View of the Peroxiredoxin Family………………………

SYSTEMS BIOLOGY ANALYSIS OF MACROPHAGE FOAM CELLS:

FINDING A NOVEL FUNCTION FOR PEROXIREDOXIN I

JAMES PATRICK CONWAY

Submitted in partial fulfillment of the requirements

for the degree of Doctor of Philosophy

Dissertation Advisor: Dr. Michael Kinter

Department of Physiology and Biophysics

School of Medicine

CASE WESTERN RESERVE UNIVERSITY

January 2007 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

______

candidate for the Ph.D. degree *.

(signed)______(chair of the committee)

______

(date) ______

*We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents

List of Tables………………………………………………………………… 4

List of Figures…………………………………………………………..…… 5

Acknowledgements………………………………………………………… 7

List of Abbreviations……………………………………………………….. 8

Abstract………………………………………………………………………. 9

Chapter 1 Introduction

1.1 Atherosclerosis: background and significance………………… 11

1.2 Macrophage biology: relevance to atherosclerosis…………… 13

1.3 Differential expression induced by oxidized LDL……………… 16

1.4 Identification of differentially-expressed mRNA and proteins.. 18

1.5 Chronic exposure models in stress adaptation……………….. 22

1.6 An overview of the peroxiredoxin family……………………….. 25

1.7 Differential expression of peroxiredoxin……………………….. 27

1.8 Regulation of peroxiredoxin activity…………………………….. 29

1.9 Summary and aims……………………………………………….. 30

1.10 Figures……………………………………………………………… 32

Chapter 2 Differential expression in a chronic-exposure

model of foam cell formation

2.1 Introduction…………………………………………………………… 35

2.2 Experimental procedure…………………………………………..… 37

2.3 Results………………………………………………………………... 45

2.4 Summary……………………………………………………………… 53

2.5 Figures………………………………………………………………… 54

Chapter 3 Antioxidant Role for Peroxiredoxin I in

Macrophage Foam Cells

3.1 Introduction…………………………………………………………… 77

3.2 Experimental procedures…………………………………………… 78

3.3 Results………………………………………………………………… 82

3.4 Summary……………………………………………………………… 88

3.5 Figures………………………………………………………………… 89

Chapter 4 Regulation of cell signaling by peroxiredoxin I

4.1 Introduction……………………………………………………………. 106

4.2 Experimental procedures……………………………………………. 107

4.3 Results…………………………………………………………………. 110

4.4 Summary……………………………………………………………..… 113

4.5 Figures………………………………………………………………… 113

Chapter 5 Discussion

5.1 Summary of results…………………………………………………… 118

5.2 Chronic exposure to oxLDL attenuates toxicity in foam cells……. 119

5.3 Differential expression induced by chronic vs. acute oxLDL

exposure………………………………………………………………. 121

5.4 Induction of peroxiredoxin I provides protective antioxidant

functionality…………………………………………………………… 131

5.5 Peroxiredoxin I regulates p38 MAPK signaling in macrophage foam

cells……………………………………………………………………. 137

5.6 Dual role of peroxiredoxin I in macrophage foam cells…...... 139

Chapter 6 Future Directions

6.1 Targeting the pro-atherogenic foam cell in atherosclerosis

prevention……………………………………………………………… 141

6.2 Functional roles for peroxiredoxin I in macrophage foam cells….. 144

6.3 A parallel role for peroxiredoxin in cancer and atherosclerosis…. 145

6.4 Figures………………………………………………………………… 147

Literature cited…………………………………………………………..……. 150

List of Tables

Table 1 Differentially expressed proteins in oxLDL-treated J774 or J774-CE

macrophages.

Table 2 Upregulated mRNA in oxLDL-treated J774 or J774-CE

macrophages.

Table 3 Downregulated mRNA in oxLDL-treated J774 or J774-CE

macrophages

Table 4 Differential expression of antioxidant response genes.

Table 5 Differential expression of immune response mRNA transcripts

List of Figures

Figure 1 Current model of foam cell and fatty streak formation.

Figure 2 Foam cell formation assayed by oil red O staining.

Figure 3 Foam cell formation assayed using DiI-labeled lipoprotein.

Figure 4 Lipid-uptake of oxLDL-treated J774 and J774-CE macrophages.

Figure 5 OxLDL-induced cytotoxicity in J774, J774-CE & J774-CE(-)

macrophages.

Figure 6 2D gel representation of the J774 macrophage proteomic analysis.

Figure 7 Subset of proteins that are significantly expressed in oxLDL-treated

J774-CE macrophages compared to oxLDL-treated J774

macrophages.

Figure 8 Identification of Prx I as a protein upregulated during macrophage

foam cell formation.

Figure 9 Western blot analysis of the time- and dose-dependent

upregulation of Prx I following exposure to oxLDL.

Figure 10 Detecting the oxidative inactivation of Prx I by 2D SDS-PAGE

Western blot analysis.

Figure 11 Effects of ethoxyquin treatment on Prx I induction.

Figure 12 Knock-down of Prx I expression by siRNA transfection.

Figure 13 Oxidant-induced cytotoxicity in macrophages with induced or

knocked-down expression of Prx I.

Figure 14 Flow cytometric histogram data demonstrating increased ROS in

macrophages with induced or knocked-down expression of Prx I.

Figure 15 Oxidant-induced ROS generation in macrophages with induced or

knocked-down expression of Prx I.

Figure 16 Prx I regulates the activation of p38 MAPK stimulated by H2O2,

ethoxyquin, and oxLDL.

Figure 17 Antioxidant activity of Prx I is not dependent on p38 MAPK activity.

Figure 18 Proposed model for the dual role of Prx I in macrophage foam cells.

Acknowledgements

This dissertation is dedicated to my parents, sisters, and Margaret, for their continuous and unconditional support. Thank you for supporting me over these

years, throughout my financial, physical, and emotional distress!

Thank you to Dr. Ken Humphries for convincing me to join the department over

martinis at Tremont’s Hi & Dri.

Thank you to Dr. Graciela Lacueva, who convinced me to stay in science when I

was ready to quit many years ago.

List of Abbreviations

CE chronic exposure

CM-H2DCFDA 5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein

diacetate acetyl ester

CVD cardiovascular disease

DAPI 4',6-diamidino-2-phenylindole, dihydrochloride

DiI 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine

perchlorate

ERK extracellular signal-regulated kinase

GAPDH glyceraldehyde-3-phosphate dehydrogenase

IPG immobilized pH gradient

JNK c-Jun N-terminal kinase

LC-MS liquid chromatography-mass spectrometry

MAPK mitogen-activated protein kinase

(Ox)LDL (oxidized) low density lipoprotein

PIP3 phosphatidylinositol 3,4,5-trisphosphate

Prx peroxiredoxin

PTEN phosphatase and tensin homolog

ROS reactive oxygen species siRNA small interfering ribonucleic acid t-BOOH tert-butyl hydroperoxide

TNBS trinitrobenzene sulfonic acid

TNF-α tumor necrosis factor alpha

Systems Biology Analysis of Macrophage Foam Cells:

Finding a novel function for peroxiredoxin I

Abstract

James Patrick Conway

Atherosclerosis is a type of cardiovascular disease that is characterized by

localized thickening of the arteries at sites referred to as atheromas. The earliest events leading to atheroma formation involve the oxidative modification of low- density lipoprotein in the arterial intima, and the uptake of oxidatively-modified low-density lipoprotein by intimal macrophages. This unregulated uptake results in lipid-laden macrophage-derived foam cells, which aggregate at potential sites of atheroma development known as fatty streaks.

We hypothesized that the transition from macrophage to foam cell is

characterized by differential expression that, as a whole, is pro-atherogenic. A

proteomic and transcriptomic analysis identified several proteins and mRNA

transcripts that were differentially-regulated following acute and chronic exposure to oxLDL, including mediators of antioxidant and immune response. Further,

chronic oxLDL exposure led to a decrease in oxLDL-induced toxicity, which

coincided with an increased antioxidant response when compared to

macrophages treated with a single exposure to oxLDL.

Data obtained from the analysis of differential expression in foam cells provided a foundation for the second stage of this project. Peroxiredoxin I was identified as a protein that is upregulated in macrophages following exposure to oxLDL, which led to the hypothesis that the upregulation of peroxiredoxin I could protect against toxicity induced by oxLDL-generated reactive oxygen species. It was determined that peroxiredoxin I can decrease oxLDL-induced toxicity when induced prior to oxLDL-exposure. This effect coincides with a decrease in reactive oxygen species, verifying the antioxidant role of peroxiredoxin I.

Additionally, alternative functionality for peroxiredoxin I was tested, and it was determined that the activation of p38 MAPK is dependent on peroxiredoxin I expression. The sum of this data suggests that peroxiredoxin I contributes to cell survival and signaling, which affects macrophage foam cell function and survival.

Chapter 1

Introduction

1.1 Atherosclerosis: Background and Significance

Since 1919, cardiovascular disease (CVD) has remained the leading cause of

death in the United States (Thom, Haase et al. 2006). In 2006, the direct and

indirect costs of CVD in this country are estimated to reach $403 billion.

Although CVD includes several types of heart and vascular diseases,

atherosclerosis is the cause of approximately three-fourths of all CVD-related

deaths. Atherosclerosis, commonly described as a thickening of the arteries, is a

disease associated with several risk factors, including elevated blood-levels of

low-density lipoprotein (LDL) (Armstrong, Cremer et al. 1986), hypertension

(Marchesi, Martignoni et al. 1996), obesity (Kortelainen and Sarkioja 1999;

Lakka, Lakka et al. 2001), cigarette smoking (Strong and Richards 1976; Vander

Zwaag, Lemp et al. 1988), and physical inactivity (Laufs, Wassmann et al. 2005).

Advanced atherosclerosis is characterized by fibrous, raised lesions that are

vulnerable to rupture, leading to thrombosis, angina, myocardial infarction and

stroke. These lesions, otherwise known as atheromas, are composed of

proliferating smooth muscle cells , recruited T-cells and macrophages, and an

extracellular lipid core (Adams and Bayliss 1976; Gaton and Wolman 1977;

Parums, Dunn et al. 1990). Prior to the appearance of the atheroma, however,

two key events are believed to be critical to the initiation of atherosclerosis

(Figure 1). First, LDL enters the arterial intima, where it has the tendency to be

retained and oxidatively-modified by cell-mediated mechanisms (Henriksen,

Mahoney et al. 1981; Morel, DiCorleto et al. 1984; Steinbrecher, Parthasarathy et

al. 1984). This modification is toxic to cells in culture, and the modification and

subsequent cytotoxicity can be prevented by the addition of antioxidants, such as

butylated hydroxytoluene, vitamin E or glutathione (Cathcart, Morel et al. 1985;

Katsetos, Herman et al. 1998). The second critical event occurs when oxidized

LDL (oxLDL) is bound and internalized by the scavenger receptors of monocyte-

derived macrophages (Parthasarathy, Young et al. 1986), which become lipid-

laden and take on an ovoid, foamy appearance (Schaffner, Taylor et al. 1980).

These so-called foam cells aggregate at fatty streaks, which represent potential

sites for advanced lesion development (Pearson, Kramer et al. 1977).

Throughout the history of atherosclerosis research, attention has focused on

the pro-atherogenic role of the macrophage-derived foam cell in lesion

development. For example, foam cell formation is associated with the upregulation of pro-inflammatory mediators, including monocyte-chemoattractant protein (MCP-1) (Nelken, Coughlin et al. 1991), interleukin-1 alpha and beta (IL-

1α and IL-1β) (Moyer, Sajuthi et al. 1991) and interleukin-6 (IL-6) (Kishikawa,

Shimokama et al. 1993). This recruitment of immune cells to the lesion contributes to the raised nature of the atheroma. Enhanced expression of growth factors is also associated with foam cells, which stimulate local proliferation of

cells, further developing the lesion (Falcone, McCaffrey et al. 1993; Ramos,

Kuzuya et al. 1998). In advanced atherosclerosis, foam cells constitutively

express matrix-metalloproteinases (MMPs), which contribute to the

destabilization and rupture of the atheroma (Galis, Sukhova et al. 1994; Galis,

Sukhova et al. 1995). The sum of this data suggests that the macrophage- derived foam cell is associated with pro-atherogenic activity throughout the development of atherosclerosis.

1.2 Macrophage Biology: Relevance to Atherosclerosis

Macrophages are a diverse cell type that depend on local environment, stage of differentiation and activation state to determine their functional role. The heterogeneous nature of a macrophage population is often overlooked when describing their role in lesion development. This section will describe how tissue macrophages progress through these phenotypic states, and how the functional capacity of the macrophage relates to its transition to the lipid-laden foam cell.

In the most basic sense, macrophages are categorized as either resident or inflammatory. Resident macrophages, also known as normal or unstimulated, are phagocytic, chemotaxic and proliferative [reviewed in (Ma, Chen et al. 2003)].

However, they have a reduced immunological role, demonstrated by negligible cytokine production and low expression of major histocompatability complex

(MHC) class II genes. Resident macrophages specialize in the phagocytic uptake of apoptotic cells and cellular debris, and are thus geared toward clearance and tissue maintenance. They also recognize Gram-negative and

Gram–positive bacteria (Freimer, Ogmundsdottir et al. 1978), as well as products of bacteria such as lipopolysaccharide (LPS) (Tizard 1971). This broad capacity for recognition is mediated by several cell-surface receptors, including the scavenger receptors that bind and internalize oxLDL. Therefore, it follows that

resident tissue macrophages are capable of forming foam cells through

scavenger-receptor mediated uptake of oxLDL, which has been demonstrated in

mouse resident peritoneal macrophages under cell culture conditions (Knight and

Soutar 1984).

However, the progression of atherosclerosis requires an inflammatory

response that recruits monocytes to the site of the developing fatty streak.

These monocytes differentiate into inflammatory macrophages, which are distinct

from the resident macrophages generated by local proliferation under steady-

state conditions. Both resident and inflammatory macrophages can transition to

activated states, depending on the extracellular factors that initiate the activation.

Prior to activation, a macrophage may become primed by exposure to low

levels of IFN-γ, IL-3, M-CSF, GM-CSF or TNF-α, and acquire a phenotype that is

distinct from both the non-active and active states (Nestel, Price et al. 1992).

Primed macrophages exhibit an enhanced expression of MHC class II and

antigen presentation proteins, while maintaining a reduced capacity for

proliferation.

Exposure to microbial products or IFN-γ in combination with other cytokines

triggers full activation, characterized by an anti-microbial, tumoricidal, pro-

inflammatory phenotype and a loss of proliferative capacity (reviewed in

(Mantovani, Sica et al. 2004)). These macrophages exhibit high NAPDH oxidase

activity, elevated production of reactive oxygen species, and secretion of TNF-α,

PGE-2, IL-1 and IL-6. This phenotype is known as classical or M1 activation, and is typically associated with the presence of T-helper 1 (Th-1) cell cytokines.

Macrophages can also follow a non-classical activation pathway, also known

as alternative or M2 activation, which is associated with T-helper 2 (Th-2)

cytokines such as IL-4, IL-5 and IL-10 (Stumpo, Kauer et al. 1999; Stumpo,

Kauer et al. 2003). These cytokines are generally considered to be anti- inflammatory and associated with the humoral response of acquired immunity, in contrast to the innate immune responses described for classical macrophage activation. Macrophages activated by this alternative pathway retain phagocytic ability and attenuate inflammatory response through IL-10 secretion (Stumpo,

Kauer et al. 1999).

What is the connection between these various phenotypic states, and the

macrophage functionality retained in foam cells? As was previously stated,

macrophages require scavenger receptor activity to transform into foam cells,

and oxLDL has been described as a trigger for macrophage activation. Activated

macrophages and macrophage-derived foam cells have each been described for

their pro-inflammatory response and increased NADPH oxidase activity

(Heinloth, Heermeier et al. 2000; Shatrov and Brune 2003). Additionally, an

increase in scavenger receptor expression is a feature common to oxLDL-

exposed macrophages (Aoyama, Fujiwara et al. 1999; Hirano, Yamashita et al.

1999) and macrophages activated by other means (Bell, Lopez-Gonzalez et al.

1994; Iwashima, Eto et al. 2000). Taken together, these features describe a

classical activation state for the macrophage-derived foam cell.

The pro-atherogenic nature of an increased inflammatory response suggests

that classical Th1 activation of macrophages promotes lesion development.

Correspondingly, one could speculate that the anti-inflammatory response generated by non-classical Th2 activation could attenuate lesion development.

Indeed, researchers have demonstrated a Th1/Th2 functional imbalance due to upregulated Th1 functionality in coronary arterial inflammation. Thus, the activation state of macrophages may play a significant role in the developing lesion by governing the balance of inflammatory response.

1.3 Differential expression induced by oxidized LDL

Although foam cell formation is typically associated with the lipid-loading of

macrophages (Henriksen, Mahoney et al. 1983), other researchers have

described oxLDL-internalization in smooth muscle cells and fibroblasts (Liu,

Ramjiawan et al. 1991; Pitas, Friera et al. 1992). The effect of oxLDL-exposure

on all cell types in the lesion environment, including in endothelial cells and T-

cells (Hamilton, Thorin et al. 1994; Huang, Ronnelid et al. 1995; Stemme, Faber

et al. 1995), have been studied. A common theme throughout this research is

the effect of oxLDL on protein expression in various cell types of the developing

lesion.

The process of foam cell formation is exacerbated by oxLDL-induced changes in protein expression. Macrophage scavenger receptors, including

CD36, SRA, macrosialin and LOX-1, are upregulated following exposure to oxLDL (Han and Nicholson 1998; Moriwaki, Kume et al. 1998; Yoshida,

Quehenberger et al. 1998). However, cholesterol efflux mechanisms are impaired by oxLDL, most likely due to an effect on lipoprotein degradation

(Roma, Bernini et al. 1992; Maor and Aviram 1994; Dhaliwal and Steinbrecher

2000). Therefore, oxLDL accelerates lipid-uptake in macrophages while

inhibiting lipid-efflux, favoring a shift toward the foam cell phenotype.

The induction of innate immune response proteins is a well-documented

effect of oxLDL-exposure in various cell types. One of the earliest findings

described the oxLDL-induced stimulation of arachidonate metabolism in

macrophages, which involved the induction of prostaglandin E2 (PGE2) (Yokode,

Kita et al. 1988). Inflammatory cytokines, including interleukin-1α (IL-1α) (Lipton,

Parthasarathy et al. 1995), IL-1β (Ku, Thomas et al. 1992), and IL-8 (Liu, Hulten et al. 1997), are also induced by exposure of macrophages to oxLDL. In oxLDL- exposed T-cells, upregulation of interferon-gamma (IFN-γ), a critical component of macrophage activation, has been reported (Huang, Ronnelid et al. 1995).

In smooth muscle cells, oxLDL-exposure may induce growth factor

production, such as fibroblast growth factor (FGF) (Ananyeva, Tjurmin et al.

1997), platelet-derived growth factor (PDGF) and its receptor (Stiko-Rahm,

Hultgardh-Nilsson et al. 1992). Other researchers have described the induction

of cell-adhesion molecules, including VCAM and ICAM, in endothelial cells

exposed to oxLDL (Khan, Parthasarathy et al. 1995).

Toxicity due to oxLDL-exposure has been documented (Cathcart, Morel et al.

1985; Katsetos, Herman et al. 1998). Consequently, the induction of an

antioxidant defense is necessary to preserve cell survival, which has been

demonstrated by exogenously-added antioxidants (Kawamura, Miyazaki et al.

2000; Schroeter, Williams et al. 2000). This endogenous defense mechanism is

demonstrated by the induction of glutamate-cysteine ligase, the rate-limiting step

in glutathione synthesis (Moellering, Levonen et al. 2002; Bea, Hudson et al.

2003). Additionally, the induction of thioredoxin reductase has been discovered

in atherosclerotic plaques and oxLDL-treated macrophages in vitro (Furman,

Rundlof et al. 2004). Large-scale analyses of gene and protein expression will continue to identify protective enzymes induced during foam cell formation.

In summation, the differential expression induced by oxLDL is decidedly pro-

atherogenic. Foam cell formation is promoted through scavenger receptor upregulation and the impairment of cholesterol efflux. A pro-inflammatory and growth-promoting response is also induced, contributing to the inflammatory nature of the developing lesion. The profound effect of oxLDL on cell phenotype is the motivation behind proteomic and gene array analyses of the macrophage- to-foam cell transition.

1.4 Identification of differentially-expressed mRNA and proteins:

Gene chips and mass spectrometry

The data reviewed in Section 1.3 was often dependent on protein-specific

antibodies used to identify suspected targets of differential expression.

Consequently, this data was produced on a small scale where differential-

expression that was not specifically assayed would be overlooked. However, the

past few years have brought key technological advances that facilitate the

identification of large sets of differentially-expressed mRNA and proteins. A

major component of this advance was the complete sequencing of the human

genome (Venter, Adams et al. 2001), which continues with the genomic

sequencing of several other species. This genomic data is used to create the

mRNA and amino acid sequence databases that have proved invaluable for

broad-scale transcriptomic and proteomic analyses. Data generated by these

means is produced in a discovery-driven manner, which indicates that the scale

of identification is not limited by prior knowledge of the system. In other words, a

complete system of differential expression can be identified at the message or

protein level, and this data will include both known, hypothesized and previously

unknown targets of differential expression. This section summarizes the

methodology behind the large-scale identification of differential expression at the

mRNA and protein level.

A transcriptomic analysis determines the differential expression of mRNA

across a number of samples using a powerful technology called gene chip or

DNA microarray analysis [see descriptions and protocols in (Amaratunga and

Cabrera 2004; Allison 2006)]. A gene chip is a small glass substrate printed with

thousands of oligonucleotides organized into probe cells. Each probe cell

contains several oligonucleotide probes synthesized to hybridize to a single

mRNA transcript. Each probe is accompanied by a negative control probe

synthesized with a single nucleotide mismatch. This collection of positive and negative probes constitute a probe cell, which is unique to each mRNA transcript in the gene chip library. A typical gene chip may include probe cells for well over

30,000 mRNA transcripts.

Approximately 200 ng of polyA mRNA are required for each sample in a gene

chip analysis. Double-stranded cDNA is synthesized from the mRNA samples,

followed by an in vitro transcription (IVT) reaction that produces biotin-labeled

cRNA. The cRNA is fragmented, and then hybridized to the gene chip for

approximately 16 hours. After appropriate washes, the gene chip is stained with

streptavidin phycoerythrin and analyzed for fluorescent intensity. The set of

unique positive and negative probes for each transcript provides data that can be

statistically analyzed to confirm the likely presence or absence of a transcript, the

relative level of expression, and the differential expression of the transcript when

compared to other samples in the set.

The strength of the gene chip analysis does not depend on the differential

expression of any one transcript, but rather the differential response of transcript

sets that are united under a particular response. While it may be easy to disregard the upregulation of a particular inflammatory mediator, for example, it is less likely that the upregulation of several pro-inflammatory mediators, coupled with the downregulation of anti-inflammatory mediators, could be relegated to chance or experimental error. The gene chip analysis relies on strength-in- numbers, but it also requires a subsequent focus on selected transcripts as further verification of differential expression results.

The union of liquid chromatography (LC) and mass spectrometry (MS) has

made the large-scale analysis of differential protein expression possible, allowing

for complete automation from sample analysis to protein identification. Many

variations on this procedure exist, ranging from the complete identification of

20 complex protein mixtures to the fully-automated excision, processing and analysis of protein bands from 2D gels. The following description represents the most commonly used procedure to date (Kinter and Sherman 2000), and is also the procedure used for the proteomic analyses in this report.

A proteomic analysis begins with a set of differentially-expressed proteins that are assembled by comparing 2D gels processed with Coomasie blue or silver stain. This set of protein bands are excised from the gel, digested with trypsin, extracted from the gel fragment and suspended in an acidic solution. In a LC-MS peptide sequencing procedure, tryptic peptides are separated in the LC stage and injected directly into the mass spectrometer as concentrated elutions. The mass spectrometer measures the mass-to-charge (m/z) ratio of each peptide, then fragments peptides of a specific, isolated m/z in a process called collision- induced dissociation (CID). Sequencing is made possible by the ability to measure peptide mass accurately enough to distinguish losses of individual amino acids in the CID spectra. For example, if a CID spectrum has neighboring peaks that differ by 57 on the m/z axis, this represents the loss of a glycine residue from the peptide during fragmentation. Each peak interval in the CID spectrum represents the next residue in the peptide sequence. Although the sequencing can be done “by hand” through visual analysis of a CID spectrum, the analysis of the full set of CID spectra is typically performed by a computer.

Specialized software is used to compare the experimental peptide sequences to sequence databases, which produces a list of probable protein identifications. In this manner, a set of peptide samples can be systematically injected, analyzed

21 and identified, limited only by the chromatography stage. Differential expression can be determined by intensity changes in gel bands. However, other methods are typically used to confirm protein identity and thoroughly analyze the magnitude of differential expression.

The differential expression of mRNA and protein in macrophage foam cells is described in Chapter 2, and the procedures used are consistent with those described above.

1.5 Chronic exposure models in stress adaptation

While acute exposure to a particular agent may stimulate a response characterized by differential expression, chronic exposure to the same agent over a period of time may alter the steady state levels of the differentially- expressed proteins, and also the acute response to the agent. Though chronic exposure to oxidants, for example, are often studied for their toxic effects, data has demonstrated that chronic, low-dose oxidant exposure may also develop a protective resistance to subsequent exposures. This section provides a brief overview of this principle, providing background for the chronic oxLDL-exposure experiments described in Chapter 2.

Ethanol catabolism produces an oxidant stress of acetaldehyde and free radicals, and antioxidants are required to reduce these toxic products.

Researchers have described diminished antioxidant capability following chronic exposure to ethanol (Grattagliano, Vendemiale et al. 1997; Bailey, Patel et al.

2001). However, other researchers have described opposite results. Eysseric et

al. demonstrated that chronic ethanol exposure results in the upregulation of

several key antioxidants in cultured astrocytes, including catalase, superoxide

dismutase and glutathione peroxidase (Eysseric, Gonthier et al. 2000). The

increased antioxidant activity coincided with decreased free radical production.

Another experiment followed the long-term exposure of rats to flavonol-rich red wine, and determined that rat kidneys show an increase in catalase and glutathione peroxidase activity in this chronic exposure model (Rodrigo, Rivera et al. 2002). Both the ethanol and non-alcoholic antioxidant constituents were responsible in part for the protective effect. Thus, while the negative effects of chronic, elevated ethanol exposure are well-documented, a protective antioxidant response may be developed over time with chronic, low-dose exposure to ethanol.

Another common model of chronic exposure involves the environmental effect

of ozone inhalation, which has been studied since the 1950’s (Stokinger, Wagner

et al. 1957). In general, these reports have linked the toxic effect of chronic

ozone exposure due to oxidant production in exposed cells, resulting in

increased lipid peroxidation (Mustafa 1990). However, some researchers have

reported that adaptive mechanisms may develop during chronic ozone exposure,

although these mechanisms were maintained only temporarily post-exposure

(Wiester, Tepper et al. 1995). For example, heat shock protein 70 (HSP 70) is

highly expressed throughout chronic ozone exposure at normal urban levels

(Wong, Bonakdar et al. 1996), and heat shock proteins are well-known for their

contribution to adaptive responses (Mizzen and Welch 1988; Derocher, Helm et

al. 1991). The induction of heat shock proteins (Loven, Leeper et al. 1985) and chronic ozone exposure (Weller, Crapo et al. 1997) have both been linked to

increases in superoxide dismutase activity, which may contribute to the adaptive

mechanism developed from sub-lethal chronic exposure.

Perhaps the most well-documented example of adaptive response is the

mechanism of ischemic preconditioning [reviewed in (Gori and Forconi 2005)]. It

has been demonstrated that short, non-lethal ischemic episodes can develop a

protective response when they occur prior to an extended ischemic event. As

with ozone exposure and other cellular stressors, heat shock proteins are

induced in response to ischemia and may play a role in the acquired protective

response (Nayeem, Hess et al. 1997; Taggart, Bakkenist et al. 1997). The

induction of HSP 70 following ischemia/reperfusion is believed to result from the

production of oxidant species (Kukreja, Kontos et al. 1994), which may lead to

the initiation of an antioxidant defense (Yamashita, Hoshida et al. 1998; Das and

Maulik 2003).

The examples described above vary greatly in their duration and mode of

exposure, however they also maintain key similarities. Each example is

undisputedly toxic at elevated levels, but may be administered at sub-lethal levels over a given period of time. This low-level chronic exposure generates oxidants that stimulate stress response proteins, antioxidants and other cytoprotective components. The induced protective response remains for a particular duration, which is dependent on the system, and may protect against a

subsequent exposure.

The parallels between the systems described above and macrophage foam

cell formation are striking. The internalization of oxLDL that leads to foam cell formation introduces oxidants to the macrophage, and various proteins are

differentially expressed in response. More importantly, foam cell formation is

reversible, which allows for continuous cycles of oxLDL internalization and efflux.

Finally, although oxLDL is cytotoxic, our knowledge of the developing atheroma

suggests that oxLDL-internalization by macrophages does not result in

immediate cell death. Therefore, a component of this project sought to

determine if a model of chronic oxLDL exposure could lead to an adaptive and protective response in macrophage foam cell formation.

1.6 An overview of the peroxiredoxin family

Peroxidase enzymes are uniquely suited to reduce products of peroxidation,

which are reactive and may inflict oxidative damage to the cell. Well-known

peroxidases include catalase, a peroxisomal enzyme capable of reducing hydrogen peroxide (Hashimoto and Hayashi 1987), and glutathione peroxidase,

which is capable of reducing lipid hydroperoxides (Ursini and Sevanian 2002). A more recently described peroxidase is peroxiredoxin (Prx), which was initially identified in yeast and soon after in mammalian species (Ishii, Yamada et al.

1993; Chae, Chung et al. 1994).

The mammalian peroxiredoxin family consists of six proteins (Prx I-VI)

expressed as unique gene products, with the capability to reduce hydrogen

peroxide, lipid hydroperoxides and peroxynitrite (Ishii, Kawane et al. 1995; Kang,

Chae et al. 1998; Bryk, Griffin et al. 2000; Kim, Lee et al. 2000). They are

divided into three categories: the typical 2-Cys peroxiredoxins (Prx I – IV),

atypical 2-Cys peroxiredoxin (Prx V) and 1-Cys peroxiredoxin (Prx VI).

The typical 2-Cys peroxiredoxins function as homodimers, although a decameric toroid structure composed of five dimers has also been determined

(Schroder, Littlechild et al. 2000). When reducing a substrate, a catalytic

cysteine (Cys51 in Prx I) of one monomer forms an intermolecular disulfide with a

172 resolving cysteine (Cys in Prx I) on the associated monomer. The disulfide is

reduced by the electron donor thioredoxin, re-activating peroxiredoxin antioxidant

capability. In humans, the typical 2-Cys peroxiredoxins share >65% protein

sequence homology, including the two conserved cysteines responsible for

peroxide reduction.

The atypical 2-Cys peroxiredoxin, Prx V, forms an intramolecular disulfide when reducing substrate, rather than an intermolecular disulfide (Seo, Kang et al.

2000). However, like Prx I-IV, Prx V relies on thioredoxin as a reducing partner.

Prx V shares approximately 10% sequence homology with the typical 2-Cys peroxiredoxins, including a conserved catalytic cysteine. Originally, Prx V was thought to function as a monomer, and its structure was determine in reduced monomer form (Declercq, Evrard et al. 2001). However, it is now believed that

Prx V forms a dimer when oxidized, despite the fact that no disulfides contribute to the dimerization (Evrard, Capron et al. 2004). Unlike the typical 2-Cys peroxiredoxins, Prx V exhibits peroxynitrite reductase activity (Dubuisson,

Vander Stricht et al. 2004).

The 1-Cys peroxiredoxin, Prx VI, is not as well-defined as the other peroxiredoxins. It is known that the conserved catalytic cysteine of Prx VI is oxidized to a sulfenic acid (Cys-SOH) as part of the reductive mechanism, which does not form a disulfide due to the unavailability of neighboring cysteines (Kang,

Baines et al. 1998). The identity of its reducing partner is still under investigation, but it is known that Prx VI is not reduced by thioredoxin (Kang, Baines et al.

1998). Increasing evidence suggests Prx VI may be uniquely suited for the reduction of phospholipid hydroperoxides (Fisher, Dodia et al. 1999; Manevich,

Sweitzer et al. 2002; Pak, Manevich et al. 2002).

The peroxiredoxins also vary by subcellular localization. Prx I and II are abundantly expressed and localized to the cytosol. Prx III is localized to mitochondria, and is well-documented in its ability to scavenge radicals produced there (Watabe, Hiroi et al. 1997; Araki, Nanri et al. 1999). Prx IV is cytosolic, but also possesses an N-terminal secretory signal sequence and has been identified in culture media (Matsumoto, Okado et al. 1999). Prx V is localized to the cytosol, mitochondria and peroxisomes (Knoops, Clippe et al. 1999). Prx VI is also localized to the cytosol, but its unique properties may distinguish it functionally from other cytosolic peroxiredoxins.

1.7 Differential expression of peroxiredoxin

The induction of peroxiredoxin at the message or protein level has been described as a response to many stimuli, including hydrogen peroxide (Kim, Lee et al. 2000; Mitsumoto, Takanezawa et al. 2001), high glucose (Morrison, Knoll et

27 al. 2004), hyperoxia (Das, Pahl et al. 2001; Kim, Kang et al. 2001), radiation

(Park, Chung et al. 2000), neurodegenerative diseases (Kim, Fountoulakis et al.

2001; Krapfenbauer, Engidawork et al. 2003), and several forms of cancer

(Chang, Jeon et al. 2001; Noh, Ahn et al. 2001; Memon, Chang et al. 2005).

Typically, the induction of peroxiredoxin is a result of some form of oxidative stress generated endogenously or introduced exogenously. Based on these results, several reports have described the protective effect of recombinantly- overexpressed peroxiredoxin and injurious effects resulting from the knock-down of peroxiredoxin expression.

Prx I is induced not only by the exogenous addition of hydrogen peroxide, but by reagents that lead to the production of hydrogen peroxide, such as thyrotropin and glucose oxidase (Ishii, Yamada et al. 1993; Kim, Lee et al. 2000). Prx I is also induced by ionizing radiation, and expression knock-down experiments have determined that this induction has a protective effect (Chen, McBride et al. 2002;

Zhang, Su et al. 2005). Ionizing radiation induces the other major cytosolic peroxiredoxin, Prx II, and blocking the expression of Prx II with antisense transfection sensitizes cells to radiation-induced toxicity (Park, Chung et al.

2000). In an experiment that follows the chronic exposure model described in

Section 1.4, researchers exposed MCF-7 breast cancer cells to chronic ionizing radiation and isolated radiation-sensitive and radiation-resistant populations.

Through a proteomic analysis, they determined that Prx II was constitutively upregulated in the radiation-resistant cells (Wang, Tamae et al. 2005). Blocking the expression of Prx II returned radiation-sensitivity to the cells. In cancer

28 treatment, therefore, elevated peroxiredoxin expression may block the intended effect of radiation treatment.

Perhaps the most intriguing feature of peroxiredoxin is its frequent identification in large-scale proteomic or gene microarray analyses. For example, a cross-section of Entrez PubMed searches indicate that approximately

14% of all publications that invoke peroxiredoxin also include a proteomic or gene chip microarray analysis (search abstracts for peroxiredoxin vs. peroxiredoxin AND (proteome OR proteomic OR gene chip)). This is in sharp contrast to catalase (0.15%) and glutathione peroxidase (0.10%). What is the significance of this observation? As is the case with most proteomic or transcriptomic analyses, the majority of these publications do not characterize peroxiredoxin any further than to report its differential expression. Thus, peroxiredoxin has generated great interest as an inducible protein without producing much mechanistic insight into its physiological role. However, due to the typical “healthy vs. disease” model of many proteomic investigations, the frequently observed upregulation of peroxiredoxin may be indicative of a major role in stress response and cell survival functions.

1.8 Regulation of peroxiredoxin activity

Inhibition of peroxiredoxin activity has been reported following phosphorylation at Thr-90, which occurs at a consensus sequence for cyclin- dependent kinases (CDKs), Thr-Pro-Lys-Lys (Chang, Jeong et al. 2002).

Although multiple CDKs phosphorylate Prx I in vitro, it was determined that Cdc2

is the likely regulatory kinase. Phosphorylation of Prx I occurs exclusively during

mitosis, possibly because active Cdc2 can localize with Prx I only during the

breakdown of the nuclear envelope. The phosphorylation and inhibition of Prx I

activity may be required to prevent thiol-reducing activity during mitosis, but little

has been done to characterize this hypothesis.

A second and well-characterized inhibitory mechanism involves the oxidative-

inactivation of the typical 2-Cys peroxiredoxins, Prx I-IV. The reducing function

of the 2-Cys peroxiredoxins is a two-step process that includes a sulfenic acid

intermediate state (Cys-SOH) en route to an intermolecular disulfide across its

homodimer structure. At elevated oxidant levels, Prx I - IV can be inactivated

through the over-oxidation of the active site cysteine to sulfinic (Cys-SO2) or sulfonic acid (Cys-SO3) (Yang, Kang et al. 2002; Woo, Kang et al. 2003). The

Cys-SO2 modification is reversed by sulfiredoxin in an ATP-dependent reductive mechanism (Chang, Jeong et al. 2004; Woo, Jeong et al. 2005). The significance of this reversible inactivation as a means to regulate intracellular peroxide levels has generated interest in peroxiredoxin as a mediator of H2O2-

regulated signaling pathways (Kwon, Lee et al. 2004; Choi, Lee et al. 2005;

Vivancos, Castillo et al. 2005).

1.9 Summary and Aims

A critical step in atheroma development is the oxidative modification of LDL

retained in the arterial intima. Macrophages internalize oxLDL and aggregate as

lipid-laden foam cells at fatty streak formations, which represent potential sites

for advanced lesion development. Several reports have described changes in

protein expression in cells exposed to oxLDL, but the contribution of this

differential expression to atheroma development is not well characterized.

The initial stage of this project was conducted according to the hypothesis

that the transition from macrophage to foam cell is characterized by a set of

differentially-expressed genes and proteins that, as a whole, are pro-atherogenic.

To aid in the investigation of this hypothesis, a model system of chronic oxLDL-

exposure was developed to compare changes in expression that occur in acute

and chronic exposure. The following aims were addressed: 1) Using proteomic

and gene microarray analyses, identify differentially-expressed proteins and

mRNA in macrophage foam cells. 2) Compare the model of chronic oxLDL-

exposure to acute exposure, identifying differential expression that is unique to a

pro-atherogenic phenotype. 3) In accordance with the discovery-driven nature of

this analysis, identify a key protein to serve as a focus for further analysis in the

subsequent stage of this project.

The analysis of differential expression led to a focus on peroxiredoxin, a

peroxidase with proposed roles in antioxidant defense, cell cycle regulation and

signal pathway modulation. The upregulation of Prx I has been described in several oxidant-stressed systems, but data describing its induction and role in macrophage foam cells was incomplete. Working under the hypothesis that the upregulation of Prx I could protect against toxicity induced by oxLDL-generated reactive oxygen species, the following aims were addressed: 1) Characterize the induction of Prx I by oxLDL, specifically identifying the upregulation of active Prx

I. 2) Analyze oxLDL-induced toxicity and ROS levels in macrophages using chemical induction and siRNA knock-down of Prx I expression. 3) Investigate a potential role for Prx I in signal pathway regulation.

The sum of these experiments created new knowledge by providing a novel picture of foam cell formation through the broad-scale analysis of differential expression. Additionally, the discovery-driven aspect of this research led to the analysis of Prx I and its role in foam cell formation, which was demonstrated to contribute both to antioxidant defense and signal-pathway regulation.

1.10 Figures

Figure 1: Current model of foam cell and fatty streak formation. Two critical

steps of early atherosclerosis are 1) the oxidative modification of low-density

lipoprotein (LDL) and 2) the internalization of oxidatively-modified LDL by intimal

macrophages. Lipid-laden macrophages, also known as foam cells, aggregate at fatty streaks (3) that may potentially develop into advanced atheromas.

Chapter 2

Differential expression in a chronic-exposure model of foam cell formation

2.1 Introduction

Although many factors contribute to the progression of atherosclerosis, foam cells appear to have a uniquely pro-atherogenic phenotype that goes beyond the lipid deposition produced by the uptake of modified LDL. The complexity of the foam cell response to oxLDL-loading makes this system an excellent subject for a systems biology approach.

Systems biology is a method of analysis that attempts to describe the

association of all components in a particular biological system. The system of interest may be a large multi-protein complex, a subcellular organelle, a defined

cell type, tissue or organ, or an entire organism. The motivation behind this

analytical method maintains that the whole is greater than the sum of its parts,

and a focus on the molecular level may overlook critical higher order interactions.

Systems biology is a discovery-driven approach, in contrast to hypothesis-

driven, because data that is obtained is not always a predicted or expected

outcome. A common example is the immunoprecipitation and identification of components of a multi-protein complex. Although some of the identified proteins may be previously identified or currently hypothesized, others may be neither known or suspected. This type of methodology is often criticized as “fishing for

data”, but it carries the positive aspect of discovering new leads for further,

focused analysis. This opportunity has motivated the increasing popularity of

proteomic investigations, which have gained an increased presence in the

literature every year over the past ten years. The research presented here

represents an ideal case study for the discovery-driven methodology. The

systems biology approach to macrophage foam cell formation presented in this

chapter created new leads that were carried through to focused, hypothesis-

driven investigations in Chapters 3 & 4.

Previous systems biology analyses of foam cell formation applied a single

oxLDL exposure to initiate differential expression. However, since

atherosclerosis is a disease primarily seen later in life, it is important to model the

long-term oxLDL exposure component of atheroma development. The reversible

nature of macrophage foam cell formation presents the possibility that

macrophages in the lesion area may undergo a chronic exposure to oxLDL

before transitioning to a fatty streak foam cell.

In the present study, differentially regulated genes and proteins were

identified in foam cells derived from J774 murine macrophages treated with

oxLDL. A chronic exposure model was developed that used repeated exposure

of the J774 cells to oxLDL over a period of several weeks to generate a derivative line referred to as J774-CE cells. This chronic exposure condition has

been characterized to determine differential mRNA and protein expression

relative to both untreated macrophages and foam cells formed by a single

oxLDL-exposure.

2.2 Experimental Procedure

Lipoprotein Preparation

Fresh human plasma was obtained from the Blood Bank of the Cleveland

Clinic, and LDL (1.019 < d < 1.063 g/ml) was isolated by differential

ultracentrifugation (Stanova, Siman et al. 1992). LDL in NaBr solution containing

0.02% EDTA was dialyzed against 0.9% NaCl, 0.02% NaN3, 0.02% EDTA (pH

7.4) and stored in the dark at 4°C (Paromov and Morton 2003). Oxidation of LDL

was achieved by dialyzing 500 μg/mL LDL in TBS (20 mM Tris pH 7.8, 150 mM

NaCl) for 24 hours in Spectra/Por dialysis tubing (MWCO 6-8kDa) (Spectrum

Laboratories, Rancho Domingues CA), followed by dialysis in TBS and 5 μM

CuSO4 for 24 hours. Oxidation was stopped by a final dialysis in TBS and 0.3 mM EDTA for 24 hours. The extent of oxidation was monitored by using the

TNBS (trinitrobenzene sulfonic acid) assay to estimate the number of free

amines. Previous investigators have shown that the apoB-100 protein of

oxidatively-modified LDL contains fewer non-oxidized histidines and lysines when compared to unmodified LDL (Sevanian, Bittolo-Bon et al. 1997). In our system, a typical Cu2+-oxidation procedure resulted in 40% modified amines, compared to

2+ 3% modified amines when LDL was dialyzed in TBS without 5 μM CuSO4. Cu -

oxidation is an established method for generating a form of modified LDL that

binds macrophage scavenger receptors and generates foam cells. Other

methods of LDL oxidation exist, such as cell-mediated enzymatic modification by

myeloperoxidase or lipoxygenase, however there is no established physiological

mechanism that explains LDL oxidation in vivo. Therefore, the Cu2+-oxidation

method is used for its established role in macrophage foam cell formation in vitro.

Cell Culture Conditions and Treatment

J774A.1 murine macrophages were obtained from American Type Culture

Collection (ATCC TIB 67) and maintained in culture media (DMEM containing

10% fetal bovine serum, 100 IU/mL penicillin and 100 μg/mL streptomycin) at

37°C in 5% CO2. Two J774 sub-lines were developed through chronic exposure to oxLDL. J774-CE macrophages were treated with 50 μg/mL oxLDL for 24 hours before each passage for a period of approximately 3 months. J774-CE(-) macrophages were derived from the J774-CE sub-line by stopping the chronic oxLDL-treatment. All cells were passed twice each week in 75 cm2 tissue

culture-treated flasks. J774-CE macrophages reserved for an experiment were

grown in a separate flask and kept free of oxLDL for 4 - 5 days prior to ensure

that the cells were no longer lipid-loaded. Foam cells were generated by adding

50 μg/mL oxLDL to culture media of J774 macrophages followed by a 48 hour

incubation.

Cytotoxicity Assay

Approximately 104 cells were seeded into 24-well tissue culture plates and

incubated overnight. Cells were treated with increasing concentrations of oxLDL

(0 to 200 μg/mL) for 48 hours. Cell survival was assayed with the CellTiter 96

Aqueous One Solution Cell Proliferation Assay (Promega, Madison WI), a

colorimetric assay which produces a soluble formazan product that is directly

proportional to the number of metabolically active cells. 200 μL of media per

sample were transferred to a 96 well plate and the absorbance at 490 nm was read in a microplate reader. Background absorbance of no-cell wells was

subtracted, and the resulting values were normalized to corresponding values for

untreated cell and expressed in % cell survival.

Lipoprotein Labeling and oxLDL–Internalization Assay

Native LDL and oxidized LDL (oxLDL) were labeled with the lipophilic tracer

1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI; Molecular

Probes, Eugene OR). DiI was maintained in the dark at room temperature as a

10 mg/mL stock in DMSO. Lipoprotein labeling was performed by adding 20 μL

of DiI stock per 1 mL lipoprotein (500 μg/mL) or TBS-vehicle (150 mM NaCl, 20

mM Tris-HCl, pH 7.8). The DiI mixtures were vortexed briefly, incubated in the

dark at 37°C overnight and centrifuged at 10,000xg for 5 minutes to pellet

unbound DiI. The supernatants were sterile-filtered with a Millex-GP filter unit

(0.2 μm pore PES; Millipore, Billerica, MA) and kept in the dark until needed.

Extent of foam cell formation was assayed by measuring DiI-labeled oxLDL

internalization. DiI-labeled LDL and free DiI-TBS vehicle were used as negative

controls. Samples were incubated with 50 μg/mL DiI-oxLDL, -LDL or vehicle for

fluorescent microscopy, or 0 – 100 μg/mL DiI-oxLDL for dose-response

experiments. Following 48 hours of incubation at 37°C in 5% CO2, DiI internalization was assayed by fluorescent microscopy or in a fluorescent plate

reader (excitation λ = 540 nm, emission λ = 565 nm, cut-off λ = 550 nm).

Background fluorescence was measured in DiI:oxLDL-incubated (0 – 100 μg/mL)

no-cell wells, and subtracted from corresponding raw data values. Results were

normalized by total number of cells, assayed by DAPI-nuclei staining (excitation

λ = 358 nm, emission λ = 461 nm, cut-off λ = 420 nm) or the CellTiter 96 cell

survival assay described previously.

2D Gel Electrophoresis

Approximately 106 cells were plated in 100 mm tissue-culture treated plates

and incubated overnight. The following day, cells were treated with 50 μg/mL oxLDL or an equivalent volume of vehicle (20 mM Tris-buffered saline, pH 7.8).

Cells were incubated for 48 hours, then harvested by scraping in DMEM and washed once in sterile PBS. Cell pellets were suspended in lysis buffer (25 mM

Tris, pH 7.5; 2.5 mM MgCl2; 0.5% SDS) and boiled for 5 minutes with occasional

vortexing. Samples were cooled to room temperature and treated with 100

μg/mL DNase I and 100 μg/mL RNase A for 15 minutes. Protein concentration

was determined using a SDS-compatible Lowry-based method (DC Protein

Assay Kit; Bio-Rad Laboratories, Hercules, CA), and sample volumes were adjusted with lysis buffer to equal concentrations. For 2D gel analysis, 1 mg total protein (typically 200 – 400 μL total cell lysate) was precipitated in acetone (80% v/v) overnight. Protein precipitate was resuspended in a rehydration buffer consisting of 7 M urea, 2 M thiourea, 1% dithiothreitol, 1% CHAPS (3-[(2- cholamidopropyl) dimethylamonio]-1-propanesulfonic acid), 1% Triton X-100 and

1% ampholytes (Bio-Rad Bio-Lyte 3/10). A Bio-Rad Protean IEF Cell was used

to rehydrate 11cm IPG strips with 200 μL samples (1 mg total protein) at 50V for

16 hours. An isoelectric focusing program (8000 V max) was applied that accumulated 35,000 V-hours over a 5 hour period, separating proteins in one dimension by net charge. The focused IPG strips were equilibrated in SDS under reducing and alkylating conditions with 6 M urea, 2% SDS, 20% glycerol containing first 100 mM dithiothreitol (reduction), then 100 mM iodoacetamide

(alkylation) for 10 minutes. Excess amounts of each reagent (2 mL per strip) were used, and the DTT solution was removed prior to addition of the iodoacetamide. The 2nd dimension of separation was achieved by subjecting the

focused IPG strip to SDS-PAGE in a 12.5% Tris-HCL gel. Gels were fixed in

50% ethanol/10% acetic acid, washed in distilled water 3 x 15 minutes and

stained overnight in Bio-Rad Gel Code Blue. Stained gels were washed in

distilled water and stored in heat-sealable bags (Kapak; Minneapolis, MN) with

2mL 5% acetic acid/10% glycerol.

Analysis of Differential Protein Expression

Differentially-expressed protein bands were determined by comparing 2D

SDS-PAGE gels representing oxLDL-treated vs. untreated cell cultures. The 4

samples of interest (untreated and oxLDL-treated J774, untreated and oxLDL-

treated J774-CE) were each represented by 3 replicate gels originating from independent cell culture experiments. Each gel was scanned with a Bio-Rad GS-

700 Densitometer and imported into Bio-Rad PDQuest 2D Analysis software.

Replicate gels were combined into groups, normalized to the total density of detected bands, and protein bands were matched across the set of 12 gels.

Resulting band intensities were an average across each replicate group, and

standard deviations were calculated based on the band intensities of the

individual gels. Over- and underexpressed protein bands were determined using

a Student’s t-test within PDQuest with significance level set to 95% (p<0.05).

The resulting band sets were visually inspected to verify band quality and the

integrity of the statistical significance.

Protein Identification by Tandem Mass Spectrometry

Protein bands from 2D SDS-PAGE gels were excised, destained in 50%

ethanol/5% acetic acid and dehydrated in acetonitrile. Gel pieces were dried in a

SpeedVac, rehydrated with 5μL trypsin solution (20 μg/mL in 50 mM ammonium bicarbonate), and incubated overnight at room temperature. Peptides were extracted with 30 μL 50% acetonitrile/10% formic acid, and these extracts were concentrated in a SpeedVac for 30 minutes to approximately 5 μL. Sample volume was adjusted to 30 μL with 1% acetic acid.

Protein digests were analyzed with a Finnigan LTQ linear ion trap mass

spectrometer system incorporating a 6.5 cm x 75 μm ID Phenomenex Jupiter

C18 reversed-phase capillary chromatography column (Phenomenex, Torrance

CA). Peptide samples were auto-injected and eluted from the column by an acetonitrile/0.05 M acetic acid gradient at a flow rate of 1 μL/min. Digests were analyzed in a data-dependent mode during which the instrument acquired a full

mass scan spectrum to determine peptide molecular weights, followed by

product ion spectra to determine amino acid sequence in successive scans.

The CID spectra produced by each tryptic digest were searched against the

National Center for Biotechnology Information (NCBI) non-redundant database

using Mascot software (Matrix Science, Boston, MA). Peptide tolerance was set

to ±2 Da allowing 1 missed cleavage, and peptide charges of +2 and +3 were

fragmented. MS/MS tolerance was set to ±1 Da. All identified proteins gave

Mascot total scores greater than 200, which was calculated from the expression

–10*Log(P) where P is the probability that the match is a random event (P<10-20).

Individual peptide scores of 40 or greater represented extensive homology, and at least 2 of these CID spectra from each analysis were visually inspected to verify the peptide sequence. Typical sequence coverage was at least 20%.

Gene Chip Analysis

Total RNA was isolated from cell samples according to manufacturer’s instructions using the Qiagen RNAeasy kit (Qiagen Inc., Valencia, CA). RNA samples were submitted to the Gene Expression Array Core Facility at Case

Western Reserve University for cRNA preparation and gene chip analysis.

Instrumentation included the Affymetrix GeneChip Instrument System with a

GeneChip Scanner 3000 and autoloader. Message RNA levels were analyzed using the GeneChip® Mouse Expression Set 430 chip set, which consists of

45,037 probe sets representing 34,323 genes. Each probe set consists of 11 unique probe pairs that are 25 nucleotides in length. Each probe pair consists of

a perfect match (PM) oligonucleotide and a single base mismatch (MM)

oligonucleotide. Thus, a probe set representing a specific gene hybridizes

mRNA to 11 different sequence segments, with a negative control for each

sequence to determine non-specific hybridization.

Statistical significance of each mRNA identification was confirmed as follows.

A Discrimination score (R) was calculated as the ratio of target-specific

hybridization to overall hybridization, where R = 1 is a perfect match with no non-

specific binding: R = (PM – MM) / (PM + MM)

Detection p-values were calculated using the One-Sided Wilcoxon’s Signed

Rank test, which ranks the probe pairs based on the separation of R from a

threshold value Tau (Tau = 0.015 for this analysis). mRNA transcripts were

denoted as “present” if the result of this statistical test was p < 0.04. For the

purposes of our analysis, mRNA transcripts were considered only if the transcript was present in the untreated sample of an underexpressed pair or in the oxLDL- treated sample of an overexpressed pair. Signal intensity also incorporated the values from each probe pair in a probe set, and was calculated using the One-

Step Tukey’s Biweight Estimate.

The statistical significance of the differential expression for each transcript

was determined by the Wilcoxon Signed Rank test. Each probe set in the

differentially-expressed sample was compared to its counterpart, and a p-value

was calculated to determine the degree of change. A value of p<0.002 denoted

an increase in transcript, and a value of p>0.998 denoted a decrease in

transcript. All of the differentially-expressed transcripts reported here were

determined to be increased or decreased in the oxLDL-treated sample of at least

one sample pair (J774 untreated/oxLDL-treated or J774-CE untreated/oxLDL-

treated) according to this statistical test.

Statistical analysis of the gene chip data was carried out in Affymetrix

GeneChip® Operating Software (GCOS). Further data analysis was preformed

in Microsoft Access and Excel, and functional categorizations were assigned

using Lucidyx Searcher (Lucidyx LLC, Cleveland OH).

2.3 Results

Development of J774-CE Macrophages

The effect of chronic oxLDL-exposure on J774 macrophages was studied by

exposing cells to 50 μg/mL oxLDL for 24 hours twice per week for 3 months - the

equivalent of 25 oxLDL-exposures. This concentration of oxLDL is consistent

with the amount of oxLDL used by other laboratories to generate foam cells in

culture. The twice-weekly exposure allowed significant uptake of lipoprotein by the cells with minimal toxicity. Each week, the cells were harvested by scraping and passaged for continued exposure. This chronic oxLDL exposure sub-line to the J774 parent line was designated as J774-CE. No differences were observed in the morphology, growth rate, or cell cycle progression of the J774-CE cells relative to the parent J774 cells. The intent of these experiments was to investigate the effects of chronic oxLDL-exposure on the two hallmarks of the oxidative hypothesis of atherosclerosis - oxLDL internalization and cytotoxicity.

Lipid Uptake in J774 and J774-CE Macrophages

The extent of foam cell formation was determined by monitoring the uptake of

lipid by the cells using two different methods - oil red O-staining of fixed cells or

DiI-labeling of the LDL and oxLDL. Results of the oil red O experiments are

shown in Figure 2 and results of the DiI-labeling are shown in Figure 3.

In the oil red O experiments (Figure 2), foam cell formation is indicated by

red-stained lipid droplets within the cell cytoplasm. Figure 2c shows the extent of

foam cell formation following incubation of cultured macrophages with 50 μg/mL

oxLDL for 48 hours. In contrast, untreated (Figure 2a) and LDL-treated (Figure

2b) cells exhibit minimal staining. The small number of lipid-droplets in LDL- treated macrophages are likely a product of cell-mediated oxidation of LDL over

the course of the incubation period. Oil red O, however, is a general neutral lipid

stain that can bind to other lipids such as triglycerides. This lack of specificity is

particularly evident, for example, when cultured primary human monocyte-

macrophages internalize significant quantities of triglyceride during incubation in

human serum-containing media and produce extensive oil red O-staining

(Garner, Baoutina et al. 1997).

Lipoprotein uptake was confirmed by monitoring the uptake of LDL and

oxLDL labeled with the fluorescent lipophilic tracer DiI (1,1’-dioctadecyl-3,3,3’,3’-

tetramethylindocarbocyanine perchlorate). Macrophages incubated with DiI-

labeled oxLDL are positive for DiI internalization (Figure 3c), in contrast to

macrophages treated with vehicle (Figure 3a) and DiI-labeled LDL (Figure 3b).

The cell nuclei were also counter-stained with the fluorescent nucleic acid stain

DAPI (4',6-diamidino-2-phenylindole, dihydrochloride). A quantitative assessment of DiI internalization was made by determining the ratio of DiI fluorescence at 565 nm relative to DAPI fluorescence at 460 nm (Figure 3d).

This experiment demonstrated negligible DiI uptake in vehicle-treated cells, while

DiI-oxLDL treated cells internalized 5-fold more lipid than DiI-LDL treated cells.

Figure 4 shows a dose-response analysis of DiI-labeled oxLDL internalization over a range of 0 to 100 μg/mL oxLDL in J774 and J774-CE macrophages. No statistically significant differences were present in oxLDL internalization when comparing the two cell lines. The general pattern of oxLDL-internalization was consistent with other experiments where internalization begins to plateau at approximately 50 μg/mL oxLDL, a typical dose used to generate foam cells in culture.

OxLDL-induced Cytotoxicity in J774 and J774-CE Macrophages

A second component of the oxidative modification hypothesis of

atherosclerosis is the cytotoxicity of oxLDL. Therefore, oxLDL-induced toxicity

was investigated in both the J774 and J774-CE macrophages. Figure 5 shows

the results of a typical cytotoxicity assay in which J774 and J774-CE

macrophages were treated with increasing concentrations of oxLDL (ranging

from 0 to 200 μg/mL) for 48 hours. Previous investigators have determined that

low doses of oxLDL induce a proliferative response in macrophages (Bjorkerud

and Bjorkerud 1996; Hamilton, Myers et al. 1999). That observation is verified by

these experiments, in which 10 and 25 μg/mL oxLDL induced proliferation in both

the J774 and J774-CE cell lines. At toxic concentrations of oxLDL, the J774

macrophages required 98 μg/mL oxLDL to reduce the cell survival to 50%,

whereas the J774-CE macrophages required 140 μg/mL to reach 50% cell

survival – a 43% increase in oxLDL dose required to produce 50% cytotoxicity.

Therefore, the chronic exposure of the J774 macrophages produces a significant

resistance to oxLDL-mediated cytotoxicity.

Additional experiments were carried out to determine the stability of this cytotoxicity-resistant phenotype. The repetitive oxLDL treatment was stopped for a population of the J774-CE cells for a period of 2 months and both cytotoxicity and lipid uptake was assayed. These cells were designated J774-CE(-). As seen in Figure 5, no difference was observed in the dose-response course of the oxLDL-mediated cytotoxicity, indicating that the J774-CE(-) cells retained the resistant phenotype of the J774-CE cells. Similarly, the pattern of DiI-labeled oxLDL uptake was also unchanged in the J774-CE(-) cells (data not shown).

Therefore, these results indicate that chronic exposure of macrophages to oxLDL does not affect lipid-uptake characteristics of foam cell formation but does produce a significant degree of resistance to oxLDL-induced cytotoxicity that is stable for at least 2 months after the oxLDL challenges are stopped. An investigation into the differential proteome and transcriptome of the J774-CE cells, in comparison to parental J774 cells, was used to begin the investigation of the proteins and genes that may contribute to this resistant phenotype.

Proteomic Analysis

The differential proteomes of the J774 and J774-CE cell lines were analyzed

to determine the effects of foam cell formation on protein expression. A series of

2D SDS-PAGE gels representing total cell lysates were run in replicate

experiments covering the pI range of 5 to 8. The images were processed to

produce a master image of protein bands (Figure 6a) and the relative amounts of

the detected proteins were determined and compared. Examples of oxLDL-

induced overexpression are shown for aldose reductase-related protein 2 (Figure

6b) and superoxide dismutase (Cu, Zn) (Figure 6c). Statistically significant

(p<0.05) over- and underexpressed bands were determined in oxLDL-treated

J774 or J774-CE macrophages relative to respective untreated controls. These

results, presented in Table 1, are denoted as the fold change in protein quantity

for 4 different comparisons designed to detect differences in protein expression

in response to oxLDL treatment. Protein expression data in Table 1 was

determined by densitometric analysis of 2D gel band intensities, with the

exception of peroxiredoxin I (band #32), which is represented by the normalized

intensity of Western blot bands using a peroxiredoxin I polyclonal antibody. The

basic position of peroxiredoxin I (pI 8.26) on a 2D gel separated over pI 5-8

results in bands that are unsuitable for densitometric analysis. A detailed

analysis of peroxiredoxin I expression and function is reported in Chapters 3 and

The primary comparison in Table 1 is the effect of oxLDL treatment on protein expression in either the J774 or J774-CE cells (columns A and B, respectively).

A total of 46 bands were overexpressed with statistical significance following

oxLDL treatment. These bands were identified by tandem mass spectrometry and found to represent 29 unique proteins. Similarly, a total of 7 bands, representing 6 unique proteins, were underexpressed with statistical significance following oxLDL treatment.

Subsequent evaluations of the protein expression data determined

differences in protein expression in J774-CE vs. J774 cells before the oxLDL

treatment (column C), in a non-stimulated state. The difference in the magnitude

of oxLDL-induced protein expression in J774-CE vs. J774 cells (column D) is

also included. Table 1 includes the gene identification number, an NCBI reference sequence (RefSeq) number, and a summary of the cellular process of the respective identified proteins.

Of particular interest are the differentially regulated proteins in J774-CE

macrophages that have significantly altered oxLDL-induced responses relative to

the response seen in the oxLDL-treated J774 cells. For example, approximately

half (25 of 52) of the differentially expressed proteins shown in Table 1 were

differentially expressed (p<0.05) in the J774-CE cells, relative to the J774 cells,

prior to any treatment. Following oxLDL treatment, 19 of these proteins had

statistically different responses in the J774-CE cells compared to the J774

parental cells. The expression patterns for this subset of proteins are shown in

Figure 7. The differences in regulation between the J774 and J774-CE cells

provide direct evidence of novel effects of chronic oxLDL treatment that are not

seen in single exposure experiments. These differences in protein expression

demonstrate the unique phenotype developed in the J774-CE cell by the chronic

oxLDL exposure regimen.

Gene Chip Analysis of J774 and J774-CE Macrophages and Foam Cells

Differential mRNA expression was analyzed in an Affymetrix GeneChip

experiment using GeneChip® mouse expression set 430 microarrays, which

contain over 45,000 unique mRNA transcripts. J774 macrophages and the J774-

CE sub-line were analyzed following oxLDL-induced foam cell formation for 48

hours and compared to untreated controls. A representative set of differentially-

expressed transcripts was assembled from mRNA that was ±4-fold differentially

expressed in oxLDL-treated J774 or J774-CE macrophages. Under this criteria,

a total of 41 transcripts were overexpressed (Table 2) and 52 transcripts were

underexpressed (Table 3). The identified transcripts were grouped according to functional class, defined by GeneOntology entries in the NCBI database as

provided by Mouse Genome Informatics. Genes that were not assigned a

function under GeneOntology were grouped as undefined. GeneOntology

process classifications are listed for each gene when assigned, or are otherwise

classified as undefined.

Further focus was placed on regulation of genes involved in antioxidant

defense by adjusting the selection criteria to transcripts that had greater than ±2-

fold differential expression in oxLDL-treated J774 or J774-CE macrophages.

Table 4 lists the 13 overexpressed genes related to antioxidant responses. It is

interesting to note that no antioxidant-related genes were underexpressed by

more than 2-fold. The genes listed in Table 4 lead directly to antioxidant production, or contribute an essential component to antioxidant activity.

Glutamate-cysteine ligase (GCL) is an example of indirect antioxidant activity,

since it catalyzes the rate-limiting step in the synthesis of the antioxidant glutathione. Overexpression of the GCL modifier subunit, which comprises one

half of the GCL heterodimer, potentially contributes to an increase in antioxidant

response. The alpha 3 isozyme of glutathione-S-transferase (GST) is

overexpressed approximately 10- and 20-fold in oxLDL-treated J774 and J774-

CE macrophages, respectively. Excluding alpha 3 GST, the remaining

antioxidant defense genes are upregulated approximately 2-fold in oxLDL-treated

J774 and J774-CE macrophages.

An additional analysis of the transcriptome data was carried out to focus on

genes involved in immune response (Table 5). For purposes of this

classification, “pro-inflammatory” genes were defined as being involved in

leukocyte recruitment or cell proliferation at a site of inflammation. This definition

includes cytokines that stimulate leukocyte production and promote leukocyte

attachment, cytokines that stimulate smooth muscle cell proliferation or

extracellular matrix production, and chemokines that promote leukocyte

chemotaxis. Four transcripts were assigned to the inflammatory response

process (TNF-α and the macrophage inflammatory protein (MIP) 1α, MIP-1α

receptor and MIP-2) by GeneOntology designations. The overexpressed TNF

receptor superfamily transcript was included in this subset of pro-inflammatory

genes for the purposes of this analysis. Additionally, the overexpressed

52 transcripts interleukin-6 (IL-6) (Choi, Kang et al. 1994; Szekanecz, Shah et al.

1994; Kaplanski, Marin et al. 2003), oncostatin M (Langdon, Kerr et al. 2000; de

Hooge, van de Loo et al. 2002; Langdon, Kerr et al. 2003) and prostaglandin- endoperoxide synthase 2 (Meade, Smith et al. 1993; Smith and DeWitt 1995;

Wang, Cao et al. 1996) were designated as pro-inflammatory in this analysis considering their links to the inflammatory response. This set of 8 pro- inflammatory transcripts were overexpressed an average of 2.5-fold and 2.4-fold in oxLDL-treated J774 and J774-CE macrophages, respectively, compared to untreated macrophages. However, these transcripts were 2.5-fold underexpressed in the untreated J774-CE sub-line compared to untreated J774 parent macrophages (steady-state comparison). Similarly, pro-inflammatory transcripts were 2.9-fold underexpressed in oxLDL-treated J774-CE sub-line macrophages compared to oxLDL-treated J774 parent macrophages (stimulated- state comparison). Therefore, although oxLDL-treatment upregulates inflammatory response in both J774 and J774-CE macrophages, expression of pro-inflammatory transcripts were decreased as a whole in the J774-CE sub-line.

2.4 Summary

A model system of macrophage foam cell formation was developed for the in vitro analysis of oxLDL-induced toxicity, oxLDL-uptake, and differential mRNA and protein expression following acute- and chronic-exposure to oxLDL (J774 and J774-CE, respectively). Though the extent of oxLDL-uptake did not change, a resistance to oxLDL-induced cytotoxicity was observed in the J774-CE

53 macrophages. These cells required a 40% higher concentration of oxLDL to achieve 50% survival in a 48-hour treatment, relative to macrophages subjected to a single oxLDL exposure.

A main feature of the differentially expressed proteome was a series of significantly upregulated antioxidant and antioxidant-related proteins in the oxLDL-exposed cells. A large proportion of these proteins (45%) were upregulated in the chronically-exposed cells prior to the oxLDL treatment, indicative of the unique phenotype produced by the chronic oxLDL-exposure.

Analysis of the transcriptome also revealed a broad increase in the expression of antioxidant and antioxidant-related proteins. In addition, the transcriptome experiments described an increased inflammatory response under conditions of both acute and chronic oxLDL-exposure. Overall, the combined functional, proteomic, and transcriptomic experiments show that macrophages respond to oxLDL by developing an oxidative stress-resistance that increases and stabilizes with chronic exposure. Further, this protective response and the increased foam cell survival that it supports amplifies their pro-atherogenic role by promoting a continued inflammatory state.

2.5 Figures

Figure 2: Foam cell formation assayed by oil red O staining. J774 macrophages exposed to 50 μg/mL LDL or oxLDL for 48 hours, or left untreated, were fixed and stained with oil red O. Few lipid droplets are stained in untreated

(A) and LDL-treated (B) macrophages, indicating an absence of significant lipid uptake. Foam cell formation in the oxLDL-exposed cells (C) is indicated by the punctate oil red O staining of lipid droplets.

Figure 3: Foam cell formation assayed using DiI-labeled lipoprotein. LDL and oxLDL (50 μg/mL) were labeled with DiI and incubated with J774 murine macrophages for 48 hours. DiI-labeled lipoprotein (red) and DAPI-stained nuclei

(blue) were analyzed separately, and images were additively combined. Vehicle- treated cells (A) showed negligible DiI fluorescence. Lipid uptake was present in

DiI:LDL-treated cells (B), but the uptake was minimal and heterogeneous.

DiI:oxLDL-treated cells (C) internalized significant quantities of lipid in a homogenous manner. Uptake was measured with a fluorescent plate reader and expressed as the ratio of DiI to DAPI emission (D). Data are expressed as mean

± one standard deviation from an average of four separate experiments. * p <

.001 vs. untreated cells. + p < .001 vs. LDL-treated cells.

Figure 4: Lipid-uptake of oxLDL-treated J774 and J774-CE macrophages.

J774 (♦) and J774-CE (□) macrophages were incubated with 0 – 100 μg/mL DiI- labeled oxLDL for 48 hours and assayed for DiI uptake. Results are expressed as the ratio of DiI uptake to normalized cell number as determined by an assay of cell survival. Data are expressed as mean ± one standard deviation from an average of four separate experiments. No statistically significant differences were found at any concentration.

Figure 5: OxLDL-induced cytotoxicity in J774, J774-CE and J774-CE(-)

macrophages. J774 (♦), J774-CE (□) and J774-CE(-) (▲) macrophages were

incubated with 0 – 200 μg/mL oxLDL for 48 hours and assayed for cell survival.

Results of each data set are normalized to untreated controls. Data are expressed as mean ± one standard deviation from an average of four separate experiments. * p < 0.05, J774-CE and J774-CE(-) vs. J774 macrophages.

Figure 6: 2D gel representation of the J774 macrophage proteomic

analysis. A 2D gel image generated by Bio-Rad PDQuest software represents the entire set of analyzed gels (4 unique samples, 3 gels per sample) (A).

Numbered bands (arrows) represent statistically significant (p < 0.05) differentially-regulated bands identified and tabulated in Table 1. Examples of oxLDL-induced overexpression are shown for aldose reductase-related protein 2

(B) and superoxide dismutase (Cu,Zn) (C).

Figure 7: Subset of proteins that are significantly expressed (p < 0.05) in oxLDL-treated J774-CE macrophages compared to oxLDL-treated J774 macrophages. Gel images were obtained with a scanning densitometer. Band intensities and statistical significance were determined using Bio-Rad PDQuest

software and are the product of triplicate gel sets. * p < 0.05, oxLDL-treated

J774-CE (4) vs. oxLDL-treated J774 (2). X p < 0.05, untreated J774-CE (3) vs.

untreated J774 (1). + p < 0.05, oxLDL-treated J774 (2) vs. untreated J774 (1).

# p < 0.05, oxLDL-treated J774-CE (4) vs. untreated J774-CE (3).

Table 1: Differentially expressed proteins in oxLDL-treated J774 or J774-CE

macrophages.

Table 2: Upregulated mRNA in oxLDL-treated J774 or J774-CE

macrophages.

Table 3: Downregulated mRNA in oxLDL-treated J774 or J774-CE

macrophages.

Table 4: Differential expression of antioxidant response genes.

Table 5: Differential expression of immune response mRNA transcripts.

Chapter 3

Antioxidant Role for Peroxiredoxin I in Macrophage Foam Cells

3.1 Introduction

The findings presented in this report focus on Prx I, a basic (pI 8.3) 22 kDa protein localized to the cytosol. Prx I is upregulated in a variety of cell types following exposure to oxidative stress. Upregulation of Prx I in response to H2O2

was first demonstrated in mouse peritoneal macrophages (Ishii, Yamada et al.

1993), and in cultured vascular smooth muscle cells following exposure to oxLDL

(Siow, Ishii et al. 1995). Further studies characterized the upregulation of Prx I

resulting from exposure to ionizing radiation (Chen, McBride et al. 2002; Lee,

Park et al. 2002), hyperoxia (Kim, Kang et al. 2001), and 4-hydroxy-2-nonenal

(Ishii, Itoh et al. 2004). Additionally, elevated levels of Prx I have been

discovered in several types of cancer, including lung (Chang, Jeon et al. 2001;

Kim, Chae et al. 2003), breast (Noh, Ahn et al. 2001) and pancreatic cancer

tissues (Shen, Person et al. 2004).

The induction of Prx I in macrophage foam cells may reduce reactive oxygen

species generated by oxLDL and decrease oxLDL-induced toxicity, provided that

Prx I is in its active form. The oxidant-induced deactivation of Prx I has been

demonstrated under specific experimental conditions, but has not been researched in macrophage foam cells. This is data describes the induction of active Prx I as a result of oxLDL-exposure, and demonstrates that an induction of

Prx I prior to oxLDL-exposure decreases cellular ROS and oxLDL-induced

toxicity.

3.2 Experimental Procedures

Materials

siRNA and siPORT amine transfection reagent were obtained from Ambion

Inc. (Austin, TX). Rabbit polyclonal antibody for Prx I was obtained from BioMol

Int. (Plymouth Meeting, PA). Rabbit polyclonal antibody for Prx I-SO3 and mouse

monoclonal antibody for GAPDH were obtained from Abcam (Cambridge, MA).

For detection of ROS, 5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein

diacetate acetyl ester (CM-H2DCFDA) was obtained from Molecular Probes

(Eugene, OR). Ethoxyquin and tert-butyl hydroperoxide were purchased from

Sigma-Aldrich (St. Louis, MO).

Lipoprotein Preparation

Fresh human plasma was obtained from the Cleveland Clinic Blood Bank,

and LDL was isolated by differential ultracentrifugation (1.019 < d < 1.063 g/ml)

(Stanova, Siman et al. 1992). This LDL preparation, in NaBr solution containing

0.02% EDTA, was dialyzed against 0.9% NaCl, 0.02% NaN3, 0.02% EDTA (pH

7.4) and stored in the dark at 4°C (Paromov and Morton 2003). Oxidatively-

modified LDL was prepared as previously described in Chapter 2 - Experimental

Procedures.

Cell Culture Conditions

J774A.1 murine macrophages were obtained from American Type Culture

Collection (ATCC TIB 67) and maintained in culture media (DMEM containing

10% fetal bovine serum, 100 IU/mL penicillin and 100 μg/mL streptomycin) at

37°C in 5% CO2. Cells were split every 2 – 3 days at ~80% confluence, and

discarded after 20 passages.

siRNA Inhibition of Prx I

Approximately 5x105 cells were plated in 60mm tissue-culture treated plates

and left to adhere overnight at 37°C in 5% CO2 in normal culture media. For

each 60mm plate, 13 μL of siPORT Amine Transfection Reagent (Ambion Inc.,

Austin, TX) was incubated with 200 μL Opti-Mem reduced-serum medium for 10

minutes. Annealed double-strand siRNA (20 μM stock) was added to a separate aliquot of 200 μL Opti-Mem to give the final desired siRNA concentration

(typically 30 nM). The transfection agent and the siRNA mixtures were combined and incubated for 10 minutes at room temperature. Media in the 60 mm culture

plates was changed to 4 mL DMEM + 10% FCS only (no antibiotics), and the

siRNA-transfection agent complexes were added drop-wise. Transfection

conditions were maintained for 16 – 24 h. The Prx1 siRNA sequence, obtained

from the online Ambion siRNA library (ID #68674), targeted exon 4 and was as

follows: sense - GGAUUAUGGAGUCUUAAAGtt, antisense -

CUUUAAGACUCCAUAAUCCtg. Scrambled and reverse siRNA sequences

were designed and used as negative controls. All siRNA were obtained in

lyophilized, annealed form, resuspended in DEPC ddH2O to a stock

concentration of 20 μM and stored at -30°C in 100 μL aliquots.

Cell Toxicity Assay

Approximately 2x104 cells were seeded into 24-well tissue culture treated

plates and incubated overnight at 37°C in 5% CO2. For oxLDL-induced toxicity,

cells were treated for up to 48 hours with 10 to 100 μg/mL oxLDL. For tert-butyl

hydroperoxide-induced toxicity, cells were treated with 10 to 200 μM tert-butyl

hydroperoxide in DMEM for 2 hours, followed by a 48 hour incubation in normal

culture media. Cell survival was assayed with the CellTiter 96 Aqueous One

Solution Cell Proliferation Assay (Promega, Madison WI) as previously described

in Chapter 2 - Experimental Procedures. Results were calculated from four

independent experiments, and experimental error is expressed as ± 1 standard

deviation.

Flow Cytometric Assay of Reactive Oxygen Species

Reactive oxygen species (ROS) were assayed using a Guava EasyCyte Flow

Cytometer and the ROS-detecting agent CM-H2DCFDA. Lyophilized CM-

H2DCFDA (50 μg) was suspended in DMSO to a stock concentration of 1 mM

and added to pre-warmed Hank’s Balanced Salt Solution without phenol red

(HBSS) to a working concentration of 10 μM. Cells were washed with HBSS, covered with the CM-H2DCFDA/HBSS solution (typically 1.5mL per well in a 6-

well plate), and incubated at 37°C/5% CO2 for 10 minutes. Cells not exposed to

CM-H2DCFDA were included to assay the autofluorescent background initiated by experimental conditions. Following the CM-H2DCFDA incubation, the media

was removed and cells were washed with HBSS, then harvested by scraping in 1

mL HBSS, centrifuged at 1000 rpm in an Eppendorf Microfuge, and washed once

with PBS. Cells were then fixed in 3.7% formalin for 15 minutes at room

temperature. The cells were pelleted by centrifugation, washed once with PBS,

and resuspended in 500 μL PBS for further analysis. For flow cytometric

analysis, 5000 events were counted within the gate parameters for each sample.

Acquisition parameters were as follows: forward gain = 2x, side scatter = 330 V, forward scatter threshold = 215, green channel= 552 V, red channel= 524 V, yellow channel = 533 V. Gate parameters were set to X1= 251, X2= 2154, Y1=

3857, Y2= 574. A 5000-count acquisition required approximately 20 μL sample and 30 seconds at a flow rate of 0.59 μL/sec. Background fluorescence measured in cells without CM-H2DCFDA was subtracted from values obtained for

the respective CM-H2DCFDA-treated samples. This background was most

significant when assaying oxLDL-treated macrophages, and ranged from 10 –

30% of the total measured fluorescence. The background for all other treatments

was less than 10%. Results are normalized with respect to ROS measured in

untreated control cells, and expressed as relative n-fold change in ROS. Final

results are calculated from an average of three independent experiments, and

experimental error is expressed as ± 1 standard deviation.

2D Western Blot Analysis of Oxidized Prx1

Approximately 106 cells were seeded onto 100 mm tissue-culture treated plates and incubated overnight. Cells were treated with oxLDL, tert-butyl

hydroperoxide or an equivalent volume of buffer (20 mM Tris-buffered saline, pH

7.8), incubated for a given time period, harvested by scraping in cold DMEM, and

washed once in sterile PBS. Cell pellets were prepared for 2D SDS-PAGE as

previously described in Chapter 2 - Experimental Procedures. The focused 2D gel was immediately incubated in Novex Tris-Glycine Transfer Buffer + 10% methanol for 10 minutes. The gel was transferred to PVDF in a Bio-Rad Criterion

Gel Transfer Chamber for 1 hour at 100V, blocked in 2% non-fat dry milk and incubated overnight at 5°C in Prx I rabbit polyclonal antibody (1:2000). Western blots were completed with a 1 hour incubation in HRP-conjugated donkey anti- rabbit secondary antibody, followed by enhanced ECL chemiluminescent detection. The membranes were stripped and re-probed with Prx I-SO2/-SO3

(oxPrx I) rabbit polyclonal antibody (1:1000). All samples were analyzed in parallel by 1D Western blot for GAPDH to confirm equal loading.

3.3 Results

OxLDL-treatment Increases Prx I Expression

Previously, our group used a proteomic approach to study changes in protein expression that occur as macrophages become loaded with oxidized LDL and become foam cells (Conway and Kinter 2005). The 2D SDS-PAGE gel shown in

Figure 8A represents a typical set of protein bands observed from the J774 murine macrophages used in these experiments. The gel covers the pI range of

3 to 10 and was obtained from a whole cell homogenate. More detailed views of two such 2D gels show a band with a significant increase in expression in the macrophages treated with 50 μg/mL oxLDL for 24 hours (Fig. 8C) compared to

untreated macrophages at the same time-point (Fig. 8B). This band was cut

from the gel, digested with trypsin, and identified by tandem mass spectrometry

as Prx I (NCBI nr accession number 547923). The oxLDL-induced upregulation

of Prx I seen in the 2D electrophoresis experiments was examined more closely

by Western blot analysis. Figure 9A shows that Prx I expression is not changed

in macrophages left untreated or treated with native LDL (50 μg/mL) for 24 or 48 hours. However, exposure of the macrophages to 50 μg/mL oxLDL results in a significant upregulation of Prx I protein, with continued increases from 24 to 48 hours. The oxLDL-induced upregulation was also dose-dependent, increasing with oxLDL concentration over a range from 10 to 50 μg/mL oxLDL (Fig. 9B).

Concentrations greater than 50 μg/mL could not be tested due to increasing toxicity of the treatment.

Additional experiments were carried out to verify that the increase in Prx I was

in a native (non-oxidized) form. Under certain oxidant-treatment conditions, the active site cysteine of Prx I can be over-oxidized to a sulfinic (SO2) or sulfonic

(SO3) acid, inactivating the reductase function of the enzyme (Yang, Kang et al.

2002). The oxidatively-inactivated forms of Prx I can be detected on a 2D SDS-

PAGE gel by an acidic band shift associated with sulfinic or sulfonic acid

formation at the affected cysteine residues, or by an antibody specific to oxidized

peroxiredoxin (Prx I-IV). Figure 10 shows a combination of 2DE and Western

blot analysis used to verify that the increased expression of Prx I results in the

functionally-active, non-oxidized form of the protein. For this experiment, the

J774 macrophages were exposed to tert-Butyl hydroperoxide (t-BOOH) (100μM)

or oxLDL (50 μg/mL) for 24 hours. t-BOOH was chosen as a positive control

based on its known ability to oxidize Prx I (Rabilloud, Heller et al. 2002).

The antibody used to detect oxidized, inactive Prx I is specific to both the

sulfonic (Cys-SO3) and sulfinic (Cys-SO2) modifications of Prx I. These forms are detected as separate bands that are differentiated by the acidity of these modifications. Accordingly, the left-most band in each panel of Figure 10

represents the Cys-SO3 modification and the center-band represents the Cys-

SO2 modification of Prx I. Both of the oxidized Prx I forms were detected by the

oxPrx I antibody (Figs. 10D, E, F), further supporting these identifications. The

active, unmodified Prx I has a calculated pI of 8.3, which corresponds to the

right-most band position.

After 24 hours of oxLDL-treatment, the active form of Prx I is upregulated by

oxLDL-treatment (Fig. 10C) with respect to the untreated Ctl macrophages (Fig.

10A). In both the t-BOOH and oxLDL treatments, the center bands representing

inactive, SO2-modified Prx I were increased, consistent with the ability of both

treatments to modify Prx I. These 2D data, combined with the data shown in

Figure 9, clearly demonstrate that the oxLDL-induced upregulation of Prx I

produces a significant increase in the active, unmodified form of the protein.

Induction and Inhibition of Prx I Protein Expression

In order to characterize the role of Prx I in foam cell survival, methods were

developed to induce and inhibit Prx I protein expression. Prx I-induction was

achieved by treatment with ethoxyquin, a small molecule capable of inducing the

expression of phase II detoxifying proteins (Buetler, Gallagher et al. 1995).

Figure 11 shows a series of Western blots used to determine changes in Prx I

expression that occur with different combinations of ethoxyquin and oxLDL

treatment. Ethoxyquin induces Prx I protein expression in a dose-dependent

manner (Fig. 11A). Figure 11B demonstrates the combined effect of ethoxyquin

pre-treatment for 24 hours, followed by oxLDL treatment for 24 hours. In

macrophages treated with 25 μg/mL oxLDL, ethoxyquin pre-treatment gave an

increase in Prx I expression compared to oxLDL-treatment alone. When treated

with 50 μg/mL oxLDL, however, the extent of Prx I upregulation is similar in

macrophages with and without ethoxyquin pre-treatment. Thus, ethoxyquin may enhance oxLDL-induced expression of Prx I under certain conditions, but this increase can be saturated at higher concentrations of oxLDL.

Conversely, Prx I expression was knocked-down by transfection with a Prx I- specific siRNA (Prx I-KD macrophages). This siRNA treatment gave an effective

reduction in Prx I expression at concentrations greater than 30 nM and did not

affect the other major cytosolic peroxiredoxin, Prx II (Fig. 12B). Negative

controls, including transfection reagent-only, scrambled and reverse sequence

siRNA did not affect the expression of Prx I or Prx II (Fig. 12B). Importantly, the

siRNA treatment also effectively inhibited the oxLDL-induced increase in Prx I

expression (Fig. 12C).

Effect of Prx I Induction and Inhibition on OxLDL-Induced Cytotoxicity

Prx I is classified as an antioxidant enzyme based on its ability to catalyze the

reduction of hydrogen peroxide and lipid hydroperoxides. Therefore, a likely

functional effect of Prx I induction would be the protection of macrophages from

the cytotoxicity of oxLDL. This cytoprotective potential was characterized with

the induction and inhibition of Prx I expression prior to oxidant exposure, using

both oxLDL and t-BOOH as probes for the protective effect. The t-BOOH

treatment was added as a model of lipid hydroperoxides, which are a component

of oxLDL (Schmitt, Negre-Salvayre et al. 1995; Lupo, Anfuso et al. 2001) and

substrates for Prx I (Konig, Lotte et al. 2003).

For these cytotoxicity experiments, four groups of macrophages were tested,

including a control group and three groups with altered Prx I expression; the

results are shown in Figure 13. The control macrophages (Ctl) were transfected with a reverse sequence siRNA that had no effect on Prx I expression. Prx I expression was knocked down after a 24 hour transfection of Prx I siRNA (Prx I-

KD) or increased with a 24 hour treatment with ethoxyquin (+Ethx). Finally, the

Prx I component of the ethoxyquin effect was tested with a combination of ethoxyquin treatment and Prx I siRNA transfection (Prx I-KD, +Ethx). The four groups of cells were then exposed to increasing concentrations of t-BOOH and cell survival was determined (Fig. 13A). Knock down of Prx I expression in the

Prx I-KD macrophages resulted in a significant decrease in cell survival following t-BOOH treatment compared to control macrophages, consistent with a sensitizing effect in the Prx I knockdown. In contrast, Prx I induction by the

ethoxyquin pre-treatment was protective and increased cell survival following t-

BOOH exposure. Finally, the protective effect produced by the ethoxyquin

treatment was partially reduced when combined with Prx I expression knockdown

(the Prx I-KD, +Ethx treatment). These data demonstrate that Prx I contributes

significantly to the ethoxyquin-induced cytoprotective response.

The pattern of cytotoxicity observed with oxLDL treatments showed an

intriguing difference (Fig. 13B). The protective effect of the ethoxyquin treatment

(+Ethx vs. Ctl) was still observed with oxLDL-induced toxicity and a component of

this protection was reversed by the Prx I knockdown (Prx I-KD, +Ethx vs. Ctl);

consistent with the effects seen in the t-BOOH exposure. Thus, the ethoxyquin-

induced upregulation of Prx I prior to the treatment with oxLDL or the oxLDL- component-model t-BOOH decreases sensitivity to oxidant-induced cytotoxicity.

In contrast to t-BOOH-exposure, however, no difference was seen in cell survival for the Prx I knockdown relative to the control macrophages (Prx I-KD vs. Ctl).

Therefore, these data show that while the upregulation of Prx I that occurs during macrophage foam cell formation can protect against selected oxidants, the non- stimulated level of Prx I in macrophages do not have measurable effects on oxLDL-induced toxicity.

As an antioxidant, the cytoprotective effect of Prx I could be associated with

its ability to reduce reactive oxygen species (ROS). To test this function, t-BOOH and oxLDL-induced ROS levels were assayed using the fluorescent ROS indicator CM-H2DCFDA to determine if changes in intracellular ROS correspond

to the changes in oxidant-induced toxicity seen with Prx I induction and inhibition.

Figure 14 shows typical raw data produced by this experiment in histogram form.

In this example, control (reverse siRNA-transfected) and Prx I-KD macrophages were exposed to 50 μM t-BOOH for 24 hours. An increase in ROS was detected by the positive shift in fluorescent intensity in macrophages with knocked-down expression of Prx I.

Figure 15 shows ROS levels measured in a variety of experimental

conditions. Treatment with either t-BOOH or oxLDL gave corresponding dose-

dependent increases in the relative ROS measurements. In Prx I-KD

macrophages, a significant increase in ROS production was observed with both

oxidants. Conversely, Prx I induction with 50 μM ethoxyquin (+Ethx) gave a significant decrease in ROS production and a component of the reduction was reversed when combined with Prx I knockdown (Prx I-KD, +Ethx). As a group, these data show that changes in ROS detection correspond directly to Prx I knockdown or induction, including the demonstration that Prx I contributes to the reduction in ROS observed with ethoxyquin treatment.

3.4 Summary

Foam cell formation initiated by exposing macrophages to oxLDL triggers the differential expression of a number of proteins. Previous 2D SDS-PAGE experiments identified Prx I as a protein upregulated by oxLDL-exposure, and

this induction was verified by Western blot analysis. Further experiments

confirmed an increase in the active functional form of Prx I.

By applying methods to either induce or inhibit Prx I protein expression, our data show that Prx I upregulation prior to foam cell formation promotes cell survival, and that the protective effect of Prx I corresponds directly to its ability to reduce reactive oxygen species. Induction of Prx I expression led to improved cell survival following treatment with oxLDL or tert-butyl hydroperoxide. The improved survival coincided with a decrease in measurable reactive oxygen species (ROS), and both the increased survival and reduced ROS were reversed by Prx I siRNA transfection.

3.5 Figures

Figure 8: Identification of Prx I as a protein upregulated during macrophage foam cell formation. J774 murine macrophages were lysed in 1%

SDS buffer and assayed for protein concentration. 1 mg total protein was acetone-precipitated, then prepared for 2D gel separation. A, 2D SDS-PAGE gel

(12.5% Tris-HCL) showing the entire pI range 3 – 10 and molecular weights of approximately 10 – 100 kDa. A band identified by LC-ESI mass spectrometry as

Prx I is labeled. B, Magnified inset from 2D gel showing Prx I in control

(untreated) macrophages, compared to C, macrophages treated with 50 μg/mL oxLDL for 24 hours.

Figure 9: Western blot analysis of the time- and dose-dependent

upregulation of Prx I following exposure to oxLDL. A, J774 murine

macrophages were left untreated (Ctl), or treated with 50 μg/mL LDL or oxLDL for 24 and 48 hours. B, Macrophages were treated with 0, 10, 25 or 50 μg/mL

oxLDL for 24 hours. In both A and B, protein was detected using a rabbit

polyclonal antibody to Prx I (upper panel) or mouse monoclonal antibody to

GAPDH as a loading control (lower panel).

Figure 10: Detecting the oxidative inactivation of Prx I by 2D SDS-PAGE

Western blot analysis. J774 murine macrophages were left untreated (Ctl) (A &

D), treated with 100 μM t-BOOH (B & E) or 50 μg/mL oxLDL (C & F) for 24 hours.

After oxidant-exposure, macrophages were collected and analyzed by 2D SDS-

PAGE. 2D gels were subjected to Western blot analysis using antibodies for Prx

I (A, B, C) or oxidatively-modified Prx I (oxPrx I) (D, E, F). The three protein bands seen in each panel correspond to, from left to right, inactive Prx I (Cys-

SO3 modification), inactive Prx I (Cys-SO2 modification), and active Prx I (non-

oxidized). The oxPrx I antibody is designed to detect both Cys-SO3 and Cys-SO2

modifications.

Figure 11: Effects of ethoxyquin treatment on Prx I induction. A,

Ethoxyquin-induced upregulation of Prx I. J774 murine macrophages were

exposed to 0 – 100 μM ethoxyquin for 24 hours, then collected and analyzed by

Western blot. B, Effect of ethoxyquin pre-treatment in the oxLDL-induced

upregulation of Prx I. Macrophages were treated with 50 μM ethoxyquin or left

untreated for 24 hours, then treated with 0, 25 or 50 μg/mL oxLDL for 24 hours.

In A and B, panels depict Western blots using antibodies for Prx I (upper panels) or GAPDH as a loading control (lower panels).

Figure 12: Knock-down of Prx I expression by siRNA transfection. A,

J774 macrophages were transfected for 24 hours with varied concentrations of siRNA targeting exon 4 of Prx I mRNA. B, Cells were not transfected (Ctl), exposed to transfection vehicle only (Veh), or transfected with scrambled (Scr), reverse (Rev), or Prx I sequence siRNA (Prx I). C, Cells were transfected with reverse sequence (Rev) or Prx I siRNA (Prx I), then exposed to 0 or 50 μg/mL oxLDL for 24 hours. In A, B, and C, panels depict Western blots using antibodies to Prx I (upper panels), Prx II (middle panels), and GAPDH (bottom panels).

Figure 13: Oxidant-induced cytotoxicity in macrophages with induced or

knocked-down expression of Prx I. J774 murine macrophages were treated

with (A), 0 – 200 μM t-BOOH or (B), 0 – 100 μg/mL oxLDL for 48 hours, and

assayed for the presence of respiring cells. Results are expressed as % cell

survival normalized to untreated controls. The data are labeled as follows: (♦)

Ctl: macrophages transfected with reverse-sequence siRNA, (◊) Prx I-KD: macrophages transfected with Prx I siRNA for 24 hours, (■) + Ethx: macrophages transfected with reverse-sequence siRNA, then treated with 50 μM ethoxyquin for

24 hours prior to oxidant-exposure, (□) Prx I-KD, + Ethx: macrophages transfected with Prx I siRNA for 24 hours, then exposed to 50 μM ethoxyquin for

24 hours. Results represent an average of three independent treatments. Error bars represent ± one standard deviation.

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Figure 14: Flow cytometric histogram data demonstrating increased ROS in macrophages with induced or knocked-down expression of Prx.

Macrophages transfected with reverse-sequence siRNA (Control) or Prx I siRNA

(Prx I-KD) were exposed to 50 μM t-BOOH for 24 hours. ROS levels were detected with CM-H2DCFDA, and raw histogram data was overlaid to demonstrate the increase in ROS that occurs when Prx I expression is knocked- down, as indicated by the positive shift in fluorescent intensity. Fluorescence was measured on the green channel of a Guava EasyCyte flow cytometer.

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Figure 15: Oxidant-induced ROS generation in macrophages with induced or knocked-down expression of Prx I. J774 murine macrophages were treated with (A), 0, 25 or 50 μM t-BOOH or (B), 0, 10 or 50 μg/mL oxLDL for

24 hours, and assayed for the generation of ROS. Results are expressed as relative ROS normalized to untreated Ctl macrophages. The data are labeled as follows: (♦) Ctl: macrophages transfected with reverse-sequence siRNA, (◊) Prx

I-KD: macrophages transfected with Prx I siRNA for 24 hours, (■) + Ethx: macrophages transfected with reverse-sequence siRNA, then treated with 50 μM ethoxyquin for 24 hours prior to oxidant-exposure, (□) Prx I-KD, + Ethx: macrophages transfected with Prx I siRNA for 24 hours, then exposed to 50 μM ethoxyquin for 24 hours. Results represent an average of three independent treatments. Error bars represent ± one standard deviation.

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Chapter 4

Regulation of Cell Signaling by Peroxiredoxin I

4.1 Introduction

The induction of the peroxiredoxin is generally related to a cellular antioxidant response. However, recent findings suggest alternative functionality for the peroxiredoxin family, including the ability to modulate various signaling pathways.

Prx II, for example, is believed to regulate peroxide levels that would otherwise inhibit phosphatases such as PTEN (Kwon, Lee et al. 2004). PTEN converts the signaling molecule PIP3 to PIP2, inhibiting the downstream activation of Akt kinase. Thus, increased Prx II activity is able to protect and support increased

PTEN activity, and prevent the downstream phosphorylation of Akt targets.

Additionally, Prx II can regulate H2O2 levels produced in response to TNF-α, and thus limit the activation of the JNK and p38 MAPKs in response to increased peroxide levels (Kang, Chang et al. 2004). One report invoking a similar role for

Prx I in intracellular signaling has also been made. In those experiments, the

Schizosaccharomyces pombe 2-Cys peroxiredoxin Tpx1, the yeast homolog for mammalian Prx I, was shown to regulate the peroxide-induced activation of Sty1, the yeast homolog for mammalian p38 (Veal, Findlay et al. 2004). The regulation is thought to occur through a direct association of Tpx1 with Sty1, forming an intermolecular disulfide that protects Sty1 from oxidant-induced inactivation

(Veal, Findlay et al. 2004). A parallel role for mammalian Prx I in p38 regulation in macrophages would be significant, considering the downstream effects of p38 activation (Jing, Xin et al. 1999; Hsu and Twu 2000).

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4.2 Experimental Procedures

Materials

siRNA and siPORT amine transfection reagent were obtained from Ambion

Inc. (Austin, TX). Rabbit polyclonal antibody for Prx I was obtained from BioMol

Int. (Plymouth Meeting, PA). Rabbit polyclonal antibodies for p38,

phosphorylated p38 (Thr180/Tyr182), Akt and phosphorylated Akt (Ser473) were obtained from Cell Signaling (Danvers, MA). Ethoxyquin and hydrogen peroxide were purchased from Sigma-Aldrich (St. Louis, MO). The p38 MAPK inhibitor

SB 203580 was obtained in a 1 mg/mL DMSO solution from EMD Biosciences

(San Diego, CA).

siRNA Inhibition of Prx I

Approximately 5x105 cells were plated in 60 mm tissue-culture treated plates

and left to adhere overnight at 37°C in 5% CO2 in normal culture media. For

each 60 mm plate, 13 μL of siPORT Amine Transfection Reagent (Ambion Inc.,

Austin, TX) was incubated with 200 μL Opti-Mem reduced-serum medium for 10

minutes. Annealed double-strand siRNA (20 μM stock) was added to a separate

aliquot of 200 μL Opti-Mem to give a final siRNA concentration of 30 nM. The

transfection agent and the siRNA mixtures were combined and incubated for 10

minutes at room temperature, then added dropwise to culture media (4 mL

DMEM + 10% FCS only; no antibiotics) and incubated for 24 hrs.

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Preparation of activated sodium orthovanadate

Sodium orthovanadate (SOV), a phosphatase inhibitor, was added to the lysis

buffer to prevent dephosphorylation during sample preparation. SOV must be

depolymerized prior to use to produce an activated stock. A 200 mM solution of

SOV was prepared and adjusted to pH 10.0, at which point the solution is yellow.

The solution was boiled until it turned colorless (approximately 10 minutes), then

cooled to room temperature. The pH is re-adjusted to 10.0 and boiled again, and

these steps are repeated until the solution remains colorless at a stabile pH of

10.0. Activated sodium orthovanadate is stored as aliquots at -20°C, and used at a dilution of 1mM.

Western Blot strip and re-probe procedure

After initial detection of antibodies, PVDF membranes were incubated in wash

buffer (10mM Tris pH 7.5, 100mM NaCl, 0.1% Tween 20) for 15 minutes at room

temperature. PVDF membranes were enclosed in heat sealable sleeves with 30

mL of stripping buffer (62 mM Tris-HCl [pH 6.8], 2% SDS, 100 mM β-

mercaptoethanol) and incubated in a 50°C water bath with agitation for 30

minutes. PVDF membranes were removed from strip buffer and washed 3 x 10

minutes in wash buffer, then blocked for 1 hour in 2% non-fat dry milk at room

temperature. After blocking, the PVDF membranes were ready for re-probe,

which was always performed by incubating with primary antibody overnight at

4°C. Western blot analysis continued according to accepted procedures.

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Membranes were not stripped more than once to prevent appreciable protein

loss.

p38 MAPK/Akt kinase signaling experiments

J774 macrophages were transfected with control (Ctl) or Prx I siRNA (Prx I-

KD) for 24 hours prior to addition of signaling stimulus. Transfection media was

aspirated and fresh culture media was added with either 100 μM H2O2, 50 μM ethoxyquin or oxLDL (0, 10, 25 or 50 μg/mL) and incubated for 24 hours. Cells were scraped and pelleted in DMEM, washed once in PBS and placed on ice.

Cell pellets were lysed in SDS buffer (25 mM Tris, pH 7.5; 2.5 mM MgCl2;

0.5% SDS) + 1 mM activated SOV and boiled for 5 minutes with occasional

vortexing. Samples were cooled to room temperature and treated with 100

μg/mL DNase I and 100 μg/mL RNase A for 15 minutes. Protein concentrations

were assayed using a SDS-compatible Lowry-based method (DC Protein Assay

Kit; Bio-Rad Laboratories, Hercules, CA), and sample volumes were adjusted with lysis buffer to equal concentrations.

Samples were separated by 10%, 12.5% or 15% Tris-HCl SDS-PAGE and

transferred to PVDF membranes. Phosphorylated p38 MAPK, phosphorylated

Akt kinase and Prx I were assayed by Western blot. PVDF membranes were

stripped and re-probed for total p38 MAPK or Akt.

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p38 MAPK inhibition

SB 203580 binds to the ATP-binding pocket of p38 MAPK, and can be used

as a competitive inhibitor of p38 MAPK function. SB 203580 (10 μM) was added to cell culture 30 minutes prior to experiment. An equivalent volume of anhydrous DMSO was added to control cultures without SB 203580. The inhibitor was left in the culture for the duration of the experiment.

4.3 Results

p38 MAPK activation stimulated by hydrogen peroxide

Prx I was tested for its ability to regulate the activation of p38 MAPK

stimulated by various methods. Control and Prx I-KD macrophages were

exposed to 100 µM H2O2 for up to 2 hours and analyzed by Western blot to

determine expression of Prx I, phosphorylated and total p38 MAPK (Fig. 16A). In

control macrophages, phosphorylation of p38 MAPK increased over time as total

Prx I and p38 MAPK remained constant. In Prx I-KD macrophages,

phosphorylation of p38 MAPK was negligible with respect to control

macrophages until 2 hours after H2O2 exposure. At 2 hours, phosphorylation of p38 MAPK was still significantly decreased in the Prx I-KD macrophages compared to controls. These data are the first confirmation that mammalian Prx I expression directly regulates p38 MAPK activation, as seen with the yeast homologs Tpx1 and Sty1 (Veal, Findlay et al. 2004).

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Effect of ethoxyquin-induced Prx I on p38 MAPK and Akt activation

This experiment sought to determine if the differential regulation of Prx I

would directly regulate the extent of p38 MAPK activation. Control and Prx I-KD

macrophages were treated with 50 µM ethoxyquin to induce Prx I expression (Fig

16B). Phosphorylation of p38 MAPK increased with ethoxyquin-induced Prx I

upregulation. In Prx I-KD macrophages, expression of Prx I was inhibited in both

the 0 and 50 μM ethoxyquin-treated macrophages, and the corresponding

phosphorylation of p38 MAPK was negligible in each. Thus, the induction of Prx

I by ethoxyquin creates conditions under which p38 MAPK activation is also

increased.

Effect of oxLDL-induced Prx I on p38 MAPK and Akt activation

The Prx I-regulated activation of p38 MAPK was tested under conditions of

macrophage foam cell formation (Fig. 16C). Control and Prx I-KD macrophages were exposed to 10, 25 or 50 μg/mL oxLDL for 24 hours. Prx I was upregulated

in a dose-dependent manner, while total p38 MAPK expression remained

constant. In control and Prx I-KD macrophages, phosphorylation of p38 MAPK

increased with oxLDL concentration. However, in Prx I-KD macrophages, phosphorylation of p38 MAPK was significantly reduced in parallel with the near-

complete inhibition of Prx I. At this time, it is not clear if this modest amount of

p38 MAPK activation is due to the small amount of residual Prx I expression or

the presence of other, non-Prx I-dependent p38 MAPK activation pathways.

Nonetheless, these data present a clear role for Prx I as a necessary component

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for maximal p38 MAPK activation, and suggest that the upregulation of Prx I is accompanied by a corresponding increase in p38 MAPK activation.

p38 MAPK activity is not required for antioxidant activity of Prx I

The observed increase in p38 MAPK activation with Prx I induction raises the

possibility that this activation is responsible for the increase in antioxidant activity,

rather then the Prx I induction, either directly or by down-stream effects. To test

this possibility, the ability of Prx I to decrease ROS levels was examined under

conditions where Prx I was induced but p38 MAPK activity was inhibited (Fig.

17). Macrophages were treated with 50 μM ethoxyquin to induce Prx I or left untreated for 24 hours. The compound SB-203580 was used to inhibit p38

MAPK, then samples were exposed to 200 μM t-BOOH for 2 hours and assayed for ROS levels. Ethoxyquin-exposed macrophages, which express increased levels of Prx I, showed the expected decrease in ROS compared the untreated controls, consistent with the antioxidant activity of the induced Prx I, but the p38

MAPK inhibition did not alter this effect. Therefore, the decrease in ROS levels that results from Prx I induction (Figs. 15 and 17) is not dependent on p38 MAPK

activity.

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4.4 Summary

This data demonstrates that activation of p38 MAPK in oxLDL-treated macrophages is dependent on the upregulation of Prx I. Reduction of Prx I expression by siRNA transfection resulted in a significant decrease in p38 MAPK activation, while the upregulation of Prx I expression with either oxLDL or ethoxyquin led to increased p38 MAPK activation. These results, in conjunction with the data presented in Chapter 3, confirm a dual-role for Prx I in macrophage foam cells that includes functionality as both an antioxidant and a regulator of oxidant-sensitive signal transduction.

4.5 Figures

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Figure 16: Prx I regulates the activation of p38 MAPK stimulated by H2O2,

ethoxyquin, and oxLDL. A, H2O2-stimulated p38 MAPK activation. Ctl

(transfected with reverse-sequence siRNA) and Prx I-KD macrophages were

exposed to H2O2 for the indicated times, collected, and prepared for Western blot

analysis. The knocked-down expression of Prx I is demonstrated by Western

blot of Prx I (upper panel). Activation of p38 MAPK by dual-phosphorylation

(middle panel) and total p38 MAPK (bottom panel) were also determined by

Western blot analysis. B, The effect of ethoxyquin-induced Prx I overexpression

on p38 MAPK activation. Ctl and Prx I-KD macrophages were exposed to 50 μM

ethoxyquin for 24 hours, or left untreated. Macrophages were collected and

analyzed by Western blot for Prx I (top panel), phosphorylated p38 MAPK (2nd from top), total p38 MAPK (3rd from top), Akt activated by phosphorylation at Ser-

473 (2nd from bottom) and total Akt (bottom panel). C, oxLDL-stimulated p38

MAPK activation. Ctl and Prx I-KD macrophages were treated with 0, 10, 25 or

50 μg/mL oxLDL for 24 hours, collected and analyzed by Western blot. Panels are identical to the listing in B.

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Figure 17: Reduction of ROS in macrophages with inhibited p38 MAPK

activity. Macrophages were treated with 50 μM ethoxyquin for 24 hours to

induce Prx I expression, or left untreated. The p38 MAPK inhibitor SB-203580

was added to half of the ethoxyquin-treated and untreated samples. All samples

were exposed to 200 μM t-BOOH for 2 hours and ROS levels were assayed by

flow cytometry. (■) Control macrophages, (□) macrophages with p38 MAPK

inhibitor added. Results represent an average of three independent experiments.

Error bars represent ± one standard deviation. * p < 0.05 compared to the corresponding value without ethoxyquin treatment (0 μM).

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Chapter 5

Discussion

5.1 Summary of Results

Early atheroma development requires the internalization of oxidatively- modified LDL by macrophages in the arterial intima, which develop into foam cells and aggregate at fatty streak formations. However, atherosclerosis is a slow-to-develop disease, beginning as early as the 2nd decade of life and continuing over many years. Thus, considering the reversible nature of foam cell formation, macrophages may potentially develop a stress-response after chronic exposure to oxLDL. We hypothesized that foam cell formation is accompanied by differential mRNA and protein expression that is ultimately pro-atherogenic, and that this differential expression is altered by prior chronic exposure to oxLDL.

The aims of this project were to 1) identify differentially-expressed mRNA and proteins in macrophage foam cells and describe their role in atheroma development, and 2) determine how chronic exposure to oxLDL can alter this differential expression, thus affecting oxLDL-uptake and oxLDL-induced toxicity.

A second stage of this project involved Prx I, a protein identified in the initial stage of this research as being upregulated in macrophages following oxLDL- exposure. The upregulation of Prx I in foam cells is intriguing because of the elevated presence of peroxiredoxin in tumors that are resistant to radiation- treatment and chemotherapy. Invoking a parallel between cancer and atherosclerosis, we hypothesized that the upregulation of Prx I in macrophage foam cells desensitizes cells to oxLDL-induced toxicity through the reduction of

118 reactive oxygen species. The aims for this portion of the project were as follows:

1) Characterize the induction of Prx I by oxLDL, specifically identifying the upregulation of active Prx I. 2) Analyze oxLDL-induced toxicity and ROS levels in macrophages using chemical induction and siRNA knock-down of Prx I expression. 3) Additionally, we investigated alternative roles for Prx I, in accordance with previous data that has described Prx I functionality that is alternative to its antioxidant capability.

The results of this project demonstrate that macrophage foam cell formation is accompanied by differential expression that, as a whole, is pro-atherogenic.

This differential expression is altered in macrophages with prior chronic-exposure to oxLDL, such that an increased antioxidant defense results in decreased oxLDL-induced toxicity. Additionally, the upregulation of Prx I is described for macrophage foam cells. Induction of Prx I expression prior to foam cell formation decreases oxLDL-induced toxicity in parallel with a reduction in reactive oxygen species. In addition to this antioxidant functionality, Prx I is described for its ability to regulate p38 MAPK signaling.

5.2 Chronic exposure to oxLDL attenuates toxicity in foam cells

A major difference that exists between the in vivo events and in vitro experiments that investigate foam cell formation is the fact that macrophages commonly used in atherosclerosis research, such as mouse peritoneal macrophages, elutriated human monocyte-macrophages, or the J774 murine cell line used here have not been previously exposed to oxLDL. In these

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experiments, a chronic exposure sub-line (J774-CE) was generated from parent

J774 macrophage cells by repeated exposure to oxLDL. The goal was to model

the long time-frame over which atherosclerosis develops, which is an intrinsic

component of the disease. Pathological examinations have shown that lipid

accumulation in the vasculature begins in the first decade of life and proceeds

steadily as one ages (Smith 1965; Smith, Evans et al. 1967). The morbidity and

mortality of the disease, however, typically does not become apparent until after

40 to 50 years. The development of the J774-CE sub-line was intended to model

in vitro any effects of this repetitive, long-term exposure.

As the cells were exposed to oxLDL, foam cell formation was validated in

oxLDL-treated J774 murine macrophages by lipid-staining experiments that were

carried out either after the lipid had accumulated in the cell (oil red O) or before

the lipid was placed on the cells (DiI). Positive staining with both stains verified

formation of foam cells. It is known that LDL in culture media with macrophages

can be oxidized to some degree by cell-mediated processes and this form of

oxLDL can be taken up by macrophages. Therefore, over the course of a 48

hour incubation period, some degree of lipid uptake is expected in LDL-treated

macrophages. While oil-red O stained droplets are apparent in the LDL-treated

cells, the occurrence is minimal in comparison to the oxLDL-treated

macrophages. The use of DiI-labeling not only verified the oil red O-staining

results but also allowed quantitative analysis of the extent of oxLDL uptake.

The chronic exposure phenotype was tested at two levels - the ability of macrophages to internalize oxLDL and the cytotoxicity of oxLDL. These tests

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were based on the key events of unregulated oxLDL accumulation through

scavenger receptors and the cytotoxicity of oxLDL described in the oxidative

modification hypothesis of atherosclerosis (Chisolm and Steinberg 2000). A

dose-response analysis of DiI-labeled oxLDL internalization revealed that there is no difference in lipid-loading between the parental J774 macrophages and the

J774-CE sub-line. Therefore, chronic exposure to oxLDL did not change the lipid

loading characteristics of foam cell formation. Cell survival experiments, however, showed that chronic exposure to oxLDL did produce a significant resistance in the cells to oxLDL-mediated cytotoxicity. Further, this difference in survival was also seen in the J774-CE(-) sub-line, indicating that the cytotoxicity resistant-phenotype is stable for at least two months.

5.3 Differential expression induced by chronic vs. acute oxLDL exposure

The availability of the J774-CE sub-line had a secondary effect of providing

an additional test of important trends in the data. Of particular interest in these experiments were the proteins and genes for which expression was not altered by the single exposure but was altered by the chronic exposure. Not only do these proteins and transcripts represent a systems biology confirmation of the importance of modeling the chronic exposure but they are also novel new observations that could not be made in the standard acute exposure experiments.

Notable proteins identified in these experiments with increased expression in

oxLDL exposed cells were a series of antioxidant proteins that react directly with

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oxidant species, including superoxide dismutase, aldose reductase-related

protein, peroxiredoxin and esterase D. Other proteins that contribute indirectly to oxidant defense through the production of antioxidant molecules or co-factors were also induced by oxLDL treatment. Biliverdin reductase B produces the antioxidant riboflavin and NADPH, a co-factor that represents reducing equivalents for enzymes such as glutathione peroxidase. The overexpression of transaldolase, an enzyme of the non-oxidative pentose phosphate pathway, also suggests an increased need for NADPH. This enzyme is a part of the pentose phosphate pathway that coordinates with glycolysis to produce varying combinations of NADPH, NADH, ATP or ribose 5-phospate. The precise balance between different components of the pathway depends on the metabolic requirements of the cell. Similarly, the overexpression of the three late-stage glycolytic enzymes (phosphoglycerate kinase, phosphoglycerate mutase and enolase) is consistent with an increased demand for ATP. The increased ATP requirement coincides with the overexpression of the ATP synthase beta subunit that was also seen. The underexpression of glutamate dehydrogenase that was observed is consistent with this pattern because it would prevent L-glutamate from entering the Kreb’s cycle and reserve it for use in the synthesis of glutathione, a co-factor used by a number of antioxidant enzymes. Glutathione is also used by glutathione transferase to detoxify reactive aldehydes that are formed by lipid and amino acid oxidation. This pattern of protein expression is consistent with an increased expression of antioxidant and antioxidant-related

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proteins in response to oxLDL treatment that would lead to the resistance of the

J774-CE cells to oxLDL-mediated cytotoxicity.

Other changes in protein expression agree with known changes in foam cell

function. For example, increased expression of enolase 1 was seen in oxLDL

treated J774 and J774-CE cells. While enolase plays a role in the final stages of

glycolysis, it has also been identified as a cell-surface receptor for plasminogen,

the zymogen of plasmin (Horwitz, Shen et al. 1997; Lopez-Alemany, Longstaff et

al. 2003). Therefore, overexpression of enolase could potentially contribute to an

increase in plasmin activity and fibrinolysis. This change is consistent with

findings that macrophages contribute to the degradation of extracellular matrix

proteins (Jones and Werb 1980), leading to thrombosis in advanced

atherosclerosis. Although an overexpressed band representing full length alpha-

enolase was identified (MW 50 kDa, pI 7.0), six additional bands were identified

as alpha-enolase at various molecular weights and isoelectric points. The bands

range from 30–50 kDa and pI 5.5-7.0, and each band is overexpressed in oxLDL-

treated J774 and J774-CE macrophages. Recent studies have identified alpha-

enolase as a target of nitration under oxidative stress conditions (Casoni, Basso et al. 2005; Xiao, Nel et al. 2005), and such a modification may lead to inhibition of activity and targeted degradation. Other proteins that have been described as targets of protein nitration were identified in the current study, including ATP- synthase beta-chain, farnesyl pyrophosphate synthase, and superoxide dismutase [Cu,Zn]. In addition, protein nitration has been implicated in the reversible inhibition of the antioxidant heme oxygenase (Kinobe, Ji et al. 2004)

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and glutathione-S-transferase (Wong, Eiserich et al. 2001; Lee and Fung 2003),

which were upregulated at the message level in this study. There is evidence

that proteins within foam cells of fatty streaks undergo extensive nitration

(Beckmann, Ye et al. 1994), but as of yet, no studies have addressed the role of

protein nitration as a regulatory mechanism in foam cell formation.

Finally, a number of the differentially expressed proteins have unclear roles in

foam cell formation. For example, L-plastin is a 64 kDa actin-binding protein

involved in integrin activation and leukocyte adhesion (Jones, Wang et al. 1998).

Although two bands identifying this protein are overexpressed, they migrate to

approximately 15 kDa and 50 kDa on the 2D gel. The molecular weights and

peptide sequencing indicate that, together, these bands represent the full-length protein, suggesting proteolysis as a regulatory mechanism. The overall effect of this proteolysis would lead to an inhibition of L-plastin activity. Perilipin and fatty- acid binding protein 5 (FABP5) bind to lipid droplets in adipocytes, and likely play a similar role in lipid-laden foam cells. Perilipin, which is overexpressed in foam cells of ruptured atheromas (Faber, Cleutjens et al. 2001), is overexpressed here in both the J774 and J774-CE foam cells. FABP5 has been detected at the message and protein level in macrophages (Tolle, Schlame et al. 2005), but as of yet has no link to atherosclerosis. The earliest research linking other fatty-acid binding proteins and atherosclerosis showed that a decrease in arterial FABP coincided with increased arterial cholesterol content and lesion development in rabbits (Van der Vieren, Le Trong et al. 1995). In a more recent study, a decrease in adipocyte FABP was linked to decreased lesion development in mice

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(ApoE -/-) (Falcone, Khan et al. 1998; Boord, Maeda et al. 2004). FABP5 in

particular was shown to be overexpressed in the culture media of differentiated

THP-1 monocyte-macrophages following oxLDL-treatment (Fach, Garulacan et

al. 2004). In this system, FABP5 is intriguing for its overexpression in oxLDL-

treated vs. untreated J774-CE macrophages, but even more for its increased

expression in untreated J774-CE cells relative to untreated J774 cells. This

increased expression of a lipid-binding protein in a non-stimulated state may

provide the J774-CE macrophages with an increased tolerance for oxLDL-

induced lipid loading. The novel implication that this protein may be linked to

lesion development is an example of the discovery element of proteomic

experiments.

Peroxiredoxin I (Prx I) was initially identified at the band positioned directly

adjacent and to the right of band #52. This band was typically upregulated

following oxLDL-treatment, however, the theoretical pI for Prx I (pI 8.26) is not

consistent with this gel location. Rather, this band location coincides with the

theoretical pI for Prx I if one cysteine is replaced with glutamate or aspartate (pI

7.66), which models the oxidatively-inactive form of peroxiredoxin I (Cys-SO2).

After combining this experimental identification with theoretical calculations, the band representing active Prx I was identified at the expected position on the 2D gel. This is a good example of how empirical and theoretical knowledge can guide a proteomic analysis to the correct and expected results.

The transcriptome of the parent J774 and J774-CE cells was investigated in a gene chip microarray experiment. The goal of these experiments was to

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complement the proteomics studies by creating a parallel dataset of differentially

expressed transcripts that respond to the oxLDL treatment. As expected, a

broad set of mRNA transcripts were up- and downregulated, but particular

interest was given to gene expression changes related to antioxidant defense

and immune response.

The overexpressed genes listed in Table 4 are involved directly in oxidant

removal or participate in an upstream event that leads to antioxidant expression.

There were no genes involved in antioxidant defense that were reduced by

oxLDL treatment. Most of the genes listed in Table 4 detoxify hydrogen peroxide

or the products of lipid peroxidation. Catalase directly reduces H2O2, and peroxiredoxin 5 reduces H2O2, lipid hydroperoxides and peroxynitrites.

Thioredoxin reductase participates in the reactivation of oxidatively-modified

proteins, such as peroxiredoxin, through its reduction of thioredoxin. Glutamate-

cysteine ligase is the rate-limiting step in the synthesis of the antioxidant

glutathione, and glutathione S-transferase conjugates glutathione to lipid

hydroperoxides in a detoxification process. Heme oxygenase 1 is an inducible

enzyme that converts heme to biliverdin. Biliverdin, and its by-product bilirubin,

are potent antioxidants that detoxify peroxyl radical and hydrogen peroxide (Ling,

Kaur et al. 1998; Minetti, Mallozzi et al. 1998).

Carbonyl reductase, which is also listed as an anti-inflammatory mediator,

specifically reduces the lipid peroxidation product 4-oxonon-2-enal (4ONE).

However, one product of this reaction is another reactive aldehyde and a component of oxLDL, 4-hydroxynon-2-enal (4HNE). (Doorn, Maser et al. 2004)

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There is currently no data that addresses a role for carbonyl reductase in the

macrophage.

Other overexpressed genes provide defense against other types of oxidants.

Esterase D (S-formylglutathione hydrolase) contributes to the removal of

formaldehyde, a toxic metabolite indicated in endothelial injury and potential

atheroma development (Boor, Trent et al. 1992; Yu and Deng 1998). NAD(P)H

dehydrogenase (quinone 1) detoxifies quinone, derived from phenolic

metabolites of benzene, and is active in macrophages (Ross, Siegel et al. 1996;

Moran, Siegel et al. 1999). Quinone oxidoreductase-like 1 may have similar

functionality, but little is know about this gene product other than its high degree

of homology to zeta-crystallin (Kim, Lee et al. 1999). However, an NCBI

nucleotide Megablast search of the mRNA sequence reveals zinc-dependent

alcohol dehydrogenase activity, which coincides with the upregulation of two other alcohol dehydrogenase genes – retinol dehydrogenase and the retinol- specific alcohol dehydrogenase 7 (Satre, Zgombic-Knight et al. 1994). These enzymes convert retinol isomers to their corresponding aldehyde retinal isomers, and produce the reducing equivalent NADH in the process (Haeseleer, Jang et al. 2002). Retinal isomers are further oxidized by aldehyde dehydrogenases to corresponding isomers of retinoic acid, including 9-cis-retinoic, a ligand for the retinoid X receptor (RXR) (Paik, Vogel et al. 2000). Activation of RXR in combination with peroxisome proliferator-activated receptor γ (PPARγ) activation can increase cholesterol efflux and modulate foam cell formation, despite triggering an increase in CD36 scavenger receptor expression (Stein, Ben-Naim

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et al. 2002; Argmann, Sawyez et al. 2003). Consequently, the upregulation of

retinol and alcohol dehydrogenases may contribute to antioxidant defense

through NADH production, and contribute to foam cell prevention by stimulating

an increase in cholesterol efflux.

An additional observation made in the analysis of the microarray data was the

change in expression of transcripts linked to the inflammatory response. The link

between inflammation and atherosclerosis has gained significant attention in recent years. Several pro-inflammatory mediators have been investigated for

their contribution to atherosclerosis, including IFN-γ (Gupta, Pablo et al. 1997),

interleukin-18 (Schonbeck, Gerdes et al. 2002), interleukin-6 and –8 (Rus, Vlaicu

et al. 1996), macrophage colony-stimulating factor (Clinton, Underwood et al.

1992) and others. Additionally, anti-inflammatory mediators such as interleukin-

10 are a focus of therapeutic applications for atherosclerosis. (Dallmann, Junker et al. 2004). Much of the attention has focused on the effect of inflammation on smooth muscle cells and endothelial cells, but less information is available about

the foam cell and its contribution to inflammation and immune response in

general. The results of the gene chip analysis in this experiment describe an intriguing balance between upregulated and downregulated mRNA transcripts involved in immune response.

A large number of overexpressed genes in Table 3 are pro-inflammatory,

including interleukin-6, macrophage inflammatory protein 1α and its receptor,

macrophage inflammatory protein 2, oncostatin M and tumor necrosis factor α.

Prostaglandin-endoperoxidase synthase 1 and 2, more commonly known as

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COX1 and COX2, both influence a downstream pro-inflammatory response.

COX1, the constitutively-expressed enzyme, is downregulated in both the J774 and J774-CE foam cells, whereas COX2, the corresponding inducible enzyme, is upregulated in both cell lines. Also contributing to the overall pro-inflammatory state is the underexpression of transcripts for the anti-inflammatory interleukin-10 and the guanylate binding proteins 1, 2 and 4. Taken as a group, these changes are consistent with a shift in pro-/anti-inflammatory balance to a distinct pro- inflammatory state.

Despite this upregulation of pro-inflammatory response, the response of the foam cell transcriptome to oxLDL-treatment showed an intriguing downregulation in other aspects of macrophage functionality. For example, oxLDL- treatment reduced the expression of a number of interferon-induced genes, including 2’-5’-oligoadenylate synthetases, interferon alpha-inducible protein, interferon gamma-inducible protein, interferon-induced proteins 35 and 44, and interferon-activated gene 203. Interferon-induced proteins often participate in the anti-pathogenic, anti-angiogenic, and cell cycle inhibitory roles of innate immunity. Another critical component of the innate immune response to pathogens are the toll-like receptors, which were also downregulated following oxLDL-treatment. In addition, genes associated with adaptive immune response were also largely downregulated. Indeed only one transcript, histocompatibility-2 complex class 1-like mRNA, was overexpressed in J774 and J774-CE foam cells. The other genes of this class were downregulated, and include genes encoding complement components, histocompatability proteins and lymphocyte

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antigen complex. The overall effect of this downregulation is a decrease in the

normal anti-pathogenic response of the macrophage following exposure to oxLDL.

In summary, this investigation has used a systems biology approach to

examine the transition of macrophages into foam cells at the protein and

message levels and has included a novel focus on macrophages that have been

chronically exposed to oxLDL. These chronically-exposed cells exhibited an

increased capacity for survival under increasing concentrations of oxLDL, but

showed no difference in lipid-loading when compared to naïve macrophages.

The functional results are consistent with the changes seen in a group of

antioxidant proteins that were upregulated at either the message or proteins

level. A subset of these proteins were overexpressed significantly more in

macrophages subjected to chronic oxLDL-exposure. The increased resistance to

oxLDL-induced cytotoxicity in the J774-CE macrophages is attributed to this

increase in antioxidant defense proteins. This elevated protective response is not dependent on the presence of oxLDL, since an increase in survival was maintained several weeks after chronic oxLDL-treatment was terminated. Our results also detected a group of pro-inflammatory transcripts that were upregulated following oxLDL-exposure, in addition to transcripts of an anti- inflammatory counter-response. However, the chronically oxLDL-exposed

macrophages presented an attenuated steady state and oxLDL-stimulated pro-

inflammatory response when compared to control macrophages. These results

support the hypothesis that chronic exposure to oxLDL produces important

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changes in foam cell function that are not seen in single oxLDL-exposure

experiments. A large number of changes in protein and mRNA expression are

produced by these treatments. As a group, these changes are consistent with both known (e.g. an enhanced inflammatory response) and novel (e.g. an increased resistance to oxLDL-mediated cytotoxicity) differences between foam cells and macrophages and between single and multiple oxLDL treatments.

5.4 Induction of Prx I provides protective antioxidant functionality

The previous data identified members of the peroxiredoxin family, a class of

peroxidases that are upregulated under various conditions of oxidative stress,

including hyperoxia (Kim, Kang et al. 2001), ischemia/reperfusion (Shau, Merino

et al. 2000), radiation (Lee, Park et al. 2002), and exposure to oxidant-generating

reagents (Siow, Ishii et al. 1995). A comparison of 2D SDS-PAGE gels showed that Prx I is upregulated in J774 murine macrophages following treatment with oxLDL under conditions that generate lipid-loaded macrophage foam cells in vitro

(Conway and Kinter 2005). Despite work by other laboratories showing the

upregulation of peroxiredoxins under various treatment conditions, studies of the

their oxLDL-induced upregulation is limited (Siow, Ishii et al. 1995; Ishii, Itoh et al.

2004). The data presented here fully demonstrate the upregulation of Prx I with

oxLDL treatment. Through Western blot analysis, upregulation of Prx I was

induced by oxLDL, but not native LDL, and both the dose-dependent and time-

dependent characteristics were determined.

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While the oxLDL-induced increase in Prx I expression is quantitatively

significant, the data does not indicate whether Prx I is in its active form.

Therefore, an additional step in the evaluation of expression was required to

demonstrate that the redox state of Prx I was consistent with the fully functional

form. The reducing function of the 2-Cys peroxiredoxins, including Prx I, is a two-step process that includes a sulfenic acid intermediate state (Cys-SOH) en route to an intermolecular disulfide across its homodimer structure. The disulfide is reduced by thioredoxin, returning functionality to peroxiredoxin. Recent attention has focused on the reversible inactivation of Prx I – IV caused by over- oxidation of the active site cysteine. In a mechanism proposed by Rhee and co- workers, the sulfenic acid intermediate can undergo further oxidation to a sulfinic

(Cys-SO2H) or sulfonic acid (Cys-SO3H) that can not be reduced by thioredoxin

(Yang, Kang et al. 2002; Woo, Kang et al. 2003). This over-oxidation step is a

particular concern for the oxidant-induced upregulation of peroxiredoxin, because

the conditions that lead to increased peroxiredoxin expression can also

oxidatively-inactivate newly expressed peroxiredoxin. The higher oxidation state

is not detectable in 1D SDS-PAGE analyzed by Western blot, because both the

active and inactive forms of peroxiredoxin run at the same position in the gel.

The modification is, however, readily apparent by the acidic shift on a 2D

Western blot of Prx I, and confirmed when the Western is re-probed using an

antibody specific to the Cys-SO2 and Cys-SO3 species (oxPrx I antibody). It

should be noted that the oxPrx I antibody identifies the oxidized species of all

typical 2-Cys peroxiredoxins (Prx I – IV). Because Prx I and Prx II are

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comparable in molecular weight, the use of this antibody on a 1D gel is not

sufficient to specifically identify inactive Prx I. However, due to a significant

difference in isoelectric point, Prx I and Prx II run at distinct locations on a 2D

SDS-PAGE gel. Therefore, the conditions of this experiment are uniquely suited

for identifying active vs. oxidatively-inactive Prx I.

While past studies have applied t-BOOH (Chevallet, Wagner et al. 2003) or

H2O2 (Yang, Kang et al. 2002) as an oxidative stress, there is currently no

evidence for the oxidative inactivation of Prx I in macrophage foam cells due to

an oxLDL treatment. The presence of active, upregulated Prx I was detected by

the combination of 2D SDS-PAGE with Western blot analysis described above.

Both untreated and oxidant-treated macrophages contain detectable amounts of modified Prx I, implying that Prx I may be modified by endogenous oxidants under normal culture conditions. The increased abundance of Cys-SO2 bands detected with both the oxLDL and t-BOOH- treatments shows that oxLDL is able to oxidized Prx I in a manner that is comparable to t-BOOH (54). More importantly, however, these 2D experiments also show that the increase in this modification is small and confirm that the oxLDL-induced upregulation of active

Prx I produces a substantial increase in the active form.

The next stage of this investigation sought to determine the functional significance of Prx I-upregulation by investigating the effects of Prx I induction on foam cell survival under conditions of t-BOOH- and oxLDL-induced cytotoxicity.

These experiments were based on the protective effects of the peroxiredoxins, including Prx I, described in other cells treated with other types of cytotoxins,

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including oxidants (Chung, Yoo et al. 2001; Chen, McBride et al. 2002; Hattori,

Murayama et al. 2003; Plaisant, Clippe et al. 2003). Our data show that inducing

the expression of Prx I with ethoxyquin increased cell survival for both t-BOOH-

and oxLDL-induced toxicity. In each case, the increased survival was attenuated

by the Prx I siRNA transfection, thus confirming a significant role of Prx I

induction in the enhanced survival.

It was notable, however, that the direct knock-down of Prx I sensitized the cells to t-BOOH-induced toxicity but had no effect on the oxLDL-induced toxicity.

There are at least two possible explanations for this difference. First, the sensitization to t-BOOH-induced toxicity by Prx I knockdown may be related to the relatively uniform nature of this oxidative stress. Lipid peroxidation products, modeled by t-BOOH, are typical substrates for peroxiredoxin. Therefore, the decrease in Prx I activity would directly diminish the metabolism of the t-BOOH and give a corresponding reduction in cell survival. Oxidatively-modified LDL, however, is a heterogeneous mixture of oxidants and oxidation by-products, including lipid hydroperoxides, aldehydes, and oxysterols (Quinn, Parthasarathy et al. 1985; Steinbrecher, Lougheed et al. 1989; Zhang, Basra et al. 1990). Prx I may aid in the reduction of lipid peroxidation products generated by or contained

in oxLDL, but other toxic components of oxLDL would remain unaffected.

Second, a number of other effective antioxidants and antioxidant enzymes are present in the untreated cells, giving an element of redundancy to the antioxidant system. In this case, the decreased antioxidant activity caused by Prx I knockdown would not be a significant loss to the overall antioxidant capability of the

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cell and would not produce a measurable change in the cytotoxicity of oxLDL.

However, the increase in Prx I expression induced prior to the oxidant exposure

appears to give a significant increase to the total antioxidant capacity, which

translates to an increase in cell survival. This positive effect of pre-induced

factors is a common theme in cytoprotection, i.e. the induction of cellular stress

response results in increased protection during a subsequent cellular stress

(Arstall, Zhao et al. 1998; Wiegant, Souren et al. 1999; Guo and Mattson 2000;

Conway and Kinter 2005).

Parallel experiments examined the ability of the differentially-expressed Prx I

to affect intracellular ROS. Inhibiting Prx I expression resulted in increased ROS

in both t-BOOH and oxLDL-treated macrophages compared to controls.

Conversely, increasing Prx I expression with ethoxyquin prior to oxidant

exposure decreased the ROS levels relative to control macrophages that were

not pre-treated with ethoxyquin. When the cells were treated Prx I siRNA prior to

ethoxyquin pre-treatment, ROS levels were increased to the levels seen in the

untreated controls.

A few details of the ROS detection, which influence the interpretation of these

results, deserve attention. First, not all oxidants may target CM-H2DCFDA for

oxidation with equal affinity. While the oxidation of dichlorofluorescin (DCFH)

analogs by H2O2 is well characterized (Ogawa, Kobayashi et al. 2004;

Diepeveen, Verhoeven et al. 2005; Tokay, Masmoudi et al. 2005), some data suggests that singlet oxygen does not oxidize DCFH to the fluorescent moiety,

dichlorofluorescein (DCF) (Bilski, Belanger et al. 2002). However, while some

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oxidants may not target DCFH analogs for oxidation, they can still produce downstream oxidants for which DCFH is a substrate. A second important

consideration involves the presence of endogenously-produced ROS in addition

to the exogenous oxidants introduced by t-BOOH and oxLDL (Fischer, von

Knethen et al. 2002; Shatrov and Brune 2003; Zmijewski, Moellering et al. 2005).

A sub-lethal oxLDL exposure induces NADPH-oxidase to produce endogenous

H2O2 (Heinloth, Heermeier et al. 2000), which has gained attention for its role in

regulating signal transduction. As a result, the ROS assayed in these

experiments include those introduced exogenously by t-BOOH and oxLDL, and

those induced endogenously as a response to cellular stress. With these

considerations in mind, there remains a direct correlation between the induction

of Prx I, the generation of ROS and the resulting cytotoxicity. These data show that the induction of Prx I prior to foam cell formation provides a protective role through its ability to reduce ROS levels generated during oxLDL-uptake. These results provide a mechanistic link between the cytoprotective effects of Prx I induction and the enzymatic activity of Prx I to metabolize, and thereby decrease, the amounts of intracellular ROS. In both cases, pre-treating the cells with ethoxyquin results in decreased oxidant-induced cytotoxicity and a corresponding decrease in oxidant-induced ROS. When the induction of Prx I is eliminated from the effect of ethoxyquin treatment, both toxicity and ROS levels increase.

Overall, these data describe an oxidant-protective role for Prx I in

macrophage-derived foam cells when the induction of Prx I occurs prior to oxLDL

exposure. Such a situation may occur if macrophages endure chronic exposure

136

to sub-lethal levels of oxLDL prior to foam cell formation, thus leading to enhanced foam cell survival (Fischer, von Knethen et al. 2002; Shatrov and

Brune 2003). Although several roles have been attributed to Prx I, this antioxidant functionality was the key to its discovery and remains the most commonly associated role (Netto, Chae et al. 1996; Peshenko, Novoselov et al.

1998). At the same time, however, the lack of a direct effect on oxLDL-induced toxicity in the Prx I-KD macrophages is also consistent with other activities for

Prx I, beyond the toxicity-protective antioxidant effects.

5.5 Prx I regulates p38 MAPK signaling in macrophage foam cells

A subsequent series of experiments were carried out to look at the role of Prx

I in oxidant-dependent signaling systems. Since the initial report describing the

ability of Prx I to associate with and inhibit c-Abl kinase activity (Wen and Van

Etten 1997), the interest in the peroxiredoxins as signal regulators has steadily

increased. Prx I has been described as a tumor suppressor due to its ability to

associate with a region of the c-Myc regulatory domain and inhibit c-Myc-

mediated transformation (Mu, Yin et al. 2002). However, most of the data

describing signal regulation by peroxiredoxin has focused on a different cytosolic

peroxiredoxin, Prx II. A role for Prx II as an endogenous ROS scavenger that

prevents the oxidant-induced deactivation of PTEN phosphatase has been

reported (Kwon, Lee et al. 2004). By positively regulating PTEN function, Prx II

prevents the downstream activation of pro-survival Akt kinase targets. Both

PTEN, and Prx II by association, are described as tumor suppressors for this

137

regulatory role. Additionally, Prx II has been shown to reduce endogenous H2O2

produced in response to TNF-alpha signaling, and thus limit the activation of the

JNK and p38 pathways while stimulating ERK activation (Kang, Chang et al.

2004). Regulation of the p38 MAPK pathway is directly linked to pro-atherogenic

factors. Exposing cells to oxLDL has been shown to activate the p38 MAPK

pathway and lead to oxLDL-induced toxicity (Jing, Xin et al. 1999). Additionally,

oxLDL-induced p38 MAPK activation has been linked to the downstream release

of the pro-inflammatory cytokines TNF-α and IL-1β (Han, Nicholson et al. 2001).

Recently, it was shown that Tpx1, the yeast homolog of Prx I, protects Sty1,

the yeast homolog of p38 MAPK, from oxidative inactivation (Veal, Findlay et al.

2004), but a similar role for Prx I has not been presented in mammalian cells.

Further, oxidative conditions that upregulate Prx I have not been studied to

determine the effect of differential Prx I expression on p38 MAPK activation.

Therefore, p38 MAPK activation was assayed to determine if the upregulation of

Prx I during macrophage foam cell formation could regulate p38 MAPK

activation.

The initial use of H2O2 to induce activation of p38 MAPK was based on the

previous results from yeast (Veal, Findlay et al. 2004). In our experiments, the

reduced expression of Prx I by siRNA knock-down coincided with a decrease in

H2O2-induced p38 MAPK phosphorylation. Conversely, ethoxyquin induction of

Prx I coincided with an increase in p38 MAPK activation that was nearly

eliminated by siRNA knock-down of Prx I expression. Finally, the Prx I-regulated

activation of p38 MAPK was studied under the conditions of macrophage foam

138

cell formation. In these experiments, exposure to oxLDL resulted in both the dose-dependent upregulation of Prx I and the activation p38 MAPK. Prx I siRNA

decreased the oxLDL-induced upregulation of Prx I and led to a highly

attenuated, but dose-dependent activation of p38 MAPK. When treated with 50

μg/mL oxLDL, activation of p38 MAPK was approximately 3-fold greater in

control macrophages compared to Prx I-KD macrophages. However, the

decrease in Prx I expression from control to Prx I-KD macrophages was much

larger than the corresponding decrease in p38 MAPK activation. This

observation contrasts with the ethoxyquin-induced activation of p38 MAPK,

where negligible activation was observed in Prx1-KD macrophages. These data

suggest that other factors may be induced during foam cell formation that

contribute to p38 MAPK activation, and are not induced by ethoxyquin-treatment.

A potential mechanism may exist where partial p38 MAPK activation occurs

when Prx I expression is limited. As a group, however, these data demonstrate

that p38 MAPK activation is largely dependent on Prx I.

5.6 Dual role of peroxiredoxin I in macrophage foam cells

Our data support a functional model in which Prx I has at least two distinct

roles ⎯ as an antioxidant enzyme and as a regulator of p38 MAPK. These data

describe the regulation of p38 MAPK activity as a function that is separate from

the antioxidant activity of Prx I. However, an alternative mechanism may exist in which the Prx I-induced activation of p38 MAPK is indirectly responsible for the increased survival and decreased ROS levels reported following Prx I induction.

139

Thus, the antioxidant functionality of Prx I was tested in the absence of p38

MAPK activity. In sets of macrophages treated with t-BOOH, ROS levels were decreased in macrophages that underwent ethoxyquin-induced Prx I- upregulation, and the inhibition of p38 activity did not affect this reduction in

ROS. Therefore, p38 MAPK activity is functionally separate from the antioxidant capacity of Prx I, implying a dual-role for Prx I in this system.

The antioxidant functionality of Prx I was demonstrated in this work to be cytoprotective. However, the antioxidant activity of Prx I may not be as much pro-survival as it is anti-necrosis. One could speculate that the antioxidant role of

Prx I helps to maintain macrophage foam cells in the atherosclerotic lesion by preventing an undesirable necrotic cell death. At the same time, Prx I also maintains the activation of p38 MAPK, providing the option for the downstream initiation of apoptosis. This concept overlaps with observations made for cancerous cells in tumors, where Prx I upregulation is well-documented. In both cancerous cells and foam cells, the cells have transformed to a pathological state where an apoptotic death may be a more beneficial result. Prolonged tumor cell survival leads to tumor growth and metastasis. Similarly, prolonged foam cell survival is pro-atherogenic by promoting a continued inflammatory response with its downstream effects. Further study of Prx I expression and function will continue to resolve the roles of this enzyme in these downstream effects.

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Chapter 6

Future Directions

6.1 Targeting the pro-atherogenic foam cell in atherosclerosis

prevention

The research presented here supports the premise that an increase in antioxidant defense is induced during foam cell formation, and this response may support foam cell survival. These results are presented as data from a single cell line, the J774 murine macrophage. Further investigation should extend into alternative macrophage cell lines to verify results obtained in the J774 cells, verifying the legitimacy of this data in other types of macrophages. Experiments involving mouse models would then expand upon this data. While a Prx I knockout (KO) mouse is available, it has not been crossed with the atheroma-prone

apoE-KO mouse. This transgenic mouse cross may confirm the in vitro data presented here, and provide new insight into the role of Prx I during lesion development.

Considering that foam cell formation is associated with pro-atherogenic

responses, one may speculate that the preservation of foam cells in the lesion

promotes the continued development of atherosclerosis. Eventually, foam cells

in the lesion die, but the current view of foam cell death describes a necrotic

event that perpetuates pro-inflammatory signaling and results in the deposition of

lipid to the developing plaque (Klurfeld 1985; Axel, Brehm et al. 1996). Thus,

data describing the response of macrophages to oxLDL suggest that both foam

cell survival and death contribute to the progression of atherosclerosis.

141

A preferred fate for the macrophage foam cell would occur prior to fatty streak

formation and through a controlled, apoptotic process. If an apoptotic response

were initiated early in the lipid-loading process, it may prevent fatty streak

formation and the pro-inflammatory response that is so critical to the

development of atherosclerosis. Researchers have demonstrated oxLDL-

induced apoptosis in cultured macrophages (Reid, Mitchinson et al. 1993),

however this response does not appear to dominate in vivo (Pratico, Iuliano et al.

1997; Arai, Shelton et al. 2005). Other researchers have demonstrated an

inhibition of apoptosis following macrophage exposure to aggregated LDL and

oxLDL (Yang, Galeano et al. 1996; Kubo, Kikuchi et al. 1997). Data presented in

this report support the inhibition of apoptosis in macrophage foam cells through the downregulation of 2’,5’-oligoadenylate synthetase mRNA (Ghosh, Sarkar et

al. 2001). When apoptosis is blocked in oxLDL-treated macrophages, a necrotic

death ensues (Salvayre, Auge et al. 2002). The sum of this data suggests that,

although oxLDL may promote apoptosis in cultured macrophages, other factors

that inhibit apoptosis may also be induced by oxLDL. This outcome is certainly

possible due to the heterogenous nature of oxLDL.

The combination of apoptotic inhibition and eventual foam cell necrosis

potentially contribute to the pro-atherogenic nature of macrophage foam cells. If

oxLDL-induced apoptosis is inhibited in macrophage foam cells in vivo, then one

might hypothesize that a reversal of apoptotic inhibition may slow fatty streak

formation, prevent lipid deposition and, perhaps most importantly, reduce the pro-inflammatory response of the foam cell. Future studies should characterize

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the inhibition of apoptosis in macrophage foam cells, and develop methods to

reverse this inhibition in the atheroma-prone apoE knockout mouse. This model

system can be used to determine the downstream effect of oxLDL-induced

apoptosis, rather than necrosis, on inflammatory response and lesion

development.

Whether oxLDL-exposed macrophages undergo apoptosis or necrosis, the

amount of internalized lipid may still pose a problem by contributing to lipid

accumulation in the arterial intima. Thus, it is critical that cellular processes for

cholesterol efflux are fully functional, allowing for the transport of internalized

cholesterol to high-density lipoprotein (HDL). The induction of cholesterol efflux

processes may limit foam cell formation (Bielicki, McCall et al. 1999), but in the

event of foam cell formation, treatments that allow for oxLDL-induced apoptosis

will prevent a pro-inflammatory response.

The oxidation of LDL, which prevents uptake by LDL receptors, may be a

process that regulates the local LDL environment by tagging excess LDL for

degradation. The macrophage response to oxLDL is likely a necessary process

for LDL clearance, but the combination of genetic and environmental factors have elevated LDL and oxLDL to pathological levels. The multiple risk factors for atherosclerosis, the slow development of the atheroma, and the differentiation between physiological and pathological responses come together to create a complex disease. A significant amount of research is focused on stabilizing advanced atheromas to prevent rupture, and subsequent heart attack and stroke.

However, future studies that focus on an early intervention at fatty streak

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formation are necessary to develop measures that prevent atheroma

development. The macrophage foam cell will be a key target for therapeutic

intervention.

6.2 Functional roles for peroxiredoxin I in macrophage foam cells

Peroxiredoxin proteins are often upregulated in oxidant-stressed systems,

and are described for their ability to reduce and detoxify peroxide-related

oxidants. The research presented in this report is consistent with past data, as it

describes the upregulation of Prx I in macrophage foam cells and demonstrates

an antioxidant functionality for Prx I through a reduction in ROS. However, the

regulation of p38 MAPK activation by Prx I was also established by the current

data, which prompted the proposal of a dual-functional role for Prx I.

In the strictest terms, the only molecular function displayed by Prx I is its

reductive capacity. Prx I forms a stable disulfide by donating an electron to an

accepting molecule. This acceptor may be a toxic and reactive oxidant, such as

hydrogen peroxide or a lipid peroxide, or it may be a free cysteine on a nearby

protein. The regulation of p38 MAPK activity by Prx I is thought to occur through

the formation of an intermolecular disulfide between Prx I and p38 MAPK (Veal,

Findlay et al. 2004), although this mechanism requires further investigation. Veal

et al propose that the intermolecular disulfide protects p38 MAPK from cysteine oxidation that would otherwise structurally modify and inactivate p38 MAPK. If

this is true, Prx I may have a similar role in the regulation of other thiol-sensitive proteins. Thus, the physiological role of Prx I becomes multi-faceted. As the

144 topic of oxidant-regulated signaling gains interest, the contribution of Prx I to these regulatory mechanisms will become more fully characterized. In addition to the regulatory targets of Prx I, researchers will need to determine the role of

Prx I upregulation and how it may predict the targets of Prx I regulation. One possibility is that base level Prx I interacts with various target proteins and endogenous oxidants to regulate steady-state signaling events. However, in a stress-activated system, Prx I may be upregulated to reduce the elevated oxidant levels and also protect key stress-activated proteins from oxidative-inactivation.

Regardless, there is much to learn about Prx I, as well as the other peroxiredoxin proteins, as research moves forward.

6.3 A parallel role for peroxiredoxin in cancer and atherosclerosis

Research publications that describe a role for peroxiredoxin in atherosclerosis are increasing, but are still few in number compared to the number of publications that examine the role of peroxiredoxin in cancer. Indeed, it was cancer-related research that generated key questions in this project by drawing parallels to peroxiredoxin and its contribution to foam cell formation. Why are peroxiredoxin proteins upregulated in so many different types of cancer, what is the functional effect of induced peroxiredoxin in tumor cells, and how does that function relate to its upregulation in macrophage foam cells?

Peroxiredoxin has been described as a tumor suppressor due to its inhibition of c-Abl kinase and c-Myc induced tumorigenesis (Mu, Yin et al. 2002). Recent evidence supporting its regulation of p38 MAPK activation is consistent with

145

tumor suppression. Data presented by us and Veal et al show that p38 MAPK activation is directly related to Prx I expression. Therefore, in cells that exhibit elevated Prx I, p38 MAPK activation can potentially be elevated. Activation of p38 MAPK has been linked to the downstream initiation of Prx I gene expression in macrophages (Hess, Wijayanti et al. 2003), as well as the posttranscriptional regulation of Prx I expression in osteoblasts (Li, Ishii et al. 2002). Accordingly, a

feed-forward mechanism may exist in which Prx I maintains p38 activity, resulting

in a continued increase in Prx I expression and a sustained p38 MAPK response.

The intent of this response may be directed toward the initiation of apoptosis,

considering the several pro-apoptotic transcription factors that are targets of p38

MAPK phosphorylation. In support of this proposal, the pro-survival Akt pathway

was not activated in our experiments following treatment with ethoxyquin or

oxLDL. Therefore, Prx I may assume the role of tumor suppressor by

contributing to a downstream apoptotic response, which may be defective in

cancer cells with elevated Prx I. A sustained upregulation of Prx I and activation

of p38 MAPK may indicate an impaired apoptotic response downstream of p38

MAPK. One potential point of defect could be p53, a substrate of p38 MAPK that

is mutated in over 50% of several human forms of cancer (Marks, Davidoff et al.

1991; Fujimoto, Yamada et al. 1992; Lee, Shew et al. 1994; Yasunobu, Hayashi

et al. 2001).

If one considers the inflammatory response of atherosclerosis to result in

what is effectively a cancer of the artery, then the upregulation of Prx I may serve the same pro-apoptotic role in macrophage foam cells. Within the scope of our

146

project, this role was not observed. However, in the lesion environment, future studies may link elevated Prx I and p38 MAPK activity to a decrease in fatty streak formation.

If Prx I functions as an antioxidant, does this cytoprotective role run contrary

to a pro-apoptotic response? That may not necessarily be the case if the

oxidative insult is strong enough to cause a necrotic cell death. A model could

be proposed in which Prx I maintains exogenously-introduced ROS levels to prevent a necrotic cell death (Fig. 18). In parallel, Prx I would protect p38 MAPK from oxidative inactivation, allowing for a pro-apoptotic pathway to progress through the p38 MAPK pathway. This response may not be observed in oxLDL- exposed macrophages due to an inhibition of apoptotic factors by specific components of oxLDL.

While this model is purely speculative, it attempts to link the various

contributions of Prx I into a unified response. Continued research will develop

this model and test the importance of peroxiredoxin, and likely other antioxidants,

in the regulation of major signal pathways.

6.4 Figures

147

Figure 18: Proposed model for the dual role of Prx I in macrophage foam cells. Foam cell formation induces Prx I expression, which maintains oxidant levels that may otherwise lead to a necrotic cell death. Additionally, Prx I regulates the activation of p38 MAPK, which may induce a pro-apoptotic pathway. The sum of these events would attenuate atherogenic progression by

reducing inflammatory response triggered by macrophage foam cell signaling

and oxidant-induced necrosis.

148

149

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