University of Southern California Dissertations and Theses

LDL protein nitration: implication for protein unfolding and mitochondrial function by p-JNK-2

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LDL protein nitration: implication for protein unfolding and mitochondrial function by p-JNK-2

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LDL PROTEIN NITRATION: IMPLICATION FOR PROTEIN UNFOLDING AND
MITOCHONDRIAL FUNCTION BY P-JNK-2

by

Ryan Thomas Littleton Hamilton

_____________________________________________________________________

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

December 2007

Copyright 2007 Ryan Thomas Littleton Hamilton

ii
DEDICATION

My parents Jack and Ellen Hamilton
I would like to thank my parents for their continued support of my education and
endeavors.
To my wonderful Wife, Tia Sabawi for all of her support of my research and
educational goals.
To my wonderful father and mother in law for all their support and commitment to my
education and research goals.
Dr. Alex Sevanian
This research is dedicated to Dr. Alex Sevanian. Dr. Alex Sevanian provided me with
guidance on LDL
-
and was unable to witness the completion of my dissertation
defense.

iii
ACKNOWLEDGEMENTS

I would like to acknowledge Dr. Enrique Cadenas for mentoring me and allowing
me to join his laboratory and continue my research project that I had started under Dr.
Alex Sevanian. I would like to acknowledge all of the work my laboratory members
and coworkers Dr. Derick Han, Dr. Allen Chang, Dr. Juliana Hwang, Dr. Jerome
Garcia, Li-Peng Yap, Philip Lam, Lulu Tang, Chen Li, Fei Yin, William Tsoi and
Joanne Lee. Each has helped me to attain my research goals. I would also like to
thank Dr. Howard Hodis for all of his input on oxidative stress and cardiovascular
research from his and my former laboratory work with Dr. Sevanian. I would also like
to thank Dr. Liana Asatryan for her input on LDL
-
analysis and Dr. Juliana Hwang for
her input on cellular treatments. I would like to also thank Dr. Roberta Brinton for
allowing me to use her laboratory fluorescent microscope and Dr. Jon Nilsen for
training and helping me with the analyses of images.
I would like to acknowledge the USC School of Pharmacy Proteomics Core for
their analysis of LDL for oxidative modifications. I would also like to acknowledge
Dr. Mario Isas and Dr. Ralf Langen for their instrumental support in CD spectroscopy
analysis. I would like to thank Dr. Tatsuya Sawamura at the University of Osaka Japan
for providing LOX- antibody for our research goals and I would like to thank Dr.
Tzung Hsiai, Dr. Mark Barr, and Dr. Adrian Correa for providing human coronary
artery slices for IHC of nitrotyrosine, CD-36, and mitochondrial localization of p-
JNK-2 data. I would further like to acknowledge Dr. Hsiai for his model of flow in

iv
bifurcations versus straight regions. I would like to thank Dr. Mohammad Alavi and
Dr. Lilian Young for their IHC work on human coronary arteries from the Hoffman
IHC core. I would also like to thank the confocal and electron microscope core at the
Doheny Eye Institute for providing the instrument and assistance to obtain p-JNK-2
co-localization with mitochondria.
Lastly, I would like to thank my committee of Dr. Enrique Cadenas, Dr. Roberta
Brinton, and Dr. Howard Hodis as well as Dr. Roger Duncan, Dr. Tzung Hsiai and Dr.
Alex Sevanian of my qualifying exam committee.

v
TABLE OF CONTENTS

DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF TABLES x
LIST OF FIGURES xi
ABBREVIATIONS xiii
ABSTRACT xvi
CHAPTER I 1
ROLE OF LDL CHOLESTEROL IN THE DEVELOPMENT
OF ATHEROSCLEROSIS 1
Introduction 1
Heart Disease 1
LDL Cholesterol 2
Mechanism to Lesion Formation 2
LDL Cholesterol Structure and Modification 4
LDL Modification by ONOO
-
6
LDL Protein Unfolding 7
OxLDL Scavenger Receptor Mediated Signaling 8
JNK Family of Protein Kinases, ROS and Mitochondrial Regulation 9
JNK as a Redox Sensitive Pathway and Lipid Peroxide Involvement 10
LDL Modification Hypothesis and OxLDL Receptor Signal
Transduction 11
Hypothesis 12
Specific Aims to Test our Hypothesis 13

CHAPTER II 17
PROTOCOLS AND PROCEEDURES USED FOR LDL MODIFICATION
AND CELLULAR EXPERIMENTS 17
Materials and Methods 17
Reagents and Chemicals 17
Isolation of In Vivo LDL, Modification of LDL and Isolation of LDL
-
18
Analysis of Modified LDL (Percent LDL
-
) 19
Analysis of LDL Modifications 19
Oxidation- 19

vi
Nitrotyrosine- 19
Lipid Peroxide Measurements 20
Analysis of Specific Sites of LDL Protein Nitration 20
CD Spectrum Analysis of Protein Structure 21
EC Culture 21
Binding and Uptake of LDL Particles 22
Immunohistochemistry Analyses of Human Coronary Arteries 22
Flow Experiments to Analyze LDL Protein Nitration 23
Measurement of Extracellular O
2
.-
Anion Formation 25
Analysis of NO
2
-
and NO
3
-
26
Quantitative RT-PCR 27
Western Blotting Analyses 28
Treatment of Bovine Aortic ECs with Modified LDL 29
JNK-2 Phosphorylation in Response to LDL Receptor Inhibition 30
Caspase-3 Activity 30
JNK Localization Studies 31
SR-A and CD-36 JNK-2 Co-localization Studies 31
Co-localization of P-Bcl-xL 32
Statistical Analysis 32

CHAPTER III 33
LDL PROTEIN NITRATION: IMPLICATIONS FOR PROTEIN
UNFOLDING 33
Abstract 33
Introduction 34
Results 37
Post-translational Modifications of In Vivo LDL
-
37
CD and Protein Post-translational Modifications 40
Characteristics of ONOO
-
-modified LDL 42
CD Analysis of ONOO
-
-treated LDL 45
SIN-1-modified LDL 47
Binding and Uptake of ONOO
-
-modified LDL 49
Discussion 52
Chemical Modifications and Structural Changes in LDL
-
52
A Functional Role for ONOO
-
in LDL
-
Formation 53
Specific Cellular Receptors for LDL
-
55

vii
CHAPTER IV 57
HEMODYNAMICS INFLUENCE VASCULAR ONOO
-
FORMATION:
IMPLICATION FOR LOW-DENSITY LIPOPROTEIN APOB-100
NITRATION 57
Abstract 57
Introduction 58
Result 60
eNOS and Nitrotyrosine Immunostaining in OSS-exposed Regions
Versus PSS-exposed Regions 60
PSS and OSS Regulated the Relative Production of O
2
−
and
.
NO
(NO
2
−
and NO
3
−
) 64
PSS and OSS Differentially Influenced the Formation of ONOO
-
68
ONOO
-
Modified Specific Protein Nitration 69
Discussion 73

CHAPTER V 80
OXLDL-R DEPENDENT JNK-2 PHOSPHORYALTION: IMPLICATION
FOR MITOCHONDRIAL REGULATION BY P-JNK-2 80
Abstract: 80
Introduction 82
Results 86
Characteristics of Differentially Modified LDL 86
Modified LDL Induced p-JNK-2 and was Suppressed by JNK
Inhibitor 89
JNK-2 Phosphorylation is SR-A and CD-36 Dependent 91
Receptor Dependent Superoxide Production 93
JNK-2 Activation by Modified LDL Ligands Co-localizes
with Mitochondria 97
CD-36 Dependent P-JNK-2 Co-localization with Mitochondria 101
SR-A Dependent p-JNK-2 Co-localization with Mitochondria 103
Modified LDL Induced p-Bcl-xL that was Inhibited by JNK
Inhibitor 107
Modified LDL Induced p-Bcl-xL that was Inhibited by Blocking
both CD-36 and SR-A 109
Differentially Modified LDL Induces P-Bcl-xL Co-localization
with mitochondria 111
Caspase-3 Activation by Differentially Modified LDL 113
Differential Activation of Caspase-3 by Different OxLDL-R 115
Discussion: 117
Modifications of LDL that are Inherently Involved in the
Phosphorylation of JNK-2 117
What OxLDL Receptors are Responsible for Modified LDL Induced
JNK-2 Phosphorylation and Mitochondrial Co-localization 118

viii
A Possible Mechanism to how JNK-2 may be Involved in Endothelial
Cell Regulation/Dysfunction 120

CHAPTER VI 123
ROBUST CD-36 STAINING AND P-JNK-2 CO-LOCALIZATION WITH
MITOCHONDRIA OF THE LUMEN AND VASA VASORUM ECS IN
BIFURCATIONS AND STRAIGHT REGIONS OF DISEASED HEARTS 123
Abstract 123
Introduction 125
Results 125
CD-36 Staining of Human Coronary Artery 125
P-JNK-2 and Cytochrome C Co-localization in ECs 130
Discussion 133
Relevance of CD-36 in ECs of Human Coronary Arteries 133
ECs had P-JNK-2 Co-localization with Mitochondria in Human
Coronary Arteries of Diseased Hearts that were also Positive for CD-36 135

CHAPTER VII 137
LDL PROTEIN UNFOLDING AS A MECHANISM OF
MITOCHONDRIAL DYSFUNCTION IN ENDOTHELIAL CELLS
MEDIATED BY P-JNK-2: INSIGHTS INTO NEW DRUG DISCOVERY 137
Conclusions 137
LDL Cholesterol and Initiation of Atherosclerosis 137
Lipid Peroxides 138
Protein Unfolding 138
Protein Oxidative Modifications 139
The Chemical Modifications Inherent in Atherosclerotic LDL
-
140
Functional Role of ONOO
-
in LDL
-
Formation 141
LDL Modified by ONOO
-
is Physiological 141
Nitrotyrosine Positive Staining of Atherosclerotic Prone Bifurcation
Versus Nitrotyrosine Negative Staining of Non-atherogenic Straight
Region 142
Role of Scavenger Receptors in Modified LDL Induced
Atherosclerosis 142
Whether JNK-2 Phosphorylation is Dependent on LOOH, LDL
Protein Unfolding or Protein Oxidative Modifications 144
JNK-2 Phosphorylation is SR-A and CD-36 OxLDL-R Dependent 144
OxLDL-R Dependent JNK-2 Phosphorylation Involves
Mitochondrial Localization 145
Protein Unfolded LDL Induces oxLDL-R Dependent Apoptosis
Signaling Through P-JNK-2 146
CD-36 Staining is Robust in both Lumen and Vasa Vasorum ECs of
Bifurcations and Straight Regions 147

ix
CD-36 Staining of ECs was Accompanied by P-JNK-2 Co-localization
with Mitochondria Confirming In Vitro Modified LDL Findings 147
Protein Unfolding of LDL as a Model for Mitochondrial Dysfunction
in Atherosclerosis 148
Future Work 149
Concluding Remarks 149
Therapeutics 152

BIBLIOGRAPHY 154

x
LIST OF TABLES

Table 1: LC/MS/MS Analyses of LDL
–
Apo-B100 Protein Modifications 39

Table 2: LC/MS/MS Analyses of ONOO
–
-modified LDL ApoB-100 44

Table 3: Relative Rates of O
2
.-
and
.
NO Production in Response to
PSS and OSS 66

Table 4: PSS and OSS Influenced LDL Protein Nitration 67

Table 5: Tyrosine nitration of LDL ApoB-100 71

Table 6: Differentially Modified LDL Dependent O
2
.-
Production 94

Table 7: Differentially Modified LDL Induced OxLDL Receptor
Dependent O
2
.-
Production 95

xi
LIST OF FIGURES

Figure 1: Overview 12

Figure 2: Overview of Specific Aim 1 13

Figure 3: Overview of Specific Aim 2 14

Figure 4: Overview Flow of Specific Aim 2 and 3 15

Figure 5: Overview of Specific Aim 2 and 3 16

Figure 6: Chemical Modifications of In Vivo LDL Subfractions 38

Figure 7: CD Spectral Analyses of In Vivo 41

Figure 8: ONOO
–
-modified LDL 43

Figure 9: CD Spectral Analyses of ONOO
–
-modified LDL 46

Figure 10: SIN-1-modified LDL 48

Figure 11: Binding of ONOO
–
-modified LDL 50

Figure 12: Uptake of ONOO
–
-modified LDL 51

Figure 13: Sites of Chemical Modification in In Vivo LDL
–

and ONOO
-
-treated LDL 54

Figure 14: Immunostaining of Coronary Arteries for eNOS
and Nitrotyrosine 62

Figure 15: mRNA Expression of BAEC under Differential Flow 65

Figure 16: MS Spectrum of a Representative Nitrated Peptide 72

Figure 17: Characteristics of Differentially Modified LDL 88

Figure 18: Modified LDL Induced Phosphorylation of JNK-2 90

Figure 19: CD-36 and SR-A Induced JNK-2 Phosphorylation and
Minimal LOX-1 and LDL-R Involvement 96

xii
Figure 20: Modified LDL Induced P-JNK-2 Co-localization
with mitochondria 99

Figure 21: JNK Inhibitor Decreases Global P-JNK-2 Levels but not Co-
localization of P-JNK-2 to Mitochondria 100

Figure-22: CD-36 Dependent p-JNK-2 Co-localization to Mitochondria 102

Figure 23: SR-A Dependent P-JNK-2 Co-localization with Mitochondria 105

Figure 24: SR-A and CD-36 Receptor Blocking and Ablation of P-JNK-2
Co-localization with Mitochondria 106

Figure 25: Differentially Modified LDL Induction of Bcl-xL
Phosphorylation and Inhibition with JNK Inhibitor 108

Figure 26: Differentially Modified LDL Induced Phosphorylation of
Bcl-xL 110

Figure 27: Differentially Modified LDL and P-Bcl-xL and
Mitochondrial Localization 112

Figure 28: Differentially Modified LDL Induced Caspase-3 Activity and
Inhibition by JNK Inhibitor 114

Figure 29: Differentially Modified LDL and OxLDL-R Dependent
Caspase-3 Activation 116

Figure 30: CD-36 Staining of Diseased Human Coronary Artery Straight
Regions 128

Figure 31: CD-36 staining of atherosclerosis prone bifurcations of
diseased coronary arteries 129

Figure 32: P-JNK-2 Co-localization with Mitochondria in Straight
Regions of Diseased Human Coronary Arteries 131

Figure 33: P-JNK-2 Co-localization with Mitochondria in the Bifurcations
of Diseased Human Coronary Arteries 132

Figure 34: Significance 151

Figure 35: Future Plausible Therapeutics 153

xiii
ABBREVIATIONS

Low density Lipoprotein LDL

very low density Lipoprotein vLDL

Total LDL tLDL

Native, normal LDL nLDL

electronegative LDL LDL
-

Circular dichroism CD

3-morphilino sydnonimine SIN-1

Peroxynitrite ONOO
-

Nitric oxide
.
NO

Superoxide O
2
.-

Hydrogen peroxide H
2
O
2

Lectin like oxidized receptor (oxLDL-R) LOX-1

Low density lipoprotein receptor LDL-R

oxidized LDL receptor oxLDL-R

oxidized LDL receptor CD-36

oxLDL-R scavenger receptor-A SR-A

oxidized LDL oxLDL

Oxidized phosphatidyl choline oxPAPC

Nicotinic acid dinucleotide phosphate NADPH

NADPH oxidase subunit 4 NOX-4

Cuprous Cu
2+

xiv

Secretory Phospholipase A2 sPLA2

Phospholipase A2 PLA2

Lipid peroxides LOOH

Bovine serum albumin BSA

Pulsatile shear stress PSS

Oscillatory shear stress OSS

Oscillatory flow OF

Pulsatile flow PF

Nitrite NO
2
-

Nitrate NO
3
-

Quantitative reverse transcriptase polymerase chain reaction qRT-PCR

Nuclear factor kappa beta NF-kb

Apolipoprotein E ApoE

Apolipoprotein B-100 ApoB-100

Stress activated protein kinase SAPK

Inducible/endothelial nitric oxide synthase i/e-NOS

High performance liquid chromatography HPLC

Liquid chromatography mass spectrometry mass spectrometry LC/MS/MS

Liquid chromatography electron ion spray mass spectrometry LC/EIS/MS/MS

Left coronary artery LCA

Right coronary artery RCA

Smooth muscle cell SMC

xv

Macrophage foam cell MFC

Endothelial cells EC

c-jun terminal kinase JNK

Reactive oxygen species ROS

Reactive nitrogen species RNS

Anti-apoptotic complex BCL-XLxl

4'-6-Diamidino-2-phenylindole (blue fluorescent probe) DAPI

Fluorescein isothiocyanate (Green probe) FITC

xvi
ABSTRACT

An elevated level of LDL cholesterol is associated with the development of
atherosclerosis and is also associated with aging. Modification of LDL particle is one
of the main contributors to the development of atherosclerosis and is elevated with
increasing plasma LDL concentrations. Modified LDL is usually composed of
LOOH/aldehydes, unfolded protein and some protein post-translational modifications.
It has been debated whether the lipid peroxides or unfolded apoB-100 protein is
important. An important pathway in atherosclerosis may be the phosphorylation of
JNK-2 in ECs. OxLDL-R CD-36 knockout macrophages which have decreased foam
cell formation and decreased JNK-2 phosphorylation as well as an ApoE and JNK-2
double knockout mouse has decreased lesion size and MFC formation. Foam cells
have increased ROS production and mitochondria are the major source of ROS and
this evidence may suggest that p-JNK-2 is involved in regulating mitochondrial
function. We hypothesize that ONOO
-
induced nitration and unfolding of apoB-100
may be a potential mechanism for modification of LDL in vivo and that this unfolded
LDL induces oxLDL-R dependent irreversible mitochondrial dysfunction in ECs to
promote atherosclerosis. The purpose of this study was to determine (a) where the
modified fraction of LDL in vivo (LDL
-
) is nitrated, (b) whether ONOO
-
produces a
particle with a similar nitration pattern and protein unfolding to in vivo LDL
-
, (c)
whether nitrotyrosine is co-localized to the bifurcation and whether OSS induces
ONOO
-
formation, (d) how differentially modified LDL induces JNK-2
phosphorylation, (e) what oxLDL receptors are involved in the phosphorylation of

xvii
JNK-2, (f) whether phospho-JNK-2 co-localizes with mitochondria and is regulating
mitochondrial function. The modified LDL fraction in vivo (LDL
-
) is nitrated in alpha
helices that are involved in protein unfolding. It was also determined that ONOO
-

treated LDL had a similar nitration and unfolding to in vivo LDL
-
. We found that
human coronary artery at the bifurcation where atherosclerosis is prevalent and OF
occurs were positive for nitrotyrosine and implicated ONOO
-
formation by a 1:1 ratio
of O
2
.-
and
.
NO production. Protein unfolding of LDL was the most important initiator
of JNK-2 phosphorylation. Upon receptor blocking, JNK-2 phosphorylation was
dependent on both CD-36 and SR-A oxLDL-R. Modified LDL dependent JNK-2
phosphorylation co-localized with mitochondria and was ablated by both SR-A and
CD-36 receptor blocking antibodies but was only minimally affected by either one
alone suggesting that both receptors are induce p-JNK-2 co-localization to
mitochondria. Phosphorylation of Bcl-xL and caspase-3 activation was blocked by
incubation with both CD-36 and SR-A receptor blocking antibodies. Human coronary
arteries in diseased hearts were robustly positive for CD-36 and p-JNK-2 co-
localization with mitochondria in ECs of the lumen and of the vasa vasorum as well as
in macrophages, MFCs and SMCs. Our findings demonstrate that ONOO
-
may be
involved in the modification of LDL in vivo, that ONOO
-
modified LDL was similar
in structure and nitration pattern to an in vivo nitrated LDL particle and that protein
unfolding is involved in the initiation of apoptosis and atherosclerosis through an
oxLDL-R dependent phosphorylation of JNK-2.

1
CHAPTER I

ROLE OF LDL CHOLESTEROL IN THE DEVELOPMENT
OF ATHEROSCLEROSIS

Introduction

Heart Disease
Atherosclerosis is the leading cause of death in developed countries.
Atherosclerosis, is a multifactorial genetic disease (Maitland-van der Zee, Klungel et
al. 2002) involving the hardening of the artery, occlusion of the artery, plaque
breakage (acute coronary syndrome) as well as thrombosis and is strongly associated
with environmental changes as a result of oxidative stress injury (Munzel, Heitzer et
al. 1997; Heitzer, Schlinzig et al. 2001). Multiple pathways are involved in the
development of atherosclerosis (hardening of the arteries). LDL cholesterol is one of
the major and most important risk factors for development of atherosclerosis.
Hypercholesterolemia in individuals has been associated with an increased risk of
cardiovascular disease (Reiner, Carlson et al. 2007). Cholesterol lowering therapies
have proven to be beneficial in reducing atherosclerosis in both hypercholesterolemia
as well as in normal individuals (Sobel 2007). These findings demonstrate the
importance of LDL cholesterol in the development of atherosclerosis. Although, LDL

2
cholesterol lowering therapy is beneficial, the mechanism of LDL induced
atherogenesis is poorly understood.

LDL Cholesterol
Our laboratory has shown that an electronegative form of LDL (LDL
-
) is
associated with the development of atherosclerosis (Sevanian, Bittolo-Bon et al.
1997). This LDL- is lipid peroxide and aldehyde rich and posseses an extensive alpha
helical unfolding into beta structure (Ursini, Davies et al. 2002; Asatryan, Hamilton et
al. 2005). In vitro modified LDL subfractions have been shown to have increased
LDL-subfractions (Asatryan, Ursini) suggesting that LDL modification may play a
role in LDL toxicity. LDL
-
has also been shown to induce MFC formation in vitro
(Sevanian, Bittolo-Bon et al. 1997) and suggests the importance of the lipid peroxide
rich and protein unfolded LDL particles in the initiation of atherosclerosis.

Mechanism to Lesion Formation
It is thought that LDL is the initiator of atherosclerosis. LDL cholesterol is
known to initiate the expression of monocyte adhesion proteins to induce an initial
inflammatory response through the activation of the NF-κB pathway (Natarajan,
Reddy et al. 2001). EC injury occurs as a result of increased inflammation and SMC
migration resulting in thrombosis and exposure of the extracellular matrix (Siegel-
Axel and Gawaz 2007). The extracellular matrix is continually remodeled by matrix
metalloproteinases as the disease progresses (Hartung, Schafers et al. 2007).

3
However, monocytes invading the ECs of the lumen differentiate into macrophages
and these macrophages begin to uptake modified/unmodified LDL particles resulting
in the formation of MFCs (Hung, Hong et al. 2006). MFCs continue to undergo
necrosis/apoptosis resulting in the formation of a necrotic core (Han, Liang et al.
2006). The necrotic core and matrix continue to expand into the lumen and begin to
demonstrate characteristics of a vulnerable plaque (Han, Liang et al. 2006). The artery
responds to this increased lumen occlusion and increase the production of
.
NO through
eNOS as well as iNOS (He 2005; Zhao, He et al. 2005). The
.
NO formed initiates
vasodilatation of the artery and results in increased blood flow to the ischemic tissues
(Chicoine, Paffett et al. 2007). However, the MFCs are producing lots of H
2
O
2
and
O
2
.-
. O
2
.-
reacts with
.
NO at the fastest rate in biology known to form ONOO
-
(Crow,
Beckman et al. 1995; Turk, Henderson et al. 2005). Nitrotyrosine formation in the
lesions is a footprint of ONOO
-
mediated oxidative stress and may also be involved in
the modification of LDL in vivo suggesting its role in atherosclerosis (Leeuwenburgh,
Hardy et al. 1997). Further foam cell formation, plaque growth and enzymatic
modification of collagen result in a vulnerable plaque that ruptures when the fibrous
cap becomes weak resulting in an acute coronary syndrome which will be followed by
thrombosis (Cheruvu, Finn et al. 2007). However, it is unknown how LDL cholesterol
is completely involved in the development of atherosclerotic lesions and is of interest
for our group. We believe that LDL protein unfolding of the apoB-100 protein is one
of the main contributors to the development of atherosclerosis.

4
LDL Cholesterol Structure and Modification
Structurally, the apoB-100 is an integral LDL liposomal surface protein that
folds into three alpha helices and two beta sheets from n-terminus to c-terminus (α1-
β1-α2-β2-α3) (Hevonoja, Pentikainen et al. 2000; Ursini, Davies et al. 2002). Both
alpha helices and beta sheets stabilize negatively charged phospholipids at the surface
of the lipid core by positively charged lysine and arginine residues as well as
stabilized by hydrophobic stacking of aromatic amino acids (Hevonoja, Pentikainen et
al. 2000). Both alpha helical and beta sheet structures can interact with the
phospholipids, fitting best within the structural constraints of phosphatidylcholine
(PC) (the main phospholipid interacting with apoB-100) and mostly interacting with
alpha helical domains (Hevonoja, Pentikainen et al. 2000). The phospholipids adjust
their position into the grooves of both alpha and beta protein structures (Hevonoja,
Pentikainen et al. 2000). The first alpha helical domain (α1) seems to be important as
an anchor for initializing the lipid protein interaction (Hevonoja, Pentikainen et al.
2000). Alpha helix two and three adopt structural changes based on the size of lipid
core through reversible electrostatic phospholipid protein interactions from the vLDL
particle (200nm) to the LDL particle (80nm) (Hevonoja, Pentikainen et al. 2000). The
particle expands or contracts up or down the phospholipid belt forming a tight
phospholipid-protein (α2, α3) interaction (Hevonoja, Pentikainen et al. 2000). The
alpha three domain crosses over the LDL-receptor (LDL-R) binding domain and may
be further involved in the control of LDL particle uptake (Hevonoja, Pentikainen et al.
2000). However, the beta sheet domains seem to be important in conferring to one

5
structure and are not involved in the unfolding of the protein whereas the alpha helices
are not (Hevonoja, Pentikainen et al. 2000; Ursini, Davies et al. 2002). It is therefore
noted from previous studies by our laboratory that alpha helical unfolding is an
important factor in LDL- formation and LDL toxicity (Ursini, Davies et al. 2002).
Structural changes in modified LDL may be the result of changes in the lipid
protein interactions of the LDL particle or in the lipid environment. Oxidation of
polyunsaturated chains in phospholipids results in a more hydrophilic chain and the
movement of oxidized acyl chains to the lipid water interface (Zhang and Kirsch
2003). Changes in modified LDL structure have been documented in several papers
published by our group, focusing on the effects of lipid modification on particle
structure and on the conformational changes inherent in apoB-100 (Sevanian and
Asatryan 2002; Ursini, Davies et al. 2002; Hwang, Ing et al. 2003; Asatryan, Hamilton
et al. 2005). Less is known about the effects of protein modification on LDL structure
and lipid interactions and most studies to date have utilized LDL modified by enzymes
or oxidized by transition metals (Eiserich, Baldus et al. 2002; Torzewski and Lackner
2006). The latter induces considerable protein fragmentation (Gieseg, Pearson et al.
2003; Torzewski and Lackner 2006) and hence is modified much more than compared
to that of minimally modified LDL (LDL
-
in vivo). However, modification of LDL
particles by ONOO
-
introduces the formation of nitrotyrosine (a zwitterion) that would
confer a more hydrophilic environment. Nevertheless phospholipid-protein
interactions may be altered by increased hydrophilicity of tyrosine and tryptophan
residues upon nitration since they are often involved in aromatic stacking. The

6
physiochemical interaction of lipids and protein should interefere with tyrosine
stacking between adjacent phospholipids as well as disrupt electrostatic interactions
between adjacent phospholipids and lysine/arginine residues resulting in an unfolded
apoB-100 protein.

LDL Modification by ONOO
-

Many groups believe that oxidation of LDL occurs in the endothelial subspace
and that this hypothesis would be consistent with a ONOO
-
dependent
oxidation/modification of LDL. LDL was found positive for nitrotyrosine in LDL
isolated from human atherosclerotic lesions (Leeuwenburgh, Hardy et al. 1997)
suggesting that ONOO
-
may be a model of LDL protein nitration and atherogenesis.
These modified LDL particles under the LDL modification hypothesis may release
back into the blood stream accounting for the modified LDL subfraction obtained
from human plasma (LDL
-
). Oxidation of specific amino acids such as cysteine,
methionine, tyrosine and tryptophan are important in ONOO
-
mediated protein
nitration/oxidation of LDL particles. In this respect, modification by ONOO
-
may
result in the formation of a particle with decreased alpha helical structure and an
increased beta structure with similar structure to an in vivo LDL
-
as described above.
Tyrosine and tryptophan residues become Zwitterions upon nitration (electrostatic
nitro group) which modify the particle’s overall hydrophobicity characteristics and
result in withdrawal from the hydrophobic phase. This should facilitate the exposure
of lysine residues which are known to be involved in the formation of an LDL-and

7
may be accounted for by originally aromatic stacked aromatic amino acids pushing
towards the hydrophilic phase and increasing alpha helical stretch resulting in the loss
of lysine/arginine electrostatic interactions with neighboring phospholipids. Oxidation
of the lipids by ONOO
-
should further support the unfolding of alpha helices by both
increasing the hydrophilicity of the phospholipids as well as from the nitrated
tyrosines. Further, oxidation of cysteine residues to cysteic acid by ONOO
-
should
also induce a more electronegative particle because of the addition of three molecular
oxygen atoms. Therefore, ONOO
-
serves as a model for LDL associated
nitration/oxidation, unfolding, and toxicity.

LDL Protein Unfolding
Both oxidative and enzymatic modifications of LDL have been shown to
induce EC injury, inflammation and monocyte adhesion proteins as well as induce
MFC formation (Natarajan, Reddy et al. 2001; Hung, Hong et al. 2006; Torzewski and
Lackner 2006). These findings support LDL modification as a mechanism of
atherosclerosis as well as a model for protein unfolding in differentially modified LDL
particles. To further support this hypothesis, these differential modifications of LDL
induce the formation of an LDL-particle with similar protein structures to an in vivo
circulating atherogenic LDL
-
particle. These findings suggest that LOOH are not
necessarily the only mechanism to LDL protein unfolding and toxicity but rather may
act synergistically with enzymatic modifications to unfold the LDL particle. We

8
further believe that protein nitration may act synergistically to LOOH to unfold the
LDL particle through both the hydrophilicity of the aromatically stacked tyrosine
residues as well as through the hydrophilicity of LOOH upon the addition of
molecular oxygen. Interestingly, these modified LDL particles (LDL
-
) are ligands for
atherogenic scavenger receptors (Rahaman, Lennon et al. 2006; Torzewski and
Lackner 2006). However, there is strong debate as to what modifications of the LDL
particle are involved in scavenger receptor mediated endocytosis as well as how these
modified LDL particles are involved in scavenger receptor mediated atherosclerosis.

OxLDL Scavenger Receptor Mediated Signaling
Scavenger receptors of modified LDL have been shown to be involved in
inflammation (Natarajan, Reddy et al. 2001), foam cell formation (Hung, Hong et al.
2006) as well as fatty streak formation (Shashkin, Dragulev et al. 2005). There are
three main scavenger receptors of modified LDL in ECs (CD-36, SR-A and LOX-1).
LOX-1 has been shown in knockout mice to have reduced infarct size as well as a
decreased lumen lesion thickness (Mehta, Sanada et al. 2007). CD-36 although
originally found in macrophages is expressed in ECs as well (Butthep, Wanram et al.
2006). CD-36 has been implicated in MFC formation (Rahaman, Lennon et al. 2006).
Interestingly, CD-36 knockout macrophages have no JNK-2 phosphorylation as
compared to control macrophages upon oxLDL treatment suggesting p-JNK-2 may be
involved (Rahaman, Lennon et al. 2006). A double knockout mouse of ApoE and
JNK-2 was found to have decreased MFC formation, oil red plaque staining, as well as

9
decreased SMC migration. This study implicated JNK-2 as model of foam cell
formation whereas the double knockout mouse of JNK-1 and ApoE had no apparent
changes in foam cell formation and lesion formation (Ricci, Sumara et al. 2004).
These findings support a CD-36 and JNK-2 dependent atherosclerotic plaque and
foam cell formation (Ricci, Sumara et al. 2004; Rahaman, Lennon et al. 2006). SR-A
is a target of JNK-2 phosphorylation (Ricci, Sumara et al. 2004) suggesting an indirect
positive feedback loop between CD-36, JNK-2 and SR-A. SR-A was one of the first
scavenger receptors determined (Shechter, Fogelman et al. 1981) and is found in ECs
(Facciponte, Wang et al. 2007). However, it is not known whether SR-A and LOX-1
are involved in the phosphorylation of JNK-2 and whether SR-A and JNK-2 may have
a positive feedback mechanism.

JNK Family of Protein Kinases, ROS and Mitochondrial Regulation
The family of JNK proteins are stress activated protein kinases that are
responsive to different forms of stress including UV (Adachi, Gazel et al. 2003), free
radicals (Dougherty, Kubasiak et al. 2002) and heat shock (Frazier, Wilson et al.
2007). Interestingly, foam cell formation is attributed to both being a lipid loaded
macrophage as well as producing massive quantities of ROS (Kritharides, Upston et
al. 1998). OxLDL treatment of ECs results in mitochondrially derived ROS
(Zmijewski, Moellering et al. 2005) and since mitochondria are the major source of
ROS (Orrenius 2007) in living cells it may be implicated that ROS production in ECs
may be a result of scavenger receptor mediated JNK-2 signaling to the mitochondria.

10
However, it is unknown as to whether JNK-2 is regulating mitochondrial function.
Our laboratory has shown that JNK-1 is able to regulate the key rate limiting enzyme
PDH e1α subunit of the citric acid cycle in cortical neurons (Zhou, Lam et al. 2007).
These neurons are therefore in an energy crisis, reducing equivalent crisis and redox
status crisis and this suggests that JNK-2 may have a role in EC dysfunction (Zhou,
Lam et al. 2007). These findings may provide insight into how JNK-2 may be
mediating MFC formation as well as EC derived ROS production. Further evidence
also suggests that JNK isoforms will regulate apoptosis through the phosphorylation
of Bcl-xL and BAD {Sunayama, 2005 #558; Tsuruta, 2004 #559; Wang, 2007 #581;
Schroeter, 2003 #145} and initiating the mitochondrial apoptosome. These
observations provide the basis for a novel mechanism of mitochondrial function and
dysfunction. The mechanism in volves oxLDL-R mediated ligand binding and
signaling to p-JNK-2 to regulate mitochondrial function.

JNK as a Redox Sensitive Pathway and Lipid Peroxide Involvement
JNK-2 may also be phosphorylated by SAPK ASK1 which is activated by
H
2
O
2
and other oxidants through a redox sensitive activation (Zhou, Lam et al. 2007).
LOOH specifically oxPAPC are known to induce NADPH oxidase activity in ECs,
macrophages and SMC (Hwang, Rouhanizadeh et al. 2006; Hsiai, Hwang et al. 2007).
Production of O
2
.-
and H
2
O
2
should induce a redox sensitive activation of JNK
isoforms. Therefore, a synergistic relationship between oxLDL-receptors and LOOH
is hypothesized. LOOH and aldehydes are also known to activate receptor mediated

11
signaling and induce inflammation and inflammatory cytokines. Lipid peroxidation is
also associated with the formation of inflammatory isoprostanes as well as non-
enzymatically formed prostaglandin-like structures (Balduzzi, Diociaiuti et al. 2004).
LDL LOOH are well established as an initiator of atherosclerosis but it is still in
debate as to whether LOOH or protein unfolding are more important in
atherosclerosis.

LDL Modification Hypothesis and OxLDL Receptor Signal Transduction
LOOH are known to be associated with the development of cardiovascular
disease through the formation of MFCs (Osterud and Bjorklid 2003). However, the
strong correlation with nitrotyrosine in lesions and nitrated LDL protein isolated from
these lesions (Hsiai, Hwang et al. 2007; Parastatidis, Thomson et al. 2007) suggests
that ONOO
-
may be involved in the formation of oxLDL and in the initiation of
atherosclerosis.

12

Hypothesis

We hypothesize that ONOO
-
induced nitration and unfolding of apoB-100 may be a
potential mechanism for modification of LDL in vivo and that this unfolded LDL
induces oxLDL-R dependent irreversible mitochondrial dysfunction in ECs to
promote atherosclerosis (Fig. 1).
ApoB-100 protein unfolding
oxLDL-R dependent mitochondrial driven apoptosis
oxLDL-R dependent phosphorylation of JNK-2
Irreversible damage to the endothelium
Atherosclerosis
nLDL
ONOO
-
LDL
-
In vitro
Aim 1
In vivo
Figure 1: Overview

13
Specific Aims to Test our Hypothesis

1) Determine that the modified LDL subfraction in vivo is nitrated and that ONOO
-

induces protein nitration and unfolding of apoB-100 (Fig. 2).

Figure 2: Overview of Specific Aim 1
nLDL
NOS
.
NO O
2
.-
ONOO
-
Protein unfolding
LOOH
Protein
oxidative
modifications
NADPH
oxidase
In vitro
LDL- in vivo,
in vitro
In vivo?
nLDL
NOS
.
NO O
2
.-
ONOO
-
Protein unfolding
LOOH
Protein
oxidative
modifications
NADPH
oxidase
In vitro
LDL- in vivo,
in vitro
In vivo?

14
2) Determine what modifications of LDL and what oxLDL-Rs are involved in JNK-2
phosphorylation in endothelial cells (Fig. 3,4).

Figure 3: Overview of Specific Aim 2
nLDL
ONOO
-
Protein unfolding
LOOH
Protein
oxidative
modifications
Mod-LDL
(LDL
-
)
CD-36
Mod-LDL
LDL
-
SR-A
P-JNK-2
sPLA2
Cu
2+
nLDL
ONOO
-
Protein unfolding
LOOH
Protein
oxidative
modifications
Mod-LDL
(LDL
-
)
CD-36
Mod-LDL
LDL
-
SR-A
P-JNK-2
sPLA2
Cu
2+

15
3) Determine how modified LDL modulates mitochondrial function
in endothelial cells (Fig. 4,5).

Figure 4: Overview Flow of Specific Aim 2 and 3
Modifiers of LDL
oxLDL-R dependent mitochondrial driven apoptosis
oxLDL-R dependent phosphorylation of JNK-2
Irreversible damage to the endothelium
Atherosclerosis
Aim 2
ApoB-100 protein unfolding
Aim 3
Modifiers of LDL
oxLDL-R dependent mitochondrial driven apoptosis
oxLDL-R dependent phosphorylation of JNK-2
Irreversible damage to the endothelium
Atherosclerosis
Aim 2
ApoB-100 protein unfolding
Aim 3

16

nLDL
ONOO
-
Protein unfolding
Protein
oxidative
modifications
Mod-LDL
(LDL
-
)
CD-36
Mod-LDL
LDL
-
SR-A
P-JNK-2
sPLA2
Cu
2+
Mitochondrial localization
P-BCLxl:BCL-2
Caspase-3
apoptosis
LOOH
nLDL
ONOO
-
Protein unfolding
Protein
oxidative
modifications
Mod-LDL
(LDL
-
)
CD-36
Mod-LDL
LDL
-
SR-A
P-JNK-2
sPLA2
Cu
2+
Mitochondrial localization
P-BCLxl:BCL-2
Caspase-3
apoptosis
LOOH
Figure 5: Overview of Specific Aim 2 and 3

17
CHAPTER II

PROTOCOLS AND PROCEDURES USED FOR LDL MODIFICATION AND
CELLULAR EXPERIMENTS

Materials and Methods

Reagents and Chemicals
ONOO
-
, monoclonal nitrotyrosine antibody, monoclonal phospho-JNK
antibody, and monoclonal p-Bcl-xL antibody were purchased from Millipore
(Billerica, MA). Bovine serum albumin (BSA), 3-morphilino-sydnonimine (Sin-1),
biotin, sPLA2 and 3'-tetramethylindocarbocyanine perchlorate (DiI), and cytochrome
C were purchased from Sigma (St Louis, Missouri). Bovine Aortic ECs were
purchased from Cell Applications, Inc. (San Diego, California). Lectin, like oxLDL-
R, was received from Dr. T. Sawamura as a kind gift (Japan). CD-36 receptor
blocking antibody, SR-A receptor blocking antibody, and LDL-R antibody were
obtained from Beckman Coultier (Fullerton, California), Serotec (Raleigh, North
Carolina), and Calbiochem (San Diego, California) respectively.

Isolation of In Vivo LDL, Modification of LDL and Isolation of LDL
-
LDL (LDL) was pooled from different human plasma (USC blood bank). LDL
isolation was performed by density gradient ultra centrifugation (L-80 XP centrifuge,
SW-41 rotor, Fullerton CA) as previously described (Hodis, Kramsch et al. 1994).

18
LDL (d=1.019-1.063) was then collected and washed with phosphate buffered saline
(PBS) several times using a Millipore (Bedford, MA) centrifugal filtering device with
a 30kDa cut-off. HPLC was used to analyze the tLDL- to the sum of the nLDL, LDL
-
,
and LDL
2-
. Concentrated LDL at 200μg/ml was then treated with the following
conditions for 30 minutes at 37
o
C: (1) ONOO
-
at 0, 10, 25, and 100 μM, (2) Sin-1 at
0, 10, 25, 50, 100, 250, 500, 1000 μM. LDL modification. Sin-1 was used to donate
both
.
NO and O
2
.-
to form ONOO
-
. Concentrated LDL was treated with the following
conditions to yield differentially modified LDL particles: (1) 200 μg/ml of LDL was
suspended in 1x PBS at 37
o
C for 1 hour; (2) 200 μg/ml of LDL was treated with
40μM of copper (Cu
2+
), the conjugated dienes were measured at 234 nm at 37
o
C for 4
hrs, and reaction was stopped by addition of 100 μM DTPA when conjugated dienes
were exponential; (3) 200 μg/ml of LDL and 100 μM ONOO
-
(Millipore, Billerica,
MA) at 37
o
C for 30 minutes and with (4) 50 ng/ml of PLA2 with 1 μg/ml bovine
serum albumin (BSA) (Sigma, St Louis, Missouri) in calcium buffer at 37
o
C for 1
hour. After treatment with PLA2 and Cu
2+
, LDL was subjected to ultracentrifugation
as described above to remove contaminating PLA2 and Cu
2+
. The concentration of
ONOO
-
stock was measured prior to the individual experiments using UV absorption
spectra (302nm) according to the manufacturer’s specification (Upstate, Lake Placid,
NY).

19
Analysis of Modified LDL (Percent LDL
-
)
In vivo LDL and modified LDL were subjected to anion exchange HPLC
(HPLC). NLDL and modified LDL (LDL
-
) were eluted by a stepwise sodium chloride
gradient. NLDL represents the non-atherogenic subfraction whereas the LDL
-

subfraction represents the atherogenic subfraction. Area under the curve was
determined for different electronegative subfractions and percent LDL
-
was
determined as the LDL
-
area divided by the sum of the area for the three subfractions
(nLDL, LDL
-
and LDL
2-
) as previously described (Sevanian, Bittolo-Bon et al. 1997).
The percent LDL
-
serves as a measure of the modified LDL as well as the unfolded
LDL subfraction (previous references to unfolding of LDL in LDL
-
(Ursini, Davies et
al. 2002; Asatryan, Hamilton et al. 2005).

Analysis of LDL Modifications
Oxidation - LDL oxidative protein modifications were assessed for in vivo
LDL subfractions (LDL
-
), in vitro ONOO
-
-treated LDL, PLA2-LDL, and Cu-LDL.
Protein oxidation was determined by a decrease in biotin labeling to the oxidized
cysteine residue. Biotin labeling was performed by incubating biotin with LDL for 1
hour at 37
o
C to label free unmodified cysteine residues.
Nitrotyrosine - Two μg of ONOO
-
-treated LDL, PLA2-LDL and Cu-LDL
were spotted on PVDF and 10 μg of in vivo LDL subfractions and BAEC treated LDL
under flow were spotted on Millipore PVDF membranes. Dot blots were performed at
a 1:3000 dilution in TBS-tween for primary nitrotyrosine antibody and 1:10000

20
dilution for anti-mouse secondary antibody. Similar procedures were performed for 4
μg of biotin-labeled LDL with a monoclonal anti biotin antibody (dilution at 1:10,000,
Sigma, St. Louis, MO) and a secondary antibody (dilution at 1:10,000). Dot Blots
were analyzed using an ECL chemiluminescence kit (Pierce, Rockford, IL), and
densitometry was performed using a Scion Image Software (Scion Corp).

Lipid Peroxide Measurements
Lipid peroxidation was measured by using the leucomethylene blue assay with
tert-butylhydroperoxide as a standard. Colorimetric assay was measured at 650 nm
after 1 hour incubation at room temperature with leucomethylene blue cocktail
mixture prepared as previously described (Asatryan, Hamilton et al. 2005).

Analysis of Specific Sites of LDL Protein Nitration
Liquid chromatography and tandem mass spectrometry (LC-MS/MS) was
performed by using a ThermoFinnigan Surveyor MS-Pump with a BioBasic-18 100
mm X 0.18 mm reverse phase capillary column. Analysis was obtained by the
ThermoFinnigan LCQ Deca XP Plus ion trap mass spectrometer equipped with a
nanospray ion source (ThermoFinnigan) that employed a 4.5 cm metal needle
(Hamilton, 950-00954) in a data-dependent acquisition mode. Electrical contact and
voltage application to the probe tip were established via the nanoprobe assembly.
Spray voltage was set at 2.9 kV and heated capillary temperature at 190
o
C. Mass
spectra were acquired in the m/z 400 – 800 range. Peptide identification was achieved

21
using Mascot 1.9 search software (Matrix Science) with confirmatory or
complementary analyses by TurboSequest as implemented in the Bioworks Browser
3.2, build 41 (ThermoFinnigan). Spectra were searched against the NCBI human
genome database, NCBI build 35. Aromatic nitration, cysteine oxidation and aromatic
hydroxylation’s were assessed using Mascot and Sequest program taking into
consideration charge and mass for in vivo LDL
-
, ONOO
-
treated LDL and LDL
subjected to flow conditions with BAEC for four hours.

CD Spectral Analysis of Protein Structure
CD (CD) allows for probing the secondary structure content of proteins. The
CD spectrum of modified LDL provides a means to determine the conformational
changes of secondary structure in apoB-100 (α-helix, anti-parallel and parallel β-
sheet, β-turn, and random coil). Different secondary structures are represented by the
spectrum between 200 and 260 nm. Deconvolution analysis using CD spectra software
(CDNN) allowed for assessment of the percent structural integrity in the LDL
subfractions of modified LDL.

Endothelial Cell Culture
Bovine aortic ECs (BAEC) between passages 5 and 9 were grown to confluent
monolayers in high glucose (4.5 g/l) DMEM (Dulbecco’s Modified Eagle’s Medium)
supplemented with 10% heat-inactivated fetal bovine serum (Gemcell, West

22
Sacramento, CA) and 100 U/ml penicillin-streptomycin (Irvine Scientific, Santa Ana,
CA), for 48 h in 5% CO
2
at 37
o
C.

Binding and Uptake of LDL Particles
Control and ONOO
–
-supplemented LDL particles were treated with 75 mg/ml
DiI overnight. DiI-labeled LDL particles were ultracentrifuged, collected, dialyzed,
and sterilized to remove the DiI particulate (Ricci, Sumara et al. 2004). BAEC were
treated with 10 µg/ml DiI-labeled LDL at 0
o
C for 90 min or at 37
o
C for 4 h for LDL
binding or uptake experiments, respectively (Ricci, Sumara et al. 2004). Cells were
also treated with 10 μg/ml DiI-labeled LDL with 200 μg/ml of excess unlabeled LDL
as a control to exclude DiI labeling of cell membranes and for LDL binding and
uptake specificity (Ricci, Sumara et al. 2004). Cells were mounted in DAPI-containing
mounting medium and visualized using an Axiom 200M Zeiss Fluorescent
Microscope (Zeiss, Thornwood, NY). Analysis was performed using a DAPI filter for
the nucleus and CY3 filter for the DiI-labeled LDL. Quantification of mean intensity
and image analysis were assessed by Slidebook software (Santa Monica, CA).

Immunohistochemistry Analyses of Human Coronary Arteries
Human coronary arteries were obtained from the explanted hearts of cardiac
transplant patients with ischemic cardiomyopathy in compliance with the Institutional
Review Board. Specific cross sections of the left and right coronary arteries were
analyzed: the lateral wall of arterial bifurcations where OSS develops and the straight

23
segments where PSS occurs (Karino 1986; Ku 1997). Monoclonal antibodies were
used for NOS isoforms (Transduction Labs) and nitrotyrosine (Upstate).
Immunostaining was performed with standard techniques in frozen vascular tissue
using biotinylated secondary antibodies and peroxidase staining. Nitrotyrosine
residues were assessed on paraffin sections after quenching of endogenous peroxidase
with 3% H
2
O
2
, proteinase K treatment for 15 min at room temperature
(Dakocytomation, Carpinteria, CA), and nonspecific adsorption with 3% BSA in PBS
for 15 min using a monoclonal anti-nitrotyrosine antibody (1:50 in PBS/1% BSA,
overnight incubation at 4°C; Zymed, Clinisciences). CD-36 immunostaining was
performed using standard techniques in frozen vascular tissue utilizing biotinylated
secondary staining and peroxidase staining. Staining of CD-36 was performed in the
same methods as described above for nitrotyrosine (CD-36 1:50 in PBS/1%BSA,
overnight at 4
o
C). Further frozen sections were analyzed for double staining of p-JNK
antibody at a 1:100 dilution and cytochrome C at a 1:50 dilution. Sections were then
incubated with a biotin-conjugated goat anti-mouse antibody (1:200 in PBS/1% BSA;
Santa Cruz Biotechnology), followed by a streptavidin-biotin-peroxidase complex (1-h
incubation at room temperature with both agents; Zymed, Clinisciences). DAB were
used as a chromogen and the sections were counterstained with hematoxylin for
visualization of intima, media, SMCs, and adventia. Furthermore, endothelial and
SMCs were stained with monoclonal antibodies specific for von Willebrand factor
(1:25 dilution) and β-actin (1:4000 dilution) (Darkocytomation, Carpintreria, CA).
Negative controls were performed by omitting the primary antibody. Positive controls

24
included brain, kidney, and lymph node tissues. Immunoreactivity to nitrotyrosine and
endothelial NOS (eNOS) was compared between the left main bifurcation and the
right coronary arteries whereas CD-36 staining and p-JNK-2 co-localization with
mitochondria was observed in both bifurcations and straight regions. A semi-
quantitative analysis was performed to determine the percentage of vascular cells
staining positive for eNOS and nitrotyrosine as well as CD-36 and p-JNK-2 co-
localization with mitochondria.

Flow Experiments to Analyze LDL Protein Nitration
Venous blood was obtained from fasting adult human volunteers under
Institutional Review Board approval from the Atherosclerosis Research Unit at the
University of Southern California. Plasma was pooled and immediately separated by
centrifugation at 1500 g for 10 min at 4°C. The technique used for separating LDL ( δ
= 1.019 to 1.063 g/mL) was similar to that described previously (Hodis, Kramsch et al.
1994; Hwang, Saha et al. 2003).
A dynamic flow system was used to deliver temporal variations in shear stress
( ∂τ/ ∂t); namely pulsatile and OSS (Nerem, Alexander et al. 1998; Papadaki 1998;
Hsiai, Cho et al. 2002). Confluent BAEC were subjected to the flow conditions in the
absence and presence of LDL at 50 μg/mL: (1) Control, using cells grown under static
conditions ( τ
ave
= 0 dyn cm
− 2
at ∂τ/ ∂t = 0), (2) PSS at a mean shear stress ( τ
ave
) of
23 dyn cm
− 2
with a temporal variation (∂τ/ ∂t) at 71 dyn cm
− 2
s
− 1
, and (3) OSS at τ
ave

= 0.02 with ∂τ/ ∂t at ± 3 dyn cm
− 2
s
− 1
. After 4 h, BAEC were collected for quantitative

25
RT-PCR and Western blots. In the absence of LDL, the culture medium was collected
to identify the differential production of O
2
−
and NO
2
−
/NO
3
−
in response to PSS and
OSS, respectively. In the presence of LDL, the culture medium was used to determine
apo-B 100 posttranslational modifications.

Measurement of Extracellular O
2
.-
Anion Formation
The production of O
2
−
from BAEC monolayers exposed to the flow conditions
as described above was measured as the O
2
.-
dismutase (SOD)-sensitive reduction of
cytochrome c (Hazen, Zhang et al. 1999; Handy and Loscalzo 2006). In each case, the
medium contained 100 μM acetylated ferricytochrome c (Sigma-Aldrich, St. Louis,
MO) (Hwang, Rouhanizadeh et al. 2006). Control samples were maintained in a cell
culture dish with media containing cytochrome c (100 μM) and incubated at 37°C.
Aliquots of culture medium (300 μl) were collected at 0, 1, 2, 3, and 4 h, and
ferricytochrome c absorbance was measured at 550 nm ( ε
550
= 2.1 × 10
4
M
− 1
cm
− 1
)
(Hwang, Wang et al. 2003). For the OSS and PSS conditions, aliquots of medium
bathing the BAEC in the flow apparatus were aspirated into a media solution
containing acetylated-ferricytochrome c (1 mM) and absorbance was measured at
550 nm at 0, 1, 2, 3, and 4 h. The specificity of reduction by O
2
−
was established by
comparing reduction rates in the presence and absence of SOD at 60 μg/ml
(Rouhanizadeh, Hwang et al. 2005). The corrected rates for SOD-inhibited
cytochrome c reduction were plotted after computing O
2
−
formation. BAEC
monolayers were treated with and without differentially modified LDL particles as

26
described above for 3 hours. O
2
.-
production was measured for receptor blocking
experiments as well for JNK inhibitor experiments. Incubation of cells with 160μM of
partially acetylated-cytochrome c was performed at 37
o
C for three hours and measured
every five minutes at 550nm in an incubated Bio-Rad 96 well micro plate reader (Bio
Rad,). O
2
.-
was measured as an initial rate of production in nmoles/min/million cells.
Cytochrome c extinction coefficient (E
550
=2.1 x 10
4
M
-1
cm
-1
) was used to determine
moles of O
2
.-
produced as measured by cytochrome c reduction. The specificity of
reduction of cytochrome C

was established by comparing reduction rates in the
presence and absence of O
2
.-
dismutase (Cu,Zn-SOD at 60 μg/ml).

Analysis of NO
2
-
and NO
3
-

Quantitative measurements of NO
2
−
and NO
3
−
were performed as an index of
global
.
NO production following methods described previously (Braman and Hendrix
1989; Van Der Vliet, Nguyen et al. 2000). NO is metabolized or decomposed via
various reactions to the metabolites as NO
2
−
and NO
3
−
, which serve as useful measures
of overall NO production and metabolism (Pietraforte, Salzano et al. 2004). Briefly,
the analytical procedure was based on acidic reduction of NO
2
−
and NO
3
−
to NO by
vanadium (III) and purging of NO with helium into a stream of ozone and detected by
an Antek 7020 chemiluminescence NO detector (Antek Instruments, Houston, TX).
At room temperature, vanadium (III) only reduced NO
2
−
, whereas NO
3
−
and other
redox forms of NO (such as S-nitrosothiols) were reduced only if the solution was
heated to 90–100°C, so that both NO
2
−
and total NO could be measured. This allowed

27
for determination of the relative levels of NO
2
−
and NO
3
−
, which might be indicative
of differential oxidative metabolic routes of NO. Quantification was performed by a
comparison with standard solutions of NO
2
−
and NO
3
−
.

Quantitative RT-PCR
Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was
performed to study whether one of the NADPH oxidase homologues, Nox4, and
eNOS were implicated in the relative production of O
2
−
and NO, respectively. After
BAEC were exposed to the flow conditions, total RNA was isolated using the RNeasy
kit (Qiagen). qRT-PCR was performed according to the recommendations of the PE
Biosystems TaqMan PCR Core Reagent Kit (Chu, Heistad et al. 2002). Equal amounts
of RNA at 0.5 μg/ μl were reverse-transcribed with rTth DNA polymerase to bring the
mixed solution to a final concentration of 1× TaqMan buffer, 5 mM MgCl
2
, 0.2 mM
dATP/dCTP/dGTP, 0.4 mM dUTP, 0.1 mM probe, 0.4 μM primers, 0.01 U/ μl
AmpErase, and 0.025 U/ μl rTth DNA polymerase. Total cDNA at 0.4 ng/ μl in 5 μl
was then transferred to the 96-well plate, followed by RT, beginning with manual
ramp rate at 50°C for 2 min, 60°C for 30 min, and then 95°C for 5 min. Next, PCR
was performed for 50 cycles at 94°C for 20 s and annealing from 58 to 65°C for 1 min
(MJ Research Opticon System). C
T
is the threshold cycle number at which the initial
amplification becomes detectable by fluorescence. ΔR
n
normalizes fluorescence.
TaqMan probes were used for added specificity and sensitivity (Walker 2002). Each
sample was tested in duplicate. Each experiment was performed 5 times. The

28
difference in C
T
values for various flow conditions versus control was used to
mathematically determine the relative difference in the level of NADPH oxides
homologue, Nox4, and eNOS mRNA expression (Walker 2002). For quantification of
relative gene expression, the target sequence was normalized to the expressed
housekeeping gene GAPDH or β-actin.

Western Blotting Analyses
Western blots were performed to determine if one of the NADPH oxidase
homologues, Nox4, was implicated in O
2
−
and NO production, respectively. BAEC
lysate was size-separated in 10% SDS Bio-Rad polyacrylamide electrophoresis gel
(Bio-Rad) and electro-transferred to a PVDF membrane (Millipore). Membranes were
blocked with 5% nonfat milk in TBS-T and probed with mouse Nox4 antibody
(generously provided by Dr. J. David Lambeth at Emory University School of
Medicine, Pathology and Laboratory Medicine). Membranes were incubated overnight
at 4°C and then probed with secondary antibody, goat anti-mouse IgG-HRP
conjugated (Bio-Rad). Chemiluminescence detection was used to visualize bands of
interest (SuperSignal, Pierce) followed by exposure to autoradiography film
(Hyperfilm MP, Amersham Pharmacia Biotech). Loading and transfer of equal
amounts of protein in each lane were verified by reprobing the membrane with a
monoclonal anti- β-actin antibody from mouse ascites fluid (1:3000 dilution, Sigma-
Aldrich). Photographic films were scanned by an imaging densitometer and quantified
using the NIH Image software program.

29
Cross section slices of coronary arteries were fixed with paraformaldehyde.
Slides were then heated to 95C overnight in ascorbate buffer to release non specific
paraformaldehyde cross linking for antibody binding and analysis. Nitrotyrosine
antibody was used at a dilution of 1:300 while CD-36 was used at a dilution of 1:50,
p-JNK-2 antibody at a dilution of 1:250, and cytochrome c at a dilution of 1:100.
Secondary antibody at a dilution of 1:1000 for nitrotyrosine antibody, CD-36 and p-
JNK-x was conjugated to a brown pigment whereas to visualize co-localization of p-
JNK-x and cytochrome C, the cytochrome c antibody was conjugated to a
blue pigment.

Treatment of Bovine Aortic ECs with Modified LDL
Bovine aortic ECs (BAEC) were treated with different concentrations of
modified LDL over various durations to assess the optimal concentrations and time
points for JNK-2 phosphorylation. Optimal LDL at a concentration of 0 mg/ml, 10
mg/ml for control, 10μg/ml of Cu
2+
-treated LDL, 10μg/ml of ONOO
-
-treated LDL,
and 10μg/ml PLA2-treated LDL were incubated with BAEC for 30 minutes.
Membranes were analyzed for phospho-JNK-2 using an antibody to phospho-JNK and
determining the MW from protein standards and β-actin (Millipore, Billerica, MA)
was used as a loading control. JNK-2 phosphorylation was inhibited by pre-treatment
of cells for 1 hour with JNK and inhibitor. Cells were then treated the same way as
described above with modified LDL.

30
JNK-2 Phosphorylation in Response to LDL Receptor Inhibition
Bovine Aortic ECs were pretreated with oxLDL and LDL receptor antibodies
at 37
o
C at 1:250 dilution; specifically, anti-LOX-1, anti-CD-36, anti-SR-A and anti-
LDL
-
R. Cells were pretreated for one hour as follows: (1) control in the absence of
anti-LDL receptors, (2) treatment of BAEC with anti-LOX-1, anti-CD-36, anti-SR-A
and anti-LDL
-
R, (3) treatment with all oxLDL/LDL receptors blocking antibodies
except for anti-LOX-1, (4) treatment with all oxLDL/LDL receptor blocking
antibodies except for anti-CD-36, (5) treatment with all oxLDL/LDL receptor
blocking antibodies except for anti-SR-A, and (6) treatment with all oxLDL/LDL
receptor blocking antibodies except for anti-LDL
-
receptor. Cells were then treated
with 10μg/ml of modified LDL as described above for 30 minutes and western
analysis performed for p-JNK-2 (Millipore, Billerica, MA). β-actin was used as a
loading control (Millipore, Billerica, MA).

Caspase-3 Activity
Caspase-3 activity was measured after the treatment of BAECs with modified
LDL at 10μg/ml for 5 hours with the above mentioned modified LDL particles.
Caspase-3 activity was measured for JNK inhibitor, and receptor blocking experiments
as described above. Cellular samples at 50μg were incubated overnight with a
caspase-3 substrate kit. After 15 hours, the total p-NA released was assessed for each
treatment condition as μM p-NA/μg protein/day.

31
JNK Localization Studies
BAEC cells were treated with and without JNK inhibitor as described above
for 30 minutes and with 10μg/ml of modified LDL particles. Cells were then
incubated with mitotracker red at a dilution of 1:10000 for ten minutes, washed three
times and fixed in -80
o
C chilled methanol for ten minutes. Slides were maintained in
a dark environment to minimize photo-bleaching. Slides were then probed with JNK
antibody at a 1:250 dilution in the cold room overnight and with FITC labeled
secondary for 1hr at room temperature. Slides were then mounted in DAPI containing
mounting medium and fluorescence measured by confocal
microscopy (Zeiss, Thornwood, NY).

SR-A and CD-36 JNK-2 Co-localization Studies
BAEC cells were pre-treated with CD-36 receptor blocking antibody, SR-A
receptor blocking antibody or with both CD-36 and SR-A receptor blocking antibodies
for 1 hour. After a 1 hour pre-treatment cells were incubated with differentially
modified LDL as ascribed above for p-JNK-2 co-localization studies and incubated for
30 minutes and analyzed for p-JNK-2 co-localization with mitochondria as
ascribed above.

32
Co-localization of P-Bcl-xL
BAEC cells were treated with 10μg/ml of differentially modified LDL
particles for 30 minutes and immunofluorescence experiments performed as described
above for p-JNK-2 at a 1:250 dilution of both primary and anti-FITC secondary.

Statistical Analysis
Data were expressed as mean ± SD and compared among separate
experiments. For comparisons between two groups, two-sample independent-groups
t-test was used. Comparisons of multiple values were made by one-way analysis of
variance (ANOVA), and statistical significance among multiple groups determined
using the Tukey test and statistical significance among multiple groups determined
using the Tukey test (for pairwise comparisons of means between control and
treatments. P-values of < 0.05 are considered statistically significant.

33
CHAPTER III

LDL PROTEIN NITRATION: IMPLICATION FOR LDL
PROTEIN UNFOLDING

Abstract
Oxidatively- or enzymatically-modified low-density lipoprotein (LDL) is
intimately involved in the initiation and progression of atherosclerosis. The in vivo
modified LDL is electro-negative (LDL
–
) and consists of peroxidized lipid and
unfolded ApoB-100 protein. This study was aimed at establishing specific protein
modification and conformational changes in LDL
–
by liquid chromatography/tandem
mass spectrometry (LC/MS/MS) and CD analyses. The functional significance of
these chemical modifications and structural changes were validated with binding and
uptake experiments to- and by bovine aortic ECs (BAEC).
The plasma LDL
–
fraction showed increased nitrotyrosine and lipid peroxide
content as well as a greater cysteine oxidation as compared with native- and tLDL.
LC/MS/MS analyses of LDL– revealed specific modifications in the apoB-100
moiety, largely involving nitration of tyrosines in the α-helical structures and β
2
sheet
as well as cysteine oxidation to cysteic acid in β
1
sheet. CD analyses showed that the
α-helical content of LDL
–
was substantially lower (∼25%) than that of nLDL (∼90%);
conversely, LDL
–
showed greater content of β-sheet and random coil structure, in
agreement with unfolding of the protein. These results were mimicked by treatment of

34
LDL subfractions with peroxyNO3- (ONOO
–
) or SIN-1: similar amino acid
modifications as well as conformational changes (loss of α-helical structure and gain
in β-sheet structure) were observed. Both LDL
–
and ONOO
–
-treated LDL showed a
statistically significant increase in binding and uptake to- and by BAEC compared to
nLDL.
It is suggested that lipid peroxidation and protein nitration may account for the
mechanisms leading to apoB-100 protein unfolding and consequential increase in
modified LDL binding and uptake to and by ECs.

Introduction
LDL (LDL) particles transport cholesterol, cholesterol esters, lipids,
phospholipids, and are involved in the maintenance of membrane fluidity (Colell,
Garcia-Ruiz et al. 2003). The LDL particle is comprised of lipid core and an apoB-100
(apoB-100) moiety. The latter assumes a pentapartite structure with alternating α-
helices and β-pleated sheets (α
1
–β
1
–α
2
– β
2
–α
3
) (Hevonoja, Pentikainen et al. 2000). α
1

anchors the protein to the lipid core; α
2
and α
3
expand and contract across the
phospholipid belt of the LDL particle to stabilize electrostatic interactions, thus
maintaining LDL protein structural integrity. β-sheets are structurally rigid and
engaged in electrostatic interactions with the phospholipids (Hevonoja, Pentikainen et
al. 2000).

35
It is widely recognized that oxidative and/or enzyme-mediated modifications
of LDL are required for the particle to acquire the inflammatory properties inherent in
the initiation and progression of atherosclerosis (Asatryan, Hamilton et al. 2005;
Torzewski and Lackner 2006). This notion is strengthened by the observation that
post-translational modifications of apoB100 are elevated in atherosclerotic lesions
(Leeuwenburgh, Hardy et al. 1997). Oxidation of LDL can be carried out by transition
metals, hemoglobin, myeloperoxidase, ceruloplasmin and ROS generated by vascular
endothelium (Osterud and Bjorklid 2003; Koller, Volf et al. 2006; Malle, Marsche et
al. 2006). The oxidative modifications render the LDL particle electronegatively
charged (LDL
–
) as compared to nLDL (nLDL) (Gomes, Alves et al. 2004; Asatryan,
Hamilton et al. 2005; Hwang, Rouhanizadeh et al. 2006). Also, LDL
–
(in vivo
oxidatively modified LDL) contains elevated level of LOOH and aldehydes that are
implicated in protein unfolding (Ursini, Davies et al. 2002). RNS, especially ONOO
-
,
generated by the vascular endothelium, NO3- apo-B-100 in LDL particles (Hazen and
Heinecke 1997; Botti, Batthyany et al. 2004; Hwang, Rouhanizadeh et al. 2006; Hsiai,
Hwang et al. 2007). Enzyme-mediated modifications of LDL –accomplished through
the action of ubiquitous hydrolytic enzymes– confer atherogenic properties to the
lipoprotein particles (Han, Momeni et al. 2003; Torzewski and Lackner 2006): s-PLA2
(Asatryan, Hamilton et al. 2005) and its free fatty acid product (Dersch, Ichijo et al.
2005), cholesteryl esterases (Torzewski and Lackner 2006), plasmin (Torzewski,
Suriyaphol et al. 2004), and matrix metalloproteinase -2 and -9 (Torzewski,
Suriyaphol et al. 2004).

36
This oxidative- and/or enzymatically-modified LDL possesses inflammatory
properties: it activates cytokines (Saad, Virella et al. 2006) and monocyte adhesion
molecules (Hsiai, Cho et al. 2003). The LDL particle is internalized by cells via the
ubiquitously expressed LDL receptor (LDL-R). Rather than binding to the LDL-R
commonly present in cells, protein unfolding in modified LDL promotes binding to
the scavenger receptors (LDL-SR) in vascular ECs (Brown, Mander et al. 2000) and to
CD-36 in macrophages (Ursini, Davies et al. 2002; Zheng, Nukuna et al. 2004).
ONOO
–
-modified LDL is recognized by macrophages, thus gaining further relevance
in endothelial dysfunction and initiation of atherosclerosis (Ferraro, Galli et al. 2003;
Yamaguchi, Matsuno et al. 2004; Hsiai, Hwang et al. 2007).
In this study, we assessed specific protein modifications and conformational
changes of in vivo oxidatively-modified LDL (LDL
–
) and ONOO
–
-treated LDL by
liquid chromatography and tandem mass spectrometry (LC/MS/MS) analyses and CD
(CD) spectra. The significance of ONOO
–
-driven modifications is underscored by the
implication of both NADPH oxidase (a source of O
2
.
–
) (Hwang, Wang et al. 2003;
Stocker and Keaney 2005; Hwang, Rouhanizadeh et al. 2006) and eNOS (a source of
.
NO) (Behr-Roussel, Rupin et al. 2000) activities in vascular endothelial dysfunction
and by the fast reaction of O
2
.
–
and
.
NO to yield ONOO
-
.

37
Results

Post-translational Modifications of In Vivo LDL
–
In vivo nLDL (nLDL) and LDL
-
were isolated from tLDL (tLDL) using anion
exchange chromatography as described in the Materials and Methods section. LDL
nitration was assessed by immunoreactivity to nitrotyrosine antibody (Fig. 6A). While
nitrotyrosine was not detectable in either nLDL or tLDL, it was prominent in the LDL
–

fraction (n = 3; P < 0.001; Fig. 6A). Oxidation of the nine free cysteine residues in the
in vivo LDL subfractions was assessed by biotin labeling of free cysteine (Fig. 6B).
LDL
–
harbored a significantly lower level of free cysteine in comparison with nLDL
and tLDL: nLDL < tLDL < LDL
–
) (n = 3, P < 0.001), thus suggesting an elevated
amount of cysteine oxidation in LDL
–
(Fig. 6B). Also worth noting was a 3-fold
increase in LOOH in LDL
–
in comparison with nLDL and
tLDL (n = 3, P < 0.001) (Fig. 6C).
LC/MS/MS analyses of in vivo LDL– revealed specific modifications in the
apoB-100 moiety (Table 1): tyrosine (Tyr), tryptophan (Trp), cysteine (Cys), and
phenylalanine (Phe) underwent nitration/oxidation in both α helices and β sheets;
namely, α
1
(Tyr
276,666,720
, Trp
583
), α
2
(Tyr
2523
), β
2
/α
3
(Phe
3969
), β
1
(Cys
1112
), α
3
(Tyr
4141
),
and β
2
(Tyr
3139,3295,3489
) (Table 1) as corroborated by the Mascot and Sequest scores as
well as analysis of peptide and peptide ions masses. Cys
1112
in β
1
sheet was oxidized to
cysteic acid.

38
Figure 6: Chemical Modifications of In Vivo LDL Subfractions.

LDL subfractions were isolated by anion exchange chromatography
as described in the Materials and Methods section and analyzed for (A)
Nitrotyrosine content, (B) Free cysteine content (biotin labeling), and
labeling, and (C) Lipid peroxide content. (n = 3, P < 0.01).
nLDL tLDL LDL
-
A
nLDL tLDL LDL
-
***
Nitrotyrosine Density a.u.
4
3
2
0
1
nLDL
tLDL LDL
-
***
Free cysteine a.u.
4
3
2
0
1
***
B
[M LP]/g apoB-100
nLDL tLDL LDL
-
**
3
2
1
0
C
nLDL tLDL LDL
-

39
______________________________________________________________________________________________________________________
Secondary Peptide Modified sequence MSc XC Δcn modification
Structure AA charge
_________________________________________________________________________________________________________________________________________________
α
1
276-287 NO
2
-Tyr
276
Y*GMVAQVTQTLK 51 2 2.8 0.4 nitrotyrosine
α
1
580-589 NO
2
-Trp
583
ILPW*EQNEQV 30 2 3.0 0.2 nitrotryptophan
α
1
655-669 NO
2
-Tyr
666
IEGNLIFDPNNY*LPK 46 2 3.3 0.4 nitrotyrosine
α
1
718-732 NO
2
-Tyr
720
ALY*WAVNQGQ 49 2 2.4 0.1 nitrotyrosine
VPDGVSK
β
1
1101-115 SO
3
H-Cys
1112
ITEVALMGH 84 2 4.9 0.5 cysteic acid
LSC*DTK
α
2
2523-2534 NO
2
-Tyr
2524
MY*QMDIQQELQR 36 2 3.0 0.3 nitrotyrosine
β
2
3137-3148 NO
2
-Tyr
3139
LPY*TIITPPLK 30 2 1.9 0.5 nitrotyrosine
β
2
3292-3311 NO
2
-Tyr
3295
VPSY*TLILPSL 70 2 5.6 0.5 nitrotyrosine
ELPVLHVPR
β
2
3481-3497 NO
2
-Tyr
3489
LSLESLTSY*FSIESSTK 62 2 4.7 0.7 nitrotyrosine
β
2
3953-3973 HO-Phe
3965
DFSAEYEEDGKF* 59 2 2.5 0.5 hydroxyphenylalanine
EGLQEWEGK
α
3
4133-4145 NO
2
-Tyr
4141
AASGTTGTY*QEWK 46 2 2.7 0.1 nitrotyrosine
___________________________________________________________________________________________________________________
Table. 1: Protein Oxidative Modifications of ApoB-100 in LDL
-
Subfraction

Mass spectrometry analysis of in vivo LDL- tabulating the secondary
structure, primary structure location, sequence, Mascot score, ion charge, Sequest
score ΔCN and type of amino acid and its modification. Modifications classified
as alpha helical or as β-sheets.

40
CD and Protein Post-translational Modifications
Previous studies have suggested that LDL
–
had significantly unfolded α-helical
structure. CD analysis was used in order to establish an association between specific
LDL protein modifications and protein structure. In vivo LDL subfractions (Fig. 7A,
B) displayed a decrease in optical rotativity at 200-260 nm from LDL
–
to tLDL to
nLDL (Fig. 7A). Increasing optical rotativity at the 220 nm valley reflects a loss in α-
helical character and an increase in β-structural components as determined by
deconvolution (Fig. 7B). Deconvolution analysis using CD spectra software (CDNN)
determined the percent structural integrity of the aforementioned components in LDL
subfractions of in vivo modified LDL. The α-helical content in nLDL (∼90%) was
largely higher than that in LDL
–
(∼25%), thus suggesting substantial protein unfolding
in the latter. These data suggest an association between α-helical nitration (Table 1),
lipid peroxidation (Fig. 6C), and protein unfolding (Fig. 7) in LDL
–
.

41

Figure 7: Circular Dichroism Spectral Analyses of In Vivo LDL Subfractions

(A) Circular dichroism spectra for different LDL subfractions were
performed as described in the Materials and Methods section. (B) Secondary
structures of LDL subfractions; data were obtained from spectra in (A), which
were deconvoluted for apoB-100 secondary structure. (n = 3, P < 0.01).
as alpha helical or as b-sheets.
% structure
100
80
60
40
20
0
Secondary structure
α-helix anti-
parallel
parallel
β-turn rndm.
coil
***
***
***
***
***
*
*
B
50
30
10
-10
-30
-50
260 245 230 215 200
A
LDL
-
tLDL
nLDL
LDL
-
tLDL
nLDL
tLDL
LDL
-
nLDL
tLDL
LDL
-
nLDL
θ (m deg)
Wavelength (nm)

42
Characteristics of ONOO
–
-modified LDL
Treatment of LDL with ONOO
–
induced tyrosine nitration in apoB-100 protein
in a dose-dependent manner (Fig. 8A; n = 3; P < 0.01). Densitometry analysis of
biotin labeling of free cysteine showed an exponential decrease in the level of free
cysteine, thus suggesting increased cysteine oxidation in response to ONOO
–

treatment (n = 3, P < 0.001) (Fig. 8B). Treatment of LDL with ONOO
–
induced a
dose-dependent accumulation of LOOH in LDL (Fig. 8C). HPLC analysis revealed a
dose-dependent linear increase in the percentage of LDL
–
in response to ONOO
–
treatment (Fig. 8D); this increase was paralleled by an increase in nitrotyrosine- (Fig.
8A) and lipid peroxide (Fig. 8C) content. Oxidized lipid-derived aldehydes were also
significantly elevated in response to ONOO
–
treatment (data not shown). It may be
surmised that both LOOH and nitrotyrosine formation are involved in ONOO
–
-
induced modification of LDL to an atherogenic form (LDL
–
).
Table 2 lists the nitration and oxidation sites in the apoB-100 protein upon
treatment with 100 µM ONOO
–
: α
1
(Tyr
103,413,666
), α
2
(Tyr
2524
), β
2
(Tyr
3490,3791
), β
2
/α
3

(Phe
3965
), and α
3
(Tyr
4088
). Neither in vivo LDL
–
(Table 1) nor ONOO
–
-treated LDL
(Table 2) showed nitration of β
1
. Similarly to in vivo LDL
–
, ONOO
–
treatment elicited
β
2
nitration at Tyr
3490
(in addition to Tyr
3791
). The phenylalanine residue between β
2

and α
3
underwent hydroxylation as also observed in in vivo LDL
–
. These data
strengthen the notion that in vivo LDL
–
may originate from ONOO
–
-driven
modifications at specific sites in the apoB-100 protein.

43
Figure 8: ONOO
–
-modified LDL

LDL samples were supplemented with different amounts of
ONOO– and analyzed for (A) nitrotyrosine, (B) free cysteine (after biotin
labeling), (C), lipid peroxides, and (D) LDL– content (percentage).
0 20 40 60 80 100 120
[M LP]/g apoB-100
20
15
10
5
0
**
**
C
m=0.034
0 20 40 60 80 100 120
Nitrotyrosine density a.u.
20
15
10
5
0
***
***
**
A
m=0.0636
0 10 25 100
ONOO
-
(μM)
Free cysteine a.u.
3
2
1
0
0 20 40 60 80 100 120
B
*
**
***
0 20 40 60 80 100 120
% LDL-
20
15
10
5
0
***
***
***
D
m=0.1582
0 10 25 100
ONOO
-
(μM)
ONOO
-
(μM)
ONOO
-
(μM)
ONOO
-
(μM)
ONOO
-
(μM)

44
______________________________________________________________________________________________________________________
Secondary Peptide Modified sequence MSc XC Δcn modification
Structure AA charge
______________________________________________________________________________________________________________________
α
1
101-110 NO
2
-Tyr
103
EVY*GFNPEGK 29 1 1.7 0.4 nitrotyrosine
α
1
401-427 NO
2
-Tyr
413
VHANPLLIDWTY* 33 2 2.6 0.3 nitrotyrosine
LVLALIPEPSAQQLR
α
1
655-669 NO
2
-Trp
666
IEGNLIFDPNNY*LPK 28 2 2.8 0.4 nitrotyrosine
α
2
2523-2534 NO
2
-Tyr
2524
MY*QMDIQQELQR 65 2 3.7 0.4 nitrotyrosine
β
2
3481-3497 NO
2
-Tyr
3490
LSLESLTSY*FSIEESSTK 20 2 0.2 2.0 nitrotyrosine
β
2
3767-3772 NO
2
-Tyr
3771
EIQIY*K 25 1 1.3 0.1 nitrotyrosine
β
2
3953-3973 HO-Phe
3965
DFSAEYEEDGKF* 30 2 3.7 0.4 hydroxyphenylalanine

EGLQEWEGK
α
3
4088-4098 NO
2
-Tyr
4088
Y*HWEHTGLTLR 28 2 2.1 0.3 nitrotyrosine
_____________________________________________________________________________________________________________________
Table 2: Protein Oxidative Modifications of ApoB-100 in LDL- Subfraction

Mass spectrometry analysis of in vivo LDL- tabulating the secondary
structure, primary structure location, sequence, Mascot score, ion charge,
Sequest score ΔCN and type of amino acid and its modification. Modifications
classified as alpha helical or as b-sheets.

45

CD Analysis of ONOO
–
-treated LDL
ONOO
–
-treated LDL (Fig. 9A) displayed a decrease in optical rotativity at
200-260 nm for LDL
–
as compared to nLDL. There was a distinctive difference in the
CD spectra for LDL subfractions at 220 nm (LDL
–
> tLDL > nLDL). As mentioned
above, increasing optical rotativity at 220 nm reflects a loss in α-helical structure and
an increase in β-sheet structure that was confirmed by CD deconvolution software
(CDNN) assessing the percent structural integrity of the different LDL subfractions
components of ONOO
–
-modified LDL (Fig. 9B). Interestingly, the percentage of
structural components in in vivo LDL
–
and that of ONOO
–
-treated LDL were
similar (Fig. 9C).

46
Figure 9: Circular Dichroism Spectral Analyses of ONOO
–
-modified LDL
tLDL was treated with 100 µM ONOO
–
as described in the Materials
and Methods section; the ONOO
–
-treated tLDL was fractionated into nLDL
and LDL
–
components and CD spectra were determined. (A) Circular
dichroism spectra of LDL subfractions. (B) Secondary structure of different
LDL subfractions. CD deconvolution software was used to determine the
changes in the structures of the LDL subfractions. (C) Comparison of
secondary structure components in in vivo LDL
–
and LDL
–
from ONOO
–
-
treated tLDL. Data taken from Fig. 2B (for in vivo LDL
–
) and Fig. 4B (LDL
–
fraction of the ONOO
–
-treated tLDL). (n = 3, P < 0.01).
50
30
10
-10
-30
-50
260 245 230 215 200
A
LDL
-
tLDL
nLDL
LDL
-
tLDL
nLDL
% structure
100
80
60
40
20
0
Secondary structure
α-helix anti-
parallel
parallel rndm.
coil
***
***
***
***
***
***
***
***
***
***
β-turn
B
tLDL
LDL
-
nLDL
tLDL
LDL
-
nLDL
Secondary structure
α-helix anti-
parallel
parallel rndm.
coil
β-turn
% structure
100
80
60
40
20
0
C
LDL
-
in vivo
LDL
-
100μM ONOO
-
θ (m deg)
Wavelength (nm)

47

SIN-1-modified LDL
Because atherosclerotic lesions have increased activities of iNOS/eNOS and
NADPH oxidase, a flux of
.
NO and O
2
.–
(to yield ONOO
–
) may be mimicked by SIN-
1. Nitrotyrosine- (Fig. 10A) and lipid peroxide (Fig. 10C) accumulation as well as the
percentage of LDL
–
formation (Fig. 10D) following incubation of LDL with SIN-1
were similar to those observed with ONOO
–
(Fig. 8). SIN-1 also induced an oxidation
of cysteine residues (Fig. 10B) greater than that obtained with ONOO
–
(Fig. 8B).

48
Figure 10: SIN-1-modified LDL

LDL particles were incubated with different amounts of SIN-1 and
analyzed for (A) nitrotyrosine, (B) free cysteine, (C) LOOH, and (D)
percentage of LDL
–
as described in the Materials and Methods section.

0 10 25 50 100 250 500 1000
0 200 400 600 800 1000 1200
Sin-1 (μM)
Sin-1 (μM)
Nitrotyrosine Density a.u.
4
3
2
0
1
***
***
**
*
A
m=0.0009
0 200 400 600 800 1000 1200
Sin-1 (μM)
4
3
2
0
1
***
***
*****
**
**
*
[M LP]/g apoB-100
C
m=0.0020
% LDL
-
20
15
10
5
0
0 200 400 600 800 1000 1200
Sin-1 (μM)
***
**
***
**
**
D
m=0.0090
0 10 25 50 100 250 500 1000
B
Sin-1 (μM)
Sin-1 (μM)
Free cysteine a.u.
4
3
2
0
1
*
*
**
**
**
**
***
0 200 400 600 800 1000 1200

49
Binding and Uptake of ONOO
–
-modified LDL
The biological significance of modified LDL was assessed with respect to LDL
binding to- (Fig. 11) and uptake by (Fig. 12) bovine aortic vascular endothelial
(BAEC) cells. Binding experiments, performed at 0°C, are shown for DiI-labeled LDL
(Fig. 11A) and DiI-labeled, ONOO
–
-treated LDL (Fig. 12B). Similar approaches with
an excess of 200 µg/ml unlabeled LDL were used to rule out DiI labeling of cell
membranes and ascertain LDL binding specificity (Fig. 11C,D). Analysis of the mean
fluorescence intensity indicated that binding of ONOO
–
-treated LDL was stronger
than that of control LDL (Fig. 11E).
Uptake experiments, performed at 37°C, are shown in Fig. 12 with a similar
approach to that in Fig. 11: DiI-labeled LDL (Fig. 12A) and DiI-labeled, ONOO
–
-
treated LDL (Fig. 12B). As with binding experiments, uptake of ONOO
–
-treated LDL
was slightly higher than that of nLDL (Fig. 12E). The stronger binding and uptake of
ONOO
–
-treated LDL may suggest alternate uptake mechanisms as well as an
increased uptake of unfolded proteins.
In the presence of a 20-fold excess of nLDL (Fig. 11C, 12C) or ONOO
–
-
treated LDL (Fig. 11D, 12D), respectively, binding and uptake were significantly
inhibited (Fig. 11E, 12E), thus suggesting that LDL is binding to- and taken up by
cells rather than nonspecific labeling of BAEC plasma membranes with DiI.

50

Figure 11: Binding of ONOO
–
-modified LDL

LDL binding experiments were performed at 0
o
C for 90 min with
the following conditions: (A) control-LDL 10μg/ml DiI-LDL, (B) ONOO
-
-
treated LDL 10μg/ml DiI-PN-LDL, (C) control-LDL 10μg/ml DiI-LDL +
200μg/ml unlabeled control-LDL, and (D) ONOO
–
-treated LDL 10μg/ml
DiI-PN-LDL + excess unlabeled ONOO
-
-treated LDL 200μg/ml. Binding
mean intensity of DiI labeled LDL was quantified using Axiom 200M
Zeiss Fluorescent Microscope (E). Experiments were performed in
triplicate and statistical significance to control-LDL and to the excess
unlabeled LDL (P < 0.01) was determined.
400
300
200
100
0
Control-LDL PN-LDL
E
DiI-LDL
DiI-LDL + Ex unlabeled LDL
C D
A B
Mean intensity DiI a.u)
**
* +++
+++

51
Figure 12: Uptake of ONOO
–
-modified LDL

LDL uptake experiments were performed at 37
o
C for 4 h with the
following conditions; (A) control-LDL 10μg/ml DiI-LDL, (B) ONOO
–
-
treated LDL 10μg/ml DiI-PN-LDL, (C) control-LDL 10μg/ml DiI-LDL +
200μg/ml unlabeled control-LDL, and (D) ONOO
–
-treated LDL 10μg/ml
DiI-PN-LDL + excess unlabeled ONOO
–
-treated LDL 200μg/ml (D). Uptake
mean intensity of DiI labeled LDL was quantified using Axiom 200M Zeiss
Fluorescent Microscope (E). Experiments were performed in triplicate and
statistical significance to control-LDL (P < 0.001) and to the excess
unlabeled LDL (P < 0.001).
400
300
200
100
0
Control-LDL PN-LDL
E
Mean intensity DiI a.u.
DiI-LDL
DiI-LDL + Ex unlabeled LDL
C D
A B
+++
+++
*
*

52
Discussion

LDL
–
may be viewed as a circulating, atherogenic form of LDL in vivo and it
harbors secondary structural changes in apoB-100 that encompass a significant loss of
α-helical structure and increase in β-sheet structure (Asatryan, Hamilton et al. 2005).
This study addresses (a) the chemical modifications and structural changes inherent in
LDL
–
formation, (b) a functional role for ONOO
–
in LDL
–
formation, and (c) the
occurrence of specific cellular receptors for LDL
–
.

Chemical Modifications and Structural Changes in LDL
–
LC/MS/MS (Table 1) and CD (Fig. 7) analyses indicated apoB-100 protein
modifications and conformational changes inherent in LDL
–
. Tyrosine nitration in
LDL
–
(Table 1) in α
1
, α
2
, and α
3
helices as well as β
2
sheets as well as cysteine
oxidation in β
1
to cysteic acid (Fig. 13) seem to assist the loss of α-helical structure in
LDL
–
and increase β-turn, parallel- and anti-parallel sheets, and random coil structures
(Fig. 7). Of note, nitration of apoB-100 occurs in the α-helical structures containing
the highest percentage of tyrosine per total amino acid residues: α
1
appears to be more
susceptible to nitrotyrosine formation, whereas β
1
seemed resistant to nitration and
susceptible to cysteine oxidation. Nitration of α-helices appears to contribute to
protein unfolding, whereas the oxidation of one of the nine free cysteines (in β
1
sheets)
may be involved in the increased electronegativity of the particle.

53
A Functional Role for ONOO
–
in LDL
–
Formation
Treatment of nLDL with either ONOO
–
(Fig. 8) or SIN-1 (Fig. 10) resulted in
extensive tyrosine nitration (Table 2; Fig. 13), accumulation of LOOH, and loss of α-
helical structure (Fig. 9) and, as a corollary, formation of LDL
–
(Fig. 8D, 10D). It may
be surmised, hence, that nitrotyrosine- and lipid peroxide accumulation are
synergistically responsible for unfolding of α-helices inherent in LDL
–
formation.

54
Secondary
Structure
Residue
N °
—NO
2
–Y
276
—NO
2
–Y
583
—NO
2
–Y
666
—NO
2
–Y
720
—SO
3
H–C
1112
—NO
2
–Y
2524
—NO
2
–Y
3295
—NO
2
–Y
3490
—NO
2
–Y
3139
—NO
2
–Y
4141
—HO–F
3965
In vivo LDL
–
ONOO
–
-LDL
—NO
2
–Y
103
—NO
2
–Y
413
—NO
2
–Y
666
—NO
2
–Y
3490
—NO
2
–Y
2524
—NO
2
–Y
3711
—NO
2
–Y
4088
—HO–F
3965
LDL-R
Binding Motif Æ 3359-3369
α
1
58-795
β
1
827-2001
α
2
2045-2587
β
2
2571-4037
α
3
4017-4515
NH
2
CO
2
H
Secondary
Structure
Residue
N °
—NO
2
–Y
276
—NO
2
–Y
583
—NO
2
–Y
666
—NO
2
–Y
720
—SO
3
H–C
1112
—NO
2
–Y
2524
—NO
2
–Y
3295
—NO
2
–Y
3490
—NO
2
–Y
3139
—NO
2
–Y
4141
—HO–F
3965
In vivo LDL
–
ONOO
–
-LDL
—NO
2
–Y
103
—NO
2
–Y
413
—NO
2
–Y
666
—NO
2
–Y
3490
—NO
2
–Y
2524
—NO
2
–Y
3711
—NO
2
–Y
4088
—HO–F
3965
LDL-R
Binding Motif Æ 3359-3369
α
1
58-795
β
1
827-2001
α
2
2045-2587
β
2
2571-4037
α
3
4017-4515
NH
2
CO
2
H
Figure 13: Site of Chemical Modifications in In Vivo LDL
–
and ONOO
–
-
treated LDL

The secondary structure of the apoB-100 is shown. The
pentapartite structure however is not drawn to scale. Data from Tables 1
and 2 were used to assign the chemical modifications in LDL
–
and ONOO
–
-treated LDL.

55
Specific Cellular Receptors for LDL
–
The aforementioned protein modifications and structural changes in LDL
–
may
suggest an LDL receptor (LDL-R)-independent mechanism for binding and uptake of
LDL
–
to and into BAEC cells; this notion is supported by the following (a) LDL
nitration coincides with the binding sites to LDL-R (encompassing amino acid
residues 3359 and 3369 in β
2
) and (b) there is evidence that ONOO
–
-treated LDL
binds to CD36 (Guy, Maguire et al. 2001). In vivo LDL
–
nitration and ONOO
–
-
modified LDL show that ONOO
–
is the most likely mechanism of protein nitration in
vivo. Protein unfolding as well as nitrotyrosine may be involved in LDL-R-
independent uptake and binding, thus strengthening the pathophysiological
significance of ONOO
–
-driven LDL modifications.
Tyrosine nitration and lipid peroxidation appear to disturb the phospholipid
belt of LDL and hydrophobic stacking of aromatic amino acids in the lipid core. The
three α-helices have the highest percentage of tyrosine residues; it may be
hypothesized that nitration of these tyrosine residues would have a synergistic affect
upon protein unfolding along with lipid peroxide formation. Addition of a nitro group
to tyrosine involves the addition of a hydrophilic moiety with a net electrostatic charge
(Zwitterion); aromatic groups are involved in hydrophobic stacking interactions in
proteins as well as in protein-lipid bilayer interface. Therefore, nitration in α-helices
is expected to interfere with hydrophobic stacking in the lipid core of the LDL particle
and possibly with the phospholipid belts of α
2
and α
3
helices, leading to protein
unfolding. Furthermore, peroxidation of lipids in LDL seem to cause unfolding of the

56
apoB-100 protein (Ursini, Davies et al. 2002). The phospholipid belt in α
2
and α
3
is
stabilized by electrostatic bonds between negatively charged phospho head groups and
positively charged lysine/arginine residues. Peroxidation of long chain poly
unstaturated fatty acyl chains of the phospholipid belts and the addition of molecular
oxygen will increase the hydrophilicity of the fatty acyl chains of phospholipids, thus
resulting in both the migration out of the lipid phase and an increasing surface area to
volume ratio of the lipid core (increased hydrophilic surface). This increased strain
would induce α
2
and α
3
to stretch and adopt a new confirmation. This mechanism
strengthens the significance of the phospholipid belts (α
2
and α
3
) in maintaining
particle protein/lipid integrity, particle structure, proper electrostatic interactions, and
aromatic stacking.
Regardless of its mechanism, these findings suggest that protein unfolding may
be the main contributor to LDL
–
-induced atherosclerosis. The extensive nitration of
apoB-100 in the α-helices and β-sheets of LDL
–
and its absence in nLDL suggest that
the latter is not susceptible to protein nitration / oxidation in vivo. In response to PLA2
and oxidation in vitro, modifications of LDL render the formation of an
electronegative subfraction with secondary structural changes similar to those of in
vivo LDL
–
. As an emergent marker for coronary artery disease, nitrotyrosine is
prominent in the atherosclerotic lesions. In this context, this study established similar
apoB-100 protein nitration patterns and secondary protein structural changes in the in
vivo circulating LDL
–
and ONOO
–
-treated LDL. Moreover, binding and uptake of the
protein unfolded fraction (LDL
–
) was higher than that of nLDL.

57
CHAPTER IV

HEMODYNAMICS INFLUENCE VASCULAR ONOO
-
FORMATION:
IMPLICATION FOR LOW-DENSITY LIPOPROTEIN
APOB-100 NITRATION

Abstract
Hemodynamics, specifically, fluid shear stress, modulates the focal nature of
atherogenesis. O
2
.-
anion (O
2
−
) reacts with NO at a rapid diffusion-limited rate to
form ONOO
-
(O
2
−
+ NO → ONOO
−
). Immunohistostaining of human coronary
arterial bifurcations or curvatures, where OSS develops, revealed the presence of
nitrotyrosine staining, a fingerprint of ONOO
-
, whereas in straight segments, where
PSS occurs, nitrotyrosine was absent. We examined vascular nitrative stress in
models of OSS and PSS. Bovine aortic endothelial cells (BAEC) were exposed to
fluid shear stress that simulates arterial blood flow: (1) PSS at a mean shear stress
( τ
ave
) of 23 dyn cm
− 2
and a temporal gradient ( ∂τ/ ∂t) at 71 dyn cm
− 2
s
− 1
, and (2) OSS
at τ
ave
= 0.02 dyn cm
− 2
and ∂τ/ ∂t = ± 3.0 dyn cm
− 2
s
− 1
at a frequency of 1 Hz. OSS
significantly up-regulated one of the NADPH oxidase subunits (NOX-4) expression
accompanied with an increase in O
2
−
production. In contrast, PSS up-regulated eNOS
expression accompanied with NO production (total NO
2
−
and NO
3
−
). To demonstrate
that O
2
−
and NO are implicated in ONOO
−
formation, we added low-density
lipoprotein cholesterol (LDL) to the medium in which BAEC were exposed to the

58
above flow conditions. The medium was analyzed for LDL apo-B-100 nitrotyrosine
by liquid chromatography electrospray ionization tandem mass spectrometry
(LC/ESI/MS/MS). OSS induced higher levels of 3-nitrotyrosine, dityrosine, and o-
hydroxyphenylalanine compared with PSS. In the presence of ONOO
−
, specific apo-
B-100 tyrosine residues underwent nitration in the α and β helices: α-1 (Tyr
144
), α-2
(Tyr
2524
), β-2 (Tyr
3295
), α-3 (Tyr
4116
), and β-2 (Tyr
4211
). Hence, the characteristics of
shear stress in the arterial bifurcations influenced the relative production of O
2
−
and
NO with an implication for ONOO
−
formation as evidenced by LDL protein nitration.

Introduction
The characteristics of shear stress, namely spatial and temporal variations,
influence the focal character of atherosclerosis (Ku 1997; Dai, Kaazempur-Mofrad et
al. 2004; Passerini, Polacek et al. 2004). Shear stress acting on ECs at arterial
bifurcations or branch points regulates both NADPH oxidase (De Keulenaer, Chappell
et al. 1998; Ziegler, Bouzourene et al. 1998) and
.
NO synthase activities (Frangos,
Huang et al. 1996; Topper, Cai et al. 1996). The former is considered a major source
of oxygen-centered radicals (i.e., O
2
.-
)(oxidative stress), whereas the latter is a source
of nitrogen-centered radicals (i.e., NO) (nitrative/nitrosative stress). Both oxidative
and nitrative stress are involved in the modification of low-density lipoprotein (LDL),
which occurs at high levels in the athero-prone regions (Berliner, Territo et al. 1990).

59
At the lateral wall of arterial bifurcations, flow separation and migrating
stagnation points create distinct characteristics of oscillating shear stress (OSS:
bidirectional net zero forward flow) (Fung 1997). In these OSS-exposed regions,
ROS (ROS) are reported to be upregulated (Zarins, Giddens et al. 1983; Ku 1997;
Passerini, Polacek et al. 2004), and OSS may account for production of O
2
−
via the
membranous NADPH oxidase (De Keulenaer, Chappell et al. 1998; Hwang, Ing et al.
2003). Furthermore, an increase in O
2
−
production has been implicated in the
oxidative modifications of LDL that plays an important role in up-regulation of
adhesion molecules and cytokines (Hwang, Saha et al. 2003). In contrast, PSS (PSS:
unidirectional and positive net forward flow) preferentially develops in the medial
wall of arterial bifurcations or the straight regions (Fung and Liu 1993). PSS down-
regulates NADPH oxidase activities and expression of adhesion molecules and
cytokines (Malek, Alper et al. 1999), providing an athero-protective mechanism to
ECs (Passerini, Polacek et al. 2004) and (Hwang, Saha et al. 2003).
Nitrotyrosine is considered to be an emergent inflammatory marker for
atherosclerosis (Shishehbor, Aviles et al. 2003). O
2
.-
anion reacts with NO at a rapid
diffusion-limited rate to form a strong oxidant ONOO
-
(O
2
−
+ NO → ONOO
−
)
(Huie and Padmaja 1993; Goldstein, Squadrito et al. 1996; Handy and Loscalzo 2006).
In the presence of ONOO
−
, the tyrosine residues of proteins undergo nitration, giving
rise to nitrotyrosine, a fingerprint for ONOO
-
(Shishehbor, Aviles et al. 2003). In the
present study, we hypothesized that the specific characteristics of shear stress
influence the formation of ONOO
-
due to the imbalance between O
2
−
and NO

60
production in the OSS-exposed regions of vasculatures, and that the presence of
ONOO
−
induces LDL apo-B-100 protein nitration. By immunohistostaining of the
explants of human coronary arteries, we demonstrated that OSS-exposed arterial
bifurcations were prevalent for nitrotyrosine formation. By a combination of dynamic
flow system (Hwang, Ing et al. 2003; Hsiai, Cho et al. 2004) and liquid
chromatography, electron ionization spray, and tandem mass spectrometry
(LC/ESI/MS/MS) (Hazen, Zhang et al. 1999; Shishehbor, Aviles et al. 2003), we
showed that OSS influenced the formation of ONOO
−
as represented by LDL protein
nitration at the tyrosine residues in the α-and β-helices.

Results

eNOS and Nitrotyrosine Immunostaining in OSS-exposed Regions Versus PSS-
Exposed Regions
Explants of human left (LCA) and right (RCA) coronary arteries were
compared for eNOS and nitrotyrosine immunostaining. In the OSS-exposed regions
(lateral wall of arterial bifurcations or curvatures), eNOS staining was absent in EC,
whereas in the PSS-exposed regions, eNOS staining was prevalent throughout the
entire EC lining the lumen (n = 3) (Fig. 14 a-c). Despite the absence of eNOS staining
in the OSS-exposed regions, eNOS staining was present in the endothelial lining of
vasa vasorum, which may be a potential source of NO needed for ONOO
−
formation.
In contrast, nitrotyrosine staining was present in the SMCs (SMC) in the OSS-exposed

61
regions, including the medial wall of arterial bifurcations and curvatures, but absent in
the PSS-exposed regions (n = 3) (Fig. 14d–f). Counterstaining with hematoxylin, von
Willebrand factor, and β-actin distinguished EC and SMC, respectively, in the lumen,
media, and/or intima.

62

Figure 14:

63
Figure 14 continued:
Figure 14: Immunostaining of Human Coronary Arteries for eNOS and
Nitrotyrosine

Immunostaining of representative sections of coronary arteries (n = 3).
(a) Endothelial cells (EC) were stained with von Willebrand factor in both the
lateral wall of left main bifurcation (OSS-exposed region) and straight regions
(PSS-exposed regions) of right coronary artery (RCA). The inserts (20×)
detailed EC lining the inner lumens. En face staining of the human left main
bifurcation was beyond the field of view at the lower magnification (10×). (b)
A section of left main bifurcation revealed that eNOS staining was absent in
the luminal EC, but was presence in both smooth muscle cells and vaso
vasorum. (c) A representative PSS-exposed section of RCA revealed that
eNOS staining was prevalent throughout the entire luminal EC. (d) Smooth
muscle cells (SMC) in the media of left main bifurcation was counterstained
with β-actin. β-Actin was observed in the intima and media of the OSS-
exposed section, suggesting SMC migration (data not shown). (e) OSS-
exposed section of the left main bifurcation revealed nitrotyrosine staining in
the media. The insert (20×) further showed nitrotyrosine staining in the SMC.
(f) A representative PSS-exposed section in RCA revealed a lack of
nitrotyrosine staining.

64
PSS and OSS Regulated the Relative Production of O
2
−
and NO (NO
2
−
and
NO
3
−
)
To demonstrate that PSS and OSS induced the relative production of O
2
−
and
NO, we used our dynamic flow system (Hsiai, Cho et al. 2003). We observed that
OSS was a stronger inducer than PSS in up-regulating Nox4 expression (Fig. 15a).
However, PSS was a stronger inducer than OSS in up-regulating eNOS mRNA at 4
and 8 h (Fig. 15b). Consequently, OSS induced a higher rate of O
2
−
production than
PSS (Table 3). OSS also induced a higher rate of NO formation accompanied with a
significantly higher ratio of O
2
−
/ NO than did PSS (Table 3), suggesting that the
imbalance in O
2
−
and NO production was likely to contribute to the presence of
nitrotyrosine in the arterial bifurcations as demonstrated by the immunostaining of
ONOO
-
(Fig. 14). Furthermore, the elevated level of total NO formation (NO
2
−
and
NO
3
−
) (Braman and Hendrix 1989; Van Der Vliet, Nguyen et al. 2000) was likely due
to the presence of ONOO
-
(Table 1).

65
Figure 15: Messenger RNA Expression of BAEC Under Differential Flow

(a) eNOS mRNA expression in response to shear stress at 4 and 8 h.
PSS (OSS) induced a sustained increase in eNOS expression by 5 ± 0.85-fold
and 4.48 ± 0.47-fold at 4 and 8 h, respectively, whereas OSS (OSS) down-
regulated eNOS expression by 2.5 ± 0.7-fold (n = 5, P < 0.05). (b) Nox4
protein expression in response to shear stress. OSS up-regulated Nox4
protein by 2 ± 0.8-fold, whereas PSS up-regulated Nox4 protein by 1.60 ±
0.7-fold at 4 h (n = 4, P < 0.05). Nox4 protein was normalized to β-actin.
Controls were performed under static conditions.

66

Control OSS PSS
dO
2
?
dt [nmoles/min/10
6
cells] 1.2 ± 0.8* 25.7 ± 5.1* 5.4 ± 4.2*
d NO/dt [nmoles/min/10
6
cells] 1.9 ± 0.3
#
37.5 ± 4.1
#
48.6 ± 5.1
#
(dO
2
?
/dt)/(d NO/dt) 0.63 0.68 0.11

Table 3: Relative Rates of O
2
−
and NO Production in
Response to PSS and OSS
The rates of O
2
−
and NO production remained
steady under the static state. OSS induced a higher rate
of O
2
−
production while PSS promoted a higher rate of
NO (total NO
2
−
and NO
3
−
) formation. OSS also induced
a higher level of NO formation and a higher ratio of
O
2
−
to
·
NO production than PSS (*,
#
P < 0.05, n = 4).

67

Control OSS PSS
3-nitrotyrosine [mmol/mol] 0.15 ± 0.08 0.17 ± 0.09* 0.09 ± 0.04*
di-tyrosine [mmol/mol] 16.0 ± 4.4 21.1 ± 3.4
#
13.0 ± 2.7
#

o-hydroxy-phenylalanine [mmol/mol] 54.5 ± 4.1 82.0 ± 4.4
+
52.0 ± 3.1
+

Table 4: PSS and OSS Influenced LDL Protein Nitration
Liquid chromatography/electron spray ionization/mass
spectrometry/mass spectrometry (LC/ESI/MS/MS) analyses of
LDL apo-B-100 revealed that OSS induced higher levels of LDL
nitrotyrosine, dityrosine, and o-hydroxyphenylalanie (o-Phe) than
PSS. Dityrosine and o-Phe appeared to be the predominant forms
of protein nitration. The differences between control and PSS
were statistically insignificant (*,
#
,
+
P < 0.05, n = 3).

68
PSS and OSS Differentially Influenced the Formation of ONOO
-

ONOO
−
reacted rather specifically with tyrosine residues in proteins to yield 3-
nitrotyrosine. To measure the production of ONOO
−
as protein-bound nitrotyrosine,
confluent BAEC were exposed to PSS and OSS in the presence of LDL at 50 μg/mL.
After shear stress exposure, LDL particles suspended in medium was collected for
apo-B-100 protein modifications. The levels of nitration in nitrotyrosine residues of
apo-B-100 were higher in response to OSS than to PSS (Table 4). LC/ESI/MS/MS
analyses demonstrated differential levels of protein modifications in response to PSS
versus OSS: [3-nitrotyrosine] [dityrosine] < [o-Phe]. Together with the
Immunohistochemistry analyses (Fig. 14) and the imbalanced production of O
2
−
and
NO (Table 3), the higher levels of apo-B-100 protein nitration in response to OSS
suggest that vascular nitrative stress is likely to develop within the lateral walls of
arterial bifurcations and curvatures. The pathophysiologic implication of d-tyrosine
and o-hydroxyphenylalanine remains to be determined.

69
ONOO
-
Modified Specific Protein Nitration
To further investigate the molecular mechanisms by which protein underwent
nitration in the presence of ONOO
−
, we performed LC/ESI/MS/MS analyses of LDL
(0.2 mg/ml) in the presence of 100 μM ONOO
−
(Table 5). The tyrosine residues of
LDL apo-B-100 were susceptible to nitration in the α-1, α-2, α-3, and β-2 helices;
specifically, α-1 (Tyr
144
), α-2 (Tyr
2524
), α-3 (Tyr
4116
), β-2 (Tyr
3295
), and β-2 (Tyr
4211
).
Relative Mascot and Sequest scores were obtained by software that demonstrated LDL
modifications. Mascot is the score obtained using matrix science software to analyze
the actual observed masses, followed by searching the database of proteins to
determine the sequence of the digested protein, whereas Sequest scores are obtained
using Thermo Finnigan (Excaliber) software to determine the most likely peptide from
actual observed peptide masses by searching a protein database (Perkins, Pappin et al.
1999). The representative MS/MS spectrum illustrates a tryptic peptide,
NLQNNAEWVYQGAIR, which was modified with tyrosine residues 14 (Fig. 16).
The Y and B ion series reflected the direction in which the mass of the ion was
observed. The Y6 ion in the MS/MS spectrum showed the presence of tyrosine
nitration as evidenced by an additional mass of 45 Da (NO
2
= 46 Da; but the
replacement of a hydrogen atom resulted in a mass of 45 Da). Further evidence for an
additional 45 Da was identified in ions Y7, Y9, Y10, Y11, Y13, B13, and B14. The
mass of the modified and unmodified peptides bears one-half the theoretical mass for
doubly charged ions and one-third of the theoretical mass for triply charged ions. A
mass difference of 22.5 Da was observed for the nitrotyrosine-containing peptide as

70
compared to the unmodified peptide. Ion mass to charge ratio (m/z) of the entire
peptide demonstrated that the observed mass of the modified peptide
NLQNNAEWVYQGAIR for the doubly charged ion was 911.23 Da, whereas the
unmodified peptide mass was 888.57 Da, consistent with the presence of nitrotyrosine.

71
AA Peptide Charge Sequence M Xcor Δcn
Y144 140-157 3 QVFLY*PEKDEPTYILNIK 42 3.2 0.328
Y2524 2523-2534 2 M*Y*QM*DIQQELQR 71 4.4 0.264
Y2524 2523-2534 2 M*Y*QM*DIQQELQR 79 4.5 0.286
Y3295 3292-3311 3 VPSY*TLILPSLELPVLHVPR 78 5.9 0.558
Y4116 4107-4121 2 NLQNNAEWVY*QGAIR 97 4.7 0.478
Y4211 4202-4213 3 FQFPGKPGIY*TR 34 2.5 0.271

Table 5: Tyrosine Nitration of LDL Apo-B-100
Six tryptic peptides were identified to have undergone
tyrosine nitration. The nitrotyrosines are denoted by asterisks.
“AA” represents modified tyrosine residue, “Peptide” the
numerical sequence of amino acid in the tryptic fragment,
“Charge” the ion precursor charge of the tryptic fragment,
“Sequence” the amino acid sequences of the tryptic fragment,
“M” the Mascot score, Xcor the Sequest score, and “ Δcn” the
difference between two closely matched peptides.

72
Figure 16: MS Spectrum of a Representative Nitrated Peptide
The tryptic peptide, NLQNNAEWVYQGAIR, revealed that Y6
ion is the largest blue peak. The molecular weight of Y6 is greater in
the modified peptide (752.4 Da) than the unmodified (707.3 Da) by
45 Da. Only representative peaks were labeled in the spectrum.

73
Discussion

This study examined whether or not spatial specific characteristics of shear
stress in the arterial bifurcations or straight segments influenced vascular ONOO
−
formation with an implication for protein nitration at the tyrosine residues.
Immunostaining of the human coronary arteries from ischemic cardiomyopathy
patients revealed nitrotyrosine staining present in the OSS-exposed arterial regions
(bifurcations or curvatures), but absent in the PSS-exposed regions (straight
segments). In contrast, eNOS staining was absent in the OSS-exposed regions, but
prevalent in the PSS-exposed regions. Next, we showed that OSS promoted an
imbalance between O
2
−
and NO formation that influenced the formation of ONOO
−
as measured by the presence of 3-nitrotyrosine, d-tyrosine, and o-
hydroxyphenylalanine in the LDL apo-B-100 protein. Finally, our LC/ MS/MS
analyses revealed specific tyrosine residue nitration in the α-and β-helices of the apo-
B-100 protein at α-1 (Tyr
144
), α-2 (Tyr
2524
), β-2 (Tyr
3295
), α-3 (Tyr
4116
),
and β-2 (Tyr
4211
).
Within the lateral walls of arterial bifurcations or curvatures, disturbed flow
(including OSS) is considered to be an inducer of oxidative stress that favors the
production of ROS through the endothelial NADPH oxidase system (Chappell, Varner
et al. 1998; De Keulenaer, Chappell et al. 1998; McNally, Davis et al. 2003). In
contrast, in the medial wall of bifurcations or straight regions where PF develops,
vascular ECs are protected from atherosclerosis (Malek, Alper et al. 1999). Temporal

74
variations in shear stress (OSS and PSS) in these regions are likely to account for the
relative expression of NADPH oxidase homologues, Nox4, and eNOS. The increased
vascular activities of NAD(P)H oxidases enhance the production of ROS, including
O
2
−.
and H
2
O
2
. O
2
−.
can inactivate
.
NO, leading to the formation of OONO
−
, thereby
lowering NO bioavailability (Irani 2000; Sowers, Epstein et al. 2001). While others
have shown the presence of eNOS staining in the mouse vasculatures (Poppa,
Miyashiro et al. 1998; Mueller, Laude et al. 2005), we revealed that eNOS staining
was absent in the OSS-exposed regions of coronary arteries in patients with ischemic
cardiomyopathy.
While eNOS expression is absent in the luminal ECs of athero-prone regions
of coronary arteries, eNOS expression is present in the luminal ECs lining the vasa
vasorum (Fig. 1b). Our observation is consistent with the previous report that reduced
.
NO release in atherosclerotic segments was accompanied by marked reduction of
immunoreactive eNOS in luminal ECs. However, ECs of vasa vasorum and the
endothelium of normal arteries remained positive for eNOS (Oemar, Tschudi et al.
1998). In the absence of disease, the vasa vasorum nurtures the outer component of
the vessel wall, and the intima is fed by oxygen diffusion from the lumen. As disease
progresses, the intima thickens, and oxygen diffusion is impaired. As a result, vasa
becomes the major source for nutrients to the vessel wall (Williams 1996; Moreno,
Purushothaman et al. 2006). Furthermore, apolipoprotein E (apo-E) ( −/ −)/low-density
lipoprotein ( −/ −) double knockout mice developed vasa vasorum in association with
advanced lesion formation (Langheinrich, Michniewicz et al. 2006). Vasa vasorum

75
plays a significant role in maintaining vessel integrity and the vasa vasorum may
contribute to the initiation and progression of different types of vascular disease in the
systemic circulation. In this context, NO production from vasa vasorum likely
attributes to the formation of ONOO
-
despite the absence of eNOS expression in the
luminal endothelium of the coronary arteries.
SMCs were stained with β-actin in the left main bifurcation and right coronary
artery (RCA) (Fig. 14d). We observed that nitrotyrosine staining is prevalent in the
OSS-exposed regions, but it was relatively sparse or absent in (Fig. 14f). This
observation is likely due to the relative straight segments of RCA from which
immunostaining for nitrotyrosine was performed. The luminal ECs in the straight
segment were known to be exposed to PF (Ku, Giddens et al. 1985), which down-
regulated NADPH oxidase subunit (NOX4) expression, but up-regulated eNOS
expression (Fig. 15). Despite the presence of eNOS staining in SMC, the relative rate
of O
2
.-
anion and
.
NO production is small in response to PSS (Table 3). In this
context, PSS-exposed regions seemed to be protected from oxidative stress, whereas
OSS-exposed regions (the bifurcations or curvatures) seemed to be prone to oxidative
stress as shown by the ONOO
-
formation (Fig. 14e).
Less eNOS is present in OSS (Fig. 15a), but
.
NO production is comparable to
PSS in (Table 3). The Griess reaction is the most frequently used analytical approach
to quantify the major metabolites of
.
NO, (i.e., NO
2
-
) and ONOO
-
(i.e. NO
3
-
). NO
2
−
is
the measurement of total usable
.
NO:
.
NO + HO → HONO → NO
2
−
+ H
+
, whereas
total NO
3
−
is the degradation product of ONOO
-
(ONOOH → NO
3
−
) or the

76
degradation product of nitrating intermediates: ONOOH → NO
2
+ HO
→ HONO
2
→ NO
3
−
.
Furthermore, the Griess reaction is specific for NO
2
-
. Analysis of NO
3
-
by this
reaction requires chemical or enzymatic reduction of NO
3
-
to NO
2
-
prior to the
diazotization reaction. Because there are numerous interferences in the analysis of
NO
2
-
and NO
3
-
in biological fluids and because there is a desire to analyze these
anions simultaneously, the Griess reaction has been repeatedly modified and
automated. In recent years, the Griess reaction has been coupled to HPLC for
postcolumn derivatization of chromatographically separated NO
2
-
and NO
3
-
.
Furthermore, there are particular analytical and pre-analytical factors that may affect
the quantitative analysis of NO
2
-
and NO
3
-
in these matrices.
We performed the analytical procedure based on acidic reduction of NO
2
−
and
NO
3
−
to NO by vanadium (III) and purging of NO with helium into a stream of
ozone and detected by an Antek 7020 chemiluminescence NO detector. At room
temperature, vanadium (III) only reduced NO
2
−
, whereas NO
3
−
and other redox forms
of NO (such as S-nitrosothiols) were reduced only if the solution was heated to 90--
100°C, so that both NO
2
−
and total NO
x
could be measured. ONOO
-
has a half-life of
about 7 day (Uppu and Pryor 1996), and it will decay to NO
2
-
. However, our
experimental samples were frozen, and then processed for NO
2
−
and heating to 90--
100°C for NO
3
−
. Thus, the presence of ONOO
-
might partly contribute to the elevated
total
.
NO production in response to OSS.

77
Previously, our lab and others have reported that the production of O
2
−

influences the capacity of endothelial monolayers to modulate oxidative modification
of LDL apo-B-100 (Hwang, Ing et al. 2003; Hwang, Saha et al. 2003), and a
significant correlation may be observed between generation of O
2
−
and the expression
of NADPH oxidase subunit, p22
phox
, or oxLDL from directional coronary atherectomy
specimens (Azumi, Inoue et al. 2002). In this study, we revealed that ONOO
-
reacted
specifically with tyrosine residues in proteins leading to the formation of nitrotyrosine.
ONOO
-
is a potent oxidant, exerting its toxic effects by undergoing redox cycling, by
interfering with signal transduction, or by becoming incorporated into the
microtubules to distort the cytoskeleton (Zhang, Xu et al. 2005). Emerging evidence
has shown that the exposure of LDL to ONOO
−
resulted in the (a) nitration of apo-B-
100 tyrosine residues (Leeuwenburgh, Hardy et al. 1997), (b) peroxidation of lipid
(Radi, Beckman et al. 1991; Shishehbor, Aviles et al. 2003), (c) depletion of lipid-
soluble antioxidants (Hogg, Darley-Usmar et al. 1993; Goss, Hogg et al. 1999), and
(d) conversion of the lipoprotein into a high uptake form for macrophages (De
Keulenaer, Chappell et al. 1998; Podrez, Schmitt et al. 1999). Thus, nitrotyrosine is
considered an emerging inflammatory marker (Shishehbor, Aviles et al. 2003).
The pathways leading to the nitration of tyrosine residues in proteins have been
extensively studied in vitro and in vivo (Ischiropoulos, Zhu et al. 1992; Eiserich,
Hristova et al. 1998; Goodwin, Gunther et al. 1998; Sampson, Ye et al. 1998; Wu,
Chen et al. 1999; Goldstein, Czapski et al. 2000; Pfeiffer, Schmidt et al. 2000; Reiter,
Teng et al. 2000; Sawa, Akaike et al. 2000; van Dalen, Winterbourn et al. 2000;

78
MacPherson, Comhair et al. 2001; Brennan, Wu et al. 2002; Thomas, Espey et al.
2002). Tyrosine nitration, a posttranslational modification of proteins through the
addition of a nitro (NO
2
) group in the meta position of tyrosine residues
(Ischiropoulos, Zhu et al. 1992), has been detected under physiological settings and in
a number of pathological states including inflammatory and septic conditions (Wu,
Chen et al. 1999; Goldstein, Czapski et al. 2000; Pfeiffer, Schmidt et al. 2000; Reiter,
Teng et al. 2000; Sawa, Akaike et al. 2000; Greenacre and Ischiropoulos 2001;
Thomas, Espey et al. 2002). At the protein levels, these modifications implicate a
number of potential consequences such as alterations in secondary structure, function,
and susceptibility to proteolysis (Wu, Chen et al. 1999; Goldstein, Czapski et al. 2000;
Pfeiffer, Schmidt et al. 2000; Reiter, Teng et al. 2000; Sawa, Akaike et al. 2000;
Greenacre and Ischiropoulos 2001; Thomas, Espey et al. 2002) as well as the behavior
of LDL particles (Ischiropoulos 1998). At the physiologic level, C57 mice undergoing
an exercise protocol, that was associated with an augmentation in shear stress,
decreased the level of protein nitration (3-nitrotyrosine level) (Young, Knight et al.
2005). In this context, protein-bound nitrotyrosine is considered to be an emergent
predictor for cardiovascular disease risk assessments (Berliner and Heinecke 1996;
Podrez, Schmitt et al. 1999; Shishehbor, Aviles et al. 2003; Paoletti, Gotto et al. 2004;
Shimada, Mokuno et al. 2004; Zheng, Nukuna et al. 2004).
The α-2 and α-3 helices of apo-B-100 have the highest content of tyrosine
residues (4.8 and 6.8% tyrosine, respectively). An aromatic amino acid tyrosine
assumes a flat and stackable structure to intercalate into the hydrophobic space

79
between adjacent negatively charged phospholipids of the α helices that are stabilized
by lysine and arginine-positive charges (Hevonoja, Pentikainen et al. 2000). The most
observed nitration of apo-B-100 occurs in the α-helical domains which likely account
for the unfolding of LDL. This LDL unfolding has been demonstrated in various LDL
modifications, including PLA2-treated LDL (Asatryan, Hamilton et al. 2005) (LDL
-
subfraction) and in vivo LDL
-
(Parasassi, Bittolo-Bon et al. 2001). ApoB-100 was
also sensitive to nitration/oxidation in the β-2 domain which is upstream from the
LDL-receptor site (3359-3369) (Hevonoja, Pentikainen et al. 2000). Furthermore,
nitration of tyrosine residues is likely to change the hydrophilic properties of the apo-
B-100 particle; rendering hydrophilic and likely disrupting hydrophobic interactions
with the outer hydrophobic lipid core. Further investigation of posttranslational
protein modification by nitration, including signaling molecules, enzymes, and
receptors, would provide insights into the biologic effects of nitrative stress in the
initiation of atherosclerosis.

80
CHAPTER V

OXLDL-R DEPENDENT JNK-2 PHOSPHORYALTION: IMPLICATION FOR
MITOCHONDRIAL REGULATION/DYSFUNCTION BY P-JNK-2

Abstract

Elevated LDL is a risk factor for atherosclerosis and the modified subfraction
in vivo (LDL
-
) is intimately involved in the development of atherosclerosis. LDL
cholesterol is known to induce MFC formation, fatty streaks, SMC migration and
endothelial dysfunction. Oxidatively modified LDL has been shown to induce JNK-2
phosphorylation, JNK-2 knockout mice have been shown to have decreased MFC
formation as well as decreased plaque size and CD-36 oxLDL-R knockout
macrophage model has decreased MFC formation as well. OxLDL is also known to
induce mitochondrial specific ROS production in ECs linking a commonality between
ROS production in MFCs and ECs. We therefore proposed that p-JNK-2 may be
intimately involved with mitochondrial function. The aim of this study was to
establish what modifications of LDL are responsible for the phosphorylation of JNK-
2, what receptors are involved, and how JNK-2 phosphorylation is regulating
mitochondrial function specifically apoptosis.

81
Modification of LDL with PLA2 (Protein unfolded LDL), Cu2+ (Protein
unfolded and lipid peroxidized) and ONOO- (Protein unfolded, lipid peroxidized and
Nitrated) all induced the phosphorylation of JNK-2. Lipid peroxide content of the
modified LDL particles increased the phosphorylation of JNK-2 but all were
significantly higher than the control-LDL suggesting protein unfolding and oxLDL-R
dependent signaling. JNK-2 phosphorylation was dependent on both CD-36 and SR-
A scavenger receptors for all modified LDL and also suggested that the minimal
amount of modified LDL in the control was also significant in activating JNK-2
phosphorylation. Immunofluorescence showed co-localization of p-JNK-2 to the
mitochondria that was significantly decreased by JNK inhibitor however p-JNK-2 was
still co-localizing with mitochondria and is downstream of oxLDL-R activity. Active
CD-36 and SR-A scavenger receptor still induced JNK-2 phosphorylation that co-
localized with mitochondria however addition of both scavenger receptor blocking
antibodies mostly ablated p-JNK-2 co-localization with mitochondria. Phospho-JNK-2
induced phosphorylation of Bcl-xL and it also co-localized to mitochondria in
differentially modified LDL. Differentially modified LDL also induced caspase-3
activity that is downstream of the p-Bcl-xl and was directly related to the JNK-2
phosphorylation. Capsase-3 activity was significantly inhibited by SP600125 as well
as significantly inhibited by both CD-36 and SR-A scavenger receptor
blocking antibodies.
These data suggest that modified LDL induces JNK-2 phosphorylation through
a protein unfolding dependent mechanism utilizing a CD-36 and SR-A dependent

82
signaling cascade. These data also suggest that JNK-2 phosphorylation is somewhat
dependent on the lipid peroxide content which may play a synergistic role in both
JNK-2 phosphorylation and in mitochondrial dysfunction. We have found that p-
JNK-2 induces an apoptotic cascade however, future work needs to be done to
determine how JNK-2 is involved in mitochondrial metabolism.

Introduction
Elevated LDL cholesterol as well as modified LDL cholesterol is intimately
associated with the initiation and progression of atherosclerosis (Jagavelu, Tietge et al.
2007). There is also a strong association between LDL cholesterol and aging (Reiner,
Carlson et al. 2007). Our laboratory has shown that the modified LDL subfraction in
vivo is protein unfolded and lipid peroxide/aldehyde rich (Sevanian, Bittolo-Bon et al.
1997). Another group has shown that apoB-100 isolated from lesions is rich for
nitrotyrosine (Leeuwenburgh, Hardy et al. 1997) and our group has also shown that
the modified LDL subfraction in vivo (LDL
-
) is Nitrated. Oxidatively or
enzymatically modified LDL cholesterol has been implicated in the initiation of
atherosclerosis (Osterud and Bjorklid 2003; Torzewski, Suriyaphol et al. 2004;
Asatryan, Hamilton et al. 2005; Dersch, Ichijo et al. 2005; Koller, Volf et al. 2006;
Malle, Marsche et al. 2006; Torzewski and Lackner 2006). However, it is in strong
debate as to what constitutes a modified LDL particle. How this particle induces
atherosclerosis? There is strong evidence suggesting that the lipid peroxide/aldehyde

83
content of LDL is involved in the regulation of inflammatory responses by activating
NADPH oxidase activity (Rouhanizadeh, Hwang et al. 2005). LOOH are also known
to have inflammatory signaling cascades that involve NF-kb signaling to inflammatory
cytokines and monocyte adhesion proteins (Natarajan, Reddy et al. 2001). These
findings are only part of the toxicity associated with modified LDL particles and
discount the responses induced by protein unfolding through binding to oxLDL
receptor’s (oxLDL-R) in vivo. It is unknown what specifically predisposes modified
LDL as a target for these scavenger receptors but most likely is the result of protein
unfolding as seen in LDL
-
induced atherosclerosis (Damasceno, Sevanian et al. 2006).
To further investigate the importance of lipid modifications and protein
modifications/unfolding we employed several strategies to modify LDL in vivo.
Enzymatically modified LDL is known to activate inflammatory
responses(Torzewski and Lackner 2006). It is also pondered as to whether enzymatic
modifications precede oxidative modifications in early lesions (Torzewski and
Lackner 2006). Our laboratory has shown that modified LDL in vivo (LDL
-
) is
associated with increased protein unfolding of apoB-100 (Ursini, Davies et al. 2002).
Unfolded proteins have been associated with multiple pathologies including
Parkinson’s, Alzheimer’s, mad cow and Huntington’s disease (Scheibel and Buchner
2006; Hachiya, Imagawa et al. 2007). Enzymatic modification of LDL by sPLA2
which is associated with coronary artery disease (Asatryan, Hamilton et al. 2005), was
able to induce a lipid modification that resulted in the loss of protein structural
integrity with similar protein structure to an in vivo LDL
-
(Asatryan, Hamilton et al.

84
2005). LDL incubated with copper is rich in protein unfolding and in LOOH
(Febbraio, Podrez et al. 2000). ONOO
-
-treated LDL is rich in LOOH, protein
unfolding and nitrotyrosine (Rahaman, Lennon et al. 2006). Each of these
modifications has select characteristics that will elucidate how modified LDL induces
atherosclerosis and it is therefore believed that different modifications on LDL may
corroborate different responses and may act synergistically. However, there is little
known as to how nitration and protein oxidative modifications on modified LDL affect
its atherogenicity. It has been shown that modified LDL activates JNK-2
phosphorylation (Rahaman, Lennon et al. 2006) and our group believes that JNK-2
phosphorylation may be involved in regulating mitochondrial function.
Our laboratory has shown that JNK-1 is involved in the regulation of
mitochondrial function in cortical neurons at the level of PDH activity (Zhou, Lam et
al. 2007). JNK-1 was able to phosphorylate the PDH complex E1α subunit and block
the formation of acetyl-Co-A leaving the mitochondria and neurons in an energy
crisis, reducing equivalent crisis and redox crisis (Zhou, Lam et al. 2007). However,
this was in neurons and was through JNK-1 and not JNK-2. Recent evidence
demonstrated that JNK-2 knockout mice had decreased MFC formation as well as
lipid oil red staining (Ricci, Sumara et al. 2004). This mouse model suggests that
there may be a role for JNK-2 in the formation of MFCs since MFCs have increased
ROS production. To further support JNK-2 as a model for mitochondrial regulation
was the finding that CD36 knockout macrophages had a decreased incidence of MFC
formation as well as had little JNK-2 phosphorylation upon oxLDL treatment

85
suggesting a role for oxLDL mediated JNK-2 phosphorylation and a possible
mechanism to foam cell formation (Rahaman, Lennon et al. 2006). These two findings
taken together suggest that oxLDL induces the phosphorylation of JNK-2 through a
scavenger receptor mediated signal transduction pathway that may result in the
regulation of the mitochondria, hence the formation of MFCs. Since MFCs are O
2
.-

and H
2
O
2
producing lipid loaded macrophages (Kritharides, Upston et al. 1998) and
that mitochondria are the major source of ROS, this finding supports JNK-2 as a
proposed mechanism of mitochondrial dysfunction. OxLDL has also been shown in
bovine aortic ECs to co-localize ROS to the mitochondria further suggesting p-JNK-2
in the dysfunction of mitochondria and ECs (Zmijewski, Moellering et al. 2005) and
these three findings together may suggest a role of JNK-2 in mitochondrially derived
ROS production. Since we were interested in endothelial dysfunction, we sought to
ask whether oxLDL induces JNK-2 phosphorylation in BAEC and whether this
activation of JNK-2 co-localizes with mitochondria. We further investigated the
possibility of JNK-2 phosphorylation by differentially modified LDL since JNK-2 is
both redox sensitive and may be CD-36 dependent. In addition we sought to
determine whether JNK-2 regualtes mitochondrial function.

86
Results

Characteristics of Differentially Modified LDL
Four LDL modification conditions were used to understand how LDL is
involved in the development of atherosclerosis. LDL was modified by the following
conditions: Control-LDL, 40μM Cu-LDL, 100μM ONOO
-
-LDL and 50ng/ml PLA2-
LDL were utilized to understand how the modifications of LDL are involved in the
development of atherosclerosis. PLA2-LDL is similar to in vivo LDL with minimal
LOOH/aldehydes but has an elevated protein unfolded subfraction (Asatryan,
Hamilton et al. 2005). Modification of LDL with Cu
2+
is known to induce protein
unfolding with large quantities of LOOH (Febbraio, Podrez et al. 2000; Sevanian,
Shen et al. 2000). Lastly, treatment with ONOO
-
should produce an LDL particle with
protein unfolding, LOOH and Nitrated tyrosine residues as we have shown in our
previous work. Therefore our aim was to determine the differential role of protein
unfolding, LOOH and nitrotyrosine in the phosphorylation of JNK-2. We show that
LDL
-
is significantly elevated for all three modifications of LDL with respect to in
vivo LDL and is associated with an unfolded LDL particle (Fig. 17A). We further
demonstrate that there is a nine-fold greater lipid peroxide content in Cu-LDL treated
LDL, that there is a three-fold greater lipid peroxide content in ONOO
-
-treated LDL
and that there is no differences between PLA2-LDL and control-LDL (Fig. 17B). We
further note that there are no significant differences in nitrotyrosine density for LDL

87
treated with copper or with PLA2 but we show that nitrotyrosine levels were
significantly greater for ONOO
-
-treated LDL (Fig. 17C). We further noted that
cysteine oxidation was elevated in ONOO
-
and Cu modified LDL (Fig. 17D). These
findings suggest that we have differentially modified LDL particles; PLA2-LDL has
protein unfolding, Cu-LDL has protein unfolding and extreme levels of LOOH, and
that ONOO
-
-LDL has elevated levels of protein unfolding, LOOH, and nitrotyrosine.
These differentially modified LDL particles will help us understand the focal nature of
JNK-2 phosphorylation, scavenger receptors and mitochondrial regulation.

88

Control-LDL Cu-LDL PN-LDL PLA2-LDL
***
***
***
% LDL-
Control-LDL Cu-LDL PN-LDL PLA2-LDL
12
8
4
0
***
***
[M LP]/g apoB-100
A B
C D C D
Control-LDL Cu-LDL PN-LDL PLA2-LDL
0
1
2
3
Free cysteine a.u.
*
Control-LDL Cu-LDL PN-LDL PLA2-LDL
0
1
2
3
Nitrotyrosine density a.u.
***
0
4
8
12
Figure 17: Characteristics of Differentially Modified LDL

Modified LDL particles were analyzed for (A) LDL- by HPLC,
(B) LOOH, (C) nitrotyrosine and (D) cysteine oxidation (n=3, P<0.05-*,
P<0.01-**, P<0.001-***)

89
Modified LDL Induced P-JNK-2 and was Suppressed by JNK Inhibitor
Since it is known that oxLDL is known to phosphorylate JNK-2 (Rahaman,
Lennon et al. 2006) and that JNK-2 knockout mice harbor decreased MFC formation
(Ricci, Sumara et al. 2004), we tested whether each of the differentially modified LDL
particles could modulate JNK-2 phosphorylation as compared to no LDL treatment
and control-LDL treatment. JNK-2 phosphorylation was significantly elevated as
compare to control for all three LDL modifications and the control was significantly
elevated to the blank (no LDL treatment) with or without JNK inhibitor (Fig. 18A).
These findings suggest that the minimal amounts of oxLDL in vivo (LDL
-
) may
activate JNK-2 phosphorylation but was not as strong as the modified LDL particles in
vitro that have 10-15 times more protein unfolded (percent LDL
-
). Lastly, the PLA2-
treated LDL had an elevated activation of JNK-2 and is purely a protein unfolded LDL
particle suggesting that the protein unfolded LDL was responsible for the activation of
JNK-2. The relationship between protein unfolding (percent LDL
-
) and JNK-2
phosphorylation was directly related (Fig. 18B). There is also a slight relationship
between the amount of lipid peroxide and the amount of JNK-2 phosphorylation (Fig.
18C) however, the control-LDL has shift in the curve and is strongly related to 10-15
times less percent LDL
-
. We also wondered how JNK-2 may be involved in MFC
formation and bovine aortic EC dysfunction. Since JNK-2 has been shown to be
involved in MFC formation we postulated that this JNK-2 activation may be oxLDL-R
dependent. Data recently has shown that JNK-2 phosphorylation is completely
ablated in CD-36 knockout mice suggesting the interplay between CD-36 and JNK-2

90
phosphorylation (Rahaman, Lennon et al. 2006). We therefore wanted to know how
different scavenger receptors and LDL-R are involved in the activation
of JNK-2.
Figure 18: Modified LDL Induced Phosphorylation of JNK-2

Modified LDL was incubated with BAEC for 30 minutes with
and without JNK inhibitor as described in the materials and methods and
western blot analysis of p-JNK-2 was determined (A). JNK-2
phosphorylation was directly related to LDL- (B) and Lipid peroxides (C)
(n=3, P<0.05-*/
+
, P<0.01-**/
++
, P<0.001-***/
+++
).

6
4
2
0
P-JNK-2 density a.u.
Blank con-LDL Cu-LDL ONOO
-
-LDL PLA2-LDL
SP600125
-
+
β-actin
β-actin
- SP600125
+ SP600125
*
***
***
**
*
***
***
***
+++
+++
+++
+++
++
A
0 4 8 12
% LDL-
B
P-JNK-2 density a.u.
0
2
4
6
0 2 4 6 8 10
C
6
4
2
0 P-JNK-2 density a.u.
μMLP/μg apoB-100
m=0.179

91
JNK-2 Phosphorylation is SR-A and CD-36 Dependent
Our laboratory has shown that the electronegative oxLDL (LDL
-
) activates
atherosclerotic responses in macrophages and in ECs (Asatryan, Ziouzenkova et al.
2003; Hwang, Rouhanizadeh et al. 2006). However, we wanted to know what
oxLDL-Rs were involved in JNK-2 phosphorylation? We obtained oxLDL-R
blocking antibodies to CD-36, SR-A and LOX-1 as well as the nLDL receptor (LDL-
R) blocking antibody. We show that LDL-R, CD-36, LOX-1 and SR-A scavenger
receptors are all present in bovine aortic ECs (Fig. 19A). We blocked different
oxLDL-R and LDL-R by six different conditions as described in the materials and
methods. LDL in vivo (control-LDL) had no significant differences between LDL-R
and LOX-1 however was significant for both SR-A and CD-36 (n=3, P<0.05) (Fig.
19B). In vivo LDL was able to induce JNK-2 phosphorylation and was dependent on
both SR-A and CD-36 and may be the direct result of in vivo LDL
-
, a scavenger
receptor ligand (Fig. 19B). These data suggest that the in vivo LDL
-
is acting
differential scavenger receptors (CD-36, and SR-A) with similar affects to the
modified ligands suggesting that protein unfolding is the dominant factor in oxLDL-R
dependent activation of JNK-2 phosphorylation. This becomes further evident when
the amount of LDL
-
is increased 6 fold (Fig. 18A). Cu
2+
, ONOO
-
and PLA2-treated
LDL all had significantly elevated levels of p-JNK-2 in both the active SR-A and CD-
36 receptor experiments further supporting that protein unfolding phosphorylates
JNK-2 (Fig. 19C-E). However, there was some slight significance for the LOX-1 but
suggests that lox-1 may be involved in activation of JNK-2 but is not a major

92
constituent to JNK-2 phosphorylation. LDL-R had no changes for all types of
modified LDL on JNK-2 activation (Fig. 19B-E). Figure 18B shows the relationship
between percent LDL
-
and phospho-JNK-2 levels in control-LDL, Cu-LDL, PN-LDL
and PLA2-LDL and these findings further corroborate a protein unfolding dependent
JNK-2 phosphorylation that is now dependent on both SR-A and CD-36 signaling
cascades. Our findings show that active SR-A and CD-36 receptors had significantly
elevated levels of JNK-2 phosphorylation compared to the blocking of all other
oxLDL/LDL-R blocking antibodies (Fig. 19D-F). Lipid peroxidized and protein
unfolded LDL (Cu-LDL) had significant JNK-2 phosphorylation to all receptors
blocked for active LOX-1 (P<0.05), and for active SR-A and CD-36 (P<0.001) but
had no significant differences for LDL-R (Fig. 20D). Lipid peroxidized, protein
unfolded and protein Nitrated LDL (ONOO
-
-LDL) had significantly elevated JNK-2
phosphorylation as well for LOX-1 (P<0.05), and for SR-A and CD-36 (P<0.01) but
had no significant differences for LDL-R (Fig. 19E). Lastly, Protein unfolded LDL
(PLA2-LDL) also had significantly elevated levels of JNK-2 phosphorylation for
LOX-1 (P<0.05), SR-A and CD-36 (P<0.001) but had no significant changes for LDL-
R blocking antibody (Fig. 19F). LOX-1 was only slightly significant in the activation
of JNK-2 to using all receptor blocking antibodies suggesting that its role in JNK
phosphorylation is minimal. These data support the findings of CD-36 mediated MFC
formation in JNK-2 knockout mice as well as support the findings that CD-36
knockout macrophages have JNK-2 phosphorylation ablated. Since our laboratory has
shown that JNK-1 in cortical neurons co-localizes with mitochondria, we wondered

93
whether JNK-2 would also co-localize with mitochondria. Our laboratory also has
shown that JNK-1 would regulate the key rate limiting enzyme of mitochondrial
function PDH by phosphorylation and inhibition of mitochondrial respiration. Other
groups have suggested that JNK regulates mitochondrial induced apoptosis by the
phosphorylation of BCL-XLxl resulting in the release of cytochrome c and the
induction of the apoptosome. We therefore wondered if JNK-2 is regulating
mitochondrial function and whether this may be the mechanism by which JNK-2
induces foam cell formation/necrotic core formation (Ricci, Sumara et al. 2004;
Rahaman, Lennon et al. 2006) as well as increased mitochondria O
2
.-
production
(Zmijewski, Moellering et al. 2005).

Receptor Dependent Superoxide Production
Extracellular Superoxide production correlated with the total amount of lipid
peroxides in the differentially protein unfolded LDL particles (Table 6). Interestingly,
the superoxide production was not affected by JNK inhibitor suggesting that the signal
is upstream of p-JNK-2 and thatp-JNK-2 is not involved in the formation of
extracellular superoxide (Table 6). However, upon addition of differential receptor
blocking, we found that SR-A and CD-36 scavenger receptors are involved in the
uptake of this modified LDL and involved in the production of superoxide and may be
the direct uptake and activation of NADPH oxidase by the increasing amount lipid
peroxides (oxPAPC) (Table 7). The production of superoxide was directly related to

94
the lipid peroxide concentration suggesting an oxPAPC dependent formation of
extracellar superoxide (data not shown).

Table 6: Differentially Modified LDL Dependent Superoxide Production

BAEC cells were pretreated with and without JNK inhibitor and
were then treated for 3 hours with differentially modified LDL and
superoxide production measured as described in the materials and methods
for control-LDL, Cu-LDL, ONOO
-
-LDL and PLA2-LDL (n=3, P<0.05-*,
P<0.01-**, P<0.001-***).
Control-LDL 0.007±0.004 0.006±0.001 0.8±0.2
Cu-LDL 0.039±0.007** 0.037±0.006** 7.8±0.9
PN-LDL 0.027±0.005** 0.025±0.005** 3.2±0.5
PLA2-LDL 0.010±0.003 0.011±0.002 0.8±0.2
μMLP/μg
apoB-100
nmoles O
2
.-
*(min)
-1
*(10
6
cells)
-1
*(μg/ml
LDL)
-1
SP600125 - +
nmoles O
2
.-
*(min)
-1
*(10
6
cells)
-1
*(μg/ml
LDL)
-1
Control-LDL 0.007±0.004 0.006±0.001 0.8±0.2
Cu-LDL 0.039±0.007** 0.037±0.006** 7.8±0.9
PN-LDL 0.027±0.005** 0.025±0.005** 3.2±0.5
PLA2-LDL 0.010±0.003 0.011±0.002 0.8±0.2
μMLP/μg
apoB-100
nmoles O
2
.-
*(min)
-1
*(10
6
cells)
-1
*(μg/ml
LDL)
-1
SP600125 - +
nmoles O
2
.-
*(min)
-1
*(10
6
cells)
-1
*(μg/ml
LDL)
-1

95

Table 7: Differentially Modified LDL Dependent Superoxide Production

BAEC cells were pretreated with and without JNK inhibitor and were
then treated for 3 hours with differentially modified LDL and superoxide
production measured as described in the materials and methods for control-
LDL, Cu-LDL, ONOO
-
-LDL and PLA2-LDL (n=3, P<0.05-*, P<0.01-**,
P<0.001-***).
α -L D L -R - + + + - +
C u -L D L 0 .0 3 6 ± 0 .0 0 2 *** 0 .0 2 0 ± 0 .0 0 3 * 0 .0 3 1 ± 0 .0 0 4 ** 0 .0 3 3 ± 0 .0 0 4 ** 0 .0 1 4 ± 0 .0 0 2 0 .0 13 ±0 . 003
α -L O X -1 - - + + + +
α -C D -3 6 - + - + + +
α -S R -A - + + - + +
L D L 0 .0 1 4 ± 0.0 01* 0 .010 ± 0 .003 0 .0 1 1 ± 0 .00 2* 0 .0 1 0 ± 0 .003* 0 .00 7 ± 0 .002 0.00 7± 0 .00 1
O N O O --L D L 0 .0 2 9 ± 0 .0 0 2 ** 0 .0 1 6 ± 0 .0 0 3 * 0 .0 2 5 ± 0 .0 0 4 * * 0 .0 2 6 ± 0 .0 0 4 * * 0 .0 1 1 ± 0 .0 0 2 0 .0 1 2 ± 0 .0 0 3
P L A 2 -L D L 0 .0 1 5 ± 0 .0 0 2 * 0 .0 1 1 ± 0 .0 0 3 0 .0 1 4 ± 0 .0 0 2 * 0 .0 1 4 ± 0 .0 0 2 * 0 .0 1 0 ± 0 .0 0 3 0 .0 0 8 ± 0 .0 0 2
nm oles O
2
.-
*(m in )
-1
*(1 0
6
ce lls )
-1
*(μ g/ m l
LD L)
-1
nm o les O
2
.-
*(m in )
-1
*(1 0
6
ce lls )
-1
*(μ g/ m l
LD L)
-1
nm oles O
2
.-
*(m in )
-1
*(1 0
6
ce lls )
-1
*(μ g/ m l
LD L)
-1
nm oles O
2
.-
*(m in )
-1
*(1 0
6
c e lls )
-1
*(μ g/ m l
LD L)
-1
nm o les O
2
.-
*(m in )
-1
*(1 0
6
ce lls )
-1
*(μ g/ m l
LD L)
-1
nm oles O
2
.-
*(m in )
-1
*(1 0
6
ce lls )
-1
*(μ g/ m l
LD L)
-1
α -L D L -R - + + + - +
C u -L D L 0 .0 3 6 ± 0 .0 0 2 *** 0 .0 2 0 ± 0 .0 0 3 * 0 .0 3 1 ± 0 .0 0 4 ** 0 .0 3 3 ± 0 .0 0 4 ** 0 .0 1 4 ± 0 .0 0 2 0 .0 13 ±0 . 003
α -L O X -1 - - + + + +
α -C D -3 6 - + - + + +
α -S R -A - + + - + +
L D L 0 .0 1 4 ± 0.0 01* 0 .010 ± 0 .003 0 .0 1 1 ± 0 .00 2* 0 .0 1 0 ± 0 .003* 0 .00 7 ± 0 .002 0.00 7± 0 .00 1
O N O O --L D L 0 .0 2 9 ± 0 .0 0 2 ** 0 .0 1 6 ± 0 .0 0 3 * 0 .0 2 5 ± 0 .0 0 4 * * 0 .0 2 6 ± 0 .0 0 4 * * 0 .0 1 1 ± 0 .0 0 2 0 .0 1 2 ± 0 .0 0 3
P L A 2 -L D L 0 .0 1 5 ± 0 .0 0 2 * 0 .0 1 1 ± 0 .0 0 3 0 .0 1 4 ± 0 .0 0 2 * 0 .0 1 4 ± 0 .0 0 2 * 0 .0 1 0 ± 0 .0 0 3 0 .0 0 8 ± 0 .0 0 2
nm oles O
2
.-
*(m in )
-1
*(1 0
6
ce lls )
-1
*(μ g/ m l
LD L)
-1
nm o les O
2
.-
*(m in )
-1
*(1 0
6
ce lls )
-1
*(μ g/ m l
LD L)
-1
nm oles O
2
.-
*(m in )
-1
*(1 0
6
ce lls )
-1
*(μ g/ m l
LD L)
-1
nm oles O
2
.-
*(m in )
-1
*(1 0
6
c e lls )
-1
*(μ g/ m l
LD L)
-1
nm o les O
2
.-
*(m in )
-1
*(1 0
6
ce lls )
-1
*(μ g/ m l
LD L)
-1
nm oles O
2
.-
*(m in )
-1
*(1 0
6
ce lls )
-1
*(μ g/ m l
LD L)
-1

96
Figure 19: CD-36 and SR-A Induced JNK-2 Phosphorylation and Minimal
LOX-1 and LDL-R Involvement

BAEC cells were pre-incubated 1 hour with receptor blocking
antibodies to CD-36, SR-A, LOX-1 and LDL-R such that only one receptor
should be active, all receptors inactive or all receptors active. Differentially
modified LDL were compared to all receptors blocked and no receptors
blocked for each condition. (A) BAEC with receptor blocking and no LDL
treatment for 30 minutes (B) BAEC with control-LDL for 30 minutes (C)
BAEC with Cu-LDL for 30 minutes, (D) BAEC with ONOO
-
-LDL for 30
minutes and (E) PLA2-LDL with BAEC for 30 minutes (n=3, P<0.05-*,
P<0.01-**, P<0.001-***).
α-LOX-1
α-CD-36
α-LDL-R
α-SR-A
A B C
D E F
*
*
** **
**
**
*
*
**
**
***
*
α-LOX-1 - - + + + +
α-CD-36 - + - + + +
α-SR-A - + + - + +
α-LDL-R - + + + - +
α-LOX-1 - - + + + +
α-CD-36 - + - + + +
α-SR-A - + + - + +
α-LDL-R - + + + - +
α-LOX-1 - - + + + +
α-CD-36 - + - + + +
α-SR-A - + + - + +
α-LDL-R - + + + - +
α-LOX-1 - - + + + +
α-CD-36 - + - + + +
α-SR-A - + + - + +
α-LDL-R - + + + - +
P-JNK-2 density a.u.
8
6
4
2
0
8
6
4
2
0
8
6
4
2
0
8
6
4
2
0
8
6
4
2
0
α-LOX-1 - - + + + +
α-CD-36 - + - + + +
α-SR-A - + + - + +
α-LDL-R - + + + - +
α-LOX-1 - - + + + +
α-CD-36 - + - + + +
α-SR-A - + + - + +
α-LDL-R - + + + - +
P-JNK-2
β-actin
P-JNK-2
β-actin
α-LOX-1 - - + + + +
α-CD-36 - + - + + +
α-SR-A - + + - + +
α-LDL-R - + + + - +
α-LOX-1 - - + + + +
α-CD-36 - + - + + +
α-SR-A - + + - + +
α-LDL-R - + + + - +
α-LOX-1 - - + + + +
α-CD-36 - + - + + +
α-SR-A - + + - + +
α-LDL-R - + + + - +
α-LOX-1 - - + + + +
α-CD-36 - + - + + +
α-SR-A - + + - + +
α-LDL-R - + + + - +
P-JNK-2
β-actin
P-JNK-2 P-JNK-2
β-actin β-actin
* *
*
P-JNK-2 density a.u.
P-JNK-2 density a.u.
P-JNK-2 density a.u.
P-JNK-2 density a.u.

97
JNK-2 Phosphorylation by Modified LDL Ligands Co-localizes with
Mitochondria
Since p-JNK-2 seems to be responsible for MFC formation (Ricci, Sumara et
al. 2004; Rahaman, Lennon et al. 2006), that mitochondria are most responsible for
ROS in living cells (Zini, Berdeaux et al. 2007) and that p-JNK-1 was shown to
regulate PDH in our laboratory (Zhou, Lam et al. 2007), we asked the question as to
whether p-JNK-2 co-localizes with mitochondria? Most p-JNK-2 is localizing to the
nucleus in the blank (No LDL) after a 30 minute incubation (Fig. 20A). Control LDL
results in a minimal translocation of p-JNK-2 from the nuclear compartment to the
cytosolic compartment with minimal mitochondrial localization (Fig. 20B).
Interestingly, Incubation of BAEC cells with each of the three differentially modified
LDL subfractions shows a global increase in p-JNK-2 levels (Cu-LDL>ONOO
-
LDL>PLA2-LDL) and that this p-JNK-2 is mostly cytosolic with mitochondrial
localization and minimal nuclear localization (Fig. 20C-E) as compared to the controls
(Fig. 20A, B). However, incubation with JNK inhibitor did not inhibit the co-
localization of JNK-2 to the mitochondria (Fig. 21B-E) but it did decrease the total
amount of p-JNK-2 in the cells suggesting it is downstream of the signal. This
suggests that oxLDL-R’s may be involved in the induction of p-JNK-2 translocation to
the mitochondria and that the JNK inhibitor inhibits down stream of the responsive
agent as well as decreases the total amount of JNK translocating to the mitochondria
and cytosol. These findings suggest a modified LDL dependent mitochondrial co-
localization of p-JNK-2 suggesting a plausible regulation/dysfunction of mitochondria

98
that potentially corroborates the findings of a decreased MFC formation and plaque
staining in CD-36 knockout macrophages (Rahaman, Lennon et al. 2006) and apoE
and JNK-2 knockout mice (Ricci, Sumara et al. 2004). Therefore we wondered
whether receptor blocking of CD-36 and SR-A would block mitochondrially co-
localized p-JNK-2.

99
Figure 20: Modified LDL Induced P-JNK-2 Co-localization with
Mitochondria

BAEC cells were treated with differentially modified LDL for 30
minutes and probed for p-JNK as described in the materials and methods
for (A) no LDL, (B) control-LDL, (C) Cu-LDL, (D) ONOO
-
-LDL, and (E)
PLA2-LDL (n=3).
A
B C
D
E

100
Figure 21: JNK Inhibitor Decreases Global P-JNK-2 Levels but not Co-
localization of P-JNK-2 to Mitochondria.

BAEC cells were pretreated 1hr with JNK inhibitor at 100mM and
then treated with modified LDL for 30 minutes as described in the
materials and methods for (A) no LDL, (B) control-LDL, (C) Cu-LDL, (D)
ONOO
-
-LDL, (E) PLA2-LDL (n=3).
A
BC
D
E

101
CD-36 Dependent P-JNK-2 Co-localization with Mitochondria
CD-36 knockout macrophages have been shown to have decreased JNK-2
phosphorylation (Rahaman, Lennon et al. 2006) and it was therefore of interest to
know if having active CD-36 affected the co-localization of JNK-2 with mitochondria.
Treatment of cells with SR-A receptor blocking antibody still resulted in the
localization of p-JNK-2 to the mitochondria for differentially modified LDL (Fig.
22C-E) and still was observed to be nuclear for no LDL treatment (Fig. 22A) and
minimally cytosolic for control-LDL (Fig. 22B) with minimal mitochondrial co-
localization. However, p-JNK-2 localization with mitochondria was not ablated
suggesting that SR-A may also be involved in the localization of p-JNK-2 to the
mitochondria (Fig. 22C-E). However, the control-LDL (Fig. 22B) had minimal
localization of p-JNK-2 with mitochondria and the BAEC treated with no LDL had
minimal cytosolic p-JNK-2 (Fig. 22B). These data suggest that CD-36 dependent
activation of p-JNK-2 may be involved in signaling p-JNK-2 to the mitochondria and
that CD-36 may also be involved.

102
Figure 22: CD-36 Dependent P-JNK-2 Co-localization to Mitochondria

BAEC cells were treated as described for figure 3 except they were pre-
treated with receptor blocking antibodies 1hr and is described in the materials and
methods for analysis of (A) no-LDL treatment, (B) control-LDL treatment, (C)
Cu-LDL treatment (D) ONOO
-
-LDL treatment and (E) PLA2 treated LDL (n=3).

A
BC
D
E

103
SR-A Dependent P-JNK-2 Localization
Since, we have found that CD-36 is involved in the activation of p-JNK-2 and
we have found that CD-36 is involved in the activation of mitochondrially derived p-
JNK-2, we wondered whether SR-A is involved in the localization of p-JNK-2 to the
mitochondria. Treatment of cells with CD-36 receptor blocking antibody resulted in
the localization of p-JNK-2 to mitochondria for differentially modified LDL (Fig.
23C-E), was still observed to be nuclear for the treatment with no LDL (Fig. 23A)
with minimal JNK localization to the cytosol and was found to be minimally co-
localized with mitochondria in the control-LDL (Fig. 23B). These findings suggest
that both CD-36 and SR-A may be involved in the co-localization of p-JNK-2 to the
mitochondria. To further confirm that both receptors are involved in the co-
localization of p-JNK-2 to the mitochondria we incubated cells with both SR-A and
CD-36 receptor blocking antibodies. We found that blocking both CD-36 and SR-A
receptors dramatically decreased the abundance of p-JNK-2 by fluorescent microscopy
and that this p-JNK-2 co-localization with mitochondria was dramatically inhibited in
differentially modified LDL treated cells (Fig. 24C-E). We further found that there
was no dramatic difference in the treatment without LDL (Fig. 24A) with mostly
nuclear localization with minimal cytosolic co-localization suggesting that the receptor
blocking antibodies were not inducing p-JNK-2 co-localization with the mitochondria.
We also noted that the control-LDL (Fig. 24B) with both receptor blocking antibodies
completely ablated most cytosolic localization and any minimal localization with
mitochondria. These findings suggest that both the SR-A and CD-36 are involved in

104
the activation of p-JNK-2 and are involved in the signaling of p-JNK-2 to the
mitochondria. We therefore have demonstrated that two oxLDL receptors may be
involved in the regulation of mitochondrial function in ECs. We therefore wondered
what p-JNK-2 may be regulating and it has been suggested that JNK isoforms are
involved in phosphorylation of Bcl-xL and thus the initiation of apoptosis.

105
Figure 23: SR-A Dependent P-JNK-2 Co-localization with Mitochondria.

BAEC cells were treated as described in the materials and methods
with pre-treatment of CD-36 receptor blocking antibody to determine SR-A
involvement in p-JNK colocalization with mitochondria for (A) no LDL
treatment, (B) control-LDL treatment, (C) Cu-LDL treatment, (D) ONOO—
LDL treatment and (E) PLA2-LDL treatment (n=3).
A
BC
D
E
A
BC
D
E

106
Figure 24: SR-A and CD-36 Receptor Blocking and Ablation of
P-JNK-2 Co-localization with Mitochondria

BAEC cells were treated the same way as described above for
both CD-36 and SR-A blocking experiments except that both
blocking antibodies were used for (A) no LDL treatment, (B) control-
LDL treatment, (C) Cu-LDL treatment, (D) ONOO—LDL treatment,
and (E) PLA2-LDL treatment.
A
BC
D
E

107
Modified LDL Induced P-Bcl-xL that was Inhibited by JNK Inhibitor
We found that modified LDL is known to induce JNK-2 phosphorylation and
wondered whether p-Bcl-xL was induced by differentially modified LDL as well as
inhibited by the use of JNK inhibitor. We found that the levels of p-Bcl-xL were
induced by all differentially modified LDL and was slightly induced by control-LDL
(Fig. 25A). We also noted that incubation with JNK inhibitor significantly reversed
the amount of p-Bcl-xL (Fig. 25A) for all differentially modified LDL particles. We
found that Cu-LDL (
*
, n=3, P<0.01), ONOO
-
-LDL (
*
, n=3, P<0.01), and PLA2-LDL
(
*
, n=3, P<0.01) had significantly elevated p-Bcl-xL as compared to treatment with no
LDL, and that JNK inhibitor still had significant elevation of p-Bcl-xL for Cu-LDL (
*
,
n=3, P<0.001), ONOO
-
-LDL (
*
, n=3, P<0.001), and PLA2-LDL (
*
, n=3, P<0.001) as
compared to treatment with JNK inhibitor for no LDL treatment (Fig. 25A). We
further found that JNK inhibitor was able to significantly inhibit p-Bcl-xL for no LDL
treatment (
+
, n=3, P<0.01), control-LDL (
+
, n=3, P<0.001), Cu-LDL (
+
, n=3, P<0.01),
ONOO
-
-LDL (
+
, n=3, P<0.001), and PLA2-LDL (
+
, n=3, P<0.01) as compared to
without JNK inhibitor (Fig. 25A). We further established that the ability of lipid
peroxides to induce p-Bcl-xL activity was somewhat similar to that of p-JNK-2 levels
suggesting that the redox sensitive pathway may also be somewhat involved. We
therefore compared the amount of p-JNK-2 and p-Bcl-xL and found that the
p-Bcl-xL and p-JNK-2 were directly related (Fig. 25B) further supporting p-JNK-2 as
a mechanism of phosphorylation of Bcl-xL and oxLDL induced apoptosis. Since we
have found that SR-A and CD-36 were involved in p-JNK-2 mitochondrial co-

108
localization we wanted to know whether p-Bcl-xL could be inhibited by blocking
these receptors.

Figure 25: Differentially Modified LDL Induction of Bcl-xL Phosphorylation and
Inhibition with JNK Inhibitor

BAEC cells were pre-treated with and without JNK inhibitor and
phosphorylation of Bcl-xL determined (A). P-Bcl-xL was directly related to p-
JKNK-2 density (B) (n=3, P<0.05-*, P<0.01-**, P<0.001-***).

blank con-LDL Cu-LDL ONOO
-
-LDL PLA2-LDL
-SP600125
+ SP600125
P-Bcl-xL density a.u.
0
1
2
3
**
**
***
***
**
**
++ +++
+++
++
++
m=0.2238 - SP600125
+ SP600125 m=0.2948
2.0
1.5
1.0
0.5
0.0
02 4 6 8
P-JNK-2 density a.u.
P-Bcl-xL density a.u.
SP600125
β-actin
β-actin
-
+
AB

109
Modified LDL Induced P-Bcl-xL that was Inhibited by Blocking both CD-36
and SR-A
Since JNK inhibitor was able to block p-Bcl-xL we further wanted to support
p-JNK-2 involvement in the activation of p-Bcl-xL as well as whether SR-A and CD-
36 scavenger receptors are involved in the phosphorylation of Bcl-xL. We found that
without receptor blocking we had an activation of p-Bcl-xL that was moderately
inhibited for both CD-36 and SR-A receptor blocking antibodies and was completely
ablated upon addition of both scavenger receptor blocking antibodies (Fig. 26A)
suggesting that modified LDL induces JNK-2 phosphorylation that co-localizes with
mitochondria and initiates the phosphorylation of Bcl-xL and the initiation of the
apoptosome. We found that Cu-LDL (
*
, n=3, P<0.01), ONOO
-
-LDL (
*
, n=3, P<0.01),
and PLA2-LDL (
*
, n=3, P<0.01) had elevated p-Bcl-xL levels in the control, that Cu-
LDL (P<0.001), ONOO
-
-LDL (
*
, n=3, P<0.05), and PLA2-LDL (
*
, n=3, P<0.01) had
significantly elevated levels of p-Bcl-xL with SR-A blocking antibody, that Cu-LDL
(
*
, n=3, P<0.01), ONOO
-
-LDL (
*
, n=3, P<0.05), and PLA2-LDL (
*
, n=3, P<0.05) had
significantly elevated levels of p-Bcl-xL with CD-36 receptor blocking antibody, and
that all three modified LDL particles were completely ablated and insignificant with
both SR-A and CD-36 receptor blocking antibodies (Fig. 26). We further found that
CD-36 or SR-A receptor blocking antibodies were able to significantly inhibit Cu-
LDL (
+
, n=3, P<0.05), ONOO
-
-LDL (
+
, n=3, P<0.05), and PLA2-LDL (
+
, n=3,
P<0.05) as compared to control (Fig. 26A). However, incubation with both receptor
blocking antibodies was able to further significantly reduce the phosphorylation of p-

110
Bcl-xL for Cu-LDL (
+
, n=3, P<0.01), ONOO
-
-LDL (
+
, n=3, P<0.001), and PLA2-LDL
(
+
, n=3, P<0.01) as compared to no receptor blocking (Fig. 26A). We also wondered
whether there was a correlation between p-JNK-2 levels and p-Bcl-xL levels for each
of the different receptor blocking experiments and found that there was indeed a direct
correlation to the activation of p-JNK-2 and the activation of p-Bcl-xL (Fig. 26B).
These findings made us wonder whether differentially modified LDL was inducing p-
Bcl-xL co-localization with mitochondria.
Figure 26: Differentially Modified LDL Induced Phosphorylation of Bcl-xL

BAEC cells were treated with differentially modified LDL and total p-
Bcl-xL measure by western blot as described in the materials and methods for
(A) with and with out JNK inhibitor, (B) with CD-36 blocking antibodies, (C)
with SR-A blocking antibodies and (D) with both SR-A and CD-36 receptor
blocking antibodies (n=3, P<0.05-*, P<0.01-**, P<0.001-***)
blank con-LDL Cu-LDL ONOO
-
-LDL PLA2-LDL
+ SR-A, CD-36
P-BCL
+ SR-A
+ CD-36
control
0
1
2
3
β-actin
control
+CD-36 antibody
+SR-A antibody
+CD-36,SR-A antibody
***
**
*
**
*
*
+
+ +
+
+
++
+++
++
Receptor
blocking
Receptor antibodies
CD-36 SR-A
--
+ -
-+
+ +
2.0
1.5
1.0
0.5
0.0
02 46
P-JNK-2 density a.u.
P-Bcl-xL density a.u.
**
**
**
P-Bcl-xL density a.u.
AB

111
Differentially Modified LDL Induces P-Bcl-xL Co-localization with
Mitochondria
We have been able to demonstrate that differentially modified LDL particles
activate both CD-36 and SR-A involvement in the phosphorylation of JNK-2, its co-
localization to mitochondria, its activation of p-Bcl-xL and wondered whether this
p-Bcl-xL was co-localized with mitochondria. We were able to show that treatment
with no LDL had minimal localization with mitochondria (Fig. 27A) where as
incubation with control-LDL (Fig. 27B) had minimal localization with mitochondria.
However, upon incubation with differentially modified LDL, p-Bcl-xL translocated
and co-localized with mitochondria (Fig. 27C-E). These findings suggest that
modified LDL is most likely initiating the apoptosis pathway through p-JNK-2
phosphorylation of Bcl-xL and is most likely dependent on SR-A and CD-36
receptors.

112
AB
C
D E
Figure 27: Differentially Modified LDL and P-Bcl-xL and Mitochondrial
Localization

BAEC cells were treated with modified LDL and p-BCL-XL
intensity measured by confocal microscopy for (A) no LDL treatment, (B)
control-LDL treatment, (C) Cu-LDL treatment, (D) ONOO
-
-LDL
treatment, and (E) PLA2-LDL treatment (n=3).

113
Caspase-3 Activation by Differentially Modified LDL
To further support the findings of BCL-XL phosphorylation by oxLDL
treatment and the initiation of apoptosis we wondered how differentially modified
LDL with and without JNK inhibitor or with different oxLDL receptor blocking
antibodies would modulate caspase-3 activity. After normalization to the cells treated
without LDL, we found that Cu-LDL (Fig. 28B) (
+
, n=3, P<0.001), ONOO
-
-LDL (
+
,
n=3, P<0.001) Fig. 28C) (
+
, n=3, P<0.01) and PLA2-LDL (
+
, n=3, P<0.05) (Fig. 28D)
treated cells all had elevated levels of caspase-3 activity as compared to control (Fig.
28A). Treatment of cells with JNK inhibitor significantly inhibited the caspase-3
activity as compared to no JNK inhibitor for control-LDL (Fig. 28A) (*, n=3, P<0.01),
Cu-LDL (Fig. 28B) (*, n=3, P<0.001), ONOO
-
-LDL (Fig. 28C) (*, n=3, P<0.001),
and PLA2-LDL (Fig. 28D) (*, n=3, P<0.001). However, the caspase-3 activity was
not significantly different from control-LDL with and without JNK inhibitor
suggesting that JNK inhibitor was able to rescue the modified LDL induced apoptosis
but was still significant for Cu-LDL (
+
, n=3, P<0.01) (Fig. 28D).

114
Figure 28: Differentially Modified LDL Induced Caspase-3 Activity and
Inhibition by JNK Inhibitor.

BAEC were treated for 5 hours with differentially modified LDL with
and without JNK inhibitor as described in the materials and total caspase-3
activity measured as percent to the blank for (A) control-LDL, (B) Cu-LDL,
(C) ONOO
-
-LDL and (D) PLA2-LDL (n=3, P<0.05-*, P<0.01-**, P<0.001-
***).
control
Cu
PN
PLA2
μM pNA/μg protein/day
0.1
0
0.2
0.3
0.4
μM pNA/μg protein/day
0.1
0
0.2
0.3
0.4
μM pNA/μg protein/day
0.1
0
0.2
0.3
0.4
μMpNA/μg protein/day
0.1
0
0.2
0.3
0.4
Cont JNK inh
**
***
***
***
++
+++
++
+
Cont JNK inh Cont JNK inh
Cont JNK inh
A B C
D

115
Differential Activation of Caspase-3 by Different OxLDL-R
We also wondered if the activation of caspase-3 is oxLDL receptor dependent.
We determined that caspase-3 activity was elevated in modified LDL treated ECs with
different scavenger receptor blocking (Fig. 29B-D) conditions as compared to the
control (Fig. 29A-D) (n=3). Active SR-A and CD-36 scavenger receptors had the
greatest caspase-3 induction whereas LOX-1 had only a slight increase in caspase-3
activity and LDL-R had no affect as compared to all oxLDL/LDL receptors blocked
(Fig. 29A-D). In comparison to all receptors blocked versus all receptors active, both
SR-A and CD-36 were similar in intensity to all receptors active as compared to active
LDL-R which was similar to having all receptors blocked (Fig. 29A-D). LOX-1
caspase-3 induction was between all receptors active and all receptors blocked (Fig.
29A-D). We further demonstrate that control-LDL, Cu-LDL, ONOO
-
-LDL and PLA2-
LDL all had activated caspase-3 activity that correlated directly to the level of JNK-2
phosphorylation suggesting p-JNK-2 as a mechanism of oxLDL induced apoptosis
through an SR-A and CD-36 dependent pathway (Fig. 29E).

116
Figure 29: Differentially Modified LDL and OxLDL-R Dependent
Caspase-3 Activation.

BAEC were treated for 5 hours with differentially modified LDL
with and without JNK inhibitor as described in the materials and total
caspase-3 activity measured as percent to the blank for (A) control-LDL,
(B) Cu-LDL, (C) ONOO
-
-LDL and (D) PLA2-LDL (n=3, P<0.05-*,
P<0.01-**, P<0.001-***).
Cu-LDL
PN-LDL
PLA2-LDL
Cont-LDL
Cu-LDL
PN-LDL
PLA2-LDL
Cont-LDL
0 1 2 3 4 5 6 7
p-JNK-2 density a.u.
μM pNA/μg protein/day
0.1
0
0.2
0.3
0.4
α-LOX-1 + - + + + -
α-CD-36 + + - + + -
α-SR-A + + + - + -
α-LDL-R + + + + - -
α-LOX-1 + - + + + -
α-CD-36 + + - + + -
α-SR-A + + + - + -
α-LDL-R + + + + - -
μMpNA/μg protein/day
μM pNA/μg protein/day
μMpNA/μg protein/day
0.1
0
0.2
0.3
0.4
0.1
0
0.2
0.3
0.4
0.1
0
0.2
0.3
0.4
A B C
μM pNA/μg protein/day
0.1
0
0.2
0.3
0.4
D E
α-LOX-1 + - + + + -
α-CD-36 + + - + + -
α-SR-A + + + - + -
α-LDL-R + + + + - -
α-LOX-1 + - + + + -
α-CD-36 + + - + + -
α-SR-A + + + - + -
α-LDL-R + + + + - -
α-LOX-1 + - + + + -
α-CD-36 + + - + + -
α-SR-A + + + - + -
α-LDL-R + + + + - -
α-LOX-1 + - + + + -
α-CD-36 + + - + + -
α-SR-A + + + - + -
α-LDL-R + + + + - -
α-LOX-1 + - + + + -
α-CD-36 + + - + + -
α-SR-A + + + - + -
α-LDL-R + + + + - -
α-LOX-1 + - + + + -
α-CD-36 + + - + + -
α-SR-A + + + - + -
α-LDL-R + + + + - -
*
**
**
**
**
**
*** ***
**
*
*
*
*** ***
***
***
***
***
*
++
++
++
+++
+++
+++
++
++
+
+++
+++
++
+
+
++
++
+++

117
Discussion

Modified LDL can be categorized into three types of modification which
include lipid modification/oxidation, protein unfolding and protein oxidative
modifications. This study addresses what modifications of LDL are inherently
involved in the phosphorylation of JNK-2, what oxLDL receptors are responsible for
modified LDL induced JNK-2 phosphorylation and its co-localization to the
mitochondria as well as a plausible mechanism into how JNK-2 may be involved in
EC regulation/dysfunction.

Modifications of LDL that are Inherently Involved in the
Phosphorylation of JNK-2
All three differentially protein unfolded LDL particles significantly induced
JNK-2 phosphorylation and control-LDL induced less significantly JNK-2
phosphorylation than did no LDL treatment which suggests that the minimal amounts
of modified LDL in vivo (LDL
-
) is probably able to induce JNK-2 phosphorylation.
Nitrotyrosine seemed to have no affect on JNK-2 phosphorylation other than the fact
that it facilitates LDL particle unfolding which is directly involved in the
phosphorylation of JNK-2. However, there was a mild relationship between the
amount of LOOH and JNK-2 phosphorylation. Interestingly, this dependence was
much less pronounced than the phosphorylation induced by the differentially unfolded
LDL particles suggesting that protein unfolding was the most important initiator of

118
JNK-2 phosphorylation. Also, the relationship between the LOOH of the control and
the three modified LDL particles versus p-JNK-2 needed a correction for the percent
LDL
-
in the control. The three protein unfolded LDL particles had similar LDL
-
levels
but the control-LDL had eight times less LDL
-
and may account for the y shift on the
axis (Fig. 18D). However, LDL protein unfolding was the most important activator of
JNK-2 phosphorylation and suggested an oxLDL-R dependent activation.

What OxLDL Receptors are Responsible for Modified LDL Induced JNK-2
Phosphorylation and Mitochondrial Co-localization?
Macrophages were shown to have decreased foam cell formation as well as
decreased JNK-2 phosphorylation in response to oxLDL in CD36 null macrophages
(Rahaman, Lennon et al. 2006). These findings suggest that JNK-2 may be involved
in increasing ROS production in macrophages since mitochondria are the main source
of ROS in the cells (Zini, Berdeaux et al. 2007). Another group showed that a double
knockout of JNK-2 and ApoE resulted in decreased MFC formation further suggesting
a JNK-2 dependent MFC formation (Ricci, Sumara et al. 2004). These two data taken
together suggested a possible ligand binding signaling cascade that would induce
JNK-2 phosphorylation and a role for JNK-2 regulation of mitochondrial function.
Our findings show that both SR-A and CD-36 are involved in the phosphorylation of
JNK-2, that LDL-R has no affect on JNK-2 phosphorylation, and that LOX-1 has a
minimal affect on JNK-2 phosphorylation. Since our laboratory has found that JNK-1
regulates cortical neuron mitochondrial function we wondered if JNK-2 would

119
regulate BAEC mitochondria (Zhou, Lam et al. 2007). After treatment of BAEC with
modified for 30 minutes with and without JNK inhibitor, we observed a marked
difference in the levels of p-JNK-2 with a minimal effect on JNK localization to
mitochondria. Treatment with LDL or modified LDL induced a movement of nuclear
p-JNK-2 to cytosolic p-JNK-2. Modified LDL treated BAEC had sparse nuclear p-
JNK-2 and mostly cytosolic and mitochondrially localized p-JNK-2. Pre-treatment of
cells with receptor blocking antibodies showed that active CD-36 and SR-A both
induced a mitochondrial localization of p-JNK-2 however pre-treatment with both
receptor blocking antibodies showed a loss in mitochondrial co-localization of p-JNK-
2 suggesting that both CD-36 and SR-A are involved in the phosphorylation of JNK
and its co-localization with mitochondria. Further evidence suggests that in ECs,
oxLDL induces mitochondrial derived ROS (Zmijewski, Moellering et al. 2005). All
of these findings support JNK-2 as a mitochondrial regulating protein. SR-A mediated
JNK-2 phosphorylation may provide a novel feedback mechanism to the findings of a
JNK-2 and ApoE double knockout mouse (Ricci, Sumara et al. 2004) where p-JNK-2
is involved in the phosphorylation of SR-A. These findings may further support CD-
36 in an indirect positive feedback mechanism with p-JNK-2 and SR-A activation
(Ricci, Sumara et al. 2004). Our laboratory has previously shown that JNK-1
regulates key PDH e1α subunit resulting in an energy crisis, a reducing equivalent
crisis, and a redox crisis (Zhou, Lam et al. 2007). These findings further support JNK-
2 as a mechanism of atherosclerosis upon ligand binding and may lead to endothelial
dysfunction through regulation of apoptosis/necrosis resulting in EC injury. Further

120
work needs to be performed to understand how p-JNK-2 changes mitochondrial
energy production, reducing equivalents and redox status.

A Possible Mechanism to how JNK-2 may be Involved in EC
Regulation/Dysfunction
Treatment of cells with receptor blocking antibodies showed that both SR-A
and CD-36 are both potent initiators of Bcl-xL phosphorylation and of caspase-3
activity that directly correlates with p-JNK-2 density in differentially modified LDL
treated cells. Our data also show that incubation of cells with JNK inhibitor
significantly ablated JNK-2 phosphorylation, phosphorylation of Bcl-xL, and
induction of caspase-3 activity supporting p-JNK-2 involvement in Bcl-xL mediated
apoptosis. P-JNK-2 has been shown to phosphorylate Bcl-xL and induce apoptosis by
many groups (Sunayama, Tsuruta et al. 2005; Wang, Pei et al. 2007). Our data
further corroborate the findings of caspase-3 activity and p-BCL-XL levels. P-BCL-
XL is known to initiate mitochondrial driven apoptosis and formation of the
apoptosome (Cui, Tashiro et al. 2006). Although this is a mechanism for apoptosis,
JNK-2 may have other mitochondrial targets that need to be further determined.
Interestingly, one may surmise that JNK-2 may be involved in ROS production in the
mitochondria since oxLDL induces mitochondrial derived ROS in ECs (Zmijewski,
Moellering et al. 2005) and that JNK-2 is implicated in ROS producing foam cells
(Ricci, Sumara et al. 2004; Rahaman, Lennon et al. 2006). To further support this
argument is the regulation of PDH by JNK-1 in cortical neurons which would be

121
proposed to decrease redox status as well as stall electron transport resulting in
electron leak to molecular oxygen to form O
2
.-
(Zhou, Lam et al. 2007). Further
studies need to be performed on JNK-2 and mitochondrial function to determine the
functional consequence that would support ROS production in BAEC as well as
support the findings of MFC formation.
Regardless of the mechanisms involved in the regulation of
mitochondria, we have shown that JNK-2 is phosphorylated through both a CD-36 and
SR-A mediated signaling cascade that is mostly dependent on protein unfolding of
LDL and may also be minimally synergistically coupled to LOOH through NADPH
oxidase activity. We also determined that modified LDL induced JNK-2
phosphorylation and co-localization with mitochondria which is dramatically reduced
by JNK inhibitor but still co-localizes with mitochondria and is dependent on both
CD-36 and SR-A receptors but is ablated when both receptors are blocked. Lastly, we
show that p-JNK-2 is involved in the activation of caspase-3 through the
phosphorylation of BCL-XL and is related to both CD-36 and SR-A signaling through
p-JNK-2. According to Ricci et al., p-JNK-2 phosphorylates and activates the SR-A
receptor (Ricci, Sumara et al. 2004). These findings with ours may provide a positive
feedback loop between SR-A and p-JNK-2 and that CD-36 may further support an
indirect positive feedback loop of both SR-A and p-JNK-2. We may have also found
a novel feedback mechanism between SR-A and p-JNK-2 that may be further
activated by CD-36 receptor activity. Our Proposed mechanism may pinpoint a key
regulatory step inatherosclerotic development. These data demonstrate the deleterious

122
effects of modified LDL on mitochondrial function and may provide several novel
therapeutics routes to reverse the adverse effects of modified LDL on EC function
thereby preventing atherosclerosis.

123
CHAPTER VI

ROBUST CD-36 STAINING AND P-JNK-2 CO-LOCALIZATION WITH
MITOCHONDRIA OF THE LUMEN AND VASA VASORUM
ENDOTHELIAL CELLS IN BIFURCATIONS AND STRAIGHT REGIONS OF
DISEASED HUMAN HEARTS

Abstract
Modified LDL is intimately involved in the initiation and progression of
atherosclerosis. Increasing evidence suggests that modified LDL initiates scavenger
receptor mediated inflammation and foam cell formation. Recent evidence suggests
that JNK-2 knockout mice lesions have decreased MFC formation and oil red staining
(plaque size). These findings suggest JNK-2 as a model of MFC formation and may
suggest JNK-2 as a mode of EC dysfunction. To further support these findings, CD-
36 knockout macrophages have decreased foam cell formation as well as the loss of p-
JNK-2 signaling upon oxLDL treatment. The aim of this study was to determine
whether human coronary arteries of diseased hearts stained positive for both CD-36
and would stain positive for mitochondrially co-localized p-JNK-2 in ECs as well as
other vascular cells.
Human coronary arteries of both bifurcations and straight regions were
robustly positive for CD-36 staining of ECs of the lumen and the vasa vasorum.
SMCs and macrophages stained positive for CD-36 as well. Interestingly, staining
mitochondria for cytochrome C and lesions with p-JNK-2 antibody, showed a

124
mitochondrial localized p-JNK-2 in both ECs of the lumen and vasa vasorum of both
the bifurcation and the straight region. SMC and macrophages were also observed to
have p-JNK-2 co-localizing with mitochondria in both the bifurcations and
straight regions.
These findings demonstrate a novel signaling to p-JNK-2 and that p-JNK-2
signal co-localizes with the mitochondria of ECs, macrophages and SMC of diseased
arteries and further supports p-JNK-2 as a novel mechanism to EC dysfunction or
MFC formation. To further support these findings are the evidence of JNK-2
knockout mice in MFC formation, CD-36 knockout macrophages with no p-JNK-2
and decreased MFC formation, increased ROS production in MFCs, mitochondria as
the major source of ROS production in cells, oxLDL treatment of ECs resulting in
mitochondrial derived ROS production, and most recently our data showing p-JNK-2
co-localizing with mitochondria.

Introduction
Lipid lowering therapy has been widely associated with decreased risk of
atherosclerosis (Sobel 2007). These observations suggest that LDL cholesterol is
intimately involved in the development of atherosclerosis (Jagavelu, Tietge et al.
2007). Modified LDL by enzymatic processes or by oxidative modification has been
associated with inflammation and the development of MFCs and plaque formation
(Osterud and Bjorklid 2003; Torzewski, Suriyaphol et al. 2004; Asatryan, Hamilton et
al. 2005; Dersch, Ichijo et al. 2005; Koller, Volf et al. 2006; Malle, Marsche et al.

125
2006; Torzewski and Lackner 2006). Furthermore, scavenger receptors of modified
LDL are involved in the signaling associated with these atherogenic changes and foam
cell formation (Rahaman, Lennon et al. 2006). Foam cells are macrophages that
become lipid loaded and produce massive quantities of ROS and the mitochondria are
the main source of cellular ROS production suggesting a mitochondrial role in foam
cell formation. It is therefore of interest to understand how oxLDL scavenger
receptors are involved in both the formation of MFCs and endothelial dysfunction.
OxLDL-R CD-36 knockout macrophages are found to have decreased MFC
formation as well as have decreased JNK-2 phosphorylation (Rahaman, Lennon et al.
2006). These findings suggest that JNK-2 may have a plausible role in MFC
formation. To further support these findings was a double knockout of ApoE and
JNK-2 mouse model was found to have both decreased MFC formation as well as
have decreased plaque formation (Ricci, Sumara et al. 2004). They further found that
JNK-2 knockout mice had decreased SR-A phosphorylation (Ricci, Sumara et al.
2004). These findings suggest that JNK-2 can further activate SR-A dependent
oxLDL uptake. Our most recent findings show that both SR-A and CD-36 are both
involved in the phosphorylation of JNK-2 in ECs suggesting a dual role of the two
receptors in mitochondrial regulation. We further concluded that the findings of
macrophage SR-A phosphorylation may provide a positive feedback mechanism to the
phosphorylation of JNK-2. We further found that p-JNK-2 co-localizes with
mitochondria suggesting a plausible role of JNK-2 signaling to the mitochondria and
the induction of foam cell formation. Also it was shown that oxLDL in ECs induces

126
mitochondrially derived ROS production (Zmijewski, Moellering et al. 2005) further
implicating a p-JNK-2 in EC dysfunction and we also found that p-JNK-2 is involved
in the induction of apoptosis by phosphorylation of BCL-XLxl and may be another
mechanism of mitochondrial regulation and necrotic core formation.
Since we have found that CD-36 and SR-A induce JNK-2 phosphorylation and
that CD-36 knockout macrophages have decreased p-JNK-2 levels and foam cell
formation (Rahaman, Lennon et al. 2006), we wondered whether CD-36 staining was
evident in ECs of diseased arteries and whether these ECs were positive for p-JNK-2
co-localization with mitochondria. These data would also support the findings of p-
JNK-2 co-localization with mitochondria upon incubation with oxLDL and specific
oxLDL receptor blocking antibodies.

Results

CD-36 staining of human coronary artery
Our previous findings have shown that CD-36 is involved in the
phosphorylation of JNK-2 in ECs and is supported by JNK-2 knockout macrophages
(Rahaman, Lennon et al. 2006), and we therefore wondered if human coronary artery
ECs of the lumen or vasa vasorum would be positive for CD-36. CD-36 staining was
prominent in both ECs of the lumen (Fig. 30B, C, 31A, C) as well as of the vasa

127
vasorum (Fig. 30A, 31B, D) for both the straight regions (Fig. 30) and bifurcation
sections (Fig. 31). These findings suggest that oxLDL may play a two fold role in
coronary artery disease by affecting blood flow and nutrients to the SMC and
surrounding tissues of the artery as well as affecting the lumen EC function resulting
in artery injury and disease. Further analysis revealed that bifurcations had much
greater CD-36 staining (Fig. 31) in both macrophages and SMC than did straight
regions (Fig. 30). However, CD-36 staining in both SMC and macrophages of the
straight regions were drastically elevated as compared to the surrounding tissue (Fig.
30). These findings further suggest a role of CD-36 induced vascular cell dysfunction
that is corroborated by JNK-2 knockout mice and CD-36 knockout macrophages
(Rahaman, Lennon et al. 2006; Ricci, Sumara et al. 2004). To further confirm that
CD-36 may play a role in EC dysfunction, we analyzed the co-localization of p-JNK-2
and mitochondria in human coronary artery sections.

128
Lumen EC
A
B
C
SMC
SMC
Macrophages
SMC
Macrophages
Vasa vasorum
Figure 30: CD-36 Staining of Diseased Human Coronary Artery Straight
Regions

IHC staining of CD-36 (brown pigment) in the straight regions of
diseased coronary arteries. CD-36 staining of coronary arteries showing SMCs
macrophages and vasa vasorum staining robustly positive for CD-36 (A),
SMC, macropages/foam cells and endothelial cells of the lumen (B,C) (n=3).

129
Figure 31: CD-36 Staining of Atherosclerosis Prone Bifurcations of
Diseased Human Coronary Arteries

IHC staining of bifurcation in diseased coronary arteries of ex vivo
transplanted hearts showing CD-36 staining of lumen endothelial cells, vasa
vasorum endothelial cells, Macrophages/foam cells and SMC. CD-36
staining of endothelial cells of the lumen, endothelial cells of the vasa
vasorum, SMC and macrophages/foam cells just downstream of the
bifurcation (A-B) and a little further downstream of the bifurcation. CD-36
staining of lumen endothelial cells, SMC and macrophages/foam cells (A),
vasa varoum and SMC (B), lumen endothelial cells and macrophages/foam
cells (C) and vasa vasorum and SMC (D) (n=3).
A
B
C
SMC
Lumen
EC
Macrophages
Vasa vasorum EC
Lumen EC
Macrophages
D
Vasa vasorum
EC
SMC
SMC

130
P-JNK-2 and Cytochrome C Co-localization in ECs
Since we previously found that oxLDL induces CD-36 dependent JNK-2
phosphorylation and that p-JNK-2 co-localizes with mitochondria upon oxLDL
treatment, we wondered if both the vasa vasorum and lumen ECs would be positive
for p-JNK-2 co-localization with mitochondria. Our findings indicate that both
straight regions (Fig. 32) and bifurcations (Fig. 33) were positive for p-JNK-2 co-
localization with mitochondria in lumen ECs (Fig. 33A, B) and vasa vasorum (Fig.
32A, B and 4C). Interestingly, these data corroborate the findings of CD-36 staining
in ECs of the lumen and vasa vasorum and strengthen the argument that CD-36
dependent JNK-2 phosphorylation results in the regulation of mitochondria and EC
dysfunction. One hallmark of MFCs is that they are lipid loaded and have increased
ROS production. Since mitochondria are the major source of ROS it is postulated that
p-JNK-2 may be involved. To further support this finding is that oxLDL induces
mitochondrially derived ROS production and that both JNK-2 knockout mice and CD-
36 knockout macrophages have decreased MFC formation linking mitochondrial
dysfunction through oxLDL induced CD-36 signaling to p-JNK-2. Lastly, p-JNK-2 is
co-localized with mitochondria in both macrophages (Fig. 32A, 33 A-C) and SMCs
(Fig. 32A-B, 33A-C) of both the straight regions and bifurcations (Fig. 32, 33). SMCs
were mitochondria rich as a positive control to cytochrome C antibody specificity
(Fig. 32, 33). These findings further support JNK-2 as a model of mitochondrial
regulation/dysfunction.

131

A
B
SMC
Vasa vasorum
EC
SMC macrophages
Figure 32: P-JNK-2 Co-localization with Mitochondria in Straight Regions
of Diseased Human Coronary Arteries

IHC staining of straight regions in diseased human coronary artery ex
vivo transplants. P-JNK staining and colocalization observed in endothelial
cells of the vasa vasorum, macrophages, and SMCs (A) as well as in
vasovasorum and SMC further upstream of the bifurcation (n=3).

132
Figure 33: P-JNK-2 Co-localization with Mitochodnria in the Bifurcations of
Diseased Human Coronary Arteries

IHC staining in the bifurcation of diseased coronary arteries of ex vivo
transplanted hearts. P-JNK-2 colocalization with mitochondria as measured by
cytochrome C and p-JNK colocalization for endothelial cells of the lumen and
macrophages/foam cells of the bifurcation (A), endothelial cells of the lumen,
SMC and macrophages/foam cells of just downstream of the bifurcation (B) and
vasa vasorum endothelial cells and SMC of just down stream of the bifurcation
(C) (n=3).
A
B
C
Lumen EC
Macrophages
Macrophages
SMC
Vasa vasorum EC
Macrophages

133
Discussion

LDL cholesterol levels and modified LDL are hallmarks of atherosclerosis
(Vohra, Murphy et al. 2006; Mehta, Sanada et al. 2007; Siegel-Axel and Gawaz 2007;
Sobel 2007). There has been much attention on scavenger receptors of oxLDL as
being atherogenic (Mietus-Snyder, Gowri et al. 2000; Nishimura, Akagi et al. 2004;
Rahaman, Lennon et al. 2006; Mehta, Sanada et al. 2007). CD-36 scavenger receptor
knockout macrophages have been shown to have decreased MFC formation as well as
loss of JNK-2 phosphorylation (Rahaman, Lennon et al. 2006). Furthermore, JNK-2
knockout mice have decreased plaque formation as well as decreased MFC formation
(Ricci, Sumara et al. 2004). Our laboratory has also shown that protein unfolded LDL
is responsible for scavenger receptor CD-36 mediated JNK-2 phosphorylation as well
as its co-localization with mitochondria. Therefore, this study was aimed to address
the relevance of CD-36 in ECs of human coronary arteries and whether these ECs had
p-JNK-2 co-localization with their mitochondria of coronary arteries in diseased hearts
that were also positive for CD-36.

Relevance of CD-36 in ECs of Human Coronary Arteries
ECs of the lumen and of the surrounding capillaries (vasa vasorum) were
heavily stained for CD-36 scavenger receptor. This was true of ECs upstream of the
bifurcation (straight regions) (Fig. 30) as well as at the bifurcation (Fig. 31) and
slightly downstream of the bifurcation (Fig. 31). Since CD-36 knockout macrophages

134
have decreased JNK-2 phosphorylation and have decreased MFC formation
(Rahaman, Lennon et al. 2006) it was postulated that the CD-36 may be signaling
JNK-2 phosphorylation and mitochondrial regulation. We have shown that oxLDL
treatment of BAEC resulted in the phosphorylation of JNK-2 and its co-localization
with mitochondria. Secondly, we found that JNK-2 was regulating mitochondrially
derived apoptosis through phosphorylation of BCL-XL. MFCs are lipid loaded and
are producing elevated levels of ROS (Zini, Berdeaux et al. 2007). Mitochondria are
the major source of ROS production in cells and these observations support the
postulation that JNK-2 may be involved in mitochondrially derived ROS production
(Zini, Berdeaux et al. 2007). To further support JNK-2 as a model for mitochondrial
derived ROS production, treatment of ECs with oxLDL was found to induce
mitochondrially derived ROS production (Zmijewski, Moellering et al. 2005).
Interestingly, the human ECs of both the lumen and that of the vasa vasorum had the
highest staining of CD-36 as compared to macrophages and SMC. Interestingly, SMC
and macrophages were positive for CD-36 scavenger receptor but were more
substantially stained in the bifurcations than in the straight regions suggesting a more
advanced disease state in the bifurcation. These findings support the hypothesis that
oxLDL may induce CD-36 dependent JNK-2 phosphorylation, co-localization to the
mitochondria and regulation/dysfunction of the mitochondria resulting in the
progression of atherosclerosis.

135
ECs had P-JNK-2 Co-localization with Mitochondria in Human Coronary
Arteries of Diseased Hearts that were also Positive for CD-36
ECs of both the bifurcation (Fig. 33) and the straight regions (Fig. 32) had co-
localization of p-JNK-2 with the mitochondria for ECs of the lumen and vasa
vasorum. These findings support previous work on oxLDL and CD-36 dependent
JNK-2 phosphorylation with co-localization to the mitochondria. Secondly, all vasa
vasorum ECs and lumen ECs were positive for CD-36 as well as for p-JNK-2 co-
localization with mitochondria suggesting that CD-36 is most likely involved in the
co-localization of JNK-2 to the mitochondria and that CD-36 may be involved in
regulating mitochondrial function. SMC cells that were positive for CD-36 also
showed some co-localization with mitochondria however, this co-localization was not
as robust as observed in ECs. SMC also have lots more mitochondria than other cells
and the co-localization may not be as obvious. Also ECs, SMC and macrophages
have p-JNK-2 that was cytosolic as was also observed upon oxLDL treatment in vitro.
To further support MFC formation as CD-36 dependent and the role of mitochondria
in MFC formation, p-JNK-2 co-localized to the mitochondria in ECs and in
macrophages that were positive for CD-36. These findings support oxLDL binding to
CD-36 scavenger receptor and initiating a p-JNK-2 cascade to the regulation of the
mitochondria and may explain the role of JNK-2 in foam cell formation (ROS
production) as observed in JNK-2 knockout mice and observed in CD-36 knockout
mice as well as oxLDL induced EC ROS production.

136
Regardless of the mechanism of JNK-2 in EC dysfunction or MFC formation,
these findings suggest a novel role of CD-36 signaling to p-JNK-2 in ECs and
macrophages that induces p-JNK-2 co-localization to the mitochondria. Not only do
these findings implicate CD-36 in arterial dysfunction at the lumen but may also
suggest the role of supporting capillaries in CD-36 dependent JNK-2 phosphorylation,
its localization to the mitochondria and the regulation of mitochondrial function.
These data suggest a novel pathway to the regulation of mitochondria in vivo as well
as a novel mechanism to EC dysfunction. These findings may suggest that a
combinational therapy of lipid lowering therapy, oxLDL antibodies and inhibitors of
anything from CD-36 and downstream may result in an increased cardiovascular
function. These findings suggest that blocking CD-36 receptor may be beneficial to
increasing mitochondrial function of most cells in atherosclerosis.

137
CHAPTER VII

LDL PROTEIN UNFOLDING AS A MECHANISM OF MITOCHONDRIAL
DYSFUNCTION IN ENDOTHELIAL CELLS MEDIATED BY P-JNK-2:
INSIGHTS INTO NEW DRUG DISCOVERY

Conclusions

LDL Cholesterol and Initiation of Atherosclerosis
LDL cholesterol is intimately involved in the initiation of atherosclerosis and is
still in strong debate as to how LDL cholesterol initiates atherosclerosis (Shen and
Sevanian 2001; Mehta, Chen et al. 2004; Sobel 2007). LDL cholesterol is known to
be modified in the endothelial subspace as well as in the extracellular space
(Leeuwenburgh, Hardy et al. 1997). LDL and modified LDL are known to get
engulfed by macrophages resulting in the formation of MFCs (lipid loaded
macrophages) and fatty streaks (Ricci, Sumara et al. 2004; Cheruvu, Finn et al. 2007).
However, modified LDL is also known to induce macrophage chemoattractant protein
and monocyte adhesion molecules upon oxLDL and LDL treatment suggesting further
evidence of the involvement of LDL in the initiation of atherosclerosis (Natarajan,
Reddy et al. 2001). Further, ECs injury and extracellular matrix exposure is one of the
initial phases in plaque formation (Han, Liang et al. 2006; Hung, Hong et al. 2006;

138
Hartung, Schafers et al. 2007; Siegel-Axel and Gawaz 2007). However, it is in strong
debate as to how the LDL particle initiates atherosclerosis. There is strong evidence
suggesting that LOOH, isoprostanes prostaglandin like peroxides (non-enzymatically
induced precursors) and aldehydes are involved in the initiation of inflammation,
endothelial toxicity and MFC formation (Balduzzi, Diociaiuti et al. 2004).

Lipid Peroxides
Specifically OxPAPC is known to induce NADPH oxidase activity
(Rouhanizadeh, Hwang et al. 2005) and results in EC, SMC, and macrophage
mediated ROS production a hallmark of MFCs. Further, 9, 4-hydroxy-nonenal and
malondialdehyde are known to induce NADPH oxidase (Berliner, Navab et al. 1995;
Mollnau, Wenzel et al. 2006), induce apoptosis and induce NK-κΒ signaling to
inflammatory cytokine production (Auge, Andrieu et al. 1996; Girotti 1998; Natarajan,
Reddy et al. 2001). LOOH are known to also evoke apoptosis and or cytoprotection
pathways (antioxidant system) (Auge, Andrieu et al. 1996; Dimmeler, Haendeler et al.
1997; Girotti 1998). However, our laboratory believes that LDL protein unfolding
may have a large impact in atherosclerosis as well and may compliment the toxicities
associated with LOOH.

Protein Unfolding
Our laboratory has demonstrated that the in vivo atherosclerotic LDL particle
LDL
-
has been associated with atherosclerotic changes in macrophages and ECs

139
(Asatryan, Ziouzenkova et al. 2003; Hwang, Rouhanizadeh et al. 2006). We have
further found that the atherogenic LDL particle is protein unfolded and lipid
peroxide/aldehydes rich (Sevanian, Bittolo-Bon et al. 1997). We therefore proposed
that protein unfolding may be involved in the initiation of atherosclerosis. Protein
oxidation is known to induce protein unfolding in many proteins as well as induce
hydrophobic residue exposure and yield sticky proteins that will aggregate and be
recognized by protein degradation systems (Bota and Davies 2001; Bota and Davies
2002). Scavenger receptor mediated uptake of oxLDL may therefore be dependent on
the unfolding of the LDL particle which then initiates its toxicity and is further
supported by the requirement for basic amino acids in LOX-1 mediated oxLDL uptake
suggesting a recognition of LDL
-
(Chen, Inoue et al. 2001). To further support protein
unfolding as a mechanism of LDL toxicity is our findings that PLA2-treated LDL
induces protein unfolded LDL particles with atherogenic properties (Asatryan,
Hamilton et al. 2005). Further enzymatic modifications of LDL have been shown to
induce atherosclerotic changes of LDL that are as potent as or more potent than lipid
peroxidized LDL particles (Torzewski and Lackner 2006). However, it is unknown
how LDL protein oxidative modifications affect the LDL particle structural integrity,
protein unfolding, and protein lipid/lipid peroxide interactions.

Protein Oxidative Modifications
LDL protein isolated from atherosclerotic lesions was shown to be positive for
nitrotyrosine by LC/EIS (Leeuwenburgh, Hardy et al. 1997). These findings implicate

140
RNS involvement in the formation of oxLDL and specifically ONOO
-
. ONOO
-
has
been further implicated in atherosclerosis under OF where atherosclerosis
preferentially occurs (Hsiai, Hwang et al. 2007). ONOO
-
formation may be the direct
result of increased NADPH oxidase activities and iNOS activities (Hsiai, Hwang et al.
2007). Further oxidation of LDL at cysteine residues would result in the formation of
a more electronegative particle as well. Lastly, LDL protein unfolding involves alpha
helical unfolding and most tyrosine residues are located in alpha helical domains
suggesting a plausible role of tyrosine nitration in the unfolding of LDL. Chapters 3
and 4 addressed (a) the chemical modifications inherent in atherosclerotic LDL
-
, (b)
functional role of ONOO
-
in LDL
-
formation, (c) whether LDL modified by ONOO
-
is
physiologically active and (d) nitrotyrosine positive staining of atherosclerotic prone
bifurcation versus nitrotyrosine negative staining of straight region.

The Chemical Modifications Inherent in Atherosclerotic LDL
-

LC/MS/MS data and CD data indicated that protein nitration located in alpha
helices inherent in LDL
-
may be involved in the unfolding LDL. Modifications of
tyrosine to nitrotyrosine in a1, a2, a3 and b2 as well as oxidation of cysteine to cysteic
acid may assist in the unfolding of the LDL particle by the loss of a-helical character
and the gain of beta structural components b-turn, parallel and anti-parallel sheets and
random coil structures. It is of note that nitration occurs in the tyrosine rich alpha
helices specifically alpha helice 1 and that b1 is resistant to nitrotyrosine but is
susceptible to cysteine oxidation. These data suggest that alpha helical nitration may

141
be involved in the unfolding of the LDL protein and that cysteine oxidation of one of
the nine free cysteine residues may be involved in the particles
increased electronegativity.

Functional Role of ONOO
-
in LDL
-
Formation
Treatment of LDL with either ONOO
-
or SIN-1 resulted in extensive nitration
of alpha helices that were similar to modifications in in vivo LDL
-
, accumulation of
LOOH, loss of alpha helical protein structural integrity, and the loss of alpha helical
structure coincided with an increased LDL
-
subfraction. It may therefore be surmised
that LOOH and alpha helical nitration may be synergistically involved in the
unfolding of LDL. Further, LDL
-
of ONOO
-
modified LDL had similar protein
structure to that of in vivo LDL
-
suggesting that the mechanism of unfolding is similar
for protein oxidative modifications as it is for other
oxidative/enzymatic modifications.

LDL Modified by ONOO
-
is Physiological
Treatment of LDL with ONOO
-
and labeling with DiI showed physiologic
activity in BAEC cells with increased uptake and binding that was inhibited by
incubation with excess unmodified LDL. ONOO
-
-treated LDL had increased uptake
suggesting the importance of different LDL and oxLDL receptors that is corroborated
by findings that ONOO
-
-treated LDL binds to CD-36 receptor (Guy, Maguire et al.

142
2001; Rahaman, Lennon et al. 2006). Further evidence suggests that modified LDL is
internalized by scavenger receptors and may account for an increased uptake.

Nitrotyrosine Positive Staining of Atherosclerotic Prone Bifurcation Versus
Nitrotyrosine Negative Staining of Non-atherogenic Straight Region
To further support LDL modification by ONOO
-
as a means of atherosclerosis,
we found that atherosclerotic prone bifurcations stained positive for nitrotyrosine
whereas the straight regions were negative for nitrotyrosine. There is further evidence
that LDL isolated from atherosclerotic lesions is positive for nitrotyrosine as well
(Leeuwenburgh, Hardy et al. 1997). Lastly, eNOS staining was positive in the straight
regions and negative in the bifurcation suggesting eNOS as an anti-atherogenic protein
system. However, nitrotyrosine staining may be more dependent on iNOS activity
since iNOS activity is an inflammatory marker and is supported by increased iNOS
activity in atherosclerosis (Deeb, Shen et al. 2006). These findings suggest that LDL
modification ONOO
-
may be involved in lesion formation and progression as well as
involved in the formation of an electronegative atherosclerotic LDL particle in vivo
with similar structure to an in vivo LDL
-
.

Role of Scavenger Receptors in Modified LDL Induced Atherosclerosis
Since ONOO
-
-treated LDL is known to be a ligand for CD-36 (Guy, Maguire
et al. 2001; Rahaman, Lennon et al. 2006), it may be postulated that ONOO
-
-treated
LDL may be involved in scavenger receptor mediated atherosclerosis. CD-36 is

143
known to be involved in development of MFCs (Rahaman, Lennon et al. 2006)
however, its involvement in ECs is less known. Recent evidence suggests that CD-36
knockout macrophages have decreased MFC formation (Rahaman, Lennon et al.
2006). Further evidence from this paper shows that p-JNK-2 levels are almost
completely ablated upon oxLDL treatment (Rahaman, Lennon et al. 2006). However,
it is unknown how different oxLDL-R are involved in the activation of JNK-2 in
macrophages or in ECs. SR-A and LOX-1 are two more scavenger receptors that are
involved in uptake of modified and atherosclerotic signaling in ECs. Lastly, the
nLDL-R may have some involvement as well. To further support p-JNK-2 as a mode
of endothelial dysfunction and MFC formation, a JNK-2 knockout mouse model had
decreased MFC formation as well as a decreased plaque size (Ricci, Sumara et al.
2004). These findings suggest that these mice may be resistant to modified LDL and
that JNK-2 may be involved in mitochondrial derived ROS. To further support EC
derived mitochondrial ROS, oxLDL treated BAEC cells had increased
mitochondrially derived ROS (Zmijewski, Moellering et al. 2005). With these animal
observations and cellular observations, chapter 5 and 6 address (a) whether JNK-2
phosphorylation is dependent on LOOH, LDL protein unfolding or protein oxidative
modifications, (b) whether JNK-2 phosphorylation is SR-A and CD-36 oxLDL-R
dependent, (c) whether oxLDL-R dependent JNK-2 phosphorylation involves
mitochondrial localization, (d) whether modified LDL induces oxLDL-R dependent
apoptosis signaling through p-JNK-2 (e) whether CD-36 staining is robust in both
lumen and vasa vasorum ECs of bifurcations and straight regions, and (f) whether CD-

144
36 staining of ECs was accompanied by p-JNK-2 co-localization with mitochondria
confirming in vitro modified LDL findings.

Whether JNK-2 Phosphorylation is Dependent on LOOH, LDL Protein
Unfolding or Protein Oxidative Modifications
We modified LDL such that we would have protein unfolded LDL (PLA2-
LDL), protein unfolded and lipid peroxidized LDL (Cu-LDL) and protein unfolded,
lipid peroxidized, protein unfolded and protein oxLDL (ONOO
-
-LDL). Each of these
types of modifications was used as a tool to determine how JNK-2 phosphorylation
may be occurring. We found that JNK-2 phosphorylation was highest in the most
lipid peroxidized LDL and decreased with decreasing LOOH. However, PLA2-LDL
still had a significantly higher level of JNK-2 phosphorylation than control-LDL and
blank suggesting that the protein unfolded LDL was the most responsible part of
modified LDL in JNK-2 phosphorylation. These findings led us to believe that
unfolded LDL is most likely receptor dependent and is supported by findings of JNK-
2 phosphorylation in CD-36 knockout macrophages (Rahaman, Lennon et al. 2006).

JNK-2 Phosphorylation is SR-A and CD-36 OxLDL-R Dependent
Since we had seen that protein unfolding was most responsible for JNK-2
phosphorylation, we hypothesized that JNK-2 phosphorylation may be dependent on
ligand binding to oxLDL-R and signal transduction from differential oxLDL-R.
Findings of protein unfolded LDL induction of oxLDL receptors is further supported

145
by basic amino acids binding to a more electronegative particle as seen in LOX-1
(Chen, Inoue et al. 2001). We used receptor blocking antibodies to CD-36, SR-A,
LOX-1 and LDL
-
R and found that most of the JNK-2 phosphorylation was dependent
on CD-36 and SR-A whereas LOX-1 had some minimal involvement and LDL-R had
no involvement in the activation of JNK-2. These findings support data found in a
CD-36 knockout macrophage that had decreased JNK-2 phosphorylation (Rahaman,
Lennon et al. 2006). We further found that SR-A is involved in the phosphorylation of
JNK-2. In the JNK-2 and ApoE double knockout mice, there was a decrease in SR-A
phosphorylation and activation suggesting a possible positive feedback mechanism as
well as an indirect feedback mechanism through the activation of JNK-2 through CD-
36 and then the activation of SR-A by JNK-2 (Ricci, Sumara et al. 2004). However,
what is the purpose of JNK-2 in inducing MFC formation or possibly
endothelial dysfunction.

OxLDL-R Dependent JNK-2 Phosphorylation Involves Mitochondrial
Localization
Since CD-36 knockout macrophages and JNK-2 knockout mice have decreased
MFC formation (Ricci, Sumara et al. 2004; Rahaman, Lennon et al. 2006), we
believed that modified LDL may be inducing p-JNK-2 co-localization with
mitochondria. To further support this hypothesis is the idea that JNK-2 is involved in
foam cell formation which are both lipid loaded and producing large quantities of ROS
as well as the fact that mitochondria are the major source of ROS production in cells

146
(Han, Liang et al. 2006; Hung, Hong et al. 2006; Hartung, Schafers et al. 2007; Siegel-
Axel and Gawaz 2007). We found that modified LDL induced p-JNK-2 and induced
its co-localization with mitochondria in BAEC. Further, we found that blocking both
CD-36 and SR-A together inhibited p-JNK-2 co-localization with mitochondria
however, did not block p-JNK-2 co-localization with mitochondria in CD-36 or SR-A
blocked cells alone. These findings suggest that two oxLDL-R’s are involved in p-
JNK-2 co-localization with mitochondria and are somehow regulating mitochondrial
function and may be a metabolism or apoptosis regulation.

Protein Unfolded LDL Induces oxLDL-R Dependent Apoptosis Signaling
Through P-JNK-2
We found that oxLDL induces JNK-2 phosphorylation as well as its
localization to the mitochondria. We wondered how p-JNK-2 may be regulating
mitochondrial function. We found that oxLDL induced p-BCL-XL that was inhibited
by JNK inhibitor suggesting that JNK-2 is most likely involved in the phosphorylation
of BCL-XL and we found that blocking SR-A and CD-36 receptors inhibited p-BCL-
XL whereas was less efficient at blocking p-BCL-XL with SR-A or CD-36 receptor
blocking alone. To further corroborate the findings of p-BCL-XL, we determined the
caspase-3 activity downstream of p-BCL-XL in the apoptosome. We found that
caspase-3 activity was inhibited by incubation of BAEC with JNK inhibitor as well as
inhibited by incubation with both SR-A and CD-36 receptor blocking antibodies alone
but was less efficient at blocking caspase-3 activity with SR-A or CD-36 blocking

147
antibodies by themselves. These findings suggest that p-JNK-2 may be involved in
the regulation of apoptosis in ECs as well as a novel mechanism by which JNK-2
regulated mitochondrial function by protein unfolded LDL through CD-36 and SR-A
scavenger receptor dependent mechanisms. We further wanted to determine whether
CD-36 is in ECs of diseased coronary arteries.

CD-36 Staining is Robust in both Lumen and Vasa Vasorum ECs of Bifurcations
and Straight Regions.
We wondered whether we could prove CD-36 involvement in ECs of diseased
human heart coronary arteries as well as confirm in vitro findings. We found that both
ECs of the lumen and the vasa vasorum of both bifurcations and straight regions were
robustly positive for CD-36. These findings helped support the hypothesis of a CD-36
dependent oxLDL EC mitochondrial dysfunction/regulation. These findings may also
further support CD-36 involvement in macrophages and SMCs as well. However, we
wanted to know whether these ECs had also p-JNK-2 co-localization
with mitochondria.

CD-36 Staining of ECs was Accompanied by P-JNK-2 Co-localization with
Mitochondria Confirming In Vitro Modified LDL Findings
Since we had robust staining of CD-36 in ECs of the lumen and vasa vasorum,
we wondered whether these same cells had p-JNK-2 co-localization with
mitochondria. We found that p-JNK-2 co-localized with mitochondria in both ECs of

148
the lumen and the vasa vasorum of both bifurcations and straight regions. These
findings suggest that our in vitro findings of oxLDL induced JNK-2 co-localization
with mitochondria may be a mechanism by which oxLDL induces mitochondrial
dysfunction/regulation in ECs. These findings also suggest a similar phenomenon in
macrophages and SMCs suggesting coronary artery disease as a mitochondrial disease
and further support the findings of JNK-2 in the formation of foam cells and
lesions (Ricci, Sumara et al. 2004; Rahaman, Lennon et al. 2006).

Protein Unfolding of LDL as a Model for Mitochondrial Dysfunction
in Atherosclerosis
Regardless of the mechanism of endothelial dysfunction, we have found that
protein unfolding of LDL induces a CD-36 and SR-A dependent activation of JNK-2
and resulted in the regulation of mitochondrial apoptosis (Fig. 34). These findings
further suggest that coronary artery disease is a disease of mitochondria and that
mitochondrial function is governed by p-JNK-2. There are many mechanisms to the
modification of LDL and the end result seems to be the formation of a protein
unfolded LDL particle that is a ligand for different oxLDL-Rs. These findings
suggest that the best mechanism to block modified LDL induced injury may be
through blocking the binding and uptake of modified LDL to scavenger receptors by
vascular cells and may be accomplished by immunization with oxLDL autoantibodies
or by direct inhibition of oxLDL-R CD-36 and/or SR-A. These findings suggest
future therapeutic targets including and not limited to protein unfolded LDL auto-

149
antibodies, CD-36 inhibitors, SR-A inhibitors, JNK inhibitors, as well as agents that
may increase mitochondrial function such as L-carnitine important in fatty acid
transport in mitochondria as well as lipoic acid to increase redox status and
energy homeostasis.

Future Work
Although we have found that p-JNK-2 induces apoptosis cascades, we want to
know how p-JNK-2 is regulating mitochondrial energy homeostasis as well as ROS
production. We know that p-JNK-1 was able to regulate key pyruvate dehydrogenase
activity in hippocampul neurons (Zhou, Lam et al. 2007). Since JNK-2 is a close
family member we wondered whether it could do the same or whether it regulates
other key mitochondrial energy, redox, reducing equivalent enzyme systems. We plan
to isolate mitochondria of bovine aortic ECs and incubate them with activated p-JNK-
2 and determine where p-JNK-2 is phosphorylating the mitoproteome.
We also want to know what is required for the upstream activation of p-JNK-2
from the two different oxLDL-Rs. Finding out how CD-36 and SR-A induce JNK-2
phosphorylation may provide some more novel therapeutic targets of mitochondrial
derived toxicity from modified LDL in vivo.

Concluding Remarks
Regardless of the mechanism of the development of atherosclerosis, we have
found a pathway that resulted in the regulation of mitochondrially derived apoptosis.

150
We also have found that the LDL particle upon modification yields a protein unfolded
particle that is a ligand for CD-36 and SR-A receptors that induce JNK-2
phosphorylation and its co-localization with mitochondria. P-JNK-2 further activates
phosphorylation of BCL-XL and induction of the apoptosome. Our findings
determined a modified LDL derived pathway to the initiation of atherosclerosis as
well as may be involved in the propagation of atherosclerosis. Since, MFCs are lipid
loaded inflammatory cells with increased ROS production and that oxLDL induces
mitochondrially derived ROS in BAEC and that JNK-2 knockout mice as well as CD-
36 knockout macrophages have decreased MFC formation, these findings suggest a
novel mechanism of mitochondrial regulation that may provide insight into the
development of atherosclerosis.

151

nLDL
ONOO
-
Protein unfolding
Protein
oxidative
modifications
Mod-LDL
(LDL
-
)
CD-36
Mod-LDL
LDL
-
SR-A
P-JNK-2
sPLA2
Cu
2+
Mitochondrial localization
P-BCLxl:BCL-2
Caspase-3
apoptosis
LOOH
nLDL
ONOO
-
Protein unfolding
Protein
oxidative
modifications
Mod-LDL
(LDL
-
)
CD-36
Mod-LDL
LDL
-
SR-A
P-JNK-2
sPLA2
Cu
2+
Mitochondrial localization
P-BCLxl:BCL-2
Caspase-3
apoptosis
LOOH
Figure 34: Significance

152
Therapeutics:

It may be postulated that usage of statins drugs will decrease the tLDL
cholesterol and that this reduction in LDL cholesterol and therefore there should be a
reduced modified LDL subfraction. Since, protein unfolded LDL has been shown to
induce the phosphorylation of JNK-2 through a protein unfolded LDL ligand
dependent binding to SR-A and CD-36, it may also be surmised that developing an
antibody against protein unfolded LDL may reverse atherosclerosis. Further, there
may also be a novel target that includes both SR-A and CD-36 oxLDL scavenger
receptors. However, this therapeutic approach may not be beneficial as for these
receptors are important in cell homeostasis. Interestingly, the use of JNK inhibitors
has been shown to fail in clinical trials. It may be surmised that these fail because
they are unable to block the initiating signal upstream of the direct phosphorylation of
JNK-2 and therefore there is still a protein unfolded LDL induction of mitochondrial
dysfunction and atherosclerosis. Lastly, since mitochondria are key players in the
apoptosis energy regulation and redox status, it may be surmised that therapeutic
approaches to improve mitochondrial function may be beneficial. Addition of both
lipoic acid and l-carnitine may improve mitochondrial function. Lipoic acid will
increase the redox status of the cell and will also induce the utilization of glucose and
other energy pathways by initiating and continually renewing the reduced fraction of
lipoic acid. Lipoic acid is also beneficial for pyruvate dehydrogenase enzymatic
activity and may improve rate limiting step to the citric acid cycle from glucose

153
metabolism. Since MFCs, ECs and smooth muscle have lipid burden in
atherosclerosis, it may be postulated that increasing fatty acid transport into the
mitochondria with l-carnitine may decrease lipid loading of vascular cells and may
decrease necrotic core size. These therapeutic approaches are summarized in figure
35. These findings may provide some novel therapeutics into drug discovery and
reduction of atherosclerosis.
Figure 35: Future Plausible Therapeutics
nLDL
ONOO
-
Protein unfolding
LOOH
Protein
oxidative
modifications
sPLA2 Cu
2+
Mod-LDL
(LDL
-
)
immunization
protein
unfolded LDL
Statins
Mod-LDL
(LDL
-
)
CD-36
Mod-LDL
(LDL
-
)
SR-A
SR-A
inhibitor
CD-36
inhibitor
P-JNK-2
JNK-2
inhibitor
I
II
III
IV
V
C
ATP
ADP
I
II
III
IV
V
C
ATP
ADP
Lipoic acid,
L-carnitine

154
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Abstract (if available)

Abstract An elevated level of LDL cholesterol is associated with the development of atherosclerosis and is also associated with aging. Modification of LDL particle is one of the main contributors to the development of atherosclerosis and is elevated with increasing plasma LDL concentrations. Modified LDL is usually composed of LOOH/aldehydes, unfolded protein and some protein post-translational modifications. It has been debated whether the lipid peroxides or unfolded apoB-100 protein is important. An important pathway in atherosclerosis may be the phosphorylation of JNK-2 in ECs. OxLDL-R CD-36 knockout macrophages which have decreased foam cell formation and decreased JNK-2 phosphorylation as well as an ApoE and JNK-2 double knockout mouse has decreased lesion size and MFC formation. Foam cells have increased ROS production and mitochondria are the major source of ROS and this evidence may suggest that p-JNK-2 is involved in regulating mitochondrial function.

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Asset Metadata

Creator Hamilton, Ryan Thomas Littleton (author)

Core Title LDL protein nitration: implication for protein unfolding and mitochondrial function by p-JNK-2

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology

Publication Date 11/16/2007

Defense Date 10/22/2007

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag apoptosis,endothelial cell dysfunction,JNK-2,LDL,mitochondrial dysfunction,nitration,OAI-PMH Harvest

Language English

Advisor Cadenas, Enrique (committee chair), Brinton, Roberta Diaz (committee member), Hodis, Howard Neil (committee member)

Creator Email rhamilto@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m927

Unique identifier UC1337323

Identifier etd-Hamilton-20071116 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-593257 (legacy record id),usctheses-m927 (legacy record id)

Legacy Identifier etd-Hamilton-20071116.pdf

Dmrecord 593257

Document Type Dissertation

Rights Hamilton, Ryan Thomas Littleton

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu