protegrins:,leukocyte,antimicrobial,peptides,that,combine,features,of,corticostatic,defensins,and,tachyplesins.!FEBS!Lett,!1993.!327(2):!p.!231?6.!

141.! Mackewicz,!C.E.,!et!al.,!alphaIDefensins,can,have,antiIHIV,activity,but,are,not,CD8,cell,antiIHIV,factors.!AIDS,!2003.!17(14):!p.!F23?32.!

142.! Rodriguez?Garcia,!M.,!et!al.,!Human,immature,monocyteIderived,dendritic,cells,produce,and,secrete,alphaIdefensins,1I3.!J!Leukoc!Biol,!2007.!82(5):!p.!1143?6.!

143.! Mak,!P.,!J.J.!Enghild,!and!A.!Dubin,!Hamster,antithrombin,III:,purification,,characterization,and,acute,phase,response.!Comp!Biochem!Physiol!B!Biochem!Mol!Biol,!1996.!115(1):!p.!135?41.!

144.! Mak,!P.,!et!al.,!Isolation,,antimicrobial,activities,,and,primary,structures,of,hamster,neutrophil,defensins.!Infect!Immun,!1996.!64(11):!p.!4444?9.!

145.! Ouellette,!A.J.,!et!al.,!Purification,and,primary,structure,of,murine,cryptdinI1,,a,Paneth,cell,defensin.!FEBS!Lett,!1992.!304(2?3):!p.!146?8.!

146.! Selsted,!M.E.!and!S.S.!Harwig,!Purification,,primary,structure,,and,antimicrobial,activities,of,a,guinea,pig,neutrophil,defensin.!Infect!Immun,!1987.!55(9):!p.!2281?6.!

147.! Selsted,!M.E.,!D.!Szklarek,!and!R.I.!Lehrer,!Purification,and,antibacterial,activity,of,antimicrobial,peptides,of,rabbit,granulocytes.!Infect!Immun,!1984.!45(1):!p.!150?4.!

148.! Tang,!Y.Q.,!et!al.,!Isolation,,characterization,,cDNA,cloning,,and,antimicrobial,properties,of,two,distinct,subfamilies,of,alphaIdefensins,from,rhesus,macaque,leukocytes.!Infect!Immun,!1999.!67(11):!p.!6139?44.!

149.! Daher,!K.A.,!et!al.,!Isolation,and,characterization,of,human,defensin,cDNA,clones.!Proc!Natl!Acad!Sci!U!S!A,!1988.!85(19):!p.!7327?31.!

150.! Valore,!E.V.!and!T.!Ganz,!Posttranslational,processing,of,defensins,in,immature,human,myeloid,cells.!Blood,!1992.!79(6):!p.!1538?44.!

151.! Arnljots,!K.,!et!al.,!Timing,,targeting,and,sorting,of,azurophil,granule,proteins,in,human,myeloid,cells.!Leukemia,!1998.!12(11):!p.!1789?95.!

152.! Cowland,!J.B.!and!N.!Borregaard,!The,individual,regulation,of,granule,protein,mRNA,levels,during,neutrophil,maturation,explains,the,heterogeneity,of,neutrophil,granules.!J!Leukoc!Biol,!1999.!66(6):!p.!989?95.!

153.! Yount,!N.Y.,!et!al.,!Rat,neutrophil,defensins.,Precursor,structures,and,expression,during,neutrophilic,myelopoiesis.!J!Immunol,!1995.!155(9):!p.!4476?84.!

154.! Panyutich,!A.V.,!et!al.,!Plasma,defensin,concentrations,are,elevated,in,patients,with,septicemia,or,bacterial,meningitis.!J!Lab!Clin!Med,!1993.!122(2):!p.!202?7.!

! 100!

155.! Flotte,!T.R.!and!C.!Mueller,!Gene,therapy,for,alphaI1,antitrypsin,deficiency.!Hum!Mol!Genet,!2011.!20(R1):!p.!R87?92.!

156.! Stakisaitis,!D.,!V.!Basys,!and!R.!Benetis,!Does,alphaI1Iproteinase,inhibitor,play,a,protective,role,in,coronary,atherosclerosis?!Med!Sci!Monit,!2001.!7(4):!p.!701?11.!

157.! Gilutz,!H.,!et!al.,!Alpha,1Iantitrypsin,in,acute,myocardial,infarction.!Br!Heart!J,!1983.!49(1):!p.!26?9.!

158.! Talmud,!P.J.,!et!al.,!Progression,of,atherosclerosis,is,associated,with,variation,in,the,alpha1Iantitrypsin,gene.!Arterioscler!Thromb!Vasc!Biol,!2003.!23(4):!p.!644?9.!

159.! Huber,!R.!and!R.W.!Carrell,!Implications,of,the,threeIdimensional,structure,of,alpha,1Iantitrypsin,for,structure,and,function,of,serpins.!Biochemistry,!1989.!28(23):!p.!8951?66.!

160.! Avanzas,!P.,!et!al.,!Markers,of,inflammation,and,multiple,complex,stenoses,(pancoronary,plaque,vulnerability),in,patients,with,nonIST,segment,elevation,acute,coronary,syndromes.!Heart,!2004.!90(8):!p.!847?52.!

161.! Panyutich,!A.V.,!et!al.,!Human,neutrophil,defensin,and,serpins,form,complexes,and,inactivate,each,other.!Am!J!Respir!Cell!Mol!Biol,!1995.!12(3):!p.!351?7.!

162.! Vaschetto,!R.,!et!al.,!Role,of,human,neutrophil,peptides,in,the,initial,interaction,between,lung,epithelial,cells,and,CD4+,lymphocytes.!J!Leukoc!Biol,!2007.!81(4):!p.!1022?31.!

163.! Boman,!H.G.,!Peptide,antibiotics,and,their,role,in,innate,immunity.!Annu!Rev!Immunol,!1995.!13:!p.!61?92.!

164.! Selsted,!M.E.!and!A.J.!Ouellette,!Mammalian,defensins,in,the,antimicrobial,immune,response.!Nat!Immunol,!2005.!6(6):!p.!551?7.!

165.! Muller,!F.M.,!C.A.!Lyman,!and!T.J.!Walsh,!Antimicrobial,peptides,as,potential,new,antifungals.!Mycoses,!1999.!42,Suppl,2:!p.!77?82.!

166.! Lehrer,!R.I.,!et!al.,!Modulation,of,the,in,vitro,candidacidal,activity,of,human,neutrophil,defensins,by,target,cell,metabolism,and,divalent,cations.!J!Clin!Invest,!1988.!81(6):!p.!1829?35.!

167.! Ganz,!T.!and!R.I.!Lehrer,!Defensins.!Curr!Opin!Immunol,!1994.!6(4):!p.!584?9.!168.! Weinberg,!A.,!S.!Krisanaprakornkit,!and!B.A.!Dale,!Epithelial,antimicrobial,peptides:,review,

and,significance,for,oral,applications.!Crit!Rev!Oral!Biol!Med,!1998.!9(4):!p.!399?414.!169.! Quayle,!A.J.,!et!al.,!Gene,expression,,immunolocalization,,and,secretion,of,human,defensinI5,

in,human,female,reproductive,tract.!Am!J!Pathol,!1998.!152(5):!p.!1247?58.!170.! Hancock,!R.E.,!Peptide,antibiotics.!Lancet,!1997.!349(9049):!p.!418?22.!171.! Zimmermann,!G.R.,!et!al.,!Solution,structure,of,bovine,neutrophil,betaIdefensinI12:,the,

peptide,fold,of,the,betaIdefensins,is,identical,to,that,of,the,classical,defensins.!Biochemistry,!1995.!34(41):!p.!13663?71.!

172.! Hancock,!R.E.,!T.!Falla,!and!M.!Brown,!Cationic,bactericidal,peptides.!Adv!Microb!Physiol,!1995.!37:!p.!135?75.!

173.! Lehrer,!R.I.,!et!al.,!Interaction,of,human,defensins,with,Escherichia,coli.,Mechanism,of,bactericidal,activity.!J!Clin!Invest,!1989.!84(2):!p.!553?61.!

174.! Quinn,!K.L.,!et!al.,!Human,neutrophil,peptides,mediate,endothelialImonocyte,interaction,,foam,cell,formation,,and,platelet,activation.!Arterioscler!Thromb!Vasc!Biol,!2011.!31(9):!p.!2070?9.!

175.! Yang,!D.,!et!al.,!Human,neutrophil,defensins,selectively,chemoattract,naive,T,and,immature,dendritic,cells.!J!Leukoc!Biol,!2000.!68(1):!p.!9?14.!

176.! Grigat,!J.,!et!al.,!Chemoattraction,of,macrophages,,T,lymphocytes,,and,mast,cells,is,evolutionarily,conserved,within,the,human,alphaIdefensin,family.!J!Immunol,!2007.!179(6):!p.!3958?65.!

! 101!

177.! Zhang,!H.,!et!al.,!Neutrophil,defensins,mediate,acute,inflammatory,response,and,lung,dysfunction,in,doseIrelated,fashion.!Am!J!Physiol!Lung!Cell!Mol!Physiol,!2001.!280(5):!p.!L947?54.!

178.! Smart,!S.J.!and!T.B.!Casale,!TNFIalphaIinduced,transendothelial,neutrophil,migration,is,ILI8,dependent.!Am!J!Physiol,!1994.!266(3!Pt!1):!p.!L238?45.!

179.! Charo,!I.F.!and!M.B.!Taubman,!Chemokines,in,the,pathogenesis,of,vascular,disease.!Circ!Res,!2004.!95(9):!p.!858?66.!

180.! Ohman,!M.K.,!et!al.,!Monocyte,chemoattractant,proteinI1,deficiency,protects,against,visceral,fatIinduced,atherosclerosis.!Arterioscler!Thromb!Vasc!Biol,!2010.!30(6):!p.!1151?8.!

181.! Gosling,!J.,!et!al.,!MCPI1,deficiency,reduces,susceptibility,to,atherosclerosis,in,mice,that,overexpress,human,apolipoprotein,B.!J!Clin!Invest,!1999.!103(6):!p.!773?8.!

182.! Riegel,!A.K.,!et!al.,!Selective,induction,of,endothelial,P2Y6,nucleotide,receptor,promotes,vascular,inflammation.!Blood,!2011.!117(8):!p.!2548?55.!

183.! Mahalingam,!S.!and!G.!Karupiah,!Chemokines,and,chemokine,receptors,in,infectious,diseases.!Immunol!Cell!Biol,!1999.!77(6):!p.!469?75.!

184.! Kanegasaki,!S.,!et!al.,!A,novel,optical,assay,system,for,the,quantitative,measurement,of,chemotaxis.!J!Immunol!Methods,!2003.!282(1?2):!p.!1?11.!

185.! Khine,!A.A.,!et!al.,!Human,neutrophil,peptides,induce,interleukinI8,production,through,the,P2Y6,signaling,pathway.!Blood,!2006.!107(7):!p.!2936?42.!

186.! Syeda,!F.,!et!al.,!Differential,signaling,mechanisms,of,HNPIinduced,ILI8,production,in,human,lung,epithelial,cells,and,monocytes.!J!Cell!Physiol,!2008.!214(3):!p.!820?7.!

187.! Chaly,!Y.V.,!et!al.,!Neutrophil,alphaIdefensin,human,neutrophil,peptide,modulates,cytokine,production,in,human,monocytes,and,adhesion,molecule,expression,in,endothelial,cells.!Eur!Cytokine!Netw,!2000.!11(2):!p.!257?66.!

188.! Sakamoto,!N.,!et!al.,!Differential,effects,of,alphaI,and,betaIdefensin,on,cytokine,production,by,cultured,human,bronchial,epithelial,cells.!Am!J!Physiol!Lung!Cell!Mol!Physiol,!2005.!288(3):!p.!L508?13.!

189.! Van!Wetering,!S.,!et!al.,!Effect,of,defensins,on,interleukinI8,synthesis,in,airway,epithelial,cells.!Am!J!Physiol,!1997.!272(5!Pt!1):!p.!L888?96.!

190.! van!Wetering,!S.,!et!al.,!Neutrophil,defensins,stimulate,the,release,of,cytokines,by,airway,epithelial,cells:,modulation,by,dexamethasone.!Inflamm!Res,!2002.!51(1):!p.!8?15.!

191.! Ihi,!T.,!et!al.,!Elevated,concentrations,of,human,neutrophil,peptides,in,plasma,,blood,,and,body,fluids,from,patients,with,infections.!Clin!Infect!Dis,!1997.!25(5):!p.!1134?40.!

192.! Zhao,!H.,!et!al.,!Acute,STIsegment,elevation,myocardial,infarction,is,associated,with,decreased,human,antimicrobial,peptide,LLI37,and,increased,human,neutrophil,peptideI1,to,3,in,plasma.!J!Atheroscler!Thromb,!2012.!19(4):!p.!357?68.!

193.! Urbonaviciene,!G.,!et!al.,!Markers,of,inflammation,in,relation,to,longIterm,cardiovascular,mortality,in,patients,with,lowerIextremity,peripheral,arterial,disease.!Int!J!Cardiol,!2012.!160(2):!p.!89?94.!

194.! Barnathan,!E.S.,!et!al.,!Immunohistochemical,localization,of,defensin,in,human,coronary,vessels.!Am!J!Pathol,!1997.!150(3):!p.!1009?20.!

195.! Higazi,!A.A.,!et!al.,!Defensin,stimulates,the,binding,of,lipoprotein,(a),to,human,vascular,endothelial,and,smooth,muscle,cells.!Blood,!1997.!89(12):!p.!4290?8.!

196.! Nassar,!H.,!et!al.,!alphaIDefensin:,link,between,inflammation,and,atherosclerosis.!Atherosclerosis,!2007.!194(2):!p.!452?7.!

197.! Quinn,!K.L.,!A.;!Han,!B.;!Zhang,!H.!,!Human,neutrophil,peptides,promote,atherogenesis,in,HNPIexpressing,ApoEI/I,mice.,,!in!Publication,in,preparation2013.!

! 102!

198.! Harrison,!D.,!et!al.,!Role,of,oxidative,stress,in,atherosclerosis.!Am!J!Cardiol,!2003.!91(3A):!p.!7A?11A.!

199.! Bonomini,!F.,!et!al.,!Atherosclerosis,and,oxidative,stress.!Histol!Histopathol,!2008.!23(3):!p.!381?90.!

200.! Schleicher,!E.!and!U.!Friess,!Oxidative,stress,,AGE,,and,atherosclerosis.!Kidney!Int!Suppl,!2007(106):!p.!S17?26.!

201.! Kougias,!P.,!et!al.,!Neutrophil,antimicrobial,peptide,alphaIdefensin,causes,endothelial,dysfunction,in,porcine,coronary,arteries.!J!Vasc!Surg,!2006.!43(2):!p.!357?63.!

202.! Porro,!G.A.,!et!al.,!Direct,and,indirect,bacterial,killing,functions,of,neutrophil,defensins,in,lung,explants.!Am!J!Physiol!Lung!Cell!Mol!Physiol,!2001.!281(5):!p.!L1240?7.!

203.! Leitinger,!N.,!Oxidized,phospholipids,as,modulators,of,inflammation,in,atherosclerosis.!Curr!Opin!Lipidol,!2003.!14(5):!p.!421?30.!

204.! Skalen,!K.,!et!al.,!Subendothelial,retention,of,atherogenic,lipoproteins,in,early,atherosclerosis.!Nature,!2002.!417(6890):!p.!750?4.!

205.! Valore,!E.V.,!et!al.,!Human,betaIdefensinI1:,an,antimicrobial,peptide,of,urogenital,tissues.!J!Clin!Invest,!1998.!101(8):!p.!1633?42.!

206.! Quinn,!M.T.,!et!al.,!Oxidatively,modified,low,density,lipoproteins:,a,potential,role,in,recruitment,and,retention,of,monocyte/macrophages,during,atherogenesis.!Proc!Natl!Acad!Sci!U!S!A,!1987.!84(9):!p.!2995?8.!

207.! de!Villiers,!W.J.!and!E.J.!Smart,!Macrophage,scavenger,receptors,and,foam,cell,formation.!J!Leukoc!Biol,!1999.!66(5):!p.!740?6.!

208.! Gerrity,!R.G.,!The,role,of,the,monocyte,in,atherogenesis:,I.,Transition,of,bloodIborne,monocytes,into,foam,cells,in,fatty,lesions.!Am!J!Pathol,!1981.!103(2):!p.!181?90.!

209.! Linton,!M.F.!and!S.!Fazio,!Class,A,scavenger,receptors,,macrophages,,and,atherosclerosis.!Curr!Opin!Lipidol,!2001.!12(5):!p.!489?95.!

210.! Ruggeri,!Z.M.,!Platelets,in,atherothrombosis.!Nat!Med,!2002.!8(11):!p.!1227?34.!211.! May,!P.,!et!al.,!The,LDL,receptorIrelated,protein,(LRP),family:,an,old,family,of,proteins,with,

new,physiological,functions.!Ann!Med,!2007.!39(3):!p.!219?28.!212.! Bajari,!T.M.,!et!al.,!LDL,receptor,family:,isolation,,production,,and,ligand,binding,analysis.!

Methods,!2005.!36(2):!p.!109?16.!213.! Llorente?Cortes,!V.!and!L.!Badimon,!LDL,receptorIrelated,protein,and,the,vascular,wall:,

implications,for,atherothrombosis.!Arterioscler!Thromb!Vasc!Biol,!2005.!25(3):!p.!497?504.!214.! Schulz,!S.,!et!al.,!Role,of,LDL,receptorIrelated,protein,(LRP),in,coronary,atherosclerosis.!Int!J!

Cardiol,!2003.!92(2?3):!p.!137?44.!215.! White?Adams,!T.C.,!et!al.,!Identification,of,coagulation,factor,XI,as,a,ligand,for,platelet,

apolipoprotein,E,receptor,2,(ApoER2).!Arterioscler!Thromb!Vasc!Biol,!2009.!29(10):!p.!1602?7.!

216.! Shen,!G.Q.,!et!al.,!An,LRP8,variant,is,associated,with,familial,and,premature,coronary,artery,disease,and,myocardial,infarction.!Am!J!Hum!Genet,!2007.!81(4):!p.!780?91.!

217.! Kim,!D.H.,!et!al.,!Human,apolipoprotein,E,receptor,2.,A,novel,lipoprotein,receptor,of,the,low,density,lipoprotein,receptor,family,predominantly,expressed,in,brain.!J!Biol!Chem,!1996.!271(14):!p.!8373?80.!

218.! Dobbing,!J.,!The,BloodIBrain,Barrier:,Some,Recent,Developments.!Guys!Hosp!Rep,!1963.!112:!p.!267?86.!

219.! Dhopeshwarkar,!G.A.!and!J.F.!Mead,!Uptake,and,transport,of,fatty,acids,into,the,brain,and,the,role,of,the,bloodIbrain,barrier,system.!Adv!Lipid!Res,!1973.!11(0):!p.!109?42.!

220.! Trommsdorff,!M.,!et!al.,!Reeler/DisabledIlike,disruption,of,neuronal,migration,in,knockout,mice,lacking,the,VLDL,receptor,and,ApoE,receptor,2.!Cell,!1999.!97(6):!p.!689?701.!

! 103!

221.! Li,!Y.,!et!al.,!Differential,functions,of,members,of,the,low,density,lipoprotein,receptor,family,suggested,by,their,distinct,endocytosis,rates.!J!Biol!Chem,!2001.!276(21):!p.!18000?6.!

222.! Korschineck,!I.,!et!al.,!Identification,of,a,novel,exon,in,apolipoprotein,E,receptor,2,leading,to,alternatively,spliced,mRNAs,found,in,cells,of,the,vascular,wall,but,not,in,neuronal,tissue.!J!Biol!Chem,!2001.!276(16):!p.!13192?7.!

223.! Riddell,!D.R.,!et!al.,!Identification,and,characterization,of,LRP8,(apoER2),in,human,blood,platelets.!J!Lipid!Res,!1999.!40(10):!p.!1925?30.!

224.! Pennings,!M.T.,!et!al.,!Platelets,express,three,different,splice,variants,of,ApoER2,that,are,all,involved,in,signaling.!J!Thromb!Haemost,!2007.!5(7):!p.!1538?44.!

225.! Desai,!K.,!et!al.,!Binding,of,apoEIrich,high,density,lipoprotein,particles,by,saturable,sites,on,human,blood,platelets,inhibits,agonistIinduced,platelet,aggregation.!J!Lipid!Res,!1989.!30(6):!p.!831?40.!

226.! Riddell,!D.R.,!A.!Graham,!and!J.S.!Owen,!Apolipoprotein,E,inhibits,platelet,aggregation,through,the,LIarginine:nitric,oxide,pathway.,Implications,for,vascular,disease.!J!Biol!Chem,!1997.!272(1):!p.!89?95.!

227.! Korporaal,!S.J.,!et!al.,!Binding,of,low,density,lipoprotein,to,platelet,apolipoprotein,E,receptor,2',results,in,phosphorylation,of,p38MAPK.!J!Biol!Chem,!2004.!279(50):!p.!52526?34.!

228.! Akkerman,!J.W.,!From,lowIdensity,lipoprotein,to,platelet,activation.!Int!J!Biochem!Cell!Biol,!2008.!40(11):!p.!2374?8.!

229.! Robertson,!J.O.,!et!al.,!Deficiency,of,LRP8,in,mice,is,associated,with,altered,platelet,function,and,prolonged,time,for,in,vivo,thrombosis.!Thromb!Res,!2009.!123(4):!p.!644?52.!

230.! Ramesh,!S.,!et!al.,!Antiphospholipid,antibodies,promote,leukocyteIendothelial,cell,adhesion,and,thrombosis,in,mice,by,antagonizing,eNOS,via,beta2GPI,and,apoER2.!J!Clin!Invest,!2011.!121(1):!p.!120?31.!

231.! Reddy,!S.S.,!et!al.,!Similarities,and,differences,in,structure,,expression,,and,functions,of,VLDLR,and,ApoER2.!Mol!Neurodegener,!2011.!6:!p.!30.!

232.! Burk,!R.F.,!et!al.,!Deletion,of,apolipoprotein,E,receptorI2,in,mice,lowers,brain,selenium,and,causes,severe,neurological,dysfunction,and,death,when,a,lowIselenium,diet,is,fed.!J!Neurosci,!2007.!27(23):!p.!6207?11.!

233.! Blake,!S.M.,!et!al.,!ThrombospondinI1,binds,to,ApoER2,and,VLDL,receptor,and,functions,in,postnatal,neuronal,migration.!EMBO!J,!2008.!27(22):!p.!3069?80.!

234.! Stockinger,!W.,!et!al.,!The,reelin,receptor,ApoER2,recruits,JNKIinteracting,proteinsI1,and,I2.!J!Biol!Chem,!2000.!275(33):!p.!25625?32.!

235.! Dumanis,!S.B.,!et!al.,!ApoE,receptor,2,regulates,synapse,and,dendritic,spine,formation.!PLoS!One,!2011.!6(2):!p.!e17203.!

236.! Ballif,!B.A.,!et!al.,!Activation,of,a,Dab1/CrkL/C3G/Rap1,pathway,in,ReelinIstimulated,neurons.!Curr!Biol,!2004.!14(7):!p.!606?10.!

237.! Mei,!S.,!et!al.,!p38,mitogenIactivated,protein,kinase,(MAPK),promotes,cholesterol,ester,accumulation,in,macrophages,through,inhibition,of,macroautophagy.!J!Biol!Chem,!2012.!287(15):!p.!11761?8.!

238.! Metzler,!B.,!et!al.,!Increased,expression,and,activation,of,stressIactivated,protein,kinases/cIJun,NH(2)Iterminal,protein,kinases,in,atherosclerotic,lesions,coincide,with,p53.!Am!J!Pathol,!2000.!156(6):!p.!1875?86.!

239.! Rahaman,!S.O.,!et!al.,!A,CD36Idependent,signaling,cascade,is,necessary,for,macrophage,foam,cell,formation.!Cell!Metab,!2006.!4(3):!p.!211?21.!

240.! Ricci,!R.,!et!al.,!Requirement,of,JNK2,for,scavenger,receptor,AImediated,foam,cell,formation,in,atherogenesis.!Science,!2004.!306(5701):!p.!1558?61.!

! 104!

241.! D'Arcangelo,!G.,!et!al.,!A,protein,related,to,extracellular,matrix,proteins,deleted,in,the,mouse,mutant,reeler.!Nature,!1995.!374(6524):!p.!719?23.!

242.! Tissir,!F.!and!A.M.!Goffinet,!Reelin,and,brain,development.!Nat!Rev!Neurosci,!2003.!4(6):!p.!496?505.!

243.! Lambert!de!Rouvroit,!C.,!et!al.,!Reelin,,the,extracellular,matrix,protein,deficient,in,reeler,mutant,mice,,is,processed,by,a,metalloproteinase.!Exp!Neurol,!1999.!156(1):!p.!214?7.!

244.! Jossin,!Y.,!et!al.,!The,reelin,signaling,pathway:,some,recent,developments.!Cereb!Cortex,!2003.!13(6):!p.!627?33.!

245.! Jossin,!Y.,!Neuronal,migration,and,the,role,of,reelin,during,early,development,of,the,cerebral,cortex.!Mol!Neurobiol,!2004.!30(3):!p.!225?51.!

246.! Hisanaga,!A.,!et!al.,!A,disintegrin,and,metalloproteinase,with,thrombospondin,motifs,4,(ADAMTSI4),cleaves,Reelin,in,an,isoformIdependent,manner.!FEBS!Lett,!2012.!586(19):!p.!3349?53.!

247.! D'Arcangelo,!G.,!et!al.,!Reelin,is,a,ligand,for,lipoprotein,receptors.!Neuron,!1999.!24(2):!p.!471?9.!

248.! Hiesberger,!T.,!et!al.,!Direct,binding,of,Reelin,to,VLDL,receptor,and,ApoE,receptor,2,induces,tyrosine,phosphorylation,of,disabledI1,and,modulates,tau,phosphorylation.!Neuron,!1999.!24(2):!p.!481?9.!

249.! Royaux,!I.,!et!al.,!Genomic,organization,of,the,mouse,reelin,gene.!Genomics,!1997.!46(2):!p.!240?50.!

250.! Rice,!D.S.,!et!al.,!The,reelin,pathway,modulates,the,structure,and,function,of,retinal,synaptic,circuitry.!Neuron,!2001.!31(6):!p.!929?41.!

251.! Kubo,!K.,!K.!Mikoshiba,!and!K.!Nakajima,!Secreted,Reelin,molecules,form,homodimers.!Neurosci!Res,!2002.!43(4):!p.!381?8.!

252.! Utsunomiya?Tate,!N.,!et!al.,!Reelin,molecules,assemble,together,to,form,a,large,protein,complex,,which,is,inhibited,by,the,functionIblocking,CRI50,antibody.!Proc!Natl!Acad!Sci!U!S!A,!2000.!97(17):!p.!9729?34.!

253.! Nakajima,!K.,!et!al.,!Disruption,of,hippocampal,development,in,vivo,by,CRI50,mAb,against,reelin.!Proc!Natl!Acad!Sci!U!S!A,!1997.!94(15):!p.!8196?201.!

254.! Tseng,!W.L.,!et!al.,!Reelin,is,a,platelet,protein,and,functions,as,a,positive,regulator,of,platelet,spreading,on,fibrinogen.!Cell!Mol!Life!Sci,!2010.!67(4):!p.!641?53.!

255.! Lutter,!S.,!et!al.,!Smooth,muscleIendothelial,cell,communication,activates,Reelin,signaling,and,regulates,lymphatic,vessel,formation.!J!Cell!Biol,!2012.!197(6):!p.!837?49.!

256.! Smalheiser,!N.R.,!et!al.,!Expression,of,reelin,in,adult,mammalian,blood,,liver,,pituitary,pars,intermedia,,and,adrenal,chromaffin,cells.!Proc!Natl!Acad!Sci!U!S!A,!2000.!97(3):!p.!1281?6.!

257.! Botella?Lopez,!A.,!et!al.,!Reelin,is,overexpressed,in,the,liver,and,plasma,of,bile,duct,ligated,rats,and,its,levels,and,glycosylation,are,altered,in,plasma,of,humans,with,cirrhosis.!Int!J!Biochem!Cell!Biol,!2008.!40(4):!p.!766?75.!

258.! Pulido,!J.S.,!et!al.,!Reelin,expression,is,upregulated,following,ocular,tissue,injury.!Graefes!Arch!Clin!Exp!Ophthalmol,!2007.!245(6):!p.!889?93.!

259.! Chen,!X.,!et!al.,!UpIregulation,of,ATP,binding,cassette,transporter,A1,expression,by,very,low,density,lipoprotein,receptor,and,apolipoprotein,E,receptor,2.!J!Biol!Chem,!2012.!287(6):!p.!3751?9.!

260.! Quinn,!K.,!et!al.,!Human,neutrophil,peptides:,a,novel,potential,mediator,of,inflammatory,cardiovascular,diseases.!Am!J!Physiol!Heart!Circ!Physiol,!2008.!295(5):!p.!H1817?24.!

261.! van!Wetering,!S.,!et!al.,!Defensins:,key,players,or,bystanders,in,infection,,injury,,and,repair,in,the,lung?!J!Allergy!Clin!Immunol,!1999.!104(6):!p.!1131?8.!

! 105!

262.! Jossin,!Y.,!et!al.,!The,central,fragment,of,Reelin,,generated,by,proteolytic,processing,in,vivo,,is,critical,to,its,function,during,cortical,plate,development.!J!Neurosci,!2004.!24(2):!p.!514?21.!

263.! Feng,!J.,!et!al.,!DownIregulation,of,DDAH2,and,eNOS,induces,endothelial,dysfunction,in,sinoaorticIdenervated,rats.!Eur!J!Pharmacol,!2011.!661(1?3):!p.!86?91.!

264.! Cai,!H.!and!D.G.!Harrison,!Endothelial,dysfunction,in,cardiovascular,diseases:,the,role,of,oxidant,stress.!Circ!Res,!2000.!87(10):!p.!840?4.!

265.! De!Keulenaer,!G.W.,!et!al.,!Oscillatory,and,steady,laminar,shear,stress,differentially,affect,human,endothelial,redox,state:,role,of,a,superoxideIproducing,NADH,oxidase.!Circ!Res,!1998.!82(10):!p.!1094?101.!

266.! Dupont,!G.P.,!et!al.,!Regulation,of,xanthine,dehydrogenase,and,xanthine,oxidase,activity,and,gene,expression,in,cultured,rat,pulmonary,endothelial,cells.!J!Clin!Invest,!1992.!89(1):!p.!197?202.!

267.! Quyyumi,!A.A.,!Endothelial,function,in,health,and,disease:,new,insights,into,the,genesis,of,cardiovascular,disease.!Am!J!Med,!1998.!105(1A):!p.!32S?39S.!

268.! Frojmovic,!M.M.,!Platelet,aggregation,in,flow:,differential,roles,for,adhesive,receptors,and,ligands.!Am!Heart!J,!1998.!135(5!Pt!2!Su):!p.!S119?31.!

269.! Sitia,!S.,!et!al.,!From,endothelial,dysfunction,to,atherosclerosis.!Autoimmun!Rev,!2010.!9(12):!p.!830?4.!

270.! Verma,!S.,!M.R.!Buchanan,!and!T.J.!Anderson,!Endothelial,function,testing,as,a,biomarker,of,vascular,disease.!Circulation,!2003.!108(17):!p.!2054?9.!

271.! Yasui,!N.,!et!al.,!Functional,importance,of,covalent,homodimer,of,reelin,protein,linked,via,its,central,region.!J!Biol!Chem,!2011.!286(40):!p.!35247?56.!

272.! Yuan,!Y.,!et!al.,!Reelin,is,involved,in,transforming,growth,factorIbeta1Iinduced,cell,migration,in,esophageal,carcinoma,cells.!PLoS!One,!2012.!7(2):!p.!e31802.!

273.! Alvarez?Dolado,!M.,!et!al.,!Thyroid,hormone,regulates,reelin,and,dab1,expression,during,brain,development.!J!Neurosci,!1999.!19(16):!p.!6979?93.!

274.! Chen,!Y.,!et!al.,!Induction,of,the,reelin,promoter,by,retinoic,acid,is,mediated,by,Sp1.!J!Neurochem,!2007.!103(2):!p.!650?65.!

275.! Botella?Lopez,!A.,!et!al.,!BetaIamyloid,controls,altered,Reelin,expression,and,processing,in,Alzheimer's,disease.!Neurobiol!Dis,!2010.!37(3):!p.!682?91.!

276.! Ma,!Y.,!et!al.,!Corticosterone,regulates,the,expression,of,neuropeptide,Y,and,reelin,in,MLOIY4,cells.!Mol!Cells,!2012.!33(6):!p.!611?6.!

277.! Bender,!R.A.,!et!al.,!Roles,of,17ssIestradiol,involve,regulation,of,reelin,expression,and,synaptogenesis,in,the,dentate,gyrus.!Cereb!Cortex,!2010.!20(12):!p.!2985?95.!

278.! Courtes,!S.,!et!al.,!Reelin,controls,progenitor,cell,migration,in,the,healthy,and,pathological,adult,mouse,brain.!PLoS!One,!2011.!6(5):!p.!e20430.!

279.! Chen,!Y.,!et!al.,!On,the,epigenetic,regulation,of,the,human,reelin,promoter.!Nucleic!Acids!Res,!2002.!30(13):!p.!2930?9.!

280.! Bird,!A.P.!and!A.P.!Wolffe,!MethylationIinduced,repressionIIbelts,,braces,,and,chromatin.!Cell,!1999.!99(5):!p.!451?4.!

281.! Lu,!R.,!et!al.,!Regulation,of,the,promoter,activity,of,interferon,regulatory,factorI7,gene.,Activation,by,interferon,snd,silencing,by,hypermethylation.!J!Biol!Chem,!2000.!275(41):!p.!31805?12.!

282.! Newell?Price,!J.,!A.J.!Clark,!and!P.!King,!DNA,methylation,and,silencing,of,gene,expression.!Trends!Endocrinol!Metab,!2000.!11(4):!p.!142?8.!

283.! Tucker,!K.L.,!Methylated,cytosine,and,the,brain:,a,new,base,for,neuroscience.!Neuron,!2001.!30(3):!p.!649?52.!

! 106!

284.! Gross,!C.M.,!et!al.,!Early,life,stress,stimulates,hippocampal,reelin,gene,expression,in,a,sexIspecific,manner:,evidence,for,corticosteroneImediated,action.!Hippocampus,!2012.!22(3):!p.!409?20.!

285.! Korcok,!J.,!et!al.,!P2Y6,nucleotide,receptors,activate,NFIkappaB,and,increase,survival,of,osteoclasts.!J!Biol!Chem,!2005.!280(17):!p.!16909?15.!

286.! Morioka,!N.,!et!al.,!The,activation,of,P2Y,receptor,in,cultured,spinal,microglia,induces,the,production,of,CCL2,through,the,MAP,kinasesINFIkappaB,pathway.!Neuropharmacology,!2013.!75C:!p.!116?125.!

287.! Krstic,!D.,!M.!Rodriguez,!and!I.!Knuesel,!Regulated,proteolytic,processing,of,Reelin,through,interplay,of,tissue,plasminogen,activator,(tPA),,ADAMTSI4,,ADAMTSI5,,and,their,modulators.!PLoS!One,!2012.!7(10):!p.!e47793.!

288.! Faurschou,!M.!and!N.!Borregaard,!Neutrophil,granules,and,secretory,vesicles,in,inflammation.!Microbes!Infect,!2003.!5(14):!p.!1317?27.!

289.! Hiemstra,!P.S.,!S.!van!Wetering,!and!J.!Stolk,!Neutrophil,serine,proteinases,and,defensins,in,chronic,obstructive,pulmonary,disease:,effects,on,pulmonary,epithelium.!Eur!Respir!J,!1998.!12(5):!p.!1200?8.!

290.! Higazi,!A.A.,!et!al.,!Defensin,modulates,tissueItype,plasminogen,activator,and,plasminogen,binding,to,fibrin,and,endothelial,cells.!J!Biol!Chem,!1996.!271(30):!p.!17650?5.!

291.! Ahn,!J.K.,!et!al.,!The,role,of,alphaIdefensinI1,and,related,signal,transduction,mechanisms,in,the,production,of,ILI6,,ILI8,and,MMPs,in,rheumatoid,fibroblastIlike,synoviocytes.!Rheumatology!(Oxford),!2013.!52(8):!p.!1368?76.!

292.! Pappas,!G.D.,!V.!Kriho,!and!C.!Pesold,!Reelin,in,the,extracellular,matrix,and,dendritic,spines,of,the,cortex,and,hippocampus:,a,comparison,between,wild,type,and,heterozygous,reeler,mice,by,immunoelectron,microscopy.!J!Neurocytol,!2001.!30(5):!p.!413?25.!

293.! Martinez?Cerdeno,!V.,!et!al.,!Reelin,immunoreactivity,in,the,adult,primate,brain:,intracellular,localization,in,projecting,and,local,circuit,neurons,of,the,cerebral,cortex,,hippocampus,and,subcortical,regions.!Cereb!Cortex,!2002.!12(12):!p.!1298?311.!

294.! Befus,!A.D.,!et!al.,!Neutrophil,defensins,induce,histamine,secretion,from,mast,cells:,mechanisms,of,action.!J!Immunol,!1999.!163(2):!p.!947?53.!

295.! K,!B.R.M.H.J.P.S.G.Z.J.,!AMPIactivated,protein,kinase,as,regulator,of,P2Y6,receptorIinduced,insulin,secretion,in,mouse,pancreatic,B,cells.!Biochemical!Pharmacology,!2013.!85:!p.!991?998.!

296.! Kottgen,!M.,!et!al.,!P2Y6,receptor,mediates,colonic,NaCl,secretion,via,differential,activation,of,cAMPImediated,transport.!J!Clin!Invest,!2003.!111(3):!p.!371?9.!

297.! Hansson,!G.K.,!Inflammation,,atherosclerosis,,and,coronary,artery,disease.!N!Engl!J!Med,!2005.!352(16):!p.!1685?95.!

298.! Eikemo,!H.,!O.F.!Sellevold,!and!V.!Videm,!Markers,for,endothelial,activation,during,open,heart,surgery.!Ann!Thorac!Surg,!2004.!77(1):!p.!214?9.!

299.! Szmitko,!P.E.,!et!al.,!New,markers,of,inflammation,and,endothelial,cell,activation:,Part,I.!Circulation,!2003.!108(16):!p.!1917?23.!

300.! Ross,!A.C.,!et!al.,!Relationship,between,inflammatory,markers,,endothelial,activation,markers,,and,carotid,intimaImedia,thickness,in,HIVIinfected,patients,receiving,antiretroviral,therapy.!Clin!Infect!Dis,!2009.!49(7):!p.!1119?27.!

301.! Knowles,!J.W.,!et!al.,!Enhanced,atherosclerosis,and,kidney,dysfunction,in,eNOS(I/I)Apoe(I/I),mice,are,ameliorated,by,enalapril,treatment.!J!Clin!Invest,!2000.!105(4):!p.!451?8.!

302.! Virdis,!A.,!et!al.,!Inducible,nitric,oxide,synthase,is,involved,in,endothelial,dysfunction,of,mesenteric,small,arteries,from,hypothyroid,rats.!Endocrinology,!2009.!150(2):!p.!1033?42.!

! 107!

303.! Nagareddy,!P.R.,!et!al.,!Increased,expression,of,iNOS,is,associated,with,endothelial,dysfunction,and,impaired,pressor,responsiveness,in,streptozotocinIinduced,diabetes.!Am!J!Physiol!Heart!Circ!Physiol,!2005.!289(5):!p.!H2144?52.!

304.! Wilcox,!J.N.,!et!al.,!Expression,of,multiple,isoforms,of,nitric,oxide,synthase,in,normal,and,atherosclerotic,vessels.!Arterioscler!Thromb!Vasc!Biol,!1997.!17(11):!p.!2479?88.!

305.! Ferdinandy,!P.,!et!al.,!Peroxynitrite,is,a,major,contributor,to,cytokineIinduced,myocardial,contractile,failure.!Circ!Res,!2000.!87(3):!p.!241?7.!

306.! Cooke,!C.L.!and!S.T.!Davidge,!Peroxynitrite,increases,iNOS,through,NFIkappaB,and,decreases,prostacyclin,synthase,in,endothelial,cells.!Am!J!Physiol!Cell!Physiol,!2002.!282(2):!p.!C395?402.!

307.! Romay?Tallon,!R.,!et!al.,!The,coexpression,of,reelin,and,neuronal,nitric,oxide,synthase,in,a,subpopulation,of,dentate,gyrus,neurons,is,downregulated,in,heterozygous,reeler,mice.!Neural!Plast,!2010.!2010:!p.!130429.!

308.! Herrmann,!G.,!G.!Mishev,!and!A.L.!Scotti,!Olfactory,bulb,interneurons,releasing,NO,exhibit,the,Reelin,receptor,ApoEr2,and,part,of,those,targeted,by,NO,express,Reelin.!J!Chem!Neuroanat,!2008.!36(3?4):!p.!160?9.!

309.! Riddell,!D.R.,!et!al.,!Localization,of,apolipoprotein,E,receptor,2,to,caveolae,in,the,plasma,membrane.!J!Lipid!Res,!2001.!42(6):!p.!998?1002.!

310.! Feron,!O.,!et!al.,!Endothelial,nitric,oxide,synthase,targeting,to,caveolae.,Specific,interactions,with,caveolin,isoforms,in,cardiac,myocytes,and,endothelial,cells.!J!Biol!Chem,!1996.!271(37):!p.!22810?4.!

311.! Youssefian,!T.,!et!al.,!Platelet,and,megakaryocyte,dense,granules,contain,glycoproteins,Ib,and,IIbIIIIa.!Blood,!1997.!89(11):!p.!4047?57.!

312.! Horn,!M.,!et!al.,!Human,neutrophil,alphaIdefensins,induce,formation,of,fibrinogen,and,thrombospondinI1,amyloidIlike,structures,and,activate,platelets,via,glycoprotein,IIb/IIIa.!J!Thromb!Haemost,!2012.!10(4):!p.!647?61.!

313.! Ahamed,!J.,!et!al.,!In,vitro,and,in,vivo,evidence,that,thrombospondinI1,(TSPI1),contributes,to,stirringI,and,shearIdependent,activation,of,plateletIderived,TGFIbeta1.!PLoS!One,!2009.!4(8):!p.!e6608.!

314.! Isenberg,!J.S.,!et!al.,!ThrombospondinI1,stimulates,platelet,aggregation,by,blocking,the,antithrombotic,activity,of,nitric,oxide/cGMP,signaling.!Blood,!2008.!111(2):!p.!613?23.!

315.! Kunapuli,!S.P.,!et!al.,!Platelet,purinergic,receptors.!Curr!Opin!Pharmacol,!2003.!3(2):!p.!175?80.!

316.! Dorsam,!R.T.!and!S.P.!Kunapuli,!Central,role,of,the,P2Y12,receptor,in,platelet,activation.!J!Clin!Invest,!2004.!113(3):!p.!340?5.!

317.! Kim,!S.!and!S.P.!Kunapuli,!P2Y12,receptor,in,platelet,activation.!Platelets,!2011.!22(1):!p.!56?60.!

318.! Hardy,!A.R.,!D.J.!Hill,!and!A.W.!Poole,!Evidence,that,the,purinergic,receptor,P2Y12,potentiates,platelet,shape,change,by,a,Rho,kinaseIdependent,mechanism.!Platelets,!2005.!16(7):!p.!415?29.!

319.! Urbanus,!R.T.,!et!al.,!Platelet,activation,by,dimeric,beta2Iglycoprotein,I,requires,signaling,via,both,glycoprotein,Ibalpha,and,apolipoprotein,E,receptor,2'.!J!Thromb!Haemost,!2008.!6(8):!p.!1405?12.!

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