the role of reelin in human neutrophil peptide- … · hypothesis: the hnp-induced endothelial...
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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
xper
imen
tal d
esig
n fo
r the
pot
entia
l the
rape
utic
effe
ct o
f CR
50 a
gain
st H
NP-
med
iate
d at
hero
gene
sis
in
vivo
.
5-w
eek
old
mal
e A
poE-/-
/HN
P(-)
Apo
E-/-/H
NP(+
)
Hig
h Fa
t Die
t
Osm
otic
Pum
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