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Macrophage Migration Inhibitory Factor and Thioredoxin in the Brain

Permanent Link: http://ufdc.ufl.edu/UFE0022514/00001

Material Information

Title: Macrophage Migration Inhibitory Factor and Thioredoxin in the Brain Implications for Hypertension
Physical Description: 1 online resource (89 p.)
Language: english
Creator: Harrison, Rachael
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: angiotensin, hypertension, mif, trx
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Previous studies from our laboratory have established that macrophage migration inhibitory factor serves as a negative regulator of the neuronal chronotropic actions of angiotensin II in normotensive rats, but not in spontaneously hypertensive rats. Furthermore, hydrogen peroxide is a known effector of angiotensin II signaling in paraventricular hypothalamic neurons and is an established inducer of macrophage migration inhibitory factor expression in the periphery. Therefore, for the first study contained in this dissertation, we hypothesized that hydrogen peroxide may be able to induce macrophage migration inhibitory factor expression in neurons, and we sought to ascertain whether normotensive and spontaneously hypertensive neurons respond differentially to hydrogen peroxide stimulation with regard to expression of this small protein. We examined the effects of hydrogen peroxide (30 ?mol/L) on macrophage migration inhibitory factor expression in neuronal cultures from normotensive (Wistar Kyoto and Sprague Dawley) and spontaneously hypertensive newborn rats. The data indicate that hydrogen peroxide induces macrophage migration inhibitory factor expression in neurons cultured from normotensive rats, but not spontaneously hypertensive rats. Lactate dehydrogenase and protein carbonyl assays suggest that 30 ?mol/L hydrogen peroxide is neither cytotoxic to the neurons, nor does it cause oxidative stress. Studies with polyethylene glycol-catalase and actinomycin D suggest that the hydrogen peroxide is acting intracellularly to increase transcription of the macrophage migration inhibitory factor gene. We conclude that macrophage migration inhibitory factor expression is regulated differentially in normotensive and spontaneously hypertensive rat neurons in response to hydrogen peroxide signaling. Furthermore, oxidative stress has become an exciting field of study with regard to neurogenic hypertension. However, it remains to be determined if oxidative stress in the brain contributes to the development of or is a consequence of this disease. The second study contained in this dissertation examines the expression and cellular localization of two important antioxidant proteins, macrophage migration inhibitory factor and thioredoxin, in the hypothalamic paraventricular nucleus of spontaneously hypertensive rats and their normotensive controls, Wistar Kyoto rats. Importantly, these studies were performed in newborn (i.e., not yet hypertensive) rat brains. Our studies establish dysregulation of expression of these two proteins in the brain of newborn rats that are destined to be hypertensive and associates them with oxidative stress that occurs well before the onset of hypertension.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Rachael Harrison.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Sumners, Colin.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022514:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022514/00001

Material Information

Title: Macrophage Migration Inhibitory Factor and Thioredoxin in the Brain Implications for Hypertension
Physical Description: 1 online resource (89 p.)
Language: english
Creator: Harrison, Rachael
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: angiotensin, hypertension, mif, trx
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Previous studies from our laboratory have established that macrophage migration inhibitory factor serves as a negative regulator of the neuronal chronotropic actions of angiotensin II in normotensive rats, but not in spontaneously hypertensive rats. Furthermore, hydrogen peroxide is a known effector of angiotensin II signaling in paraventricular hypothalamic neurons and is an established inducer of macrophage migration inhibitory factor expression in the periphery. Therefore, for the first study contained in this dissertation, we hypothesized that hydrogen peroxide may be able to induce macrophage migration inhibitory factor expression in neurons, and we sought to ascertain whether normotensive and spontaneously hypertensive neurons respond differentially to hydrogen peroxide stimulation with regard to expression of this small protein. We examined the effects of hydrogen peroxide (30 ?mol/L) on macrophage migration inhibitory factor expression in neuronal cultures from normotensive (Wistar Kyoto and Sprague Dawley) and spontaneously hypertensive newborn rats. The data indicate that hydrogen peroxide induces macrophage migration inhibitory factor expression in neurons cultured from normotensive rats, but not spontaneously hypertensive rats. Lactate dehydrogenase and protein carbonyl assays suggest that 30 ?mol/L hydrogen peroxide is neither cytotoxic to the neurons, nor does it cause oxidative stress. Studies with polyethylene glycol-catalase and actinomycin D suggest that the hydrogen peroxide is acting intracellularly to increase transcription of the macrophage migration inhibitory factor gene. We conclude that macrophage migration inhibitory factor expression is regulated differentially in normotensive and spontaneously hypertensive rat neurons in response to hydrogen peroxide signaling. Furthermore, oxidative stress has become an exciting field of study with regard to neurogenic hypertension. However, it remains to be determined if oxidative stress in the brain contributes to the development of or is a consequence of this disease. The second study contained in this dissertation examines the expression and cellular localization of two important antioxidant proteins, macrophage migration inhibitory factor and thioredoxin, in the hypothalamic paraventricular nucleus of spontaneously hypertensive rats and their normotensive controls, Wistar Kyoto rats. Importantly, these studies were performed in newborn (i.e., not yet hypertensive) rat brains. Our studies establish dysregulation of expression of these two proteins in the brain of newborn rats that are destined to be hypertensive and associates them with oxidative stress that occurs well before the onset of hypertension.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Rachael Harrison.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Sumners, Colin.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022514:00001


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467f8bb8d42d38ff9171713ffc42cc83
7df7c97004909edc0dde73f0e7b32b9709286e2f







MACROPHAGE MIGRATION INHIBITORY FACTOR AND THIOREDOXIN IN THE
BRAIN: IMPLICATIONS FOR HYPERTENSION




















By

RACHAEL ANNE HARRISON


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2008


































2008 Rachael Anne Harrison



































To my husband, Brad. Your constant support and encouragement have made this possible.









ACKNOWLEDGMENTS

I must begin by acknowledging my husband. He has stood by me and held me up through

the most difficult of times. Without his constant humor and perspective, there would often have

been little laughter through the tears.

Secondly, I must thank my mentor. He has encouraged me when I did not believe in

myself, and picked me up to put one foot in front of the other when I wanted to falter. Without

him, I most certainly would not have made it through graduate school. I have learned from him

important lessons, not just about science, but about life and about my own capabilities. I can

never thank him enough for his service as my mentor.

I must also thank my committee members for their service. Their guidance has proven

invaluable, and their advice was always given generously, patiently, and kindly. I acknowledge

my lab mates for their assistance with technical issues and their constant companionship. I

consider them esteemed colleagues and also friends.

Lastly, I thank my family and friends. My experiences with them have made me into the

person I am today, and everything that I can be proud in my life of is a result of their support and

encouragement.









TABLE OF CONTENTS


page

A CK N O W LED G M EN T S ...................................................... ..............................................

L IS T O F T A B L E S ............. ..... ............ ................. ............................ ............... 7

LIST OF FIGURES ................................. .. ..... ..... ................. .8

A B S T R A C T ............ ................... ............................................................ 10

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ............................................................12

Characteristics of Macrophage Migration Inhibitory Factor...............................................12
Introduction to the Thioredoxin Family of Proteins................ ....... ...............16
Characteristics and Functions of Thioredixon 1: A Comparative Study .............................17
Macrophage Migration Inhibitory Factor and Thioredoxin in the Brain............................. 21
The Brain Renin-Angiotensin System ........................................................ ............. 23
Reactive Oxygen Species, Neuronal Function, and Hypertension..................................25
Macrophage Migration Inhibitory Factor and Thioredoxin as Negative Regulators of
Angiotensin II in the Central Nervous System ....................................... ............... 28

2 INDUCIBLE EXPRESSION OF MACROPHAGE MIGRATION INHIBITORY
F A C T O R IN N E U R O N S ............................................................................ .....................36

Intro du action ................... .......................................................... ................ 3 6
M materials and M methods ................................... ... .. .......... ....... ...... 37
R results ................................ ...... ........ . ............. ...... .... .... .......................... 39
Hydrogen Peroxide Stimulates an Increase in Macrophage Migration Inhibitory
Factor in Neurons Cultured from Normotensive Rats, but not Spontaneously
H ypertensive R ats. .................. ... .. ..... .... ....................... .... ......... ........... .... 39
Hydrogen Peroxide Increases Macrophage Migration Inhibitory Factor mRNA
Expression in Primary Neurons Through a Specific Intracellular Action .................41
The Increase in Macrophage Migration Inhibitory Factor Levels Observed in the
Presence of Hydrogen Peroxide Involves Increased Synthesis of Macrophage
M igration Inhibitory Factor mRNA ................................................................ ....... 42
D isc u ssio n ................... ........................................................... ................ 4 3

3 MACROPHAGE MIGRATION INHIBITORY FACTOR AND THIOREDOXIN IN
THE BRAIN AND OXIDATIVE STRESS ........................................ ....................... 55

Introduction ................. ......................................... ............................55
M materials and M methods ................................... ... .. .......... ....... ...... 57
R e su lts ............... ........ .................. .............. .................................... 5 9









Macrophage Migration Inhibitory Factor and Thioredoxin Expression is Lacking in
Spontaneously Hypertensive Rat Hypothalamus.....................................................59
Newborn Spontaneously Hypertensive Rats Exhibit More Oxidative Stress in the
Hypothalamus and Brainstem than Wistar Kyoto Rats. .........................................60
Macrophage Migration Inhibitory Factor is Absent from Paraventricular Nucleus
Neurons of Spontaneously Hypertensive Rats.........................................................61
D isc u ssio n ................... ........................................................... ................ 6 1

4 CONCLUSIONS AND FUTURE DIRECTIONS ...................................... ............... 72

Inducible Expression of Macrophage Migration Inhibitory Factor in Neurons and
A ngiotensin II ........................................... ...... .... .............. .......... ............ .73
Mechanisms of Redox Regulation of Macrophage Migration Inhibitory Factor
E expression .............................. ..... .......... .............. ............................... 74
Dysregulation of Macrophage Migration Inhibitory Factor and Thioredoxin Expression
in the Hypothalamus of Spontaneously Hypertensive Rats..............................................76
Oxidative stress in the Hypothalamus of Spontaneously Hypertensive Rats.........................77
Physiological Im plications........ ................................................................ ....... ..... 78

L IST O F R E F E R E N C E S ............................................... ................................. .........................79

B IO G R A PH IC A L SK E T C H .............................................................................. .....................89










LIST OF TABLES


Table


2-1 Hydrogen peroxide (30 [tmol/L) does not elicit cytotoxic effects in primary neuronal
cultures......... ........................ ..................................... .......... ...... 52


page









LIST OF FIGURES


Figure page

1-1 Features of macrophage migration inhibitory factor and thioredoxin. ............................ 31

1-2 Catalytic mechanism of the thioredoxin system. .......................... ........................32

1-3 The renin-angiotensin system ................................................ ............................... 33

1-4 Angiotensin II-induced reactive oxygen species production.........................................34

1-5 Model of possible interactions between angiotensin II signaling and macrophage
m migration inhibitory factor........................................................... .. ............... 35

2-1 Hydrogen peroxide increases macrophage migration inhibitory factor mRNA levels
in primary neuronal cultures from normotensive rats, but not spontaneously
hypertensive rats .................. ............. .................... ........... 47

2-2 Hydrogen peroxide increases macrophage migration inhibitory factor protein levels
in prim ary neuronal cultures. ...... ........................... .......................................... 48

2-3 Glucose oxidase increases macrophage migration inhibitory factor mRNA levels in
prim ary neuronal cultures ......................................................................... ...................49

2-4 Hydrogen peroxide does not increase macrophage migration inhibitory factor mRNA
expression in prim ary glial cell cultures. .......................................................................... 50

2-5 Hydrogen peroxide acts intracellularly to elicit an increase in macrophage migration
inhibitory factor expression in primary neuronal cultures..............................................51

2-6 30 tmol/L hydrogen peroxide does not alter protein carbonyl formation.........................53

2-7 Hydrogen peroxide-induced increases in macrophage migration inhibitory factor
expression involve a transcriptional event.................................................................. 54

3-1 Single nucleotide polymorphisms of the putative macrophage migration inhibitory
factor promoter in spontaneously hypertensive rats .................................. ............... 65

3-2 Macrophage migration inhibitory factor and thioredoxin mRNA expression is
reduced in spontaneously hypertensive rat hypothalamus...............................................66

3-3 Macrophage migration inhibitory factor and thioredoxin protein expression is lower
in newborn spontaneously hypertensive rat hypothalamus ............................................67

3-4 Macrophage migration inhibitory factor and thioredoxin protein in newborn
b rain stem ............ ......... .. ..................................................................... 6 8









3-5 Protein carbonyl concentration is greater in spontaneously hypertensive rat
hypothalam us than in W istar Kyoto ............................................................................ 69

3-6 Spontaneously hypertensive rat paraventricular nucleus neurons contain less
m acrophage m igration inhibitory factor ........................................ ........................ 70

3-7 Thioredoxin cellular distribution is normal in spontaneously hypertensive rat
paraventricular nucleus ...................... .................... .. .. .......................71









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

MACROPHAGE MIGRATION INHIBITORY FACTOR AND THIOREDOXIN IN THE
BRAIN: IMPLICATIONS FOR HYPERTENSION

By

Rachael Anne Harrison

August, 2008

Chair: Colin Sumners
Major: Medical Sciences Physiology and Pharmacology

Previous studies from our laboratory have established that macrophage migration

inhibitory factor serves as a negative regulator of the neuronal chronotropic actions of

angiotensin II in normotensive rats, but not in spontaneously hypertensive rats. Furthermore,

hydrogen peroxide is a known effector of angiotensin II signaling in paraventricular

hypothalamic neurons and is an established inducer of macrophage migration inhibitory factor

expression in the periphery. Therefore, for the first study contained in this dissertation, we

hypothesized that hydrogen peroxide may be able to induce macrophage migration inhibitory

factor expression in neurons, and we sought to ascertain whether normotensive and

spontaneously hypertensive neurons respond differentially to hydrogen peroxide stimulation with

regard to expression of this small protein.

We examined the effects of hydrogen peroxide (30 [mol/L) on macrophage migration

inhibitory factor expression in neuronal cultures from normotensive (Wistar Kyoto and Sprague

Dawley) and spontaneously hypertensive newborn rats. The data indicate that hydrogen peroxide

induces macrophage migration inhibitory factor expression in neurons cultured from

normotensive rats, but not spontaneously hypertensive rats. Lactate dehydrogenase and protein

carbonyl assays suggest that 30 [mol/L hydrogen peroxide is neither cytotoxic to the neurons,









nor does it cause oxidative stress. Studies with polyethylene glycol-catalase and actinomycin D

suggest that the hydrogen peroxide is acting intracellularly to increase transcription of the

macrophage migration inhibitory factor gene. We conclude that macrophage migration inhibitory

factor expression is regulated differentially in normotensive and spontaneously hypertensive rat

neurons in response to hydrogen peroxide signaling.

Furthermore, oxidative stress has become an exciting field of study with regard to

neurogenic hypertension. However, it remains to be determined if oxidative stress in the brain

contributes to the development of or is a consequence of this disease. The second study

contained in this dissertation examines the expression and cellular localization of two important

antioxidant proteins, macrophage migration inhibitory factor and thioredoxin, in the

hypothalamic paraventricular nucleus of spontaneously hypertensive rats and their normotensive

controls, Wistar Kyoto rats. Importantly, these studies were performed in newborn (i.e., not yet

hypertensive) rat brains. Our studies establish dysregulation of expression of these two proteins

in the brain of newborn rats that are destined to be hypertensive and associates them with

oxidative stress that occurs well before the onset of hypertension.









CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW

Characteristics of Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) (Figure 1-1A) is a small (12.5 kDa), highly

conserved protein with nearly ubiquitous tissue distribution that was originally described in 1966

as a soluble factor that was expressed by T cells in delayed type hypersensitivity and inhibited

the random migration of macrophages.1-3 The factor behind this activity was not cloned and

identified as MIF until 1989,1 and, when MIF was originally characterized, it was designated a

lymphokine (later commuted to the more modern and inclusive term "cytokine"). Structurally,

MIF does not have any notable domains4 (though it does have some enzymatic motifs that will

be discussed in detail later), and it is still not clear whether the MIF homotrimer, the entity

identified in all of the structural studies, is the functionally relevant form. On the contrary,

several studies suggest that the dimer or the monomer may be responsible for the actions of MIF

in vivo.5-7

Today, determining a functional niche for MIF is far more complicated than it was 40

years ago. It has been suggested that MIF is a cytokine, a chemokine, a hormone, and an enzyme,

and the arguments for all of these designations are compelling.8 9 Clearly MIF has many,

sometimes seemingly disparate, biological and cellular functions. Physiologically, MIF plays

modulatory roles in the immune, endocrine, and nervous systems.10 Pathologically, when

dysregulated, MIF contributes to a wide range of inflammatory disorders and plays a pivotal role

in tumor biology.11, 12

MIF seems to be somewhat of a "moon-lighting" protein in that its functions can be

divided into two basic categories. It is not yet clear, but we hypothesize that this may depend on

whether it is secreted to initiate signal transduction (cytokine-like) or remains in the intracellular









space (enzymatic, perhaps antioxidant). Much of the traditional literature focuses on the pro-

inflammatory, cytokine-like function of MIF. MIF is considered to be rather an orphan cytokine,

or perhaps the first discovered member of a new cytokine family, since it bears no similarity to

other cytokine families,1 nor does its receptor, a complex consisting of CD74 and CD44. CD74 is

necessary for MIF binding, and CD44 appears to responsible for initiating intracellular signaling

cascades.13

Yet, clearly MIF can function as a pro-inflammatory mediator, and the overwhelming

majority of the literature supports this notion. It can be secreted, presumably via a leaderless

pathway,14 by many types of immune, endocrine, and parenchymal cells,15' 16 and it is well-

established that secreted MIF is a critical player in inflammatory conditions such as sepsis,

rheumatoid arthritis, asthma, cystic fibrosis, atherosclerosis, and glomerulonephritis to name

only a few.17-20 It should be noted that MIF appears to be acting as a chemokine, signaling via

chemokine receptors rather than it's recently-identified (and assumed to be canonical)

CD74/CD44 receptor complex, in the instance of atherosclerosis.9

However, MIF is distinct from other traditional cytokines in many interesting ways.

Evidence suggests that it is constitutively expressed and exists in pre-formed pools in the cytosol

of many immune and non-immune cells.15'21 In contrast, traditional cytokines are usually

produced de novo, with a lag time for transcription and translation, when induced.22 Furthermore,

as previously mentioned, the MIF peptide does not appear to contain a leader sequence, exists as

an entity free of secretary vesicles in the cytoplasm of cells, and is most likely secreted in a

regulated fashion by a non-traditional route, rather than going through a traditional secretary

pathway. Exactly how MIF is secreted is not yet fully understood, but the limited evidence

available suggests it is most likely via a transporter belonging to the ATP-binding cassette









(ABC) family.14 MIF is also unique in that its secretion is induced by glucocorticoids, and it

plays a critical role as a circulating counter-regulator of the immunosuppressive effects of

glucocorticoids. MIF is able to promote inflammatory responses despite physiological levels of

serum glucocorticoids, and it has been suggested that permitting inflammatory responses when

appropriate in the face of glucocorticoids which have many homeostatic functions, aside from

their immunosuppressive actions is precisely the intended purpose and physiological role of

secreted MIF. Concordant with this suggested anti-glucocorticoid role, under normal conditions

glucocorticoid-induced MIF secretion follows a bell-shaped dose-response curve, with MIF

secretion peaking at physiological glucocorticoid levels, and recent studies suggest that MIF

fluctuates in a circadian rhythm that is correlated to serum glucocorticoid levels in rodents.23

Aside from its classification as a cytokine, in recent years, a revolutionary group of

investigators have discovered that MIF is also an enzyme, exhibiting a thiol-protein

oxidoreductase (TPOR) activity that lies between amino acid residues 57 and 60.24 Accordingly,

it has recently been suggested that MIF be re-classified as not only a cytokine, but also a member

of the thioredoxin (Trx) family of antioxidant proteins due to the fact that it contains this TPOR

motif, an identifying characteristic of Trx family members.25 However, it should be noted that

there are cysteine-dependent, redox-active enzymes that are not considered members of the Trx

family, as well. Peroxiredoxins, enzymes that contribute to the regulation of cellular signaling by

scavenging and, therefore, controlling the intracellular levels of hydrogen peroxide an

important signaling molecule at the expense of reducing equivalents donated by Trx, are one

such example.26 Should future investigations and further clarification of the functions and

characteristics of MIF (which has yet to be crystallized as a monomer or homodimer) determine

that it is not a Trx-family member, it may be possible that MIF, like peroxiredoxin 1, is the









canonical member of a new family of TPOR-dependent enzymes (and, perhaps, these enzymes

could be assigned to a larger family designated simply as the TPOR family, as was suggested by

Thiele and Bernhagen in their recent review on the enzymatic activities of MIF). This would

hardly be surprising, given the fact that researchers working on the secreted, cytokine-like

function of MIF have proposed that the same could be said of MIF as a cytokine. Efforts to

assign it to a cytokine family have failed, yet the evidence clearly shows that it can serve this

function.

Nonetheless, assuming the proposition that MIF belongs in the Trx family is correct, as

one would expect, there is limited evidence that the production of MIF can be influenced by the

redox status of the cell.25 Because of this proposed dichotomous nature of MIF, many authors

have come to refer to it affectionately as a "cytozyme" or "redoxkine". Members of the

thioredoxin family are essential to maintaining redox balance in the intracellular environment by

scavenging of reactive oxygen species (ROS), act as electron donors for reducing enzymes, and

they may also influence signaling pathways and the activity of other proteins by reducing critical

cysteines, as will be discussed in detail later; and the available evidence, though tentative

compared to the body of literature concerning these functions of Trx, is ever-mounting and

suggests that MIF is no exception in this regard.25'27

To reconcile these seemingly disparate functions of MIF, great minds in the MIF field

suggested several years ago that perhaps whether MIF acts as a pro-inflammatory cytokine or an

antioxidant protein may depend on the concentration of MIF in the tissue, with relatively low

concentrations acting as a pro-inflammatory mediator and higher concentrations serving an

antioxidant function.28 While the circumstances that determine whether MIF fulfills its cytokine

or TPOR-based enzymatic function remain a mystery, a growing body of evidence indicates that









the simplest explanation is that something as simple as localization may be the determining

factor. Perhaps, when MIF is needed as a pro-inflammatory mediator, it is secreted and signals

via its canonical or chemokine receptors. To further complicate matters, it has been suggested

that, even when secreted MIF is acting in its cytokine capacity, the TPOR motif of MIF might be

involved in mediating its association with the CD74 portion of its receptor complex.25

Conversely, in the absence of a stimulus that would promote secretion, MIF remains in the

cytoplasm of the originating cell, and studies from many laboratories, including ours, suggest

that it then acts as a TPOR protein and probable member of the Trx family, influencing redox-

regulated targets and perhaps even scavenging ROS.

Introduction to the Thioredoxin Family of Proteins

Thioredoxin (Trx) (Figure 1-1B) was first discovered in the 1960s coincidentally,

around the same time as MIF as a hydrogen donor for deoxyribonucleotide synthesis in E.

coli.29 Contrary to the long history in prokaryotes, interest in the eukaryotic Trxs is a relatively

new matter, taking shape over the past couple of decades. The following sections will focus on

mammalian Trxs, specifically Trxl, referred to here after simply as "Trx". As the characteristics

of Trx are discussed, I will point out ways in which Trx and MIF are alike to support the

argument that MIF may be a Trx family member but also ways in which MIF and Trx may be

different in order to present a balanced perspective. Interestingly, in some ways, MIF exhibits

characteristics more similar to glutaredoxin (Grx), the other major member of the Trx family.

When appropriate, similarities between MIF and Grx will also be discussed.

Before launching into a detailed discussion of Trx and its many interesting features, in

the interest of perspective, a brief review of Trx systems in mammals is warranted. There are two

distinct isoforms of Trx in mammals. Trxl is usually found in the cytoplasm of cells, 27 but can

be induced to localize to the nucleus or be exported from the cell under the correct stimuli.30' 31









Trx2 is a mitochondrial protein.32 The active site of Trxs becomes oxidized, forming a disulfide

bond between the active site cysteines, over the course of catalysis (Figure 1-2). They are

reduced, returning to the active form, (usually at the expense of NADPH) by Trx reductases

(TrxR), large selenocysteine enzymes with active sites consisting of the amino acid sequence

Gly-Cys-SeCys-Gly. There are 3 types of TrxRs: TrxR1 (cytoplasmic), TrxR2 (mitochondrial),

and the testis-specific thioredoxin glutathione reductase.27 The thioredixon 1 gene (TXN1) is

well-characterized and contains two overlapping promoters exhibiting elements for basal and

inducible regulation of Trx expression. Interestingly, Spl seems to be important in the basal

regulation of the Trx gene,33 and a recent publication has shown the same to be true for MIF.34

An important inducible cis element of the Trx gene that, so far, appears to be lacking in the

regulation of the MIF gene is the antioxidant response element (ARE), which is particularly

well-studied because it is responsible for the induction of Trx under conditions of oxidative

stress.30,35

Glutaredoxins (Grxs) are the other major well-known members of the Trx family. They

are further-removed from the purposes of this discussion than Trx, so will be reviewed in much

more brevity. They share some functions with Trx enzymes, but also have some independent

functions. Mammalian cells contain 3 Grx isoforms: the dithiol-mechanism Grxs known as Grxl

(cytosolic) and Grx2 (mitochrondrial), and the monothiol-mechanism Grx5 (named as such due

to homology to yeast Grx5, appears to be mitochondrial). Oxidized Grxs are reduced by

glutathione, which is then reduced by glutaredoxin reductases at the expense ofNADPH. Grx2 is

interesting in that it can also be reduced by TrxRs.27

Characteristics and Functions of Thioredixon 1: A Comparative Study

Trx family members have a characteristic CXXC motif (Figure 1-1), with X being any

amino acid, which is responsible for the redox enzymatic function of these proteins. Trx's active









site contains a Cys-Gly-Pro-Cys motif that was identified when the protein was first sequenced

in 1968.36 MIF follows this rule; as mentioned above it shares this identifying characteristic,

exhibiting a Cys-Ala-Leu-Cys motif in its active site. Importantly, MIF also shares some

additional conserved residues with other Trx family members: a phenylalanine that is 5 to 7

residues upstream of the N-terminal Cys of the CALC motif and a leucine/valine (a leucine in the

case of MIF) an additional 2 to 3 residues in the N-terminal direction.25

Though the structure of the MIF monomer is vaguely similar to the Trx monomer, one

important characteristic of the Trx family that MIF is lacking is the "thioredoxin fold" structural

motif, consisting of 4 beta sheets and 3 alpha helices in most family members. Trx contains this

basic thioredoxin fold, plus an additional beta sheet and alpha helix at the N-terminus.37 In

contrast, MIF is structurally more like bacterial tautomerases and human D-dopachrome

tautomerase, and MIF has a tautomerase enzymatic motif at its N-terminus, in-depth discussion

of which is beyond the scope of these studies. The importance of the tautomerase activity of MIF

is still unclear and remains controversial, as an in vivo substrate has yet to be identified.

Furthermore, there is little sequence homology between MIF and tautomerases, despite their mild

structural similarities.25 However, it is worth noting that MIF is certainly far from the only Trx-

like protein that has been discovered but may not fit perfectly into the "Trx mold". There are

many, usually tissue- or organelle-specific, proteins that contain CXXC motifs but deviate

somewhat from the classical Trx characteristics, yet are still considered to be Trx family

members; some of which are redox active, some of which are not.27

Nonetheless, there are hundreds of available solved Trx structures, and they reveal some

important characteristics of reduced and oxidized Trx. Trx undergoes some conformational

changes upon reduction that involve hydrogen bonds in the active site. These changes can affect









the binding of Trx to other proteins.38' 39 It has been argued that MIF may share this feature of

altered conformational states (and, perhaps, altered binding activity) depending on its redox

status.25 40 Biochemical studies of MIF indicate that none of its 3 cysteines form an

intermolecular disulfide bond, but that the cysteines of the CALC motif form an intramolecular

bond under the correct conditions (as would be expected of a 2-Cys mechanism Trx protein).24

Despite the available evidence to the contrary, the limited crystal structures of the MIF

homotrimer that are available seem to indicate some spatial constraints on the location of the two

cysteines of the CALC motif, with one lying at the N-terminus of a beta sheet and the other

located in the preceding loop, which would make an intermolecular disulfide unlikely. However,

as Thiele and Bernhagen pointed out in their excellent review on the TPOR activities of MIF, it

bears noting that the crystal structures currently available for MIF were obtained from solutions

containing MIF at unnaturally high concentrations under reducing conditions. It is possible that

this encouraged aggregation of MIF and pushed the equilibrium of the various oligomers towards

the homotrimer species.25 Interestingly, it would be appropriate to mention here that, like MIF,

Trx can form dimers (though this claim is controversial and the physiological consequences are

unknown)41,42 and that Grx2 (a Trx family member) can utilize iron-sulfur clusters to form a

bridge that effectively "homodimerizes" the molecule, rendering it enzymatically inactive.43 It

may, therefore, be possible that the formation of oligomers of these redox active enzymes could

serve as regulators of their redox activity, and perhaps even sensors of oxidative stress. Despite

the conclusions drawn from the 3D structures of the MIF homotrimer, biochemical solution

studies on MIF utilized MIF at more physiological concentrations and re-folded MIF under

oxidizing conditions, and the resulting MIF was biologically and redox active. From this, the

authors concluded that perhaps MIF, like Trx, undergoes major conformational changes during









catalysis that would be more permissive of intramolecular disulfides and have not yet been

detected by the limited crystal structures of MIF that are currently available.25

Furthermore, regarding interesting regulatory and structural characteristics of Trx, this

enzyme has at least 3 cysteines that lie outside the active site and are not involved directly in

catalysis: Cys62, Cys69, and Cys73. Cys62 and 69 can form an intramolecular disulfide bond

that is not reducible by TrxR and seems to decrease the rate of reduction of the active site by

TrxR, which may regulate recycling of Trx to the reduced state.44' 45 Cys73 also seems to be

important in regulating the functions of Trx. It can undergo reversible s-nitrosylation, s-

glutathionylation, or disulfide formation. The s-nitrosylation appears to be important in Trx's

ability to modulate caspase 3 activity.46 The glutathionylation of Cys73 renders the enzyme

inactive.47 Most intriguingly, Cys73 can form an intramolecular disulfide bond with the Cys73 of

another Trx monomer, leading to the formation of a Trx homodimer.48 Dimerization, as

previously mentioned, is reminiscent of MIF, though the function of dimerization of either

molecule remains a mystery.

Though little is known about and intense investigation still surrounds the possible

mechanisms of MIF catalysis and recycling, the mechanisms governing the catalysis and

reduction of Trx and most of its other family members are well-understood. Trx utilizes its active

site cysteines to catalyze the reduction of disulfides via a dithiol mechanism (in contrast to the

monothiol mechanism that can be utilized by Grxs). The initial binding of Trx to its targets is

governed by a hydrophobic area around the active site. Next, the thiolate of the N-terminal

cysteine attacks the target disulfide, which results in a mixed disulfide intermediate. This mixed

disulfide between Trx and its target is then reduced by the C-terminal thiolate of the Trx active









site. The result is a disulfide bond between the cysteines of the Trx CXXC motif, which, as

mentioned previously, is reduced by TrxR at the expense of NADPH.

Finally, regarding the functions of the Trx molecule, it is most well known as a redox

enzyme, but Trx also has functions that are presumed to be non-redox related, or at least are not

involved in what is classically thought of as redoxx regulation of cellular function". We now

know that Trx, like MIF, is a multi-functional molecule, serving at times in many capacities:

thiol redox control of transcription factors and enzymes, as an electron donor for many reductive

enzymes and peroxiredoxins, as a ROS scavenger and absolutely crucial antioxidant. Other,

more recently identified functions of Trx include a cytokine/chemokine-like function (when

secreted, interestingly similar to MIF in this regard) and roles in the regulation of protein folding,

apoptosis (like MIF, which negatively regulates p53-mediated apoptosis), and NO metabolism.27

Macrophage Migration Inhibitory Factor and Thioredoxin in the Brain

It has been demonstrated that MIF is present in the cell bodies and processes of both

central and peripheral neurons. In 1998, Bacher et al. assessed MIF distribution in the brain of

male sprague dawley rats utilizing various techniques such as immunohistochemistry and in situ

hybridization. They demonstrated the presence of MIF mRNA and protein in neurons of the

cortex, hippocampus, cerebellum, pons, and hypothalamus. The authors also reported a diffuse

MIF signal localizing to glial cells throughout the brain.49

Several laboratories, including our own, have investigated the possible functions of CNS-

localized MIF. The aforementioned study showed that MIF can play an inflammatory role in the

CNS when its expression and secretion is induced upon central administration of LPS. However,

Bucala points out that the high constitutive expression levels of MIF in neurons argue for other,

non-inflammatory, physiological functions in the brain.10 Nishio et al. found that MIF may play

a role in regeneration of peripheral nerves.50 Another group has suggested that MIF participates









in detoxifying products of catecholamine metabolism, perhaps serving a neuroprotective

function.51 Also, the glucocorticoid-antagonism function of secreted MIF may be important in

protecting hippocampal neurons from glucocorticoid-induced atrophy in situations when these

steroids are elevated, such as chronic stress.10, 49 MIF may also have important functions in

modulating the release of cytokines and nitric oxide in the brain.52' 53 Finally, as will be discussed

later in detail, MIF can serve as a negative regulator of the central chronotropic actions of

angiotensin II (Ang II).54

The localization and functions of Trx in the brain are better understood than is the case

for MIF. This is due to the Trx system's status as one of the most important antioxidant defense

systems in neurons, which are highly metabolically active causing them greater exposure to

ROS than many other cell types. Moreover, neurons usually exhibit lower levels of other

important antioxidants, such as glutathione (GSH), than other tissues. Hence, the Trx system

may play a larger role in antioxidant defense in the CNS than it does systemically.55 Regarding

basal expression, Trx is found in neurons throughout the brain, while being mostly absent from

glial cells. A notable exception is glial cells of white matter.2

However, under conditions of acute stress, such as ischemia, glial cells are the main source

of induced Trx expression and secretion, consistent with their supportive role towards neurons.56'

57 Experiments in culture and in vivo show that secreted Trx can have neuroprotective effects.

Infusion of Trx, systemically, protects the brain from ischemic events in rodents.58 Furthermore,

when Trx secretion is induced from astrocytomas in cultures, the resulting conditioned medium

can be utilized to promote the survival of neuronal cultures in the absence of serum.27 Trx may

even be an indirect neurotrophic factor, mediating downstream effects of nerve growth factor

(NGF).59 However, dysregulation of the Trx system has been implicated in several









neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and CNS

malignancies.2 Finally, as has been indicated for MIF and will be discussed in detail later,

studies from our laboratory suggest that an intracellular function of Trx may be to negatively

regulate the central chronotropic actions of Ang II.60

The Brain Renin-Angiotensin System

The brain renin-angiotensin system (RAS) is of interest and particularly important due to

its implications in the development of essential hypertension. Essential hypertension, also known

as primary hypertension, is often simply defined as persistent high blood pressure with no known

cause, and sometimes with the added caveat that no preexisting renal disease is present. Kaplan

goes further to describe primary hypertension as "the elevation of blood pressure seen in younger

people which has a genetic foundation and is shaped by many environmental factors", while

making the distinction that the common form of hypertension seen in the elderly is usually

isolated systolic hypertension, which "reflects a stiffness of the proximal, capacitance vessels".

He further adds that essential hypertension usually reflects an "increase in cardiac output and

functional (i.e., reversible) constriction of peripheral resistance vessels."61 Despite the fact that

decades of research into the pathogenesis of this disease have not yet revealed the exact etiology,

it is widely held that the brain plays a critical role in the development and maintenance of

essential hypertension. Specifically, it is commonly believed that dysregulation of sympathetic

outflow and alterations in baroreflex function are the primary avenues by which the CNS

contributes to essential hypertension.62 Much of the evidence for the involvement of the CNS in

the pathogenesis of this disease derives from the most-studied animal model of essential

hypertension, the spontaneously hypertensive rat (SHR). However, before the evidence

concerning this model is presented, a modest review of the components of the RAS is required.









All aspects of the RAS (Figure 1-3) have been identified in the brain, but they have not

all been localized to the same cell. Accordingly, it is still not understood exactly how Ang II, the

octapeptide mediating the pressor effects of the brain RAS, is generated in the brain and, hence,

the subject remains controversial. Nonetheless, there is available evidence suggesting that it is,

indeed, generated behind the blood-brain barrier.63 64 The RAS is a cascade that originates with

the peptide angiotensinogen, which is cleaved by renin. The resulting peptide, angiotensin I, is

cleaved by angiotensin converting enzyme (ACE), to the pressor peptide angiotensin II (Ang II).

Ang II can then be cleaved into smaller peptides that have, just recently, been identified as

having independent functions, but the details are beyond the scope of this discussion.

Once Ang II is generated, it then goes on to bind to either of its cognate receptors, the type

I (AT1R) or type II (AT2R) receptor, both of which are G-protein coupled 7 transmembrane-

spanning receptors. The AT1R mediates the pressor effects of Ang II and is the predominant

isoform expressed in the brain. The contribution of the AT2R to blood pressure regulation

remains unknown, though, in a broad sense, the actions of the two receptor sub-types are thought

to be antithetical in nature. Significantly, functional studies utilizing receptor blockade strategies

indicate the presence of the AT1R in several cardiovascular regulatory regions of the brain,

including the paraventricular nucleus (PVN) of the hypothalamus. The PVN is thought to play a

critical role in integrating relevant afferent and humoral signals and adjusting endocrine and

autonomic responses accordingly.62

Studies on the over-activity of the brain RAS in SHR, the most commonly-used model of

neurogenic hypertension, abound and are reviewed in excellent detail by Veerasingham and

Raizada.62 Therefore, they will be only briefly touched upon here. In pre-hypertensive animals,

Tamura et. al. have observed increased brain angiotensinogen in SHR.65 Ruiz et. al.









demonstrated higher levels of renin-like activity in areas important to cardiovascular control such

as the NTS and the hypothalamus in SHR compared to control Wistar Kyoto (WKY) rats.66 Ang

II content was found to be increased in the PVN of SHR.67 Many studies have demonstrated

greater Ang II binding and increased AT1R mRNA in most of the areas of the brain thought to

be important to fluid balance, control of the cardiovascular system, and/or sympathetic tone such

as the SFO, MnPO, PVN, NTS, and RVLM.68-70

Studies observing the action of centrally-applied Ang II have concluded, as would be

expected when taking all of these observations into account, a greater pressor response in SHR

compared to WKY. Studies using pharmacological inhibitors of the RAS have provide further

evidence that overactivity of the RAS is involved in hypertension in SHR. For example,

losartan, an AT1R inhibitor, injected into the lateral ventricle decreases blood pressure in SHR,

but not in normotensive animals. Studies utilizing antisense gene targeting that reduce levels of

angiotensinogen and the AT1R decrease BP in SHR as well, but not in WKY. Finally, transgenic

studies over-expressing parts of the RAS in the brains of normotensive animals show that

hyperactivity of the brain RAS in normotensive animals is sufficient to cause hypertension in

these models. Considering all of these points, Veerasingham and Raizada conclude that the

increase in brain RAS activity precedes or parallels the development of hypertension in SHR. It

is not known exactly how all of these increases in brain RAS activity contribute to hypertension,

but it is currently commonly held that they result in increased sympathetic vasomotor tone.62

Reactive Oxygen Species, Neuronal Function, and Hypertension

Dr. Robin Davisson, one of the leaders in the neuronal ROS field, has recently published

an extensive and excellent review on the neuropathogenesis of hypertension and oxidative

stress,7 therefore the concepts will be only briefly reviewed here. For reference, the proposed

pathway for ROS generation in the brain is briefly illustrated in Figure 1-4.









Recently, the fields of redox signaling and neurogenic hypertension have been speedily

merging, and the collision has produced several significant studies establishing a firm argument

for the involvement of ROS in physiological Ang-II neuronal signaling and oxidative stress in

the pathogenesis of hypertension. For example, the Davisson lab has published a study

demonstrating that the ability of peripheral Ang II acting on the subfomical organ (SFO), a

circumventricular organ that is well-established in the central pressor actions of Ang II and

which sends efferents to the PVN,72 to induce hypertension is mediated by intracellular

superoxide.73 This group has further determined that scavenging of superoxide in the SFO

abrogates physiological responses to intracerebroventricular (ICV) Ang II in mice.74 Other labs

have substantiated these studies utilizing ICV tempol, a superoxide dismutase (SOD) mimetic

that scavenges superoxide, to prevent ICV Ang II-induced increases in blood pressure and

sympathetic activity.75 76 On the molecular level, in vitro studies have conclusively shown that

Ang II regulates neuronal firing by a pathway involving superoxide generation and modulation

of potassium currents.77 Finally, a recent provocative study from our laboratory has indicated

oxidative stress in the development of hypertension by showing that over-expression of MIF in

the PVN can attenuate the development of high blood pressure in SHR, and this ability of MIF

depends on the TPOR motif and, therefore presumably, MIFs catalytic redox activity.78

Likewise, advances in understanding the mechanisms by which ROS affect neuronal

behavior are steadily progressing. Davisson names at least three ways that ROS can modulate

neuronal activity: regulating ion channels, affecting transcription factor activity, and modulating

intracellular nitric oxide (NO) levels.71 Ang II-induced ROS have been shown to affect both

calcium and potassium channels in neurons. Specifically, superoxide may open calcium channels

directly, stimulating L-type calcium current, and close potassium channels, inhibiting delayed









rectifier potassium current77'79, 80 Ang II-derived ROS are also implicated in MAP kinase-

mediated neuronal activation, though the details of how MAPKs act in this regard are yet

undiscovered.81

ROS are also implicated in the modulation of gene expression in neurons by activating

various transcription factors. One such example is activator protein 1 (AP-1), a dimer of c-jun

and c-fos. The available evidence so far indicates that Ang II-mediated activation of AP-1 via

ROS depends upon the MAPK family. The likely MAPK candidates for redox-sensitive Ang II-

mediated activation of AP-1 are JNK and FRK.82-84 Another significant example of a redox-

sensitive transcription factor regulated by Ang II-induced ROS is nuclear factor KB (NFKB). Like

AP-1, NFKB binds to DNA as a dimer, the most common consisting of p50 and p65.82 Also

similar to AP-1 is the likelihood that Ang II-based redox regulation of NFKB is mediated by

redox-responsive MAPKs, most likely JNK and p38.82 Some very thought-provoking features of

NFKB redox-sensitive regulation in the periphery include activation by increased levels of

oxidized Trx82 and Ang II-induced hydrogen peroxide.85 Furthermore, a significant recent study

showed that peripheral inhibition of NFKB in young SHR prevents the development of

hypertension.86 Davisson suggests the intriguing hypothesis that redox-dependent modulation of

NFKB in cardiovascular control regions of the brain could also be involved in hypertension,

given that many aspects of Ang II signaling in the periphery are mimicked in neurons.7

Finally, ROS can affect neuronal behavior by interacting with nitric oxide (NO). Though

the role of NO with regard to sympathetic outflow is controversial, it is generally thought to be

sympathoinhibitory. Ang II-induced superoxide decreases NO availability, directly, by reacting

with NO to form peroxiynitrite and indirectly, by down-regulating neuronal nitric oxide synthase

(nNOS) in cardiovascular control regions such as the PVN.87 88 Davisson concludes, therefore,









that these studies clearly demonstrate that impairment of NO availability is at least one of the

mechanisms through which Ang II increases sympathetic drive.7

Macrophage Migration Inhibitory Factor and Thioredoxin as Negative Regulators of
Angiotensin II in the Central Nervous System

MIF is of keen interest to our lab and the field of blood pressure regulation due to its

ability to serve as a negative regulator of the neuronal actions of angiotensin II (Ang II).

Specifically, Ang II up-regulates MIF in neurons cultured from normotensive rat hypothalamus

and brainstem,89 and increased intracellular levels of MIF protein exert a negative regulatory

action over the neuronal chronotropic effects of Ang II.54 Similar interactions between MIF and

Ang II are observed in the rat brain in vivo. For example, CNS injection of Ang II increases MIF

expression in the paraventricular nucleus (PVN) of the hypothalamus, an area that has a key role

in regulating sympathetic outflow and hypothalamus/pituitary (HPA) axis activity. The increased

levels of intracellular MIF in PVN sympathetic regulatory neurons serve to blunt the increases in

discharge of these cells elicited by Ang II and the increases in blood pressure produced by CNS-

injected Ang II.90 Furthermore, it is apparent that MIF's ability to negatively regulate the actions

of Ang II is mediated by its TPOR activity and, possibly, via scavenging of ROS (Figure 1-5).54,

90

These findings became even more important when considering the fact that Ang II fails to

increase MIF expression in neurons cultured from the hypothalamus of spontaneously

hypertensive rats (SHR), or in the PVN of SHR.91 In addition, experiments from our laboratory

indicate that neurons in the PVN of SHR are devoid of immunoreactive MIF.78 However,

intracellular application of exogenous MIF into SHR hypothalamic neurons in culture can

depress the neuronal chronotropic action of Ang II in these cells, an effect mediated by the

TPOR activity of MIF. Thus, while MIF has the potential to depress the chronotropic action of









Ang II in SHR neurons, it is unlikely that this regulatory mechanism occurs since Ang II does

not up-regulate this protein in SHR neurons.91 By extrapolation, it is possible that a lack of this

MIF regulatory mechanism contributes to the hyper-responsiveness to Ang II in the PVN of SHR

and the consequent high blood pressure observed in these animals. This idea is borne out by

studies from our laboratory which indicate that long-term viral-mediated over expression of MIF

in the PVN of young SHR attenuates the development of high blood pressure in these animals.78

Based on the above, it is of major interest to understand the intracellular mechanisms that

control MIF expression in normal rat neurons, and to identify the defects that are responsible for

a lack of MIF expression in SHR neurons. To this end, we have been investigating possible

mechanisms for inducible expression of MIF in CNS neurons, which will be explored in detail in

the following chapters.

Finally, our hypothesis that MIF acts via it's TPOR activity to negatively regulate Ang II's

effects on neuronal currents lead us to investigate if other TPOR-containing proteins, namely

Trx, could also exert a negative-regulatory influence over the central actions of Ang II. Given

that ROS are now identified as important mediators of Ang II actions in neurons and that MIF's

TPOR activity appears to be a negative regulator of Ang II, we believe that TPOR-containing

proteins may, perhaps, represent a general mechanism whereby Ang II sensitivity can be

modulated in neurons. Indeed, in a recent study, we found that Trx, like MIF, is increased in

neurons in response to Ang II signaling. Furthermore, like MIF, Trx is able to prevent Ang II-

stimulated increases in delayed-rectifier potassium current, and this activity of Trx is dependent

on the action of its TPOR motif.60 Taking all of the above interactions between MIF, Trx, and

Ang II into consideration, we have also endeavored to ascertain the levels and distribution of

these important TPOR-containing proteins in areas pertinent to sympathetic activity of pre-









hypertensive SHR and WKY brains and to correlate this data with the oxidative status of these

tissues. The results and implications of these studies follow.











Tautomerase


TPOR
CALC
57-60


115
115


TPOR
CGPC
32-35


-C
104


Figure 1-1. Features of A) macrophage migration inhibitory factor and B) thioredoxin. The
TPOR motif of each protein is marked in green. N indicates the N-terminus of the
peptide, C indicates the C-terminus. Note that the MIF peptide begins at amino acid
2, as the N-terminal proline is cleaved off during post-translational processing. The
N-terminal tautomerase domain of MIF is also noted.








NADPH + H TR-S2 Trx-(SH)2 Protein-S2


NADP+ TR-(SH)2 Trx-S2 Protein-(SH)2


Figure 1-2. Catalytic mechanism of the thioredoxin system. Arrows indicate the direction of each
reaction. The catalytic mechanism of MIF is not known, but may be similar in nature.
TR = thioredoxin reductase, Trx = thioredoxin, (SH)2 = thiols (reduced form), S2 =
disulfide (oxidized form).








Angiotensinogen
4 Renin
Angiotensin I


ACE 2


Angiotensin 1-9


SIACE
Angiotensin 1-7

/
Mas-R


ACE2


SACE
Angiotensin II n Ang III1

Ang IV

'I,


AT1R


AT2R


IRAP


Vasoconstriction
Increase SNA
Increase Blood Pressure
Maintain ECFV
Growth
Induction of MIF


Vasodilation Memory retention
Natriuresis & retrieval
Anti-Growth
Apoptosis, Differentiation
Cerebroprotection
Cardioprotection


Figure 1-3. The renin-angiotensin system. Simplified schematic of the components of the RAS.
The components relevant to this work are depicted in red.


Vasodilation









































Figure 1-4. Angiotensin II-induced reactive oxygen species production. Ang II stimulates the
AT1R, which initiates intracellular signaling events, such as protein kinase C (PKC)
activation, ultimately leading to the assembly and activation of the NADPH oxidase
complex at the cell membrane. NADPH oxidase generates superoxide, which is
metabolized into hydrogen peroxide by superoxide dismutase (SOD). Catalase is one
of several enzymes that can detoxify peroxide after it has served its signaling purpose.



































Figure 1-5. Model of possible interactions between angiotensin II signaling and macrophage
migration inhibitory factor. Proposed negative regulatory mechanisms of MIF are
shown. MIF may negatively regulate the actions of Ang II directly through
scavenging of ROS second messengers or binding to the intracellular face of the
AT1R, leading to desensitization. It is also possible that MIF may indirectly affect the
receptor or Ang II signaling mediators by interacting with other signaling and
regulatory proteins.









CHAPTER 2
INDUCIBLE EXPRESSION OF MACROPHAGE MIGRATION INHIBITORY FACTOR IN
NEURONS

Introduction

Chapter 1 discusses the importance of understanding the mechanisms that control inducible

expression of MIF due to its recently-established role as a negative feed-back regulator of the

chronotropic actions of Ang II in neurons. The observation that MIF expression can be induced

in response to Ang II signaling in neurons, and that it is not in neurons of pre-hypertensive SHR,

indicates that the central actions of MIF could possibly be implicated in the development of

hypertension and poses some important questions. What downstream-mediators of Ang II and

the AT1R might be inducing MIF expression in CNS neurons? Furthermore, as established in

chapter 1, it appears that the AT1R is uncoupled from the control of MIF expression in SHR

neurons. Therefore, it follows to inquire whether these potential mediators perform the same in

normotensive and SHR rat neurons.

Our first goal in these studies was to establish a candidate downstream mediator of Ang II

that induces MIF expression in neurons. Accordingly, it is now well known that Ang II, acting

via the AT1R, induces ROS production in neurons by activating NADPH oxidase.92 This leads to

superoxide production which should be metabolized to hydrogen peroxide (H202) by superoxide

dismutases.93 As expected, many studies have concluded that H202 is a product of Ang II

signaling in many cell types, including neurons.94 Furthermore, it has been demonstrated that

H202 can cause induction of MIF expression in peripheral tissues.95 Finally, it has been shown,

recently, that the human MIF promoter is regulated by Spl and CREB,34 two redox-sensitive

transcription factors,96 97 indicating that MIF expression might be sensitive to the redox

environment of the cell. Therefore, we hypothesized that H202 may be, at least in part,

responsible for inducible expression of MIF in CNS neurons in response to Ang II signaling.









Furthermore, we sought to determine whether H202 possesses the same abilities with respect to

inducing MIF expression in normotensive rat and SHR neurons.

In the present study, our aim was to determine whether H202 can cause up-regulation of

MIF expression in CNS neurons and whether MIF is differentially regulated by ROS in neurons

cultured from SHR and WKY rats. Together, the data presented here demonstrate that H202 can

cause inducible expression of MIF in CNS neurons cultured from normotensive, but not SHR,

newborn rats.

Materials and Methods

Animals. For our experiments, we utilized adult Sprague-Dawley (SD), Wistar Kyoto

(WKY), and Spontaneously Hypertensive (SHR) rats as breeders to produce rat pups that were

used for the production of neuronal cultures. The adult breeder rats were purchased from Charles

River Farms (Wilmington, MA). All experimental procedures were approved by the University

of Florida Institutional Animal Care and Use Committee.

Materials. Dulbecco's modified Eagle's medium (DMEM) was obtained from Invitrogen

(Grand Island, NY). Plasma-derived horse serum (PDHS), Fetal Bovine Serum (FBS), 30%

stabilized H202 solution, PEG-catalase, Actinomycin D, B-actin monoclonal antibody, and

secondary HRP-conjugated antibodies were obtained from Sigma (St. Louis, MO). Cells-to-

cDNA II kits were purchased from Ambion (Austin, TX). Primers for MIF and 18S for real-time

RT-PCR were obtained from Applied Biosystems (Foster City, CA). Glucose Oxidase was

purchased from Calbiochem (San Diego, CA). CytoTox96 Non-Radioactive Cytotoxicity Assays

were purchased from Promega (Madison, WI). Rat MIF antibody was purchased from Torrey

Pines Biolabs (East Orange, NJ).









Preparation of neuronal cultures and tissues. Neuronal cultures were prepared from the

hypothalamus and cortex of newborn rats as described previously.98 Cultures were grown in

DMEM containing 10% PDHS for a further 12-16 days before use.

Preparation of glial cultures. Glial cultures were derived from the aforementioned

neuronal cultures (which are -90% neuronal, 10% astrocytes). Neurons were killed by

incubating the dishes in 100 mM KC1 at room temperature for 5 minutes. The KC1 solution was

replaced with DMEM containing 10% FBS to encourage cell proliferation and cultures were

monitored until -80% confluency. The DMEM/FBS medium was then replaced with

DMEM/PDHS medium and glial cultures were cultured for at least a further 7 days to parallel

the feeding/utilization schedule of the corresponding neuronal cultures.

Analysis of MIF mRNA in cultures. cDNA was produced from neuronal cultures with the

Cells-to-cDNA II kit, which allows reverse transcription without RNA purification. Levels of

MIF mRNA were quantified by real-time RT-PCR as described previously54. Data were

normalized to 18S rRNA.

Analysis of MIF protein in cultures. Neuronal cultures were lysed in Laemmli Sample

Buffer (Biorad, Hercules, CA) and 10 ug of total protein was loaded on the gel. Transfer to a

PVDF membrane (Biorad, Hercules, CA) was performed at 75 V for 1.5 hours in Towbin Buffer.

Western blots were carried out and analyzed as detailed previously.89

Determination of Cytotoxicity. Neuronal cultures were treated as indicated and medium

was collected from each well. Replicates were pooled, and then samples were centrifuged at 4

degrees Celsius and 5000 rpm for 5 minutes to pellet any particulate matter or unattached cells.

Supernatant was then utilized (50 uL) according to the protocol provided with the CytoTox96

kit.









Determination of Protein Carbonyl Content. Neuronal cultures (12 wells of a standard

24-well plate, equal to approximately 6 million cells) were treated as indicated, and protein

extracts were prepared in MES buffer as instructed. Protein carbonyl content was determined

according to kit protocol utilizing the Protein Carbonyl Assay Kit from Cayman Chemical (Ann

Arbor, Michigan).

Cell Treatments. For experiments involving application of H202, a stock solution of H202

in water was diluted 100-fold into the culture medium to the final dose indicated.

For experiments involving PEG-Catalase and Glucose Oxidase, the enzyme was dissolved

in sterile DPBS then diluted 100-fold into the culture medium to the final dose indicated.

For experiments involving Actinomycin D (Act D), the Act D was dissolved in DMSO to

give a concentration of 1 mmol/L. It was then diluted 1000-fold into the culture medium to give

a final dose of 1 itmol/L.

Data Analysis. Results are expressed as mean + SEM. Statistical significance was

evaluated with the use of a 1-way ANOVA, followed by a Newman-Keuls test to compare

individual means. Differences were considered significant at P<0.05.

Results

Hydrogen Peroxide Stimulates an Increase in Macrophage Migration Inhibitory Factor in
Neurons Cultured from Normotensive Rats, but not Spontaneously Hypertensive Rats.

Our first goal was to establish whether H202 contributes to inducible expression of MIF

mRNA in primary neuronal cultures from normotensive rats and SHR. To test this idea, primary

neuronal cultures prepared from newborn SD and WKY rats and SHR were incubated with H202

(30 tmol/L; 1, 2 or 3 hr.), a cell-permeant source of ROS. This was followed by analysis of MIF

mRNA expression via real time RT-PCR in neurons from all three genetic strains and analysis of

MIF protein levels in SD rat neurons. The results indicate that H202 elicits an increase in MIF









mRNA that is statistically significant within one hour in neurons from both of the normotensive

rat strains (Figure2-1, A and B). Conversely, the same dose of H202 was unable to elicit any

significant increase in neurons cultured from SHR throughout the 3 hour time period observed

(Figure2-1C). As expected, H202 also caused a time-dependant increase in MIF protein in SD rat

cultures (Figure 2-2).

To confirm these results, primary neuronal SD rat cultures were also incubated with

glucose oxidase. Glucose oxidase produces H202 via oxidation of glucose in the cell-culture

medium. Primary neuronal cultures were treated with 0.5 mU glucose oxidase for 1 and 5 hours.

The results show that glucose oxidase-derived H202 produces an increase in MIF mRNA levels

that is significant by 5 hours (Figure 2-3).

Finally, because the neuronal cultures used here contain a small (<10%) number of glial

cells, we investigated whether H202-induced increases in MIF mRNA were restricted to neurons

or if the effects could also be observed in glial cells. To this end, glial cultures that were devoid

of neurons were incubated with 1, 10, 30, or 50 itmol/L H202 for one hour, the time point at

which the increase in MIF reaches significance in the corresponding neuronal cultures, and MIF

levels ascertained by real time RT-PCR. The results demonstrate that H202 is unable to stimulate

an increase in MIF mRNA levels in glial cultures (Figure 2-4). Later time points were briefly

investigated in pilot studies, and also showed now effect (data not shown). Collectively, these

results confirm our hypotheses that MIF mRNA and protein expression can be regulated by ROS

in primary neurons.









Hydrogen Peroxide Increases Macrophage Migration Inhibitory Factor mRNA Expression
in Primary Neurons Through a Specific Intracellular Action.

Because ROS at high concentrations can cause a generalized stress response in cells, we

sought to determine if the stimulatory effect of H202 on MIF mRNA levels in neurons is a

specific intracellular action of H202, rather than a non-specific response to oxidative stress.

First, in order to determine if the effects of H202 on MIF mRNA levels in neurons were

due to an intracellular action of H202, we utilized PEG-catalase as an intracellular inhibitor of

H202 signaling. Catalase is an enzyme of very high activity that quickly and efficiently

metabolizes H202 into water and molecular oxygen. The PEG-conjugation of the enzyme not

only renders it cell-permeant, but also particularly stable once inside the cell.16 Neuronal cultures

were pre-treated with PEG-catalase for 24 hours. The medium was then removed and replaced

with fresh conditioned medium, and the cells were stimulated with H202 (30 .imol/L) for one

hour. The results show that scavenging the exogenously applied H202 in the intracellular

environment prevents an increase in MIF mRNA levels, indicating that the exogenously applied

H202 acts intracellularly to increase MIF levels (Figure 2-5).

Next, we investigated whether the dose of H202 used for our studies was cytotoxic to the

neuronal cultures. Primary SD rat neurons were incubated with H202 (30 imol/L) for 1 and 3

hours, followed by analysis of lactate dehydrogenase (LDH) within the cell culture medium. The

LDH assay measures the activity of LDH released into the medium by dying or dead cells. The

results indicate that the dose of H202 utilized for all experiments in this study is not cytotoxic to

primary neuronal cultures. However, higher doses of H202 (e.g. 100 ilmol/L, utilized as a

positive "killing" control) are, indeed, cytotoxic to the neuronal cultures (Table 2-1).

Furthermore, we sought to determine whether a 30 .imol/L dose of H202 represents a state

of oxidative stress to our neuronal cultures by measuring protein carbonyl content, an accepted









indicator of intracellular oxidative stress. Primary SD neuronal cultures were treated with 30 or

100 (utilized as a positive control) pmol/L H202 for 1 hour (i.e., the earliest time point of

significant MIF induction in the presence of H202) and protein carbonyl content measured. The

data indicate that protein carbonyl content is not significantly different in control versus 30

pmol/L H202-treated neurons (Figure 2-6). Collectively, these results demonstrate that the

effects of H202 on MIF mRNA levels in primary neurons are a specific, intracellular signaling

action of H202 and not the result of a generalized oxidative stress response.

The Increase in Macrophage Migration Inhibitory Factor Levels Observed in the Presence
of Hydrogen Peroxide Involves Increased Synthesis of Macrophage Migration
Inhibitory Factor mRNA.

Since real time RT-PCR measures steady-state mRNA levels, and steady-state mRNA

levels represent the summation of both synthesis and degradation, we investigated whether the

increase observed in steady-state MIF mRNA levels in the presence of H202 was due to an

increase in transcription. To this end, we utilized actinomycin D (Act D), a general inhibitor of

mRNA synthesis. Treatment of neuronal cultures with 1 imol/L Act D reveals that MIF mRNA

is stable in neurons for at least 6 hours, with half-life not yet reached by 8 hours (data not

shown). This observed stability of MIF mRNA in our experimental conditions is similar to that

reported previously in other cell types.99 Neuronal cultures were pretreated with 1 imol/L Act D

for one hour and then stimulated with 30 imol/L H202 for 1 hour. MIF mRNA levels were then

ascertained by real time RT-PCR. The data indicate that inhibiting transcription concomitantly

with H202 stimulation prevents an increase in MIF mRNA levels (Figure 2-7). This result

suggests that the effects of H202 on MIF mRNA levels in neurons are primarily due to increased

MIF mRNA synthesis









Discussion

To our knowledge, this study represents the first demonstration that ROS can regulate the

expression of MIF in CNS neurons. Furthermore, we have shown that this effect of H202

involves intracellular events that are specific to neurons, and the data suggest that the increase in

MIF involves de novo transcription. Finally, the observation that hydrogen peroxide fails to elicit

an increase in MIF in neurons cultured from SHR, in contrast to their normotensive controls,

provides support for the contention that the MIF gene responds in a specific and regulated

fashion to redox signaling.

In this study, we selected hydrogen peroxide as our ROS donor for many reasons. First, we

were aiming to study a reactive oxygen species that is downstream of Ang II and the AT1R in

neurons, and it has already been established that Ang II is capable of producing intracellular

H202 in many cell-types, including neurons, and that this H202 has significant physiological

effects (e.g. influencing sympathetic activity in the brain).94 Second, it is readily cell permeant,

with exogenously-applied H202 establishing equilibrium across the cell membrane on average

within minutes.100 Finally, in the realm of possibilities of ROS donors, it is relatively stable

since, while it is a ROS, it is not a free-radical. Simply stated, addition of H202 to the culture

medium is the easiest and most reliable way to manipulate the intracellular redox environment,

and it was desirable to manipulate intracellular ROS levels in the absence of the myriad,

confounding possible signaling actions for these proof-of-principle studies.

Interestingly, the LDH, protein carbonyl, and PEG-catalase experiments (Table 2-1, Figure

2-6, and Figure 2-5, respectively) support the notion that exogenously-applied H202 functions as

a signaling agent in our neuronal cultures, rather than a mediator of cell death and/or oxidative

stress. This signaling function would be only natural for intracellular ROS produced in response

to a physiological ligand (i.e., Ang II), but it may seem unusual for the exogenous dosage









utilized, 30 imol/L, considering that many in vitro studies conclude that this dosage, added

exogenously to cell lines in culture, should result in an approximately 3 .imol/L intracellular

concentration at equilibrium. This intracellular concentration might constitute an environment of

oxidative stress, since in vitro studies suggest that the highest observed intracellular

concentration of H202 generated for signaling purposes in mammalian cells is 0.7 imol/L.93

However, there are some important mitigating factors to consider in the context of our neuronal

cultures. Our cultures consist of primary cells, which evidence suggests produce less endogenous

H202 than transformed cell lines. Therefore, it has been suggested the application of more

exogenous H202 is required to oxidatively stress primary cells than transformed cells in culture.93

Furthermore, our cultures, while mostly neuronal in nature, always contain a small portion of

glial cells. Neurons, microglia, astrocytes, and oligodendrocytes all have the capacity to detoxify

H202 with varying efficiencies. Astrocytes and oligodendrocytes, in particular, are remarkably

efficient at detoxifying extracellular peroxide. In vitro studies reveal that H202 added to the

medium of astrocyte cultures has a half-life of only a few minutes. Oligodendrocytes follow at a

slightly slower rate, though their over-all capacity to detoxify H202 exceeds that of astrocytes.101

Therefore, it is highly possible that the exogenous H202 is rapidly detoxified when added to the

medium of our cultures, and, consequently, the neurons are not exposed to doses high enough to

create oxidative stress in the intracellular environment. Moreover, the knowledge that a bolus

application of H202 can be rapidly detoxified by many of the cell types in our cultures prompted

us to perform the experiments utilizing glucose oxidase, which represents a more chronic means

of administering exogenous H202, to confirm our results.

Our results raise some important questions as to the mechanism by which H202 is inducing

MIF expression in CNS neurons. It is now widely recognized that H202, like nitric oxide, may be









a readily-diffusible small molecule that acts as a signaling agent. Indeed, in prokaryotes and

yeast, systems that sense and signal in response to H202 are well-characterized.93 In higher

mammals, redox signaling is emerging as a very important and complicated field, and many of

the signaling pathways affected by ROS are still under investigation. Nevertheless, it is

becoming clear that several kinase pathways are modulated by ROS and the activity of many

transcription factors is subject to redox regulation.102-104 For example, it has already been

demonstrated that the MAP kinase, p38, is sensitive to Ang II-based ROS signaling in vascular

smooth muscle cells and neurons.81, 105 Further, it has been shown in many settings that

transcription factors such as AP-1, SP-1, CREB, and NFKB are sensitive to redox regulation,106-
108 and binding sites for these transcription factors have been identified in the promoter of the

human MIF gene.109 Furthermore, a recent publication examining the constitutive and inducible

expression of the MIF promoter has demonstrated, for the first time, that SP-1 and CREB are

important transcriptional regulators of the MIF gene.34 Experiments to determine if these

transcription factors may be the mediators of redox regulation of the MIF gene in neuronal

cultures are planned for the near future in our laboratory.

This study is provocative and physiologically significant because, as we have established

in prior reports, MIF is up-regulated in neurons in response to Ang II signaling via the AT1R.

MIF then serves, either directly or indirectly, as a negative regulator of the chronotropic actions

of Ang II in neurons that lie along key sympathetic and neuroendocrine pathways in the brain

such as the PVN.54, 89,90 Our studies strongly suggest that MIF may act in this regard by

scavenging ROS,54 as do some other proteins that contain TPOR motifs (e.g. thioredoxin,

peroxiredoxins),27 but the exact mechanism is still under investigation. In this way, we speculate









that MIF may serve as a way for neurons to regulate their sensitivity to Ang II, especially Ang II-

based ROS production and their downstream effects.

Furthermore, we have previously shown that Ang II does not induce MIF expression in

PVN neurons of SHR, in contrast to their normotensive controls, and we have hypothesized that

this lack of MIF induction may contribute to the hyper-sensitivity of these neurons to Ang II,91

and perhaps even the development and/or maintenance of hypertension in these animals. This

idea is born out by current studies in our laboratory showing that viral-mediated over-expression

of MIF in the PVN of young SHRs attenuates the development of hypertension.78 We believe

that in SHR the AT1R is uncoupled from the signaling pathway that induces MIF in

normotensive animals. Therefore, in the present study, we have sought to investigate signaling

pathways that are downstream of the AT1R in neurons, namely ROS signaling. Indeed, we have

found that our initial hypothesis regarding ROS and MIF expression is correct. This information

serves as a strong basis on which to build further investigation into the actions of ROS in

normotensive animals regarding Ang II signaling and MIF expression.

Intriguingly, a recent publication has shown that H202 produced in the PVN in response to

Ang II may play a role in regulating sympathetic activity.94 Accordingly, it is tempting to

visualize a feed-back loop such that Ang II causes H202 production in the PVN, which stimulates

MIF production, subsequently feeding back to decrease the sensitivity of the neuron to Ang II

and, perhaps, influencing the central sympathetic and/or neuroendocrine actions of Ang II.

Exactly how MIF is providing this negative feedback is still unknown and remains the subject of

intense investigation in our laboratory.














*x ** **


Time (Hours)
Time (Hours)


z
i 2.0-
U)
o 1.5-

Z 1.0'
E
u. 0.5-

0.0.


z
i 2.0-

oo 1.5-

Z 1.0.
E
u. 0.5-
-


**


1 2
Time (Hours)


Time (Hours)

Figure 2-1. Hydrogen peroxide increases macrophage migration inhibitory factor mRNA levels
in primary neuronal cultures from normotensive rats, but not spontaneously
hypertensive rats. A) SD, B) WKY, and C) SHR neuronal cultures were incubated
with either vehicle (H20) or 30 imol/L H202 for 1, 2, or 3 hours, followed by
analysis of MIF mRNA levels as described in the materials and methods. Means + SE
(n = 7 for SD, n = 5 for WKY, n = 6 for SHR) of the ratio of MIF mRNA to 18S
rRNA at each time point are shown. *P < 0.05 vs. control, **P < 0.01 vs. control


z
i 2.0-
U_
0o 1.5-

Z 1.04
E
u. 0.5-
-








A
2.00-

1.75-

1.50-

u. 1.25-

1.00 -

0.75-
0 1 2 3
Time (Hours)


B


MIF, 12.5 kDa

p-Actin, 42 kDa


Hours 0 1 2 3



Figure 2-2. Hydrogen peroxide increases macrophage migration inhibitory factor protein levels
in primary neuronal cultures. SD rat neuronal cultures were incubated with either
vehicle (H20) or 30 imol/L H202 for 1, 2, or 3 hours, followed by Western Blot
analysis of MIF protein as described in the materials and methods. A) Data are means
+ SE (n = 3) of the ratio of MIF protein to 8-actin at each time point. *P < 0.01 vs.
control. B) Representative Western Blot showing the effects of hydrogen peroxide on
MIF levels.








2.0-

z


Co
S1.5-


1.0-
z

E 0.5-
LL


0.0 -
Control 1 hour 5 hours



Figure 2-3. Glucose oxidase increases macrophage migration inhibitory factor mRNA levels in
primary neuronal cultures. SD rat neuronal cultures were incubated with either
vehicle (DPBS) or 0.5 mU glucose oxidase for 1 and 5 hours, followed by analysis of
MIF mRNA levels as described in the materials and methods. Bar graphs shown here
are means + SE (n = 4) of the ratio of MIF mRNA to 18S rRNA at each time point.
*P < 0.05 vs. control.








2.0-

z
W 1.5-
(U)
Co
1.0
z

E 0.5-
LL.


0.0 ,
Control 1 LM 10 4M 30 4M 50 4M

Hydrogen Peroxide Dose

Figure 2-4. Hydrogen peroxide does not increase macrophage migration inhibitory factor
mRNA expression in primary glial cell cultures. SD rat glial cultures were incubated
with either vehicle (H20) or 1-50 .imol/L H202 for Ihour, followed by analysis of
MIF mRNA levels as described in materials and methods. Bar graphs shown here are
means + SE (n = 3) of the ratio of MIF mRNA to 18S rRNA at each concentration.
Statistical analysis showed no significance at any time point observed.












< II
Z 2.0- 1.5
C 1.1.
E




LL 0.5-

0.0 0.
C 1.0 ::4: < <,

lev Clv NIP NC I

o 0





MIF, 12.5 kDa a f a -a


B-Actin, 42 kDa -


Co0 oV 0





Figure 2-5. Hydrogen peroxide acts intracellularly to elicit an increase in macrophage migration
inhibitory factor expression in primary neuronal cultures. SD rat neuronal cultures
were incubated with either vehicle (DPBS) or 100 U of PEG-catalase for 24 hours.
Medium was changed to fresh conditioned medium taken from age-matched, naive
neuronal cultures. Cultures were then incubated with either vehicle (H20) or 30
pmol/L H202 for 1 hour followed by analysis of MIF mRNA and protein levels as
described in materials and methods. A) Bar graphs shown are means SE (n = 6) of
the ratio of MIF mRNA to 18S rRNA under each treatment condition. *P < 0.001. B)
Bar graphs shown are means SE (n = 3) of the ratio of MIF protein to 8-actin
protein under each treatment condition. *P < 0.01. Western blot shown in lower panel
is representative of the 3 experiments quantified in B.










Table 2-1. Hydrogen peroxide (30 imol/L) does not elicit cytotoxic effects in primary neuronal
cultures. SD rat neuronal cultures were incubated with either vehicle (H20) or 30
pmol/L H202, or 100 pmol/L H202 for 1 or 3 hours. Culture medium was collected
and subjected to an LDH cytotoxicity assay as detailed in the materials and methods.
Means SE (n = 4) at each time point are shown. *P < 0.001 vs. control.
30 fLmol/L Hydrogen Peroxide 100 fLmol/L Hydrogen Peroxide
% Survival at 1 hour 100 2.00 95 4.57
% Survival at 3 hours 98 0.75 *63 4.30








5-
O
c.


O 4
0

E 2




O 0-

SControl 30 gM H202 100 M H202


Figure 2-6. 30 tmol/L hydrogen peroxide does not alter protein carbonyl formation. SD rat
neuronal cultures were incubated with either vehicle (H20), 30, or 100 .mol/L H202
for 1 hour. Cell lysates were collected and assayed for protein carbonyl content as
indicated in materials and methods. Bar graphs shown here are means + SE (n = 10
for control and 30 imol/L H202, n = 3 for 100 imol/L H202). *P < 0.01.








2.0-


1.5-



1.0-



0.5-


0.


I


-I I I


Act. D


Act. D +
H202


Figure 2-7. Hydrogen peroxide-induced increases in macrophage migration inhibitory factor
expression involve a transcriptional event. SD rat neuronal cultures were incubated
with Actinomycin D (1 imol/L) for 1 hour. The cultures were then incubated with
either vehicle (H20) or 30 imol/L H202 for 1 hour followed by analysis of MIF
mRNA levels as described in materials and methods. Bar graphs shown here are
means + SE (n = 5) of the ratio of MIF mRNA to 18S rRNA.


v









CHAPTER 3
MACROPHAGE MIGRATION INHIBITORY FACTOR AND THIOREDOXIN IN THE
BRAIN AND OXIDATIVE STRESS

Introduction

Recently, interest in the relationship between neurogenic hypertension and oxidative stress

has been gaining momentum. Studies in adult SHR rats have demonstrated that oxidative stress

is critically involved in the neurogenic hypertension observed in this model and in the related

stroke-prone spontaneously hypertensive rat (SHRSP) model.110-112 However, these studies have

mostly focused on the role of oxidative stress in the adult SHR. Studies examining oxidative

stress in any area of the brains of pre-hypertensive (i.e., newborn or very young) animals are

lacking.

Coincidentally, and in agreement with the aforementioned studies, a recent publication

from our laboratory has shown that the localization of MIF is dysregulated in adult SHR brains.

Specifically, though absolute levels of MIF mRNA and protein appear normal in adult SHR,

immunostaining reveals that MIF expression is lacking in neurons of the PVN. Furthermore, this

powerful study determined that the lack of basal MIF expression in neurons is most likely

involved in the development of hypertension in SHR, since long-term, viral-mediated over-

expression of MIF in the PVN of young SHRs with mildly elevated blood pressure was able to

significantly attenuate the development of robust hypertension in these animals over the 12 week

period studied. Notably, the ability ofMIF over-expression in the PVN to attenuate the

development of hypertension in SHR appears to be redox-dependent, since a mutated form of

MIF carrying a substitution in the TPOR motif was unable to recapitulate the effects of wild-type

MIF.78 This finding is not surprising, given that oxidative stress has already been observed in

adult SHR brains and the evidence is ever-mounting that MIF is a Trx family member, sensitive

to and most likely participating in redox homeostasis.









Merging together the studies concerning 1) oxidative stress and neurogenic hypertension in

adult SHR and 2) our studies on the role of neuronal MIF in the development of hypertension in

SHR leads us to ask some important questions. We already know that absolute MIF levels do not

differ between adult SHR and WKY PVNs. Instead, the cellular distribution of MIF seems to be

the major difference, and this difference contributes to the disparate blood pressures normally

observed in these two genetic strains.78 Accordingly, are MIF and/or Trx expression and

distribution dysregulated in the brains of pre-hypertensive, newborn SHRs? The results of

studies in chapter 1 demonstrating disparate inducible expression prompted us to sequence the

putatitive MIF promoter and gene in SHRs and WKYs, revealing SNP's located 5' to

transcriptional start, suggesting to us that examination of basal expression may be in order

(Figure 3-1). However, a more detailed analysis of the MIF promoter in rats is necessary to begin

to understand the implications of any possible mutations. Also, our studies do suggest that MIF

is lacking specifically in neurons of adult SHRs and that this lack contributes to the development

of hypertension in these animals. However, it has not yet been established when the distribution

of MIF becomes abnormal in these animals (i.e., pre- or post-hypertension).

Furthermore, like their adult counterparts, do newborn SHRs exhibit oxidative stress in

cardiovascular-relevant regions of the brain? Again, we do not yet know if the oxidative stress

observed in adult animals is the cause or a consequence of their hypertension. Therefore, it is

important to establish the situation with regard to MIF and Trx in pre-hypertensive animals. If

MIF and/or Trx are, indeed, lacking or redistributed in newborns, then it follows that oxidative

stress may be present long before hypertension becomes evident. Establishing the timing of the

development of oxidative stress in the brains of these animals with respect to the progression of

hypertension would be a considerable contribution to this exciting new field of research.









To begin addressing these questions, the comparative studies in this chapter will aim to 1)

determine relative quantities of MIF and Trx in the hypothalamus and brainstem of newborn

(i.e., pre-hypertensive) SHR and WKY rats, 2) determine the cellular localization of MIF and

Trx in the PVN of newborn SHR and WKY rats, and 3) ascertain relative levels of oxidative

stress in the hypothalamus and brainstem of newborn SHR versus WKY. Our decision to

examine the hypothalamus and brainstem is based on the fact that they are rich in cardiovascular-

relevant nuclei such as the PVN, the RVLM, and the NTS. Trx is being examined along with its

relative MIF due to the similarities between the two molecules concerning regulation of neuronal

firing (i.e., effects on delayed rectifier current), and ability to negatively regulate the central

actions of Ang II.60, 113 Furthermore, Trx is more established as an important regulator of the

intracellular redox environment, so a correlation between Trx dysregulation and oxidative stress

is even more likely than is the case for MIF.

Materials and Methods

Analysis of MIF and Trx mRNA. Relevant brain areas were dissected from newborn rat

brains and placed in RNAlater (Ambion, Austin, TX) at 4 degrees celsius overnight. They were

then placed at -20 degrees celcius until processing. RNA was isolated from the tissues utilizing

the RNAeasy minikit (Qiagen, Valencia, CA). cDNA was produced from the RNA samples

utilizing the iScript cDNA synthesis kit (Biorad, Hercules, CA). Real time was performed using

commercially available primers and universal taqman 2x PCR master mix (Applied Biosystems,

Foster City, CA). Results are expressed as a ratio of MIF or Trx mRNA to 18s rRNA.

Analysis of MIF and Trx protein. Relevant brain regions were dissected from newborn

rat brains and placed in cold boiling buffer consisting of 4% SDS, 0.25 M Tris HC1 (pH 6.8),

10% glycerol, and 2% 1-mercaptoethanol. Tissues were homogenized in this buffer by

sonication, followed by boiling at 100 degrees celcius for 3 minutes. The lysates were









centrifuged at 14,000 RPM and 4 degrees celcius for 5 minutes. Supernatants were collected and

utilized for western blotting. Concentrations were determined by Bradford Assay. 10 ug of total

protein was loaded onto a 15% gel for SDS-PAGE, then transferred to a PVDF membrane

(Biorad, Hercules, CA) at 75 V for 1.5 hours. After a brief rinse in PBS-T (PBS containing

0.05% Tween-20), membranes were blocked in PBS-T containing 10% non-fat milk for at least

one hour at room temperature. Primary antibodies were applied overnight at 4 degrees celcius.

MIF (Torrey Pines, East Orange, NJ) and Trx (Chemicon International, Temecula, CA)

polyclonal antibodies were diluted in PBS-T/1% milk. MIF antibody was diluted at 1:1000, Trx

antibody at 1:10,000. Beta-actin monoclonal antibody (Sigma, St. Louis, MO) was diluted at a

concentration of 1:100,000 in PBS-T/5% milk. The next day, membranes were washed in PBS-T

for 1 x 10 minutes and 4 x 5 minutes, then incubated in secondary antibody at a 1:30,000 dilution

(MIF), 1:50,000 dilution (Trx), or 1:90,000 (beta-actin) in 2% milk/PBS-T for one hour at room

temperature. Secondary HRP-conjugated antibodies (anti-mouse and anti-rabbit) were purchased

from Sigma (St. Louis, MO). Chemiluminescence detection was carried out according to

instructions with the Western Lighting kit from PerkinElmer (Boston, MA).

Immunostaining of brain sections for MIF and Trx. Brains were placed in

Formaldehyde Fresh (Fisher, Waltham, MA) overnight for fixation. They were then transferred

to 70% ethanol and left overnight for partial dehydration and further fixation. The brains were

then subjected to dehydration and impregnation with paraffin. After imbedding in paraffin, 5

micron sections were cut and subjected to immunostaining. Following rehydration, slides were

rinsed and incubated in TBS-T (TBS containing 0.05% tween) for at least 5 minutes, followed by

a blocking step consisting of TBS-T and 1.5% horse serum for at least 1 hour (blocking serum

was "matched" to secondary antibodies, which are donkey). Excess blocking solution was wiped









from the slides and primary antibodies were applied for an overnight, 4 degrees celcius

incubation. MIF and Trx antibodies (same as described in "analysis of MIF and Trx protein"

section) were used at a 1:200 dilution, NeuN and GFAP antibody (Chemicon International,

Temecula, CA) at a 1:400 dilution. Primary and secondary antibodies were diluted in PBS

containing 10% goat serum. The next day, after a brief rinse in TBS-T, slides were rinsed for 2 x

10 minutes. Secondary antibody (donkey anti-mouse and donkey anti-rabbit) was applied for 1

hour. Alexa Flour 488 and Alexa Flour 594 (Invitrogen, Grand Island, NY) were utilized at a

dilution of 1:500. After a brief rinse in TBS-T, slides were rinsed for 2 x 10 minutes, then

mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and viewed

and photographed on an Olympus microscope.

Measurement of protein carbonyl content. Hypothalamus and brainstem were dissected

from newborn rat brains and homogenized in 1 ml of ice cold 50 mM MES buffer, pH 6.7,

containing 1 mM EDTA (Sigma, St. Louis, MO) by sonication. Lysates were then centrifuged at

10,000 g for 15 minutes at 4 degrees celsius. Supernatant was collected and frozen at -80 degrees

celcius until utilization. Assay was performed exactly according to the protocol provided with

the Protein Carbonyl Assay kit (Cayman Chemical, Ann Arbor, Michigan).

Results

Macrophage Migration Inhibitory Factor and Thioredoxin Expression is Lacking in
Spontaneously Hypertensive Rat Hypothalamus.

Our first goal was to establish the situation with respect to the expression of MIF and Trx

in the hypothalamus and brainstem of SHR and WKY newborn (i.e., pre-hypertensive) rats. To

determine MIF and Trx mRNA expression, newborn rat brains were dissected, and relevant

regions were homogenized and subjected to RNA extraction. The RNA was then reverse

transcribed, and the resulting cDNA utilized for real-time PCR as described in materials and









methods. The results (Figure 3-2A) show that there is a statistically significant lack of MIF and

Trx mRNA in the hypothalamus of newborn SHRs compared to their WKY controls.

In contrast, when subjected to the same measurement, there was not a statistically

significant decrease in the brainstems of SHRs compared to WKY. However, there was a trend

toward a slight decrease (Figure 3-2B).

Our second goal concerning MIF and Trx expression was to determine whether the

protein levels paralleled the mRNA data (i.e., whether the protein is significantly decreased in

the hypothalamus of SHR). Newborn hypothalamus and brainstem were subjected to protein

extraction as described in the materials and methods, and western blotting was utilized to

determine relative protein levels. The results indicate that MIF and Trx protein is significantly

reduced in the hypothalamus, which parallels the mRNA data (Figure 3-3, A and B). In contrast

to the mRNA data, Trx protein appeared to be reduced in the brainstem (Figure 3-4). To further

clarify the significance of the lack of these proteins, oxidative stress in these two tissues was

measured next.

Newborn Spontaneously Hypertensive Rats Exhibit More Oxidative Stress in the
Hypothalamus and Brainstem than Wistar Kyoto Rats.

An important aim of this study was to investigate whether SHR newborns exhibit oxidative

stress in cardiovascular-relevant regions of the brain, similar to their adult counterparts. To this

end, we utilized a protein carbonyl kit (as described in materials and methods) to assess protein

carbonyl levels, one of the several accepted indicators of intracellular oxidative stress.114 Protein

was extracted from newborn SHR and WKY hypothalamus and brainstem and subjected to this

colorimetric assay. The results indicate that SHR have a statistically significant greater level of

protein carbonyl content in the hypothalamus than WKY (Figure 3-5A). SHR also exhibit a

higher level of protein carbonyl concentration in the brainstem, though the difference is not as









great or significant (Figure 3-5B). Since the hypothalamus, in contrast to the brainstem, appears

to exhibit greater levels of oxidative stress in SHR, our further studies focused on the

hypothalamus.

Macrophage Migration Inhibitory Factor is Absent from Paraventricular Nucleus Neurons
of Spontaneously Hypertensive Rats.

Since MIF distribution appears to be altered (i.e., lacking in neurons) in the PVN of adult

SHR, we were eager to determine whether MIF and Trx were also lacking in neurons in the pre-

hypertensitve newborns. Newborn SHR and WKY brains were formalin-fixed and paraffin-

embedded, and 5 micron sections of the PVN were then subjected to Trx or MIF

immunostaining. Sections were also stained with NeuN, to identify neurons, or GFAP, to

identify astrocytes. The results indicate that, similar to what is found in adult animals, MIF is

lacking in neurons of newborn SHR PVN compared to WKY. Similar to what we have found in

adults, MIF is mostly localized to astrocytes in the SHR PVN (Figure 3-6). In contrast, Trx

localization was similar between WKY and SHR PVN (Figure 3-7).

Discussion

To our knowledge, the studies in this chapter represent the first demonstration of

oxidative stress in newborn SHR hypothalamus. Furthermore, these studies are the first to show

reduced expression of MIF and Trx expression in newborn SHR hypothalamus, which contains

the important sympathetic and neuroendocrine-regulating nucleus, the PVN. Finally, the

observations contained herein demonstrate that the lack of neuronal MIF previously seen in adult

PVN can also be found in newborn (i.e., pre-hypertensive) SHR. In contract, cellular localization

of Trx was found to be similar between SHR and WKY PVNs.

Our findings that MIF and Trx are lacking in the hypothalamus of newborn SHR are

significant for several reasons. Trx is a well-established redox-homeostasis enzyme that is









essential for maintaining a proper redox balance in neurons. Further, the evidence is steadily

mounting that MIF has important redox balancing and/or signaling roles in the brain. A lack of

both of these enzymes is suggestive of oxidative stress or altered redox signaling. Indeed, the

results of our protein carbonyl studies suggest an association between the lower levels of these

TPOR enzymes and a dysregulated redox balance in the hypothalamus. Based on the

observations made here, we cannot conclude decisively that the lack of these proteins is solely

responsible for the altered redox environment observed in SHR hypothalamus. However,

combining them with our previous study supplementing MIF in the PVN of pre-hypertensive

SHR and preventing the development of robust hypertension in a TPOR-dependent fashion78 is

certainly suggestive that the lack of these TPOR enzymes in the hypothalamus leads to oxidative

stress or some other form of redox signaling dysfunction and, consequently, contributes to the

development of hypertension in these animals. One important experiment that is yet to be

performed, and would further support this proposed cause and effect relationship, would be to

over-express MIF or Trx in the PVN of pre-hypertensive SHR and determine whether the

attenuation of hypertension is accompanied by a reduction in oxidative stress in the relevant

brain areas. These studies would also further clarify whether MIF and Trx are redundant or have

slightly divergent functions in this brain area.

One important and confounding question these studies raise is: why are these antioxidant

enzymes paradoxically not up-regulated in the face of oxidative stress? We are not the first group

to observe a lack of Trx induction in the presence of oxidative stress. Tanito et. al. reported in

2004 that hypertensive SHR and severely hypertensive SPSHR exhibited a lack of Trx in the

heart, vasculature, and kidney that correlated to the severity of their hypertension.115

Furthermore, they reported dysfunctional Trx induction in response to Ang II signaling in









peripheral blood mononuclear cells of hypertensive rats. The authors concluded, therefore, that a

genetic mechanism is the likely responsible for the lack of Trx that they observed. Similarly, the

results of the studies contained in chapter 1 showing a dysfunctional induction of MIF in

response to hydrogen peroxide treatment in SHR neurons prompted us to sequence the putative

MIF promoter and MIF gene in SHR and WKY. Several single-nucleotide polymorphisms were

identified (Figure 3-1), but an in-depth study is required to determine if they are important in

basal or inducible MIF expression. Likewise, a sequencing study of the Trx promoter and gene in

SHR and WKY is certainly warranted and necessary to determine if genetic alterations are

responsible for the lack of Trx in newborn SHR hypothalamus and the apparently dysfunctional

inducible expression (i.e., lack of induction in an oxidative stress situation) of this protein.

Our findings concerning an abnormal absence of MIF in neurons of the PVN in newborn

SHR further support an argument for genetic alterations in hypertensive animals and our recently

developing model of dysregulated TPOR enzymes in the brain as a cause, rather than an effect,

of hypertension. If the lack of MIF could only be observed in adult, hypertensive rats, then this

would suggest an epigenetic alteration that is a consequence of high blood pressure. Yet, our

studies presented here argue that this is not the case, since we have found the same phenomenon

in newly-born SHRs that have not yet experienced elevated blood pressure. However,

antioxidant enzymes from several families, including glutathione peroxidase, superoxide

dismutase (SOD), and catalase, have been reported to exhibit abnormally low activity in SHR in

several tissues.116' 117 The fact that SHR exhibit such a wide-spread dysfunction in antioxidant

systems could suggest a catastrophic failure in global oxidative signaling and redox regulation.

In addition to sequencing and promoter studies, perhaps we should consider that the mutation is

not to be found in the promoters of these antioxidant enzymes but in the promoter of an up-









stream, common mediator(s) that controls) expression of redox-balancing proteins and systems.

Accordingly, a prime candidate that should be investigated in future studies is the transcription

factor Nrf2, which binds to the antioxidant response element (ARE) and increases expression of

many antioxidant enzymes.118-120 An intriguing study by Zhu et. al. showed that Nrf2-deficient

mice have dysregulated basal and inducible expression of several antioxidant and detoxifying

enzymes and small molecule antioxidants, including SOD, glutathione, glutathione S-transferase,

glutathione reductase, and catalase in cardiac fibroblasts.121 Though not examined in Zhu's

study, other publications have shown that thioredoxin expression is clearly regulated by Nrf2, as

well.122, 123 Experiments have not yet been done to determine whether Nrf2 also regulates MIF

expression. Disappointingly, no groups have yet studied blood pressure regulation or

sympathetic nerve activity in Nrf2-deficient mice. Nevertheless, studies such as the

aforementioned make it easy to imagine that perhaps an indirect global regulator of redox

balance such as Nrf2 is playing a role in the lack of TPOR enzymes we are observing in newborn

SHR brains. Clearly, extensive further study involving genetics, epigenetics, and signal

transduction is needed before it can be determined exactly what sort of abnormality is

responsible for the lack of MIF and Trx in the hypothalamus of SHR.










AP-1 Sp-1 USF NFKB
SII I I I I I I
-2kb -1kb


Figure 3-1. Single nucleotide polymorphisms of the putative macrophage migration inhibitory
factor promoter in spontaneously hypertensive rats. Those identified in color are
potential binding sites for the transcription factors indicated. Note that all 4 are redox
sensitive transcription factors.








Hypothalamus


I IMIF
= Trx


A
1.5-


z
S1.0-
Co

Z 0.5-
E

0.0-






B
1.5-


z
S1.0-
Co

z 0.5-
E

n0.


Brainstem


I IMIF
= Trx


_


A
#e


Figure 3-2. Macrophage migration inhibitory factor and thioredoxin mRNA expression is
reduced in spontaneously hypertensive rat hypothalamus. mRNA was isolated from
the A) hypothalamus and B) brainstem of newborn WKY and SHR and relative
expression levels were ascertained by reverse transcription and real-time PCR as
described in materials and methods. Means + SE (n = 10 for each tissue) of the ratio
of MIF or Trx mRNA to 18S rRNA are shown. *P < 0.0001 vs. WKY.


(A'
e <^


SAt
e <^


-I


A





Cj










MIF Trx
A B
1.5- 4.5-
4.0-
S3.5-
1.0- 3.0-
p 2.5-
.LL 2.0-
0.5- 1.5-
1.0-
0.5-
0.0- 0.0
WKY SHR WKY SHR



MIF 12.5 kDa o d IlW 4M -b W eM 4W

Trx 12 kDa, g *4 4D a 44 I Q*

fI-actin 42 kDa so A O f



WKY SHR



Figure 3-3. Macrophage migration inhibitory factor and thioredoxin protein expression is lower
in newborn spontaneously hypertensive rat hypothalamus. Total protein was extracted
from hypothalamus of newborn WKY and SHR and relative expression was
determined by western blotting as described in materials and methods. Means + SE (n
= 6 for each tissue) of the ratio of A) MIF or B) Trx protein to p-actin are shown.
Bottom panel contains representative western blots. *P < 0.05 vs. WKY.










MIF Trx
A B
1.5- 7.5-


U U


5 0.5- 2.5-
I--


0.0 0.0
WKY SHR WKY SHR


MIF 12.5 kDa aIeI 0I aIl b a & l i 4ea e s 41D

Trx 12 kDa e IaI I II I e e I aI

fI-actin 42 kDa e ao m 0 I


WKY SHR



Figure 3-4. Macrophage migration inhibitory factor and thioredoxin protein in newborn
brainstem. Total protein was extracted from the brainstem of newborn WKY and
SHR and relative expression was determined by western blotting as described in
materials and methods. Means + SE (n = 6 for each tissue) of the ratio of A) MIF or
B) Trx protein to p-actin are shown. Bottom panel contains representative western
blots *P < 0.05 vs. WKY.









Hypothalamus


WKY SHR


Brainstem


I1


WKY SHR


Figure 3-5. Protein carbonyl concentration is greater in spontaneously hypertensive rat
hypothalamus than in Wistar Kyoto. Total protein was extracted from A)
hypothalamus and B) brainstem of newborn WKY and SHR and carbonyl levels
measured as described in materials and methods. Means + SE (n = 6 for WKY, n = 6
for SHR) of the protein carbonyl concentration of each tissue measured are shown. *P
< 0.05 vs. WKY.








69









































W660





3V 03V


Figure 3-6. Spontaneously hypertensive rat paraventricular nucleus neurons contain less
macrophage migration inhibitory factor. Brains from newborn A) WKY and B and C)
SHR rats were subjected to immunostaining for MIF, NeuN (a neuronal marker), and
GFAP (an astroglial marker). Bars in lower right represent a scale of 50 microns.
Areas of interest are marked with arrows. D and E) are Ig controls. Sections pictured
are representative of 4 animals from each genetic rat strain.


SHR:





















B




Figure 3-7. Thioredoxin cellular distribution is normal in spontaneously hypertensive rat
paraventricular nucleus. Brains were removed from newborn A) WKY and B) SHR
rats, fixed, and paraffin-embedded as described in materials and methods. 5 micron
sections of the hypothalamus were subjected to immunostaining for Trx and NeuN (a
neuronal marker). Bars in lower right represent a scale of 50 microns. Selected areas
of co-localization are marked with arrows. For controls, refer to D and E of Figure 3-
6. Sections pictured are representative of 4 animals from each genetic rat strain.









CHAPTER 4
CONCLUSIONS AND FUTURE DIRECTIONS

Over the past several years, many laboratories, including our own, have made substantial

contributions to our understanding of how ROS signaling and oxidative stress contribute to

neural control of blood pressure in the physiological state and hypertension, respectively.

Progress is being steadily made in this field, but there is still much we do not understand about

redox signaling in the brain and its contribution to blood pressure regulation. The studies

contained in this dissertation are important because they contribute small steps toward

understanding the larger picture with regard to redox homeostasis in the brain and neurogenic

hypertension.

From the studies reported in chapter 1, we have been able to make advances in

understanding possible pathways that regulate inducible expression of MIF in neurons. Further,

by concluding that MIF expression can be sensitive to the intracellular redox environment of the

cell, we have contributed greatly to the paradigm shift that is presently occurring in categorizing

MIF as a TPOR enzyme and potential member of the Trx family. Finally, these studies provide

direction, in the form of ROS signaling, as a basis for future studies examining the inducible

expression of MIF in normotensive animals in response to Ang II stimulation of neurons. Also,

the knowledge that ROS do not signal appropriately in SHR neurons, at least with regard to MIF

expression, lends further direction to future studies that endeavor to determine why the AT1R is

uncoupled from inducible MIF expression in SHR.

The studies contained in chapter 2 present compelling evidence that oxidative imbalance

is present in SHR hypothalamus prior to hypertension and that a lack of MIF and Trx may be at

least partly responsible. At the very least, there is an association between reduced levels of these

important antioxidant enzymes and markers of oxidative stress in the hypothalamus of SHR.









Further, a very telling and important finding of these studies is the lack of MIF in neurons of the

PVN of pre-hypertensive SHR, indicating that this loss of MIF is not a consequence of

developing or advanced hypertension, but perhaps it may be a contributor to the cause. This

likelihood becomes even more compelling when coupled with our recent studies on MIF

supplementation in the PVN and reduction of blood pressure in SHR. Given the fact that, due to

the efforts of our laboratory, MIF and, to a lesser extent, Trx is now a known negative

regulator of the central actions of Ang II, and that it is well-established that SHR suffer over-

activity of and hypersensitivity to the brain RAS, we can now see a much clearer picture of how

a lack of MIF and/or Trx may lead to enhanced ROS signaling and/or oxidative stress in

important cardiovascular brain nuclei and how this may contribute to the development of

hypertension in SHR. Many hypertensive patients exhibit increased sympathetic nervous

activity,124 and there is a constant influx of new evidence showing that ROS are primary

effectors through which Ang II mediates its central actions, including effects on the sympathetic

nervous system.71 Therefore, dysfunction of important redox-regulating systems in the brain is

becoming a more likely contributor to the complex story of hypertension all the time. Our results

in the preceding chapters pave the way for a number of exciting new studies and inspire many

important questions, which will be explored and discussed in the following sections.

Inducible Expression of Macrophage Migration Inhibitory Factor in Neurons and
Angiotensin II

A very critical question that remains unanswered by our studies is whether hydrogen

peroxide is, indeed, the down-stream mediator that induces MIF expression in neurons of

normotensive animals. In order to answer this question, a suitable model will have to be

established. Areas of the brain that are rich in AT1R should be dissected and cultured together in

order to receive a robust induction of MIF when stimulated with Ang II. In the past, we usually









accomplished this by culturing the hypothalamus with the brainstem isolated from newborns.

However, in recent months, our laboratory has been developing a technique for culturing PVN

neurons isolated from adult rats, a very technically challenging feat. The use of these neurons as

a model for studying Ang II and AT1R signaling would be even more desirable. Regardless of

whether newborn or adult neuronal cultures are utilized, the experiments detailed in chapter 1

involving PEG-catalase should be repeated with these cultures, except Ang II stimulation should

be used in the place of hydrogen peroxide application. If one could interrupt Ang II-mediated up-

regulation of MIF expression in the cultures with PEG-catalase treatment, this would provide the

necessary evidence needed to determine that Ang II-mediated ROS signaling, specifically

hydrogen peroxide, is the pathway through which Ang II induces MIF in neurons. It would also

be interesting to repeat the aforementioned experiments utilizing PEG-SOD, in order to show

conclusively that superoxide is the source of the intracellular hydrogen peroxide, as we surmise.

These studies could, theoretically, be extrapolated to in vivo studies utilizing viral-

mediated over expression of SOD and catalase in pertinent brain areas, followed by Ang II

application and study of MIF expression. Other labs have successfully used SOD and tempol in

the brain to interrupt Ang II signaling,7476 so this seems to be a very plausible way of

determining whether Ang II-induced ROS signaling is the mechanism by which MIF expression

is up-regulated in the PVN in vivo.

Mechanisms of Redox Regulation of Macrophage Migration Inhibitory Factor Expression

Another important question that remains to be answered is that of the down-stream

mediators that are sensing and responding to changes in the intracellular redox environment and,

ultimately, altering the expression of MIF. The most sensible way to initially embark on these

studies would be to clone the putative MIF promoter from normotensive animals, make strategic

deletions and recombine with a reporter system (such as luciferase). The constructs would then









be transfected into a neuronal cell line (such as SH-SY5Y, known to be Ang II-sensitive)125, 126

and stimulated with hydrogen peroxide and/or Ang II to investigate which areas of the MIF

promoter are redox-sensitive. As we have seen in the discussion of chapter 1, many transcription

factors (TFs) are sensitive to redox-signaling, so identifying the cis-acting elements of the MIF

promoter that are responsible for redox-sensitive increases in MIF transcription would be the

first step to identifying which redox-regulated signaling pathways are inducing MIF up-

regulation. Candidate signaling pathways could then be determined in a "back-tracking" manner

using the abundant information available concerning which TFs mediate effects of particular

pathways.

Once areas of the MIF promoter are identified that mediate induction of MIF both in

response to Ang II and hydrogen peroxide, the focus can be placed on these sequences and which

TFs may bind there. The cells can then be stimulated with Ang II or hydrogen peroxide and ChIP

assays at the native MIF promoter performed for the relevant TFs to ascertain if, indeed, their

binding at the MIF promoter is increased under stimulated conditions. The final piece of

evidence needed to confirm which TFs are mediating the effects of ROS at the MIF promoter are

mutational studies. The MIF promoter-luciferase recombinant constructs could be mutated via

PCR to ablate the relevant TF-binding sites and tested for their sensitivity to hydrogen peroxide

and Ang II stimulation. ChIP assays should also be performed in Ang II and hydrogen peroxide-

treated primary neurons in order to confirm that the effects seen are not specific to the particular

cell line selected for the studies.

Once the promoter studies have begun to identify TFs and candidate signaling pathways

that may be regulating Ang II- and ROS-induced MIF expression in neurons, these pathways can

be further clarified utilizing pharmacological interventions and the aforementioned recombinant









reporter constructs. Furthermore, the results of these studies will serve as a basis for comparison

and may help identify the defects in Ang II signaling with respect to MIF induction in SHR.

Dysregulation of Macrophage Migration Inhibitory Factor and Thioredoxin Expression in
the Hypothalamus of Spontaneously Hypertensive Rats

The studies contained in chapter 3 highlight the importance of determining the cause of the

lower expression levels of MIF and Trx in the newborn SHR hypothalamus. As the future

directions concerning this endeavor were previously explored in the discussion of chapter 3, they

will not be recapitulated here.

One major question that remains to be answered is that concerning the loss of MIF in

neurons of the PVN in SHR, which can be observed in both pre- and post-hypertensive animals.

There are several possibilities that must be further explored. For example, is the MIF gene

simply not transcribed in these cells? Is MIF being transcribed, but not translated properly? If

MIF is being transcribed and translated in neurons, is it then being abnormally degraded? If this

is not the case, perhaps MIF is being improperly secreted from neurons so that its presence

cannot be detected in these cells by immunostaining? The most reasonable way to begin

addressing this issue is to determine whether MIF mRNA can be detected in these neurons. This

would determine if the root cause most likely lies in a loss of MIF transcription. In this instance,

the application of single-cell RT-PCR, a technique we have recently developed in our laboratory,

would yield the most specific information. Application of other commonly-used techniques is

complicated by the fact that PVN and hypothalamic cultures consist of a heterogenous

population of cells, so determining the situation specifically in neurons is rather difficult. Cells

could be cultured from the PVN of SHR and WKY and neurons screened for the presence of

MIF using single-cell RT-PCR. Another technique that would be useful for detecting MIF

mRNA and confirming the single-cell RT-PCR results is in situ hybridization. As the









immunostaining data indicates, we should be able to easily detect MIF mRNA in neurons

cultured from WKY. If MIF mRNA cannot be detected in the SHR cultures, this would argue

strongly that the problem is a transcriptional issue. However, we cannot rule out the possibility

of a stability problem with MIF mRNA in these cells. Obviously, further experiments would be

needed to clarify the situation. Addressing this question would be a time-consuming and difficult

undertaking, but the information obtained would merit the investment.

Contrary to observations in newborn hypothalamus, absolute MIF levels are unchanged in

the heterogenous population of cells in the PVN of adult SHR.78 One possible explanation for

this difference is that the situation in the PVN may be different from that in the rest of the

hypothalamus. Another possibility is that MIF is upregulated in glial cells of adult SHR PVN to

somehow compensate for the loss of MIF in neurons. Investigating this possibility will be very

difficult, for reasons relating to the heterogeneity of cells already mentioned, but the most

practical way to begin addressing it would be to culture the PVN of SHR and WKY, enrich for

astroglia as previously described,127 and ascertain basal levels of MIF in these primary glial

cultures, comparing mRNA and protein between WKY and SHR via real time RT-PCR and

ELISA, respectively. Increased levels of MIF in the SHR cultures would suggest that MIF is

upregulated in astroglia in SHR and this is why absolute levels appear normal in the PVN, even

when expression is lost in neurons.

Oxidative stress in the Hypothalamus of Spontaneously Hypertensive Rats

The studies in chapter 3 indicate that oxidative stress is present in the hypothalamus of

newborn SHRs. The redox environment represents a summation of oxidant-producing systems

and antioxidant systems. Therefore, oxidative stress could result from either a loss of antioxidant

function and/or an increase in ROS producing systems. Our studies indicate that there may be a

lack of antioxidant enzymes, such as those of the Trx family, in SHR hypothalamus. By









extrapolation, it would be useful to investigate whether there is a lack of other important

antioxidant systems, such as the glutathione system in this tissue. Conversely, the expression and

activity of oxidant-producing systems should also be investigated. For example, NADPH oxidase

is currently considered to be one of the most important regulated enzymatic sources of ROS in

neurons.71 128 It would be prudent to investigate whether this system is up-regulated in the brain

of SHR, which could contribute to an imbalanced redox environment.

Physiological Implications

Ultimately, the goal of future studies should be to investigate the physiological

implications of improper redox signaling and redox-regulated cellular functions in hypertension.

Previous studies from our lab have already determined that neuronal MIF supplementation can

have a significant effect on the development of hypertension in SHR over time. However, one

very important outcome that remains to be determined is whether MIF supplementation in PVN

neurons of SHR and the resulting reduction in blood pressure are accompanied by a reduction in

oxidative stress in this tissue. Since it has been demonstrated in several instances that ROS can

affect neuronal function by modulating neuronal firing,71 another important outcome to measure

in the context of MIF supplementation in SHR PVN would be sympathetic drive. Previous

experiments published by our laboratory78 should be repeated and these important parameters

determined to further complete the picture of how a lack of MIF contributes to the

neuropathogenesis of hypertension.









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BIOGRAPHICAL SKETCH

Rachael Harrison was born in 1980 in Jacksonville, FL. She has resided in Florida all of

her life and has wanted to become a scientist since she was a very small child. Rachael's career

aspirations ranged from marine biology to being a science teacher through her junior high school

and high school years. Rachael began to think seriously about a career in biomedical research in

her later high school years, and pursued a degree in molecular biology and microbiology (with an

emphasis on human health and disease) from the college of Health and Public Affairs at the

University of Central Florida, where she graduated with honors and went on to graduate school

at the University of Florida, College of Medicine. Rachael joined the Physiology and Functional

Genomics department under the supervision of Dr. Colin Sumners in fall 2004, two years after

she entered graduate school in 2002. In Dr. Sumners' laboratory, her research has focused on

redox biology and neurogenic hypertension. She will receive her doctorate in August of 2008

and go on to a career in cancer research at the Sunnybrook Health Sciences Center in Toronto,

Ontario.





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1 MACROPHAGE MIGRATION IN HIBITORY FACTOR A ND THIOREDOXIN IN THE BRAIN: IMPLICATIONS FOR HYPERTENSION By RACHAEL ANNE HARRISON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Rachael Anne Harrison

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3 To my husband, Brad. Your constant support an d encouragement have made this possible.

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4 ACKNOWLEDGMENTS I m ust begin by acknowledging my husband. He has stood by me and held me up through the most difficult of times. Without his constant humor and perspective, there would often have been little laughter through the tears. Secondly, I must thank my mentor. He has en couraged me when I did not believe in myself, and picked me up to put one foot in front of the other when I wanted to falter. Without him, I most certainly would not have made it through graduate school. I have learned from him important lessons, not just about science, but about life and about my own capabilities. I can never thank him enough for hi s service as my mentor. I must also thank my committee members for their service. Their guidance has proven invaluable, and their advice was always given generously, patiently, and kindly. I acknowledge my lab mates for their assistance with technical issues and their constant companionship. I consider them esteemed colleagues and also friends. Lastly, I thank my family and friends. My e xperiences with them have made me into the person I am today, and everything that I can be pr oud in my life of is a re sult of their support and encouragement.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT...................................................................................................................................10 CHAP TER 1 INTRODUCTION AND LITERATURE REVIEW..............................................................12 Characteristics of Macrophage Migration Inhibitory Factor ..................................................12 Introduction to the Thioredoxin Family of Proteins...............................................................16 Characteristics and Functions of Thioredixon 1: A Com parative Study................................ 17 Macrophage Migration Inhi bitory Factor and Thioredoxin in the Brain ................................ 21 The Brain Renin-Angiotensin System....................................................................................23 Reactive Oxygen Species, Neurona l Function, and Hypertension ......................................... 25 Macrophage Migration Inhibito ry Factor and Thioredoxin as Negative Regulators of Angiotensin II in the Central Nervous System ................................................................... 28 2 INDUCIBLE EXPRESSION OF MACROPHAGE MIGRATION INHIBITORY FACTOR I N NEURONS....................................................................................................... 36 Introduction................................................................................................................... ..........36 Materials and Methods...........................................................................................................37 Results.....................................................................................................................................39 Hydrogen Peroxide Stimulates an Increas e in Macrophage Migr ation Inhibitory Factor in Neurons Cultured from Norm otensive Rats, but not Spontaneously Hypertensive Rats........................................................................................................39 Hydrogen Peroxide Increases Macrophage Migration Inhibitory Factor m RNA Expression in Primary Neurons Thro ugh a Specific Intracellular Action................... 41 The Increase in Macrophage Migration Inhi b itory Factor Levels Observed in the Presence of Hydrogen Peroxide Involves Increased Synthesis of Macrophage Migration Inhibitory Factor mRNA............................................................................. 42 Discussion...............................................................................................................................43 3 MACROPHAGE MIGRATION INHIBITORY FACT OR AND THIOREDOXIN IN THE BRAIN AND OXIDATIVE STRESS........................................................................... 55 Introduction................................................................................................................... ..........55 Materials and Methods...........................................................................................................57 Results.....................................................................................................................................59

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6 Macrophage Migration Inhibito ry Factor and Thioredoxin Expression is L acking in Spontaneously Hypertensive Rat Hypothalamus......................................................... 59 Newborn Spontaneously Hypertensive Rats Exhibit More Oxidative S tress in the Hypothalamus and Brainstem th an Wistar Kyoto Rats............................................... 60 Macrophage Migration Inhibito ry Factor is Absent from Paraventricular Nucleus Neurons of Spontaneously Hypertensive Rats............................................................. 61 Discussion...............................................................................................................................61 4 CONCLUSIONS AND FUTURE DIRECTIONS................................................................. 72 Inducible Expression of Macrophage Migra tion Inhibitory F actor in Neurons and Angiotensin II.....................................................................................................................73 Mechanisms of Redox Regulation of Macr ophage Migration I nhibitory Factor Expression ...........................................................................................................................74 Dysregulation of Macrophage Migration Inhibi tory Factor and Thioredoxin Expression in the Hypothalam us of Spontan eously Hypertensive Rats................................................ 76 Oxidative stress in the Hypothalamus of Spontaneously Hypertensive Rats ......................... 77 Physiological Implications..................................................................................................... .78 LIST OF REFERENCES...............................................................................................................79 BIOGRAPHICAL SKETCH.........................................................................................................89

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7 LIST OF TABLES Table page 2-1 Hydrogen peroxide (30 mol/L) does not el icit cytotoxic effects in prim ary neuronal cultures...............................................................................................................................52

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8 LIST OF FIGURES Figure page 1-1 Features of macrophage migrati on inhibitory factor and thioredoxin. ..............................31 1-2 Catalytic mechanism of the thioredoxin system................................................................ 32 1-3 The renin-angiotensin system............................................................................................ 33 1-4 Angiotensin II-induced reactive oxyg en species production............................................. 34 1-5 Model of possible interactions betw een angiotensin II signaling and m acrophage migration inhibitory factor.................................................................................................35 2-1 Hydrogen peroxide increases macrophage m igration inhibitory factor mRNA levels in primary neuronal cultures from norm otensive rats, but not spontaneously hypertensive rats.............................................................................................................. ..47 2-2 Hydrogen peroxide increases macrophage m igration inhibitory f actor protein levels in primary neuronal cultures.............................................................................................. 48 2-3 Glucose oxidase increases macrophage mi gration inhibitory f actor mRNA levels in primary neuronal cultures.................................................................................................. 49 2-4 Hydrogen peroxide does not increase m acrophage migra tion inhibitory factor mRNA expression in primary glial cell cultures............................................................................ 50 2-5 Hydrogen peroxide acts in tracellularly to elicit an incr ease in macrophage migration inhibitory factor expression in primary neuronal cultures................................................. 51 2-6 30 mol/L hydrogen peroxide does not alter protein carbonyl formation......................... 53 2-7 Hydrogen peroxide-induced increases in m acrophage migration inhibitory factor expression involve a transcriptional event......................................................................... 54 3-1 Single nucleotide polymo rphism s of the putative macr ophage migration inhibitory factor promoter in spontan eously hypertensive rats.......................................................... 65 3-2 Macrophage migration inhibitory f actor and thioredoxin m RNA expression is reduced in spontaneously hype rtensive rat hypothalamus................................................. 66 3-3 Macrophage migration inhi bitory factor and thioredoxin protein expression is lower in newborn spontaneously hypertensive rat hypothalam us............................................... 67 3-4 Macrophage migration inhibitory f actor and thioredoxin protein in newborn brainstem ............................................................................................................................68

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9 3-5 Protein carbonyl concentration is greater in spontaneously hypertensive rat hypothalam us than in Wistar Kyoto.................................................................................. 69 3-6 Spontaneously hypertensive rat parave ntricular nucleus neurons contain less m acrophage migration inhibitory factor............................................................................ 70 3-7 Thioredoxin cellular distribution is no rm al in spontaneously hypertensive rat paraventricular nucleus......................................................................................................71

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10 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MACROPHAGE MIGRATION IN HIBITORY FACTOR A ND THIOREDOXIN IN THE BRAIN: IMPLICATIONS FOR HYPERTENSION By Rachael Anne Harrison August, 2008 Chair: Colin Sumners Major: Medical Sciences Physiology and Pharmacology Previous studies from our laboratory have established that macrophage migration inhibitory factor serves as a negative regulator of the ne uronal chronotropic actions of angiotensin II in normotensive rats, but not in spontaneously hypertensive rats. Furthermore, hydrogen peroxide is a known effector of angiotensin II signaling in paraventricular hypothalamic neurons and is an es tablished inducer of macrophage migration inhibitory factor expression in the periphery. Therefore, for the first study contained in this dissertation, we hypothesized that hydrogen peroxide may be able to induce macrophage migration inhibitory factor expression in neurons, and we sought to ascertain whether normotensive and spontaneously hypertensive neurons respond differentially to hydroge n peroxide stimulation with regard to expression of this small protein. We examined the effects of hydrogen per oxide (30 mol/L) on macrophage migration inhibitory factor expression in neuronal cultur es from normotensive (Wistar Kyoto and Sprague Dawley) and spontaneously hypertensive newborn ra ts. The data indicate that hydrogen peroxide induces macrophage migration inhibitory f actor expression in ne urons cultured from normotensive rats, but not spontaneously hyperten sive rats. Lactate dehyd rogenase and protein carbonyl assays suggest that 30 m ol/L hydrogen peroxide is neith er cytotoxic to the neurons,

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11 nor does it cause oxidative stress. Studies with polyethylene glycol-cat alase and actinomycin D suggest that the hydrogen peroxide is acting intracellularly to in crease transcription of the macrophage migration inhibitory f actor gene. We conclude that m acrophage migration inhibitory factor expression is regulated di fferentially in normotensive a nd spontaneously hypertensive rat neurons in response to h ydrogen peroxide signaling. Furthermore, oxidative stress has become an exciting field of study with regard to neurogenic hypertension. However, it remains to be determined if oxidative stress in the brain contributes to the development of or is a c onsequence of this disease. The second study contained in this dissertation examines the expr ession and cellular localiz ation of two important antioxidant proteins, macrophage migration inhibitory factor and thioredoxin, in the hypothalamic paraventricular nucleus of spontane ously hypertensive rats and their normotensive controls, Wistar Kyoto rats. Importantly, these studies were perf ormed in newborn (i.e., not yet hypertensive) rat brains. Our studies establish dysregula tion of expression of these two proteins in the brain of newborn rats th at are destined to be hypertensi ve and associates them with oxidative stress that occurs well before the onset of hypertension.

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12 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Characteristics of Macrophage Migration Inhibitory Factor Macrophage m igration inhibitory factor (MIF) (Figure 1-1A) is a small (12.5 kDa), highly conserved protein with nearly ubiquitous tissue distri bution that was origin ally described in 1966 as a soluble factor that was expressed by T cells in delayed type hypersensitivity and inhibited the random migration of macrophages.1-3 The factor behind this activity was not cloned and identified as MIF until 1989,1 and, when MIF was originally characterized, it was designated a lymphokine (later commuted to the more modern and inclusive term cytokine). Structurally, MIF does not have any notable domains4 (though it does have some en zymatic motifs that will be discussed in detail later), and it is still not clear whether the MIF homotrimer, the entity identified in all of the structural studies, is the functionally relevant form. On the contrary, several studies suggest that the dimer or the monomer may be res ponsible for the actions of MIF in vivo.5-7 Today, determining a functional niche for MI F is far more complicated than it was 40 years ago. It has been suggested that MIF is a cytokine, a chemokine, a hormone, and an enzyme, and the arguments for all of these designations are compelling.8, 9 Clearly MIF has many, sometimes seemingly disparate, biological and cellular functions. Physiologically, MIF plays modulatory roles in the immune, en docrine, and nervous systems.10 Pathologically, when dysregulated, MIF contributes to a wide range of inflammatory diso rders and plays a pivotal role in tumor biology.11, 12 MIF seems to be somewhat of a moon-lighti ng protein in that its functions can be divided into two basic categories. It is not yet clear, but we hypothesize that this may depend on whether it is secreted to initiate signal transduction (cytokine-like) or remains in the intracellular

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13 space (enzymatic, perhaps antioxidant). Much of the traditional literature focuses on the proinflammatory, cytokine-like function of MIF. MIF is considered to be rather an orphan cytokine, or perhaps the first discovered member of a new cy tokine family, since it bears no similarity to other cytokine families,1 nor does its receptor, a complex c onsisting of CD74 and CD44. CD74 is necessary for MIF binding, and CD44 appears to responsible for initiating intracellular signaling cascades.13 Yet, clearly MIF can function as a pro-in flammatory mediator, and the overwhelming majority of the literature suppor ts this notion. It can be secret ed, presumably via a leaderless pathway,14 by many types of immune, e ndocrine, and parenchymal cells,15, 16 and it is wellestablished that secreted MIF is a critical player in inflammatory conditions such as sepsis, rheumatoid arthritis, asthma, cystic fibrosis, atherosclerosis, and glom erulonephritis to name only a few.17-20 It should be noted that MIF appears to be acting as a chemokine, signaling via chemokine receptors rather than its recen tly-identified (and assumed to be canonical) CD74/CD44 receptor complex, in the instance of atherosclerosis.9 However, MIF is distinct from other trad itional cytokines in many interesting ways. Evidence suggests that it is const itutively expressed and exists in pre-formed pools in the cytosol of many immune and non-immune cells.15, 21 In contrast, traditional cytokines are usually produced de novo with a lag time for transcrip tion and translation, when induced.22 Furthermore, as previously mentioned, the MIF peptide does not appear to contain a lead er sequence, exists as an entity free of secretory vesicles in the cyto plasm of cells, and is most likely secreted in a regulated fashion by a non-traditional route, ra ther than going through a traditional secretory pathway. Exactly how MIF is secreted is not yet fully understood, but the limited evidence available suggests it is most likely via a transporter belong ing to the ATP-binding cassette

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14 (ABC) family.14 MIF is also unique in th at its secretion is induced by glucocorticoids, and it plays a critical role as a ci rculating counter-regulator of the immunosuppressive effects of glucocorticoids. MIF is able to promote inflamma tory responses despite ph ysiological levels of serum glucocorticoids, and it has been suggested that permitting inflamma tory responses when appropriate in the face of glucocorticoids wh ich have many homeostatic functions, aside from their immunosuppressive actions is precisely the intended pur pose and physiological role of secreted MIF. Concordant with this suggested anti-glucocorticoid role, under normal conditions glucocorticoid-induced MIF secretion follows a bell-shaped dos e-response curve, with MIF secretion peaking at physiological glucocorticoid levels, and recent studies suggest that MIF fluctuates in a circadian rhythm that is correl ated to serum glucocorticoid levels in rodents.23 Aside from its classification as a cytokine, in recent years, a revolutionary group of investigators have discovered that MIF is also an enzyme, exhibiting a thiol-protein oxidoreductase (TPOR) activity that lie s between amino acid residues 57 and 60.24 Accordingly, it has recently been suggested that MIF be re-classified as not only a cytokine, but also a member of the thioredoxin (Trx) family of antioxidant proteins due to the f act that it contains this TPOR motif, an identifying characteristic of Trx family members.25 However, it should be noted that there are cysteine-dependent, redox-active enzyme s that are not considered members of the Trx family, as well. Peroxiredoxins, enzymes that cont ribute to the regulation of cellular signaling by scavenging and, therefore, cont rolling the intrace llular levels of hydrogen peroxide an important signaling molecule at the expense of reducing equivalent s donated by Trx, are one such example.26 Should future investigations and furt her clarification of the functions and characteristics of MIF (which has yet to be cr ystallized as a monomer or homodimer) determine that it is not a Trx-family member, it may be possible that MIF, like peroxiredoxin 1, is the

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15 canonical member of a new family of TPOR-dep endent enzymes (and, perhaps, these enzymes could be assigned to a larger family designated simply as the TPOR family, as was suggested by Thiele and Bernhagen in their recent review on the enzymatic activities of MIF). This would hardly be surprising, given the fact that res earchers working on the se creted, cytokine-like function of MIF have proposed that the same could be said of MIF as a cytokine. Efforts to assign it to a cytokine family have failed, yet th e evidence clearly shows that it can serve this function. Nonetheless, assuming the proposition that MIF belongs in the Trx family is correct, as one would expect, there is limite d evidence that the production of MIF can be influenced by the redox status of the cell.25 Because of this proposed dichotomous nature of MIF, many authors have come to refer to it affectionately as a cytozyme or redoxkine. Members of the thioredoxin family are essential to maintaining redox balance in the intr acellular environment by scavenging of reactive oxygen sp ecies (ROS), act as electron donors for reducing enzymes, and they may also influence signaling pathways and the activity of other proteins by reducing critical cysteines, as will be discussed in detail late r; and the available evidence, though tentative compared to the body of literature concerning these functions of Trx, is ever-mounting and suggests that MIF is no exception in this regard.25, 27 To reconcile these seemingly disparate functi ons of MIF, great minds in the MIF field suggested several years ago that perhaps whether MIF acts as a proinflammatory cytokine or an antioxidant protein may depend on the concentration of MIF in the tissue, with relatively low concentrations acting as a proinflammatory mediator and high er concentrations serving an antioxidant function.28 While the circumstances that determine whether MIF fulfills its cytokine or TPOR-based enzymatic function remain a my stery, a growing body of evidence indicates that

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16 the simplest explanation is that something as simple as localization may be the determining factor. Perhaps, when MIF is needed as a pro-inflammatory mediator, it is secreted and signals via its canonical or chemokine receptors. To fu rther complicate matters, it has been suggested that, even when secreted MIF is acting in its cytokine capacity, the TPOR motif of MIF might be involved in mediating its association with the CD74 portion of its receptor complex.25 Conversely, in the absen ce of a stimulus that would promote secretion, MIF remains in the cytoplasm of the originating ce ll, and studies from many laborat ories, including ours, suggest that it then acts as a TPOR protein and proba ble member of the Trx family, influencing redoxregulated targets and perhaps even scavenging ROS. Introduction to the Thioredoxin Family of Proteins Thioredoxin (Trx) (F igure 1-1B) was first di scovered in the 1960s coincidentally, around the same time as MIF as a hydrogen do nor for deoxyribonucleotide synthesis in E. coli.29 Contrary to the long history in prokaryotes, interest in the e ukaryotic Trxs is a relatively new matter, taking shape over the past couple of decades. The following sections will focus on mammalian Trxs, specifically Trx1, referred to here af ter simply as Trx. As the characteristics of Trx are discussed, I will point out ways in which Trx and MIF are alike to support the argument that MIF may be a Trx family member but also ways in which MIF and Trx may be different in order to present a balanced perspective. Interesti ngly, in some ways, MIF exhibits characteristics more similar to glutaredoxin (Grx ), the other major member of the Trx family. When appropriate, similarities between MIF and Grx will also be discussed. Before launching into a deta iled discussion of Trx and its many interesting features, in the interest of perspective, a br ief review of Trx systems in mammals is warranted. There are two distinct isoforms of Trx in mammals. Trx1 is usually found in the cytoplasm of cells, 27 but can be induced to localize to the nucleus or be exported from th e cell under the correct stimuli.30, 31

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17 Trx2 is a mitochondrial protein.32 The active site of Trxs beco mes oxidized, forming a disulfide bond between the active site cystei nes, over the course of catal ysis (Figure 1-2). They are reduced, returning to the active form, (usually at the expense of NADP H) by Trx reductases (TrxR), large selenocysteine enzymes with ac tive sites consisting of the amino acid sequence Gly-Cys-SeCys-Gly. There are 3 types of TrxR s: TrxR1 (cytoplasmic), TrxR2 (mitochondrial), and the testis-specific thioredoxin glutathione reductase.27 The thioredixon 1 gene (TXN1) is well-characterized and contains two overlapping promoters exhi biting elements for basal and inducible regulation of Trx expression. Interesti ngly, Sp1 seems to be important in the basal regulation of the Trx gene,33 and a recent publication has shown the same to be true for MIF.34 An important inducible cis element of the Trx gene that, so far, appears to be lacking in the regulation of the MIF gene is the antioxidant response element (ARE), which is particularly well-studied because it is responsible for the induction of Trx under conditions of oxidative stress.30, 35 Glutaredoxins (Grxs) are the other major well-known members of the Trx family. They are further-removed from the purpo ses of this discussion than Trx, so will be reviewed in much more brevity. They share some functions with Trx enzymes, but also have some independent functions. Mammalian cells contain 3 Grx isofor ms: the dithiol-mechanism Grxs known as Grx1 (cytosolic) and Grx2 (mitochrondrial), and the monothiol-mechanism Grx5 (named as such due to homology to yeast Grx5, appears to be m itochondrial). Oxidized Grxs are reduced by glutathione, which is then reduced by glutaredoxin reductases at the expense of NADPH. Grx2 is interesting in that it can also be reduced by TrxRs.27 Characteristics and Functions of Th ioredixon 1: A Comparative Stu dy Trx family members have a characteristic C XXC motif (Figure 1-1), with X being any amino acid, which is responsible for the redox enzymatic function of these proteins. Trxs active

PAGE 18

18 site contains a Cys-Gly-Pro-Cys motif that was identified when the protein was first sequenced in 1968.36 MIF follows this rule; as mentioned above it shares this iden tifying characteristic, exhibiting a Cys-Ala-Leu-Cys motif in its activ e site. Importantly, MIF also shares some additional conserved residues with other Trx fam ily members: a phenylalanine that is 5 to 7 residues upstream of the N-terminal Cys of the CALC motif and a le ucine/valine (a leucine in the case of MIF) an additional 2 to 3 re sidues in the N-terminal direction.25 Though the structure of the MIF monomer is vaguely similar to the Trx monomer, one important characteristic of the Tr x family that MIF is lacking is the thioredoxin fold structural motif, consisting of 4 beta sheets and 3 alpha heli ces in most family members. Trx contains this basic thioredoxin fold, plus an additional beta sheet and alpha helix at the N-terminus.37 In contrast, MIF is structurally more like b acterial tautomerases and human D-dopachrome tautomerase, and MIF has a tautomerase enzymatic motif at its N-terminus, in-depth discussion of which is beyond the scope of these studies. The importance of the tautom erase activity of MIF is still unclear and remains controversial, as an in vivo substrate has yet to be identified. Furthermore, there is little se quence homology between MIF and ta utomerases, despite their mild structural similarities.25 However, it is worth noting that MI F is certainly far from the only Trxlike protein that has been discove red but may not fit perfectly in to the Trx mold. There are many, usually tissueor organell e-specific, proteins that c ontain CXXC motifs but deviate somewhat from the classical Trx characteristics, yet are still considered to be Trx family members; some of which are redox active, some of which are not.27 Nonetheless, there are hundreds of available solved Trx structures, and they reveal some important characteristics of reduced and oxidized Trx. Trx undergoes some conformational changes upon reduction that involve hydrogen bonds in the active site. Thes e changes can affect

PAGE 19

19 the binding of Trx to other proteins.38, 39 It has been argued that MI F may share this feature of altered conformational states (and, perhaps, altered binding activity ) depending on its redox status.25, 40 Biochemical studies of MIF indicate that none of its 3 cysteines form an intermolecular disulfide bond, but th at the cysteines of the CALC motif form an intramolecular bond under the correct conditions (as would be e xpected of a 2-Cys mechanism Trx protein).24 Despite the available evidence to the contrar y, the limited crystal structures of the MIF homotrimer that are available seem to indicate some spatial constraints on the location of the two cysteines of the CALC motif, with one lying at the N-terminus of a be ta sheet and the other located in the preceding loop, which would make an intermolecular disulfide unlikely. However, as Thiele and Bernhagen pointed out in their ex cellent review on the TPOR activities of MIF, it bears noting that the crystal stru ctures currently available for MI F were obtained from solutions containing MIF at unnaturally high concentrations under reducing c onditions. It is possible that this encouraged aggregation of MIF and pushed th e equilibrium of the various oligomers towards the homotrimer species.25 Interestingly, it would be appropriate to mention here that, like MIF, Trx can form dimers (though this claim is cont roversial and the physiolo gical consequences are unknown)41, 42 and that Grx2 (a Trx family member) can utilize iron-sulfur clusters to form a bridge that effectively homodimerizes th e molecule, rendering it enzymatically inactive.43 It may, therefore, be possible that the formation of oligomers of these redox active enzymes could serve as regulators of their re dox activity, and perhaps even sens ors of oxidative stress. Despite the conclusions drawn from the 3D structures of the MIF homotrimer, biochemical solution studies on MIF utilized MIF at more physiolo gical concentrations and re-folded MIF under oxidizing conditions, and the resu lting MIF was biologically and redox active. From this, the authors concluded that perhaps MIF, like Trx, undergoes major conformational changes during

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20 catalysis that would be more pe rmissive of intramolecular disu lfides and have not yet been detected by the limited crystal structures of MIF that are currently available.25 Furthermore, regarding interesting regulatory and structural characteristics of Trx, this enzyme has at least 3 cysteines that lie outside the active site and are no t involved directly in catalysis: Cys62, Cys69, and Cys73. Cys62 and 69 can form an intramolecular disulfide bond that is not reducible by TrxR and seems to decr ease the rate of reduction of the active site by TrxR, which may regulate recycli ng of Trx to the reduced state.44, 45 Cys73 also seems to be important in regulating the f unctions of Trx. It can undergo reversible s-ni trosylation, sglutathionylation, or disulfide formation. The s-n itrosylation appears to be important in Trxs ability to modulate caspase 3 activity.46 The glutathionylation of Cys73 renders the enzyme inactive.47 Most intriguingly, Cys73 can form an intr amolecular disulfide bond with the Cys73 of another Trx monomer, leading to the formation of a Trx homodimer.48 Dimerization, as previously mentioned, is reminiscent of MIF, though the function of dimerization of either molecule remains a mystery. Though little is known about and intense investigation still surrounds the possible mechanisms of MIF catalysis and recycling, the mechanisms governing the catalysis and reduction of Trx and most of its other family me mbers are well-understood. Trx utilizes its active site cysteines to catalyze the reduction of disulfides via a dithiol mechanism (in contrast to the monothiol mechanism that can be utilized by Grxs). The initial binding of Trx to its targets is governed by a hydrophobic area aro und the active site. Next, the thiolate of the N-terminal cysteine attacks the target disulfide, which resu lts in a mixed disulfide intermediate. This mixed disulfide between Trx and its target is then redu ced by the C-terminal thiolate of the Trx active

PAGE 21

21 site. The result is a disulfid e bond between the cysteines of the Trx CXXC motif, which, as mentioned previously, is reduced by Tr xR at the expense of NADPH. Finally, regarding the functions of the Trx molecule, it is most well known as a redox enzyme, but Trx also has functions that are presum ed to be non-redox related, or at least are not involved in what is classically thought of as redox regulatio n of cellular function. We now know that Trx, like MIF, is a multi-functional molecule, serving at times in many capacities: thiol redox control of transcripti on factors and enzymes, as an el ectron donor for many reductive enzymes and peroxiredoxins, as a ROS scavenge r and absolutely crucial antioxidant. Other, more recently identified functions of Trx in clude a cytokine/chemokine-like function (when secreted, interestingly similar to MIF in this rega rd) and roles in the regula tion of protein folding, apoptosis (like MIF, which nega tively regulates p53-mediated apoptosis), and NO metabolism.27 Macrophage Migration Inhibitory Factor and Thioredoxin in the Brain It has been dem onstrated that MIF is pres ent in the cell bodies and processes of both central and peripheral ne urons. In 1998, Bacher et al assessed MIF distribution in the brain of male sprague dawley rats utilizing various techniques such as immunohistochemistry and in situ hybridization. They demonstrated the presence of MIF mRNA a nd protein in neurons of the cortex, hippocampus, cerebellum, pons, and hypotha lamus. The authors also reported a diffuse MIF signal localizing to glia l cells throughout the brain.49 Several laboratories, including our own, have investigated the possi ble functions of CNSlocalized MIF. The aforementioned study showed that MIF can play an inflammatory role in the CNS when its expression and secretion is induced upon central administration of LPS. However, Bucala points out that the high cons titutive expression levels of MI F in neurons argue for other, non-inflammatory, physiological functions in the brain.10 Nishio et al. found that MIF may play a role in regeneration of peripheral nerves.50 Another group has suggested that MIF participates

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22 in detoxifying products of catecholamine meta bolism, perhaps serving a neuroprotective function.51 Also, the glucocorticoid-antagonism functi on of secreted MIF may be important in protecting hippocampal neurons fr om glucocorticoid-induced atr ophy in situations when these steroids are elevated, such as chronic stress.10, 49 MIF may also have important functions in modulating the release of cytokine s and nitric oxide in the brain.52, 53 Finally, as will be discussed later in detail, MIF can serve as a negative re gulator of the central chronotropic actions of angiotensin II (Ang II).54 The localization and functions of Trx in the brain are better underst ood than is the case for MIF. This is due to the Trx systems status as one of the most important antioxidant defense systems in neurons, which are highly metabolically active causing them greater exposure to ROS than many other cell types. Moreover, neur ons usually exhibit lo wer levels of other important antioxidants, such as glutathione (GSH ), than other tissues. Hence, the Trx system may play a larger role in antioxidant de fense in the CNS than it does systemically.55 Regarding basal expression, Trx is found in neurons throughout the brain, while being mostly absent from glial cells. A notable exception is glial cells of white matter.27 However, under conditions of acute stress, such as ischemia, glial cells are the main source of induced Trx expression and secretion, consis tent with their supportiv e role towards neurons.56, 57 Experiments in culture and in vivo show that secreted Trx can have neuroprotective effects. Infusion of Trx, systemically, protects the brain from ischemic events in rodents.58 Furthermore, when Trx secretion is induced from astrocytomas in cultures, the resu lting conditioned medium can be utilized to promote the surivival of neuronal cultures in the absence of serum.27 Trx may even be an indirect neurotrophic factor, media ting downstream effects of nerve growth factor (NGF).59 However, dysregulation of the Trx sy stem has been implicated in several

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23 neurodegenerative diseases such as Alzheimers disease, Parkinsons disease, and CNS malignancies.27 Finally, as has been indicated for MIF and will be discussed in detail later, studies from our laboratory suggest that an intracellular function of Trx may be to negatively regulate the central chronot ropic actions of Ang II.60 The Brain Renin-Angiotensin System The brain renin-angiotensin system (RAS) is of interest and particul arly important due to its implications in the development of essentia l hypertension. Essential hypertension, also known as primary hypertension, is often simply define d as persistent high bl ood pressure with no known cause, and sometimes with the added caveat that no preexisting renal disease is present. Kaplan goes further to describe primary hypertension as the elevation of blood pressure seen in younger people which has a genetic foundation and is sh aped by many environmental factors, while making the distinction that the common form of hypertension seen in the elderly is usually isolated systolic hypertension, which reflects a stiffness of the proxima l, capacitance vessels. He further adds that essential hypertension usua lly reflects an increas e in cardiac output and functional (i.e., reversible) constric tion of peripheral resistance vessels.61 Despite the fact that decades of research into the pathogenesis of this disease have not yet re vealed the exact etiology, it is widely held that the brain plays a critic al role in the developm ent and maintenance of essential hypertension. Specifically, it is commonly believed that dysregulation of sympathetic outflow and alterations in baroreflex func tion are the primary avenues by which the CNS contributes to essential hypertension.62 Much of the evidence for the involvement of the CNS in the pathogenesis of this disease derives from the most-studied animal model of essential hypertension, the spontaneously hypertensive rat (SHR). However, before the evidence concerning this model is presented, a modest re view of the components of the RAS is required.

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24 All aspects of the RAS (Figur e 1-3) have been identified in the brain, but they have not all been localized to the same cell. Accordingly, it is still not understood exactly how Ang II, the octapeptide mediating the pressor effects of the brain RAS, is generated in the brain and, hence, the subject remains controversial. Nonetheless, th ere is available evidence suggesting that it is, indeed, generated behind the blood-brain barrier.63, 64 The RAS is a cascade that originates with the peptide angiotensinogen, which is cleaved by renin. The resulti ng peptide, angiotensin I, is cleaved by angiotensin converting enzyme (ACE), to the pressor peptide angiotensin II (Ang II). Ang II can then be cleaved into smaller peptides that have, just recent ly, been identified as having independent functions, but the details are beyond the scope of this discussion. Once Ang II is generated, it then goes on to bind to either of its cogna te receptors, the type I (AT1R) or type II (AT2R) receptor, both of which are G-protein coupled 7 transmembranespanning receptors. The AT1R me diates the pressor effects of Ang II and is the predominant isoform expressed in the brain. The contribution of the AT2R to blood pressure regulation remains unknown, though, in a broad sense, the actions of the two receptor sub-types are thought to be antithetical in nature. Significantly, functiona l studies utilizing recept or blockade strategies indicate the presence of the AT 1R in several cardiovascular regulatory regions of the brain, including the paraventricular nu cleus (PVN) of the hypothalamus. The PVN is thought to play a critical role in integrating relevant afferent and humoral signals and adjusting endocrine and autonomic responses accordingly.62 Studies on the over-activity of the brain RAS in SHR, the most commonly-used model of neurogenic hypertension, abound and are reviewed in excellent detail by Veerasingham and Raizada.62 Therefore, they will be only briefly touched upon here. In pre-hypertensive animals, Tamura et. al. have observed increased brain angiotensinogen in SHR.65 Ruiz et. al.

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25 demonstrated higher levels of reni n-like activity in areas important to cardiovascular control such as the NTS and the hypothalamus in SHR comp ared to control Wist ar Kyoto (WKY) rats.66 Ang II content was found to be increased in the PVN of SHR.67 Many studies have demonstrated greater Ang II binding and increased AT1R mRNA in most of the areas of the brain thought to be important to fluid balance, c ontrol of the cardiovascular system and/or sympathetic tone such as the SFO, MnPO, PVN, NTS, and RVLM.68-70 Studies observing the action of centrally-applied Ang II have concluded, as would be expected when taking all of these observations into account, a greater pr essor response in SHR compared to WKY. Studies using pharmacological inhibitors of the RAS have provide further evidence that overactivity of the RAS is involved in hypertension in SHR. For example, losartan, an AT1R inhibitor, injected into the lateral ventricle decreases blood pressure in SHR, but not in normotensive animals. Studies utilizin g antisense gene targeti ng that reduce levels of angiotensinogen and the AT1R decr ease BP in SHR as well, but not in WKY. Finally, transgenic studies over-expressing parts of the RAS in the brains of normotensive animals show that hyperactivity of the brain RAS in normotensive animals is sufficient to cause hypertension in these models. Considering all of these points, Veerasingham and Raizada conclude that the increase in brain RAS activity precedes or para llels the development of hypertension in SHR. It is not known exactly how all of th ese increases in brain RAS activ ity contribute to hypertension, but it is currently commonly held that they result in increased sympathetic vasomotor tone.62 Reactive Oxygen Species, Neuronal Function, and Hypertension Dr. Robin Davisson, one of the leaders in th e neuronal ROS field, has recently published an extensive and excellent review on the neuropathogenesis of hypertension and oxidative stress,71 therefore the concepts will be only briefly reviewed here For reference, the proposed pathway for ROS generation in the brain is briefly illustrated in Figure 1-4.

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26 Recently, the fields of redox signaling and ne urogenic hypertension have been speedily merging, and the collision has produced several significant studies establ ishing a firm argument for the involvement of ROS in physiological A ng-II neuronal signaling and oxidative stress in the pathogenesis of hypertension. For example, the Davisson lab ha s published a study demonstrating that the ability of peripheral A ng II acting on the subfornical organ (SFO), a circumventricular organ that is well-establishe d in the central presso r actions of Ang II and which sends efferents to the PVN,72 to induce hypertension is mediated by intracellular superoxide.73 This group has further determined that scavenging of superoxide in the SFO abrogates physiological responses to intr acerebroventricular (ICV) Ang II in mice.74 Other labs have substantiated these studies utilizing ICV tempol, a superoxide di smutase (SOD) mimetic that scavenges superoxide, to prevent ICV A ng II-induced increases in blood pressure and sympathetic activity.75, 76 On the molecular level, in vitro studies have conclusively shown that Ang II regulates neuronal firing by a pathway in volving superoxide generation and modulation of potassium currents.77 Finally, a recent provocative study from our laboratory has indicated oxidative stress in the development of hyperten sion by showing that over-expression of MIF in the PVN can attenuate the devel opment of high blood pressure in SHR, and this ability of MIF depends on the TPOR motif and, therefore presumably, MIFs catalytic redox activity.78 Likewise, advances in unde rstanding the mechanisms by which ROS affect neuronal behavior are steadily progressi ng. Davisson names at least thr ee ways that ROS can modulate neuronal activity: regu lating ion channels, affect ing transcription factor activity, and modulating intracellular nitric oxide (NO) levels.71 Ang II-induced ROS have b een shown to affect both calcium and potassium channels in neurons. Spec ifically, superoxide may open calcium channels directly, stimulating L-type calci um current, and close potassiu m channels, inhibiting delayed

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27 rectifier potassium current77, 79, 80 Ang II-derived ROS are also implicated in MAP kinasemediated neuronal activation, though the details of how MAPKs act in this regard are yet undiscovered.81 ROS are also implicated in the modulation of gene expression in neurons by activating various transcription factors. On e such example is activator protein 1 (AP-1), a dimer of c-jun and c-fos. The available evidence so far indicates that Ang II-mediated activation of AP-1 via ROS depends upon the MAPK fam ily. The likely MAPK candidate s for redox-sensitive Ang IImediated activation of AP-1 are JNK and FRK.82-84 Another significant example of a redoxsensitive transcription factor regulated by Ang II-induced ROS is nuclear factor B (NF B). Like AP-1, NF B binds to DNA as a dimer, the most common consisting of p50 and p65.82 Also similar to AP-1 is the likelihood th at Ang II-based redox regulation of NF B is mediated by redox-responsive MAPKs, most likely JNK and p38.82 Some very thought-provoking features of NF B redox-sensitive regulation in the periphery include activation by in creased levels of oxidized Trx82 and Ang II-induced hydrogen peroxide.85 Furthermore, a significant recent study showed that periphe ral inhibition of NF B in young SHR prevents the development of hypertension.86 Davisson suggests the intriguing hypothe sis that redox-depende nt modulation of NF B in cardiovascular control regions of the brain could also be involved in hypertension, given that many aspects of Ang II signaling in the periphery are mi micked in neurons.71 Finally, ROS can affect neuronal behavior by interacting with nitric oxide (NO). Though the role of NO with regard to sy mpathetic outflow is controversial, it is generally thought to be sympathoinhibitory. Ang II-induced superoxide decreases NO availa bility, directly, by reacting with NO to form peroxiynitrite and indirectly, by down-regulating neuronal nitric oxide synthase (nNOS) in cardiovascular contro l regions such as the PVN.87, 88 Davisson concludes, therefore,

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28 that these studies clearly demonstrate that impair ment of NO availability is at least one of the mechanisms through which Ang II increases sympathetic drive.71 Macrophage Migration Inhibitory Factor a nd Thioredoxin as Negative Regulators of Angiotensin II in the Central Nervous System MIF is of keen interest to our lab and the field of blood pressure regulation due to its ability to serve as a negative regulator of th e neuronal actions of angiotensin II (Ang II). Specifically, Ang II up-regulates MIF in neurons cultured from normotensive rat hypothalamus and brainstem,89 and increased intracellular levels of MIF protein exert a negative regulatory action over the neuronal chr onotropic effects of Ang II.54 Similar interactions between MIF and Ang II are observed in the rat brain in vivo For example, CNS injection of Ang II increases MIF expression in the paraventricular nucleus (PVN) of the hypothalamus, an area that has a key role in regulating sympathetic outflow and hypothalamu s/pituitary (HPA) axis activity. The increased levels of intracellular MIF in P VN sympathetic regulatory neurons serve to blunt the increases in discharge of these cell s elicited by Ang II and the increase s in blood pressure produced by CNSinjected Ang II.90 Furthermore, it is apparent that MIFs ability to negatively regulate the actions of Ang II is mediated by its TPOR activity a nd, possibly, via scavenging of ROS (Figure 1-5).54, 90 These findings became even more important when considering the fact that Ang II fails to increase MIF expression in neurons cultu red from the hypothalamus of spontaneously hypertensive rats (SHR), or in the PVN of SHR.91 In addition, experiments from our laboratory indicate that neurons in the PVN of SHR are devoid of immunoreactive MIF.78 However, intracellular application of exogenous MIF into SHR hypothalamic neurons in culture can depress the neuronal chronotropic action of Ang II in these cells an effect mediated by the TPOR activity of MIF. Thus, while MIF has the potential to depress th e chronotropic action of

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29 Ang II in SHR neurons, it is unlik ely that this regulatory mech anism occurs since Ang II does not up-regulate this protein in SHR neurons.91 By extrapolation, it is poss ible that a lack of this MIF regulatory mechanism contributes to the hyp er-responsiveness to Ang II in the PVN of SHR and the consequent high blood pressure observed in these animals. This idea is borne out by studies from our laboratory which indicate that long-term viral-mediated over expression of MIF in the PVN of young SHR attenuates the developmen t of high blood pressure in these animals.78 Based on the above, it is of major interest to understand the intracellu lar mechanisms that control MIF expression in normal rat neurons, and to identify the defects that are responsible for a lack of MIF expression in SHR neurons. To this end, we have been investigating possible mechanisms for inducible expression of MIF in CNS neurons, which will be explored in detail in the following chapters. Finally, our hypothesis that MIF ac ts via its TPOR activity to negatively regulate Ang IIs effects on neuronal currents lead us to investigate if other TP OR-containing proteins, namely Trx, could also exert a negative-regulatory influence over the central ac tions of Ang II. Given that ROS are now identified as important mediat ors of Ang II actions in neurons and that MIFs TPOR activity appears to be a negative regulat or of Ang II, we believe that TPOR-containing proteins may, perhaps, represent a general mechanism whereby Ang II sensitivity can be modulated in neurons. Indeed, in a recent study, we found that Trx, like MIF, is increased in neurons in response to Ang II signaling. Furthermor e, like MIF, Trx is able to prevent Ang IIstimulated increases in delayed -rectifier potassium current, and th is activity of Trx is dependent on the action of its TPOR motif.60 Taking all of the above intera ctions between MIF, Trx, and Ang II into consideration, we have also endeavor ed to ascertain the leve ls and distribution of these important TPOR-containing proteins in ar eas pertinent to sympathetic activity of pre-

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30 hypertensive SHR and WKY brains a nd to correlate this data with the oxidative status of these tissues. The results and implicati ons of these studies follow.

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31 Figure 1-1. Features of A) m acrophage migration inhibitory factor and B) thioredoxin. The TPOR motif of each protein is marked in green. N indicates the N-terminus of the peptide, C indicates the C-terminus. Note that the MIF peptide begins at amino acid 2, as the N-terminal proline is cleaved off during post-translational processing. The N-terminal tautomerase doma in of MIF is also noted. 2 115 57-60 CALC TPOR Tautomerase N CA 1 104 32-35 CGPC TPOR N CB

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32 Figure 1-2. Catalytic mechanism of the thioredoxin system. Arrows indicate the direction of each reaction. The catalytic mechanis m of MIF is not known, but may be similar in nature. TR = thioredoxin reductase Trx = thioredoxin, (SH)2 = thiols (reduced form), S2 = disulfide (oxidized form). NADPH + H+NADP+TR-S2TR-(SH)2 Trx-(SH)2Trx-S2Protein-S2Protein-(SH)2

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33 Angiotensin I-7 Angiotensin I-9 ACE ACE2 AT1R AT2R Mas-R IRAP AngiotensinogenReninAngiotensin IACEAngiotensin II Ang III Ang IVACE 2 NEP Vasoconstriction Increase SNA Increase Blood Pressure Maintain ECFV Growth Induction of MIF Vasodilation Natriuresis Anti-Growth Apoptosis, Differentiation Cerebroprotection Cardioprotection Memory retention & retrieval Vasodilation Angiotensin I-7 Angiotensin I-9 ACE ACE2 AT1R AT2R Mas-R IRAP AngiotensinogenReninAngiotensin IACEAngiotensin II Ang III Ang IVACE 2 NEP Vasoconstriction Increase SNA Increase Blood Pressure Maintain ECFV Growth Induction of MIF Vasodilation Natriuresis Anti-Growth Apoptosis, Differentiation Cerebroprotection Cardioprotection Memory retention & retrieval Vasodilation Figure 1-3. The renin-angiotensin system. Simplified schematic of the components of the RAS. The components relevant to this work are depicted in red.

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34 Figure 1-4. Angiotensin II-indu ced reactive oxygen species pr oduction. Ang II stimulates the AT1R, which initiates intracellular signaling events, such as protein kinase C (PKC) activation, ultimately leading to the assembly and activation of the NADPH oxidase complex at the cell membrane. NADPH oxidase generates superoxide, which is metabolized into hydrogen peroxide by supe roxide dismutase (SOD). Catalase is one of several enzymes that can detoxify peroxide after it has served its signaling purpose. O2 SOD H2O2H2O Catalase N A D P H O x i d a s e AT1R Ang II N O X p 2 2 p 4 7 p 6 7 p 4 0r a c PKC

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35 Figure 1-5. Model of possible in teractions between angiotensin II signaling and macrophage migration inhibitory factor. Proposed negative regulator y mechanisms of MIF are shown. MIF may negatively regulate the actions of Ang II directly through scavenging of ROS second messengers or bi nding to the intracellular face of the AT1R, leading to desensitization. It is also possible that MIF may i ndirectly affect the receptor or Ang II signaling mediators by interacting with other signaling and regulatory proteins. N AD P H O x i d a s e AT1R Ang II NOX p22 p47 p67 p40rac PKC O2 SOD H2O2 Nucleus MIF Transcriptional modulation Signaling Proteins MIF Scavenging ROS? Binding to the AT1R? Indirect effects? Signaling Proteins Reducing disulfides?N AD P H O x i d a s e AT1R Ang II NOX p22 p47 p67 p40rac PKC O2 SOD H2O2 Nucleus MIF Transcriptional modulation Signaling Proteins Signaling Proteins MIF Scavenging ROS? Binding to the AT1R? Indirect effects? Signaling Proteins Signaling Proteins Reducing disulfides?

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36 CHAPTER 2 INDUCIBLE EXPRESSION OF MACROPHAGE MIGRATION INHI BITORY FACTOR IN NEURONS Introduction Chapter 1 discusses the importance of understand ing the m echanisms that control inducible expression of MIF due to its recently-established role as a negative feed-back regulator of the chronotropic actions of Ang II in neurons. The observation that MIF expr ession can be induced in response to Ang II signaling in neurons, and that it is not in neurons of pre-hypertensive SHR, indicates that the central actions of MIF could possibly be imp licated in the development of hypertension and poses some important questions. What downstream-mediators of Ang II and the AT1R might be inducing MIF expression in CNS neurons? Furt hermore, as es tablished in chapter 1, it appears that the AT 1R is uncoupled from the cont rol of MIF expression in SHR neurons. Therefore, it follows to inquire whethe r these potential mediators perform the same in normotensive and SHR rat neurons. Our first goal in these studies was to estab lish a candidate downstr eam mediator of Ang II that induces MIF expression in neurons. Accordingly, it is no w well known that Ang II, acting via the AT1R, induces ROS production in neurons by activating NADPH oxidase.92 This leads to superoxide production which should be metabolized to hydrogen peroxide (H2O2) by superoxide dismutases.93 As expected, many studies have concluded that H2O2 is a product of Ang II signaling in many cell types, including neurons.94 Furthermore, it has been demonstrated that H2O2 can cause induction of MIF e xpression in peripheral tissues.95 Finally, it has been shown, recently, that the human MIF promoter is regulated by Sp1 and CREB,34 two redox-sensitive transcription factors,96, 97 indicating that MIF expression mi ght be sensitive to the redox environment of the cell. Ther efore, we hypothesized that H2O2 may be, at least in part, responsible for inducible expression of MIF in CNS neurons in response to Ang II signaling.

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37 Furthermore, we sought to determine whether H2O2 possesses the same abilities with respect to inducing MIF expression in normo tensive rat and SHR neurons. In the present study, our aim was to determine whether H2O2 can cause up-regulation of MIF expression in CNS neurons a nd whether MIF is differentially regulated by ROS in neurons cultured from SHR and WKY rats Together, the data presente d here demonstrate that H2O2 can cause inducible expression of MIF in CNS neur ons cultured from normotensive, but not SHR, newborn rats. Materials and Methods Animals. Fo r our experiments, we utilized adult Sprague-Dawley (SD), Wistar Kyoto (WKY), and Spontaneously Hypertensive (SHR) rats as breeders to produce rat pups that were used for the production of neuronal cultures. The a dult breeder rats were purchased from Charles River Farms (Wilmington, MA). All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee. Materials. Dulbecco's modified Eagle's me dium (DMEM) was obtained from Invitrogen (Grand Island, NY). Plasma-derived horse serum (PDHS), Fetal Bovine Serum (FBS), 30% stabilized H2O2 solution, PEG-catalase, Actinomycin D, B-actin monoclonal antibody, and secondary HRP-conjugated antibodies were obtained from Sigma (St. Louis, MO). Cells-tocDNA II kits were purchased from Ambion (Austin, TX). Primers for MIF and 18S for real-time RT-PCR were obtained from App lied Biosystems (Foster City, CA). Glucose Oxidase was purchased from Calbiochem (San Diego, CA). CytoTox96 Non-Radioactive Cytotoxicity Assays were purchased from Promega (Madison, WI). Rat MIF antibody was purchased from Torrey Pines Biolabs (East Orange, NJ).

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38 Preparation of neuronal cultures and tissues. Neuronal cultures were prepared from the hypothalamus and cortex of newborn rats as described previously.98 Cultures were grown in DMEM containing 10% PDHS for a further 12 days before use. Preparation of glial cultures. Glial cultures were derived from the aforementioned neuronal cultures (which are ~90% neuronal, 10% astrocytes ). Neurons were killed by incubating the dishes in 100 mM KCl at room temperature for 5 minutes. The KCl solution was replaced with DMEM containing 10% FBS to en courage cell proliferation and cultures were monitored until ~80% confluency. The DMEM/FBS medium was then replaced with DMEM/PDHS medium and glial cultures were culture d for at least a further 7 days to parallel the feeding/utilization schedule of the corresponding neuronal cultures. Analysis of MIF mRNA in cultures. cDNA was produced from neuronal cultures with the Cells-to-cDNA II kit, which allows reverse tran scription without RNA pu rification. Levels of MIF mRNA were quantified by real-time RT-PCR as described previously54. Data were normalized to 18S rRNA. Analysis of MIF protein in cultures. Neuronal cultures were lysed in Laemmli Sample Buffer (Biorad, Hercules, CA) and 10 ug of total protein was loaded on the gel. Transfer to a PVDF membrane (Biorad, Hercules CA) was performed at 75 V for 1.5 hours in Towbin Buffer. Western blots were carried out and analyzed as detailed previously.89 Determination of Cytotoxicity. Neuronal cultures were treated as indicated and medium was collected from each well. Replicates were pooled, and then samples were centrifuged at 4 degrees Celsius and 5000 rpm for 5 minutes to pellet any particul ate matter or unattached cells. Supernatant was then utilized (50 uL) accordi ng to the protocol provided with the CytoTox96 kit.

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39 Determination of Protein Carbonyl Content. Neuronal cultures (12 wells of a standard 24-well plate, equal to approximately 6 million cells) were treated as indicated, and protein extracts were prepared in MES buffer as instructed. Protein carbonyl content was determined according to kit protocol utilizing the Protein Carbonyl Assay Kit from Cayman Chemical (Ann Arbor, Michigan). Cell Treatments. For experiments invol ving application of H2O2 a stock solution of H2O2 in water was diluted 100-fold into the culture medium to the final dose indicated. For experiments involving PEG-Catalase and Glucose Oxidase, the enzyme was dissolved in sterile DPBS then diluted 100-fold into th e culture medium to the final dose indicated. For experiments involving Actinomycin D (Act D), the Act D was dissolved in DMSO to give a concentration of 1 mmol/L. It was then d iluted 1000-fold into the culture medium to give a final dose of 1 mol/L. Data Analysis. Results are expressed as mean SEM. Statistical significance was evaluated with the use of a 1-way ANOVA, followed by a NewmanKeuls test to compare individual means. Diffe rences were considered significant at P <0.05. Results Hydrogen Peroxide Stimulates an Increase in Macrophage Migra tion Inhibitory Factor in Neurons Cultured from Normotensive Rats, but not Spontaneously Hypertensive Rats. Our first goal was to establish whether H2O2 contributes to induci ble expression of MIF mRNA in primary neuronal cultures from normotens ive rats and SHR. To test this idea, primary neuronal cultures prepared from newborn SD and WKY rats and SHR were incubated with H2O2 (30 mol/L; 1, 2 or 3 hr.), a cell-permeant source of ROS. This was follo wed by analysis of MIF mRNA expression via real time RT-PCR in neurons from all three genetic strains and analysis of MIF protein levels in SD rat neur ons. The results indicate that H2O2 elicits an increase in MIF

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40 mRNA that is statistically significant within one hour in neurons from both of the normotensive rat strains (Figure2-1, A and B) Conversely, the same dose of H2O2 was unable to elicit any significant increase in neurons cultured from SHR throughout the 3 hour time period observed (Figure2-1C). As expected, H2O2 also caused a time-dependant in crease in MIF protein in SD rat cultures (Figure 2-2). To confirm these results, primary neuronal SD rat cultures were also incubated with glucose oxidase. Glucose oxidase produces H2O2 via oxidation of glucos e in the cell-culture medium. Primary neuronal cultures were treated with 0.5 mU glucose oxidase for 1 and 5 hours. The results show that glucose oxidase-derived H2O2 produces an increase in MIF mRNA levels that is significant by 5 hours (Figure 2-3). Finally, because the neuronal cultures used he re contain a small (< 10%) number of glial cells, we investigated whether H2O2-induced increases in MIF mRNA were restricted to neurons or if the effects could also be observed in glial cells. To this end, glial cultures that were devoid of neurons were incubated with 1, 10, 30, or 50 mol/L H2O2 for one hour, the time point at which the increase in MIF reaches significance in the corresponding neuronal cultures, and MIF levels ascertained by real time RT-PCR. The results demonstrate that H2O2 is unable to stimulate an increase in MIF mRNA levels in glial cultur es (Figure 2-4). Later time points were briefly investigated in pilot studies, and also showed now effect (dat a not shown). Collectively, these results confirm our hypotheses th at MIF mRNA and pr otein expression can be regulated by ROS in primary neurons.

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41 Hydrogen Peroxide Increases Macrophage Migrat ion Inhibitory F actor mRNA Expression in Primary Neurons Through a Sp ecific Intracellular Action. Because ROS at high concentrations can cause a generalized stress response in cells, we sought to determine if the stimulatory effect of H2O2 on MIF mRNA levels in neurons is a specific intracellu lar action of H2O2, rather than a non-specific re sponse to oxidative stress. First, in order to determine if the effects of H2O2 on MIF mRNA levels in neurons were due to an intracellular action of H2O2, we utilized PEG-catalase as an intracellular inhibitor of H2O2 signaling. Catalase is an enzyme of very high activity that qui ckly and efficiently metabolizes H2O2 into water and molecular oxygen. Th e PEG-conjugation of the enzyme not only renders it cell-permeant, but also particularly st able once inside the cell.16 Neuronal cultures were pre-treated with PEG-catalase for 24 hours. The medium was then removed and replaced with fresh conditioned medium, and th e cells were stimulated with H2O2 (30 mol/L) for one hour. The results show that scavenging the exogenously applied H2O2 in the intracellular environment prevents an increa se in MIF mRNA levels, indica ting that the exogenously applied H2O2 acts intracellularly to increase MIF levels (Figure 2-5). Next, we investigated whether the dose of H2O2 used for our studies was cytotoxic to the neuronal cultures. Primary SD rat neurons were incubated with H2O2 (30 mol/L) for 1 and 3 hours, followed by analysis of lactate dehydrogena se (LDH) within the cell culture medium. The LDH assay measures the activity of LDH released into the medium by dying or dead cells. The results indicate that the dose of H2O2 utilized for all experiments in this study is not cytotoxic to primary neuronal cultures. Ho wever, higher doses of H2O2 (e.g. 100 mol/L, utilized as a positive killing control) are, indeed, cytotoxic to the neuronal cultures (Table 2-1). Furthermore, we sought to determine whether a 30 mol/L dose of H2O2 represents a state of oxidative stress to our neur onal cultures by measuring protei n carbonyl content, an accepted

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42 indicator of intracellular oxidative stress. Primary SD neuronal cultures were treated with 30 or 100 (utilized as a positiv e control) mol/L H2O2 for 1 hour (i.e., the earliest time point of significant MIF induction in the presence of H2O2) and protein carbonyl content measured. The data indicate that protein car bonyl content is not significantly different in control versus 30 mol/L H2O2-treated neurons (Figure 2-6). Collectiv ely, these results demonstrate that the effects of H2O2 on MIF mRNA levels in primary neurons are a specific, intracellular signaling action of H2O2 and not the result of a genera lized oxidative stress response. The Increase in Macrophage Migration Inhibitory Factor Levels Obs erved in the Presence of Hydrogen Peroxide Involves Increase d Synthesis of Macrophage Migration Inhibitory Factor mRNA. Since real time RT-PCR measures steadystate mRNA levels, and steady-state mRNA levels represent the summation of both synthesis and degradation, we investigated whether the increase observed in steady-state MIF mRNA levels in the presence of H2O2 was due to an increase in transcription. To this end, we util ized actinomycin D (Act D) a general inhibitor of mRNA synthesis. Treatment of neuronal cultures with 1 mol/L Act D reveals that MIF mRNA is stable in neurons for at l east 6 hours, with half-life not yet reached by 8 hours (data not shown). This observed stability of MIF mRNA in our experimental conditions is similar to that reported previously in other cell types.99 Neuronal cultures were pret reated with 1 mol/L Act D for one hour and then stimulated with 30 mol/L H2O2 for 1 hour. MIF mRNA levels were then ascertained by real time RT-PCR. The data indica te that inhibiting transcription concomitantly with H2O2 stimulation prevents an increase in MI F mRNA levels (Figure 2-7). This result suggests that the effects of H2O2 on MIF mRNA levels in neurons are primarily due to increased MIF mRNA synthesis

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43 Discussion To our knowledge, this study represents the firs t dem onstration that ROS can regulate the expression of MIF in CNS neurons. Furthermore, we have shown that this effect of H2O2 involves intracellular events that are specific to neurons, and the data suggest that the increase in MIF involves de novo transcription. Finally, the observation that hydrogen peroxide fails to elicit an increase in MIF in neurons cultured from SHR, in contrast to their normotensive controls, provides support for the contention that the MIF gene responds in a specific and regulated fashion to redox signaling. In this study, we selected hydr ogen peroxide as our ROS donor for many reasons. First, we were aiming to study a reactive oxygen species th at is downstream of Ang II and the AT1R in neurons, and it has already been established that Ang II is capab le of producing intracellular H2O2 in many cell-types, includ ing neurons, and that this H2O2 has significant physiological effects (e.g. influencing sympat hetic activity in the brain).94 Second, it is readily cell permeant, with exogenously-applied H2O2 establishing equilibrium across the cell membrane on average within minutes.100 Finally, in the realm of possibilities of ROS donors, it is relatively stable since, while it is a ROS, it is not a fr ee-radical. Simply stated, addition of H2O2 to the culture medium is the easiest and most reliable way to manipulate the intrace llular redox environment, and it was desirable to manipulate intracellula r ROS levels in the absence of the myriad, confounding possible signaling actions for these proof-of-principle studies. Interestingly, the LDH, protein carbonyl, a nd PEG-catalase experiments (Table 2-1, Figure 2-6, and Figure 2-5, respectively) support the notion that exogenously-applied H2O2 functions as a signaling agent in our neuronal cu ltures, rather than a mediator of cell death and/or oxidative stress. This signaling function would be only na tural for intracellular ROS produced in response to a physiological ligand (i.e ., Ang II), but it may seem unusual for the exogenous dosage

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44 utilized, 30 mol/L, considering that many in vi tro studies conclude that this dosage, added exogenously to cell lines in culture, should resu lt in an approximately 3 mol/L intracellular concentration at equilibrium. This intracellular co ncentration might constitu te an environment of oxidative stress, since in vitro studies suggest that the hi ghest observed intracellular concentration of H2O2 generated for signaling purposes in mammalian cells is 0.7 mol/L.93 However, there are some important mitigating fact ors to consider in the context of our neuronal cultures. Our cultures consist of primary ce lls, which evidence sugge sts produce less endogenous H2O2 than transformed cell lines. Th erefore, it has been suggest ed the application of more exogenous H2O2 is required to oxidatively stress primary cells than transformed cells in culture.93 Furthermore, our cultures, while mostly neurona l in nature, always c ontain a small portion of glial cells. Neurons, microglia, astrocytes, and oli godendrocytes all have the capacity to detoxify H2O2 with varying efficiencies. Astrocytes and o ligodendrocytes, in particular, are remarkably efficient at detoxifying extracellular peroxide. In vitro studies reveal that H2O2 added to the medium of astrocyte cultures has a half-life of only a few minutes. Oligodendrocytes follow at a slightly slower rate, though their over-all capacity to detoxify H2O2 exceeds that of astrocytes.101 Therefore, it is highly po ssible that the exogenous H2O2 is rapidly detoxified when added to the medium of our cultures, and, consequently, the neurons are not exposed to doses high enough to create oxidative stress in the intracellular envi ronment. Moreover, the knowledge that a bolus application of H2O2 can be rapidly detoxified by many of th e cell types in our cultures prompted us to perform the experiments utilizing glucose oxidase, which represents a more chronic means of administering exogenous H2O2, to confirm our results. Our results raise some important questions as to the mechanism by which H2O2 is inducing MIF expression in CNS neurons. It is now widely recognized that H2O2, like nitric oxide, may be

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45 a readily-diffusible small molecule that acts as a signaling agent. Inde ed, in prokaryotes and yeast, systems that sense and signal in response to H2O2 are well-characterized.93 In higher mammals, redox signaling is emerging as a very important and complicated field, and many of the signaling pathways affect ed by ROS are still under inves tigation. Nevertheless, it is becoming clear that several kinase pathways are modulated by ROS and the activity of many transcription factors is su bject to redox regulation.102-104 For example, it has already been demonstrated that the MAP kinase, p38, is sensi tive to Ang II-based ROS signaling in vascular smooth muscle cells and neurons.81, 105 Further, it has been shown in many settings that transcription factors such as AP-1, SP-1, CREB, and NF B are sensitive to redox regulation,106108 and binding sites for these transcription factors have been identified in the promoter of the human MIF gene.109 Furthermore, a recent publication examining the constitutive and inducible expression of the MIF promoter has demonstrat ed, for the first time, that SP-1 and CREB are important transcriptional regulators of the MIF gene.34 Experiments to determine if these transcription factors may be the mediators of redox regulation of the MIF gene in neuronal cultures are planned for the near future in our laboratory. This study is provocative and physiologically sign ificant because, as we have established in prior reports, MIF is up-regulated in neur ons in response to Ang II signaling via the AT1R. MIF then serves, either directly or indirectly, as a negative regul ator of the chronotropic actions of Ang II in neurons that lie along key sympathe tic and neuroendocrine pa thways in the brain such as the PVN.54, 89, 90 Our studies strongly suggest that MIF may act in this regard by scavenging ROS,54 as do some other proteins that contain TPOR motif s (e.g. thioredoxin, peroxiredoxins),27 but the exact mechanism is still under inves tigation. In this way, we speculate

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46 that MIF may serve as a way for neurons to regula te their sensitivity to A ng II, especially Ang IIbased ROS production and their downstream effects. Furthermore, we have previously shown that Ang II does not indu ce MIF expression in PVN neurons of SHR, in contrast to their normotensive controls and we have hypothesized that this lack of MIF induction may contribute to th e hyper-sensitivity of th ese neurons to Ang II,91 and perhaps even the development and/or mainte nance of hypertension in these animals. This idea is born out by current studies in our laboratory showing that viralmediated over-expression of MIF in the PVN of young SHRs atte nuates the development of hypertension.78 We believe that in SHR the AT1R is uncoupled from the signaling pathway that induces MIF in normotensive animals. Therefore, in the presen t study, we have sought to investigate signaling pathways that are downstream of the AT1R in neurons, namely ROS signaling. Indeed, we have found that our initial hypothesis regarding ROS and MIF expression is correct. This information serves as a strong basis on which to build furt her investigation into the actions of ROS in normotensive animals regarding An g II signaling and MIF expression. Intriguingly, a recent pub lication has shown that H2O2 produced in the PVN in response to Ang II may play a role in re gulating sympathetic activity.94 Accordingly, it is tempting to visualize a feed-back loop such that Ang II causes H2O2 production in the PVN, which stimulates MIF production, subsequently feeding back to de crease the sensitivity of the neuron to Ang II and, perhaps, influencing the central sympathe tic and/or neuroendocrine actions of Ang II. Exactly how MIF is providing this negative feedb ack is still unknown and remains the subject of intense investigation in our laboratory.

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47 Figure 2-1. Hydrogen peroxide in creases macrophage migration inhibitory factor mRNA levels in primary neuronal cultures from norm otensive rats, but not spontaneously hypertensive rats. A) SD, B) WKY, and C) SHR neuronal cultures were incubated with either vehicle (H2O) or 30 mol/L H2O2 for 1, 2, or 3 hours, followed by analysis of MIF mRNA levels as describe d in the materials and methods. Means SE (n = 7 for SD, n = 5 for WKY, n = 6 for SHR) of the ratio of MIF mRNA to 18S rRNA at each time point are shown. *P < 0.05 vs. control, **P < 0.01 vs. control A 0 1 2 3 0.0 0.5 1.0 1.5 2.0 2.5** *Time (Hours)MIF mRNA/18s rRNAA 0 1 2 3 0.0 0.5 1.0 1.5 2.0 2.5** *Time (Hours)MIF mRNA/18s rRNA 0 1 2 3 0.0 0.5 1.0 1.5 2.0 2.5* **Time (Hours)MIF mRNA/18s rRNAB 0 1 2 3 0.0 0.5 1.0 1.5 2.0 2.5* **Time (Hours)MIF mRNA/18s rRNAB 0 1 2 3 0.0 0.5 1.0 1.5 2.0 2.5Time (Hours)MIF mRNA/18s rRNAC 0 1 2 3 0.0 0.5 1.0 1.5 2.0 2.5Time (Hours)MIF mRNA/18s rRNAC

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48 Figure 2-2. Hydrogen peroxide in creases macrophage migration inhi bitory factor protein levels in primary neuronal cultures. SD rat neurona l cultures were incubated with either vehicle (H2O) or 30 mol/L H2O2 for 1, 2, or 3 hours, followed by Western Blot analysis of MIF protein as described in the materials a nd methods. A) Data are means SE (n = 3) of the ratio of MIF protein to -actin at each time point. *P < 0.01 vs. control. B) Representative Western Blot showing the e ffects of hydrogen peroxide on MIF levels. B 0 1 2 3 0.75 1.00 1.25 1.50 1.75 2.00*Time (Hours)MIF/ -actinA

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49 Figure 2-3. Glucose oxidase incr eases macrophage migration inhibitory factor mRNA levels in primary neuronal cultures. SD rat neuronal cultures were incubated with either vehicle (DPBS) or 0.5 mU glucose oxidase for 1 and 5 hours, followed by analysis of MIF mRNA levels as described in the mate rials and methods. Bar graphs shown here are means SE (n = 4) of the ratio of MIF mRNA to 18S rRNA at each time point. *P < 0.05 vs. control. Control 1 hour 5 hours 0.0 0.5 1.0 1.5 2.0*MIF mRNA/18s rRNA

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50 Figure 2-4. Hydrogen peroxide does not increase macrophage mi gration inhibitory factor mRNA expression in primary glial cell cultures. SD rat glial cultures were incubated with either vehicle (H2O) or 1-50 mol/L H2O2 for 1hour, followed by analysis of MIF mRNA levels as described in materials and methods. Bar graphs shown here are means SE (n = 3) of the ratio of MI F mRNA to 18S rRNA at each concentration. Statistical analysis showed no signi ficance at any time point observed. Control 1 M 10 M 30 M 50 M 0.0 0.5 1.0 1.5 2.0Hydrogen Peroxide DoseMIF mRNA/18s rRNA

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51 Figure 2-5. Hydrogen peroxide acts intracellularly to elicit an in crease in macrophage migration inhibitory factor expression in primary ne uronal cultures. SD ra t neuronal cultures were incubated with either vehicle (DPBS) or 100 U of PEG-catalase for 24 hours. Medium was changed to fresh conditioned medium taken from age-matched, nave neuronal cultures. Cultures were then in cubated with either vehicle (H2O) or 30 mol/L H2O2 for 1 hour followed by analys is of MIF mRNA and protein levels as described in materials and methods. A) Bar graphs shown are means SE (n = 6) of the ratio of MIF mRNA to 18S rRNA unde r each treatment condition. *P < 0.001. B) Bar graphs shown are means SE (n = 3) of the ratio of MIF protein to -actin protein under each treatment condition. *P < 0.01. Western blot shown in lower panel is representative of the 3 experiments quantified in B. AB C o ntr o l 2O2H PEG-Catalase + P E GC a t a l a s e2O2H 0.0 0.5 1.0 1.5 2.0 2.5 *MIF mRNA/18s rRNA Control 2O2H PE GC a t a l a s e + PE G -Catalase2O2H 0.0 0.5 1.0 1.5 2.0 *MIF/ -actin MIF, 12.5 kDa -Actin, 42 kDaC o n t r o l H2O2P E G C a t a l a s e P E G C a t a l a s e + H2O2

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52 Table 2-1. Hydrogen peroxide ( 30 mol/L) does not elicit cytot oxic effects in primary neuronal cultures. SD rat neuronal cultures were incubated with either vehicle (H2O) or 30 mol/L H2O2, or 100 mol/L H2O2 for 1 or 3 hours. Culture medium was collected and subjected to an LDH cytotoxicity assa y as detailed in the materials and methods. Means SE (n = 4) at each time poi nt are shown. *P < 0.001 vs. control. 30 mol/L Hydrogen Peroxide 100 mol/L Hydrogen Peroxide % Survival at 1 hour 100 2.00 95 4.57 % Survival at 3 hours 98 0.75 *63 4.30

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53 Figure 2-6. 30 mol/L hydrogen peroxide does not alter protein carbonyl formation. SD rat neuronal cultures were incuba ted with either vehicle (H2O), 30, or 100 mol/L H2O2 for 1 hour. Cell lysates were collected a nd assayed for protein carbonyl content as indicated in materials and methods Bar graphs shown here are means SE (n = 10 for control and 30 mol/L H2O2, n = 3 for 100 mol/L H2O2). *P < 0.01. Control 30 M H202 100 M H202 0 1 2 3 4 5*Protein Carbonyl Concentration (nmol/mL)

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54 Figure 2-7. Hydrogen peroxide-i nduced increases in macrophage migration inhibitory factor expression involve a transcriptional event. SD rat neuronal cultures were incubated with Actinomycin D (1 mol/L) for 1 hour. The cultures were then incubated with either vehicle (H2O) or 30 mol/L H2O2 for 1 hour followed by analysis of MIF mRNA levels as described in materials and methods. Bar graphs shown here are means SE (n = 5) of the ra tio of MIF mRNA to 18S rRNA. Act. D Act. D + 0.0 0.5 1.0 1.5 2.0 H2O2MIF mRNA/18s rRNA

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55 CHAPTER 3 MACROPHAGE MIGRATION IN HIBITORY FACT OR A ND THIOREDOXIN IN THE BRAIN AND OXIDATIVE STRESS Introduction Recently, interest in th e relationship between neurogenic hypertension and oxidative stress has been gaining momentum. Studies in adult SHR rats have demonstrated that oxidative stress is critically involved in the neurogenic hyperten sion observed in this model and in the related stroke-prone spontaneously hype rtensive rat (SHRSP) model.110-112 However, these studies have mostly focused on the role of oxidative stress in the adult SHR. Studies examining oxidative stress in any area of the brains of pre-hypert ensive (i.e., newborn or very young) animals are lacking. Coincidentally, and in agreement with the aforementioned studies, a recent publication from our laboratory has shown that the localization of MIF is dys regulated in adult SHR brains. Specifically, though absolute levels of MIF mRNA and protein appear normal in adult SHR, immunostaining reveals that MIF expression is lacking in neurons of the PVN Furthermore, this powerful study determined that the lack of basal MIF expressi on in neurons is most likely involved in the development of hypertension in SHR, since long-term, viral-mediated overexpression of MIF in the PVN of young SHRs with mildly elevated blood pressure was able to significantly attenuate th e development of robust hypertension in these animals over the 12 week period studied. Notably, the ability of MIF over-expression in the PVN to attenuate the development of hypertension in SHR appears to be redox-dependent since a mutated form of MIF carrying a substitution in the TPOR motif was unable to recapitu late the effects of wild-type MIF.78 This finding is not surprisi ng, given that oxidative stress has already been observed in adult SHR brains and the evidence is ever-mounti ng that MIF is a Trx family member, sensitive to and most likely participa ting in redox homeostasis.

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56 Merging together the studies concerning 1) oxidative stress and neurogenic hypertension in adult SHR and 2) our studies on the role of neur onal MIF in the developm ent of hypertension in SHR leads us to ask some important questions. We already know that absolute MIF levels do not differ between adult SHR and WKY PVNs. Instead, the cellular distribution of MIF seems to be the major difference, and this difference contribu tes to the disparate blood pressures normally observed in these two genetic strains.78 Accordingly, are MIF a nd/or Trx expression and distribution dysregulated in the brains of pre-hypertensive, newborn SHRs? Th e results of studies in chapter 1 demonstra ting disparate inducible expressi on prompted us to sequence the putatitive MIF promoter and gene in SHRs and WKYs, revealing SNPs located 5 to transcriptional start, suggesting to us that ex amination of basal expression may be in order (Figure 3-1). However, a more detailed analysis of the MIF promoter in rats is necessary to begin to understand the implications of any possible mutations. Also, our studies do suggest that MIF is lacking specifically in neurons of adult SHRs and that this lack contributes to the development of hypertension in these animals. However, it has not yet been established when the distribution of MIF becomes abnormal in these animal s (i.e., preor post-hypertension). Furthermore, like their adult counterparts, do newborn SHRs exhibi t oxidative stress in cardiovascular-relevant regions of the brain? Again, we do not yet know if the oxidative stress observed in adult animals is the cause or a cons equence of their hyperten sion. Therefore, it is important to establish the situation with regard to MIF and Trx in pre-hyp ertensive animals. If MIF and/or Trx are, indeed, lack ing or redistributed in newborns, then it follows that oxidative stress may be present long before hypertension becomes evident. Estab lishing the timing of the development of oxidative stress in the brains of these animals with respect to the progression of hypertension would be a considerable contributi on to this exciting new field of research.

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57 To begin addressing these questions, the compar ative studies in this chapter will aim to 1) determine relative quantities of MIF and Trx in the hypothalamus and brainstem of newborn (i.e., pre-hypertensive) SHR and WKY rats, 2) determine the cellular localization of MIF and Trx in the PVN of newborn SHR and WKY rats, and 3) ascertain relative levels of oxidative stress in the hypothalamus and brainstem of newborn SHR versus WKY. Our decision to examine the hypothalamus and brainstem is based on th e fact that they are ri ch in cardiovascularrelevant nuclei such as the PVN, the RVLM, and the NTS. Trx is being examined along with its relative MIF due to the similarities between the two molecules concerning regulation of neuronal firing (i.e., effects on delayed re ctifier current), and ability to negatively regulate the central actions of Ang II.60, 113 Furthermore, Trx is more establishe d as an important regulator of the intracellular redox environment, so a correlation between Trx dys regulation and oxidative stress is even more likely than is the case for MIF. Materials and Methods Analysis of MIF and T rx mRNA. Relevant brain areas were dissected from newborn rat brains and placed in RNAlater (Ambion, Austin, TX) at 4 degrees celsius overnight. They were then placed at -20 degrees celcius until processing. RNA was isolated from the tissues utilizing the RNAeasy minikit (Qiagen, Valencia, CA). cDNA was produced from the RNA samples utilizing the iScript cDNA synthe sis kit (Biorad, Hercules, CA). Real time was performed using commercially available primers and universal ta qman 2x PCR master mix (Applied Biosystems, Foster City, CA). Results are expressed as a ratio of MIF or Trx mRNA to 18s rRNA. Analysis of MIF and Trx protein. Relevant brain regions were dissected from newborn rat brains and placed in cold boiling buffer consisting of 4% SDS, 0.25 M Tris HCl (pH 6.8), 10% glycerol, and 2% -mercaptoethanol. Ti ssues were homogenized in this buffer by sonication, followed by boiling at 100 degrees celcius for 3 minutes. The lysates were

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58 centrifuged at 14,000 RPM and 4 degrees celcius for 5 minutes. Supernatants were collected and utilized for western blotting. C oncentrations were determined by Bradford Assay. 10 ug of total protein was loaded onto a 15% gel for SDS-PA GE, then transferred to a PVDF membrane (Biorad, Hercules, CA) at 75 V for 1.5 hours. Af ter a brief rinse in PBS-T (PBS containing 0.05% Tween-20), membranes were blocked in PBST containing 10% non-fat milk for at least one hour at room temperature. Primary antibodies were applied overnight at 4 degrees celcius. MIF (Torrey Pines, East Ora nge, NJ) and Trx (Chemicon International, Temecula, CA) polyclonal antibodies were dilu ted in PBS-T/1% milk. MIF an tibody was diluted at 1:1000, Trx antibody at 1:10,000. Beta-actin monoclonal antibody (Sigma, St. Louis, MO) was diluted at a concentration of 1:100,000 in PBS-T/5% milk. The next day, membranes were washed in PBS-T for 1 x 10 minutes and 4 x 5 minut es, then incubated in secondary antibody at a 1:30,000 dilution (MIF), 1:50,000 dilution (Trx), or 1:90,000 (beta-actin) in 2% milk/PBS-T for one hour at room temperature. Secondary HRP-conj ugated antibodies (anti-mouse and anti-rabbit) were purchased from Sigma (St. Louis, MO). Chemilumines cence detection was carried out according to instructions with the Western Lighting kit from PerkinElmer (Boston, MA). Immunostaining of brain sections for MIF and Trx. Brains were placed in Formaldehyde Fresh (Fisher, Waltham, MA) overni ght for fixation. They were then transferred to 70% ethanol and left overnight for partial dehydration and further fi xation. The brains were then subjected to dehydration and impregnati on with paraffin. After imbedding in paraffin, 5 micron sections were cut and subjected to im munostaining. Following rehydration, slides were rinsed and incubated in TBS-T (TBS containing 0.05% tween) fo r at least 5 minutes, followed by a blocking step consisting of TB S-T and 1.5% horse serum for at least 1 hour (blocking serum was matched to secondary antibodies, whic h are donkey). Excess blocking solution was wiped

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59 from the slides and primary antibodies were applied for an overnight, 4 degrees celcius incubation. MIF and Trx antibodies (same as described in analysis of MIF and Trx protein section) were used at a 1:200 dilution, NeuN and GFAP antibody (Chemicon International, Temecula, CA) at a 1:400 dilution. Primary a nd secondary antibodies were diluted in PBS containing 10% goat serum. The next day, after a brief rinse in TBST, slides were rinsed for 2 x 10 minutes. Secondary antibody (donkey anti-mouse and donkey anti-rabbit) was applied for 1 hour. Alexa Flour 488 and Alexa Flour 594 (Invitr ogen, Grand Island, NY) were utilized at a dilution of 1:500. After a brief rinse in TBS-T, slides were rinsed for 2 x 10 minutes, then mounted in Vectashield mounti ng medium (Vector Laboratories, Burlingame, CA) and viewed and photographed on an Olympus microscope. Measurement of protein carbonyl content. Hypothalamus and brainstem were dissected from newborn rat brains and homogenized in 1 ml of ice cold 50 mM MES buffer, pH 6.7, containing 1 mM EDTA (Sigma, St. Louis, MO) by sonication. Lysates were then centrifuged at 10,000 g for 15 minutes at 4 degrees celsius. Supernatant was collected and frozen at -80 degrees celcius until utilization. Assay wa s performed exactly according to the protocol provided with the Protein Carbonyl Assa y kit (Cayman Chemical, Ann Arbor, Michigan). Results Macrophage Migration Inhibitory Factor a nd Thioredoxin Expression is Lackin g in Spontaneously Hypertensive Rat Hypothalamus. Our first goal was to establish the situation wi th respect to the expression of MIF and Trx in the hypothalamus and brainstem of SHR and WKY newborn (i.e., prehypertensive) rats. To determine MIF and Trx mRNA expression, newbor n rat brains were dissected, and relevant regions were homogenized and subjected to RNA extraction. The RNA was then reverse transcribed, and the resulting cDNA utilized for real-time PCR as described in materials and

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60 methods. The results (Figure 3-2A) show that there is a statistically significant lack of MIF and Trx mRNA in the hypothalamus of newborn SH Rs compared to their WKY controls. In contrast, when subjected to the same measurement, there was not a statistically significant decrease in the brains tems of SHRs compared to WK Y. However, there was a trend toward a slight decrease (Figure 3-2B). Our second goal concerning MIF and Trx e xpression was to determine whether the protein levels paralleled the mRNA data (i.e., wh ether the protein is sign ificantly decreased in the hypothalamus of SHR). Newborn hypothalamus and brainstem were subjected to protein extraction as described in the materials and methods, and western blotting was utilized to determine relative protein levels. The results i ndicate that MIF and Trx pr otein is significantly reduced in the hypothalamus, which parallels the mR NA data (Figure 3-3, A and B). In contrast to the mRNA data, Trx protein appeared to be re duced in the brainstem (Figure 3-4). To further clarify the significance of the lack of these prot eins, oxidative stress in these two tissues was measured next. Newborn Spontaneously Hypertensive Rats E xhibit More Oxidative Stress in the Hypothalamus and Brainstem than Wistar Kyoto Rats. An important aim of this study was to invest igate whether SHR newborns exhibit oxidative stress in cardiovascular-relevant regions of the br ain, similar to their adult counterparts. To this end, we utilized a protein carbonyl kit (as described in materials and methods) to assess protein carbonyl levels, one of the several accepted indicators of intracellu lar oxidative stress.114 Protein was extracted from newborn SH R and WKY hypothalamus and brai nstem and subjected to this colorimetric assay. The results indicate that SHR have a statistically significant greater level of protein carbonyl content in the hypothalamus th an WKY (Figure 3-5A). SHR also exhibit a higher level of protein carbonyl concentration in the brainstem, though the difference is not as

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61 great or significant (Figure 3-5B ). Since the hypothalamus, in cont rast to the brainstem, appears to exhibit greater levels of oxidative stress in SHR, our further studies focused on the hypothalamus. Macrophage Migration Inhibitory Factor is Ab sent from Paraventricular Nucleus Neurons of Spontaneously Hypertensive Rats. Since MIF distribution appears to be altered (i.e., lacking in neurons) in the PVN of adult SHR, we were eager to d etermine whether MIF a nd Trx were also lacking in neurons in the prehypertensitve newborns. Newborn SHR and WKY brains were fo rmalin-fixed and paraffinembedded, and 5 micron sections of the P VN were then subjected to Trx or MIF immunostaining. Sections were also stained w ith NeuN, to identify neurons, or GFAP, to identify astrocytes. The results in dicate that, similar to what is found in adult animals, MIF is lacking in neurons of newborn SHR PVN compared to WKY. Similar to what we have found in adults, MIF is mostly localized to astrocytes in the SHR PVN (Figure 3-6). In contrast, Trx localization was similar between WKY and SHR PVN (Figure 3-7). Discussion To our knowledge, the studies in this chap ter represent the first dem onstration of oxidative stress in newborn SHR hypothalamus. Furthermore, these studies are the first to show reduced expression of MIF and Trx expression in newborn SHR hypothalamus, which contains the important sympathetic and neuroendocrine -regulating nucleus, the PVN. Finally, the observations contained herein demo nstrate that the lack of neurona l MIF previously seen in adult PVN can also be found in newborn (i.e., pre-hyperte nsive) SHR. In contra ct, cellular localization of Trx was found to be simila r between SHR and WKY PVNs. Our findings that MIF and Trx are lacki ng in the hypothalamus of newborn SHR are significant for several reasons. Tr x is a well-established redox-homeostasis enzyme that is

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62 essential for maintaining a prope r redox balance in neurons. Furt her, the evidence is steadily mounting that MIF has important redox balancing and/or signaling ro les in the brain. A lack of both of these enzymes is sugges tive of oxidative stress or a ltered redox signaling. Indeed, the results of our protein carbonyl studies suggest an association between the lower levels of these TPOR enzymes and a dysregulated redox ba lance in the hypotha lamus. Based on the observations made here, we cannot c onclude decisively that the lack of these proteins is solely responsible for the altered redox environment observed in SHR hypothalamus. However, combining them with our previous study suppl ementing MIF in the PVN of pre-hypertensive SHR and preventing the development of robust hypertension in a TPOR-dependent fashion78 is certainly suggestive that the lack of these TPOR enzymes in the hypothalamus leads to oxidative stress or some other form of redox signaling dys function and, consequently, contributes to the development of hypertension in these animals. On e important experiment that is yet to be performed, and would further support this proposed cause and effect relationship, would be to over-express MIF or Trx in the PVN of prehypertensive SHR and determine whether the attenuation of hypertension is accompanied by a reduction in oxidative stress in the relevant brain areas. These studies would also further clarify whether MI F and Trx are redundant or have slightly divergent functions in this brain area. One important and confounding question these st udies raise is: why are these antioxidant enzymes paradoxically not up-regula ted in the face of oxidative st ress? We are not the first group to observe a lack of Trx inducti on in the presence of oxidative stre ss. Tanito et. al. reported in 2004 that hypertensive SHR and severely hyperten sive SPSHR exhibited a lack of Trx in the heart, vasculature, and kidne y that correlated to the se verity of their hypertension.115 Furthermore, they reported dysfunctional Trx induction in response to Ang II signaling in

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63 peripheral blood mononuclear cells of hypertensive rats. The authors concluded, therefore, that a genetic mechanism is the likely responsible for th e lack of Trx that they observed. Similarly, the results of the studies contai ned in chapter 1 showing a dys functional induction of MIF in response to hydrogen peroxide trea tment in SHR neurons prompted us to sequence the putative MIF promoter and MIF gene in SHR and WKY. Several single-nucleotide polymorphisms were identified (Figure 3-1), but an in-depth study is required to determine if they are important in basal or inducible MIF expressi on. Likewise, a sequencing study of the Trx promoter and gene in SHR and WKY is certainly warranted and necessa ry to determine if ge netic alterations are responsible for the lack of Trx in newborn SHR hypothalamus and the apparently dysfunctional inducible expression (i.e., lack of induction in an oxidative stre ss situation) of this protein. Our findings concerning an abnormal absence of MIF in neurons of the PVN in newborn SHR further support an argument for genetic alterations in hyperten sive animals and our recently developing model of dysregulated TP OR enzymes in the brain as a cause, rather than an effect, of hypertension. If the lack of MIF could only be observed in adult, hyperten sive rats, then this would suggest an epigene tic alteration that is a consequence of high blood pressure. Yet, our studies presented here argue that this is not the case, since we have found the same phenomenon in newly-born SHRs that have not yet e xperienced elevated bl ood pressure. However, antioxidant enzymes from several families, in cluding glutathione peroxidase, superoxide dismutase (SOD), and catalase, have been reported to exhibit abnormally low activity in SHR in several tissues.116, 117 The fact that SHR exhibit such a wide-spread dysfunction in antioxidant systems could suggest a catastrophic failure in global oxidative signa ling and redox regulation. In addition to sequencing and promoter studies, pe rhaps we should consider that the mutation is not to be found in the promoters of these anti oxidant enzymes but in the promoter of an up-

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64 stream, common mediator(s) that control(s) expre ssion of redox-balancing proteins and systems. Accordingly, a prime candidate that should be investigated in future studies is the transcription factor Nrf2, which binds to th e antioxidant reponse element (ARE ) and increases expression of many antioxidant enzymes.118-120 An intriguing study by Zhu et. al showed that Nrf2-deficient mice have dysregulated basal and inducible expression of several antioxi dant and detoxifying enzymes and small molecule antioxidants, includi ng SOD, glutathione, glutathione S-transferase, glutathione reductase, and cat alase in cardiac fibroblasts.121 Though not examined in Zhus study, other publications have show n that thioredoxin expression is clearly regulated by Nrf2, as well.122, 123 Experiments have not yet been done to de termine whether Nrf2 also regulates MIF expression. Disappointingly, no gr oups have yet studied blood pressure regulation or sympathetic nerve activity in Nrf2-deficient mice. Nevertheless, studies such as the aforementioned make it easy to imagine that perhaps an indirect globa l regulator of redox balance such as Nrf2 is playing a role in the lack of TPOR enzymes we are observing in newborn SHR brains. Clearly, extensive further study involving genetics epigenetics, and signal transduction is needed before it can be dete rmined exactly what sort of abnormality is responsible for the lack of MIF a nd Trx in the hypothalamus of SHR.

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65 Figure 3-1. Single nucleotide poly morphisms of the putative macr ophage migration inhibitory factor promoter in spontaneously hyperten sive rats. Those iden tified in color are potential binding sites for th e transcription factors indicated. Note that all 4 are redox sensitive transcription factors. -1kb -2kb AP-1 Sp-1 USF NF B

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66 Figure 3-2. Macrophage migration inhibitory factor and thioredoxin mRNA expression is reduced in spontaneously hypertensive ra t hypothalamus. mRNA was isolated from the A) hypothalamus and B) brainstem of newborn WKY and SHR and relative expression levels were ascertained by re verse transcription a nd real-time PCR as described in materials and methods. Means SE (n = 10 for each tissue) of the ratio of MIF or Trx mRNA to 18S rR NA are shown. *P < 0.0001 vs. WKY. A B Hypothalamus WKY SH R W K Y SHR 0.0 0.5 1.0 1.5MIF Trx *mRNA/18s rRNA Brainstem WKY SH R W K Y S HR 0.0 0.5 1.0 1.5MIF Trx mRNA/18s rRNA

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67 Figure 3-3. Macrophage migration i nhibitory factor and thioredoxin protein expression is lower in newborn spontaneously hypertensive rat hypothalamus. Total protein was extracted from hypothalamus of newborn WKY a nd SHR and relative expression was determined by western blotting as described in materials and methods. Means SE (n = 6 for each tissue) of the ratio of A) MIF or B) Trx protein to -actin are shown. Bottom panel contains representative western blots. *P < 0.05 vs. WKY. WKY SHR 0.0 0.5 1.0 1.5*MIF/ -actin WKY SHR 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5*TRX/ -actin MIF 12.5 kDa -actin42 kDa WKY SHR Trx 12 kDaMIF Trx AB

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68 Figure 3-4. Macrophage migration inhibitory factor and thioredoxin protein in newborn brainstem Total protein was extracted from the brainstem of newborn WKY and SHR and relative expression was determined by western blotting as described in materials and methods. Means SE (n = 6 for each tissue) of the ratio of A) MIF or B) Trx protein to -actin are shown. Bottom panel c ontains representative western blots *P < 0.05 vs. WKY. WKY SHR 0.0 0.5 1.0 1.5MIF/ -actin WKY SHR 0.0 2.5 5.0 7.5*TRX/ -actin WKY SHR Trx 12 kDa MIF 12.5 kDa -actin42 kDaMIF Trx AB

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69 Figure 3-5. Protein carbonyl concentration is greater in spont aneously hypertensive rat hypothalamus than in Wistar Kyoto. Total protein was extracted from A) hypothalamus and B) brainstem of new born WKY and SHR and carbonyl levels measured as described in materials and me thods. Means SE (n = 6 for WKY, n = 6 for SHR) of the protein carbonyl concentra tion of each tissue measured are shown. *P < 0.05 vs. WKY. A B Hypothalamus WKY SHR 0 1 2 3 4*Protein Carbonyl (nmol/ml) Brainstem WKY SHR 0 1 2 3 4Protein Carbonyl (nmol/ml)

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70 Figure 3-6. Spontaneously hypertensive rat para ventricular nucleus neurons contain less macrophage migration inhibitory factor. Br ains from newborn A) WKY and B and C) SHR rats were subjected to immunostaining for MIF, NeuN (a neuronal marker), and GFAP (an astroglial marker). Bars in lo wer right represent a scale of 50 microns. Areas of interest are marked with arrows. D and E) are Ig controls Sections pictured are representative of 4 animals from each genetic rat strain. A B WKY 50.0 m 50.0 m 50.0 m SHR 3V3V3V 3V 3V 3V MIF NeuN Merge Merge NeuN MIFC3V 3V 3V MIF GFAP Merge SHR 50.0 m 50.0 m 50.0 m A B WKY 50.0 m 50.0 m 50.0 m 50.0 m 50.0 m 50.0 m SHR 3V3V3V 3V 3V 3V MIF NeuN Merge Merge NeuN MIFC3V 3V 3V MIF GFAP Merge SHR 50.0 m 50.0 m 50.0 m 50.0 m 50.0 m 50.0 m D3V 3V Rabbit Mouse WKY Rabbit Mouse 3V 3V SHRE D3V 3V Rabbit Mouse WKY Rabbit Mouse 3V 3V SHRE

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71 Figure 3-7. Thioredoxin cellular di stribution is normal in spont aneously hypertensive rat paraventricular nucleus. Brains were removed from newborn A) WKY and B) SHR rats, fixed, and paraffin-embedded as desc ribed in materials and methods. 5 micron sections of the hypothalamus were subjected to immunostaining for Trx and NeuN (a neuronal marker). Bars in lower right repr esent a scale of 50 microns. Selected areas of co-localization are marked with arrows. For controls, refer to D and E of Figure 36. Sections pictured are representative of 4 animals from each genetic rat strain. A B Trx NeuN Merge Trx NeuN Merge WKY SHR 3V 3V 3V 3V 3V 3VA B Trx NeuN Merge Trx NeuN Merge Trx NeuN Merge Trx NeuN Merge WKY SHR 3V 3V 3V 3V 3V 3V

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72 CHAPTER 4 CONCLUSIONS AND FUTURE DIRECTIONS Over the past several years, m any laboratorie s, including our own, ha ve made substantial contributions to our understanding of how ROS signaling and oxidative stress contribute to neural control of blood pressure in the phys iological state and hype rtension, respectively. Progress is being steadily made in this field, but there is still much we do not understand about redox signaling in the brain and its contribution to blood pres sure regulation. The studies contained in this dissertation are important because they contribute small steps toward understanding the larger picture with regard to redox homeostasi s in the brain and neurogenic hypertension. From the studies reported in chapter 1, we have been able to make advances in understanding possible pathways th at regulate inducible expressi on of MIF in neurons. Further, by concluding that MIF expression can be sensitiv e to the intracellular re dox environment of the cell, we have contributed greatly to the paradigm shift that is presently occurring in categorizing MIF as a TPOR enzyme and potential member of the Trx family. Finall y, these studies provide direction, in the form of ROS signaling, as a basis for future studies examining the inducible expression of MIF in normotensive animals in response to Ang II stimulation of neurons. Also, the knowledge that ROS do not signal appropriately in SHR neurons, at least with regard to MIF expression, lends further direction to future studies that endeavor to determine why the AT1R is uncoupled from inducible MIF expression in SHR. The studies contained in chapter 2 presen t compelling evidence that oxidative imbalance is present in SHR hypothalamus prior to hypertensi on and that a lack of MIF and Trx may be at least partly responsible. At the ve ry least, there is an associati on between reduced levels of these important antioxidant enzymes and markers of oxidative stress in the hypothalamus of SHR.

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73 Further, a very telling and important finding of these studies is the lack of MIF in neurons of the PVN of pre-hypertensive SHR, indicating that this loss of MIF is not a consequence of developing or advanced hypertensi on, but perhaps it may be a contributor to the cause. This likelihood becomes even more compelling when coupled with our recent studies on MIF supplementation in the PVN and reduction of blood pr essure in SHR. Given the fact that, due to the efforts of our laboratory, MIF and, to a lesser extent, Trx is now a known negative regulator of the central actions of Ang II, and th at it is well-established that SHR suffer overactivity of and hypersensitivity to the brain RAS, we can now see a much clearer picture of how a lack of MIF and/or Trx may lead to enhan ced ROS signaling and/or oxidative stress in important cardiovascular brain nuclei and how this may contribute to the development of hypertension in SHR. Many hypertensive patie nts exhibit increased sympathetic nervous activity,124 and there is a constant influx of ne w evidence showing that ROS are primary effectors through which Ang II mediates its cent ral actions, including eff ects on the sympathetic nervous system.71 Therefore, dysfunction of important re dox-regulating system s in the brain is becoming a more likely contributor to the complex story of hypertension all the time. Our results in the preceding chapters pave the way for a number of exciting new studies and inspire many important questions, which will be explored and discussed in the following sections. Inducible Expression of Macrophage Migrat ion Inhibitory Factor in Neurons and Angiotensin II A very critical question that remains unanswered by our studies is whether hydrogen peroxide is, indeed, the down-stream mediator that induces MIF expr ession in neurons of normotensive animals. In order to answer th is question, a suitable model will have to be established. Areas of the brain that are rich in AT1R should be di ssected and cultured together in order to receive a robust induction of MIF when stimulated with Ang II. In the past, we usually

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74 accomplished this by culturing the hypothalamus with the brainstem isolated from newborns. However, in recent months, our laboratory has been developing a technique for culturing PVN neurons isolated from adult rats, a very technica lly challenging feat. The use of these neurons as a model for studying Ang II and AT1R signaling would be even more desirable. Regardless of whether newborn or adult neurona l cultures are utilized, the expe riments detailed in chapter 1 involving PEG-catalase should be repeated with these cultures, except An g II stimulation should be used in the place of hydrogen peroxide applic ation. If one could interrupt Ang II-mediated upregulation of MIF expression in the cultures with PEG-catalase tr eatment, this would provide the necessary evidence needed to determine that Ang II-mediated ROS signaling, specifically hydrogen peroxide, is the pathway through which Ang II induces MIF in neurons. It would also be interesting to repeat the aforementioned experiments utilizing PEG-SOD, in order to show conclusively that superoxide is the source of the intracellular hydrogen per oxide, as we surmise. These studies could, theoretically, be extrapolated to in vivo studies utilizing viralmediated over expression of SOD and catalas e in pertinent brain areas, followed by Ang II application and study of MIF expr ession. Other labs have successf ully used SOD and tempol in the brain to interrupt Ang II signaling,74-76 so this seems to be a very plausible way of determining whether Ang II-induced ROS signali ng is the mechanism by which MIF expression is up-regulated in the PVN in vivo Mechanisms of Redox Regulation of Macrophage Migra tion Inhibitory Factor Expression Another important question that remains to be answered is that of the down-stream mediators that are sensing and responding to changes in the in tracellular redox environment and, ultimately, altering the expression of MIF. The most sensible way to initially embark on these studies would be to clone the put ative MIF promoter from normotensive animals, make strategic deletions and recombine with a reporter system (such as luciferase). The constructs would then

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75 be transfected into a neuronal cell line (suc h as SH-SY5Y, known to be Ang II-sensitive)125, 126 and stimulated with hydrogen peroxide and/or Ang II to investigate wh ich areas of the MIF promoter are redox-sensitive. As we have seen in the discussion of chapter 1, many transcription factors (TFs) are sensitive to redox-signaling, so identifying the cis-acting elements of the MIF promoter that are responsible for redox-sensitiv e increases in MIF tran scription would be the first step to identifying whic h redox-regulated signaling pathways are inducing MIF upregulation. Candidate signaling path ways could then be determined in a back-tracking manner using the abundant information available concer ning which TFs mediate e ffects of particular pathways. Once areas of the MIF promoter are identifi ed that mediate induction of MIF both in response to Ang II and hydrogen peroxide, the fo cus can be placed on these sequences and which TFs may bind there. The cells can then be stimul ated with Ang II or hydrogen peroxide and ChIP assays at the native MIF promoter performed for the relevant TFs to ascertain if, indeed, their binding at the MIF promoter is increased unde r stimulated conditions. The final piece of evidence needed to confirm which TFs are mediati ng the effects of ROS at the MIF promoter are mutational studies. The MIF promot er-luciferase recombinant constructs could be mutated via PCR to ablate the relevant TF-binding sites and tested for their sensitiv ity to hydrogen peroxide and Ang II stimulation. ChIP assays should also be performed in Ang II and hydrogen peroxidetreated primary neurons in order to confirm that the effects seen ar e not specific to the particular cell line selected for the studies. Once the promoter studies have begun to id entify TFs and candidate signaling pathways that may be regulating Ang IIand ROS-induced MIF expression in neurons, these pathways can be further clarified utilizing pharmacological interventions and the aforementioned recombinant

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76 reporter constructs. Furthermore, the results of these studies will serve as a basis for comparison and may help identify the defects in Ang II si gnaling with respect to MIF induction in SHR. Dysregulation of Macrophage Migration Inhibi tory Factor and Thioredoxin Expression in the Hypoth alamus of Spontaneously Hypertensive Rats The studies contained in chapte r 3 highlight the importance of determining the cause of the lower expression levels of MIF and Trx in the newborn SHR hypothalamus. As the future directions concerning this endeavor were previously explored in the discussion of chapter 3, they will not be recapitulated here. One major question that remains to be answer ed is that concerning the loss of MIF in neurons of the PVN in SHR, which can be observed in both preand post-hypertensive animals. There are several possibilities that must be fu rther explored. For example, is the MIF gene simply not transcribed in these cells? Is MIF be ing transcribed, but not tr anslated properly? If MIF is being transcribed and translated in neurons, is it then being abnormally degraded? If this is not the case, perhaps MIF is being improperly secreted from neurons so that its presence cannot be detected in these cells by immunosta ining? The most reasonable way to begin addressing this issue is to determine whether MI F mRNA can be detected in these neurons. This would determine if the root cause most likely lies in a loss of MIF transcription. In this instance, the application of single-cell RT-PCR, a techniqu e we have recently developed in our laboratory, would yield the most specific information. App lication of other commonly-used techniques is complicated by the fact that PVN and hypotha lamic cultures consist of a heterogenous population of cells, so determining the situation specifically in neur ons is rather difficult. Cells could be cultured from the PVN of SHR and WKY and neurons screened for the presence of MIF using single-cell RT-PCR. Another techniqu e that would be useful for detecting MIF mRNA and confirming the singl e-cell RT-PCR results is in situ hybridization. As the

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77 immunostaining data indicates, we should be able to easily detect MIF mRNA in neurons cultured from WKY. If MIF mRNA cannot be detected in th e SHR cultures, this would argue strongly that the problem is a tr anscriptional issue. However, we cannot rule out the possibility of a stability problem with MIF mRNA in these cells. Obviousl y, further experiments would be needed to clarify the situati on. Addressing this ques tion would be a time-consuming and difficult undertaking, but the information obt ained would merit the investment. Contrary to observations in newborn hypothalamus, absolute MIF levels are unchanged in the heterogenous population of cel ls in the PVN of adult SHR.78 One possible explanation for this difference is that the situation in the PVN may be different from that in the rest of the hypothalamus. Another possibility is that MIF is upregulated in glial cells of adult SHR PVN to somehow compensate for the loss of MIF in neurons Investigating this possibility will be very difficult, for reasons relating to the heterogene ity of cells already mentioned, but the most practical way to begin addressi ng it would be to culture the PVN of SHR and WKY, enrich for astroglia as previously described,127 and ascertain basal levels of MIF in these primary glial cultures, comparing mRNA and protein between WKY and SHR via real time RT-PCR and ELISA, respectively. Increased leve ls of MIF in the SHR cultures would suggest that MIF is upregulated in astroglia in SHR and this is why ab solute levels appear normal in the PVN, even when expression is lost in neurons. Oxidative stress in the Hypothalamus of Spontaneously Hypertensive Rats The studies in chapter 3 indicate that oxidati ve stress is present in the hypothalamus of newborn SHRs. The redox environment represents a summation of oxidant-producing systems and antioxidant systems. Therefore, oxidative stress could result from either a loss of antioxidant function and/or an increase in ROS producing systems. Our studies indicate that there may be a lack of antioxidant enzymes, such as those of the Trx family, in SHR hypothalamus. By

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78 extrapolation, it would be useful to investigate whether there is a lack of other important antioxidant systems, such as th e glutathione system in this ti ssue. Conversely, the expression and activity of oxidant-producing systems should also be investigated. For example, NADPH oxidase is currently considered to be one of the most important regulated enzyma tic sources of ROS in neurons.71, 128 It would be prudent to inve stigate whether this system is up-regulated in the brain of SHR, which could contribute to an imbalanced redox environment. Physiological Implications Ultimately, the goal of future studies s hould be to investigate the physiological implications of improper redox signaling and red ox-regulated cellular func tions in hypertension. Previous studies from our lab have already de termined that neuronal MIF supplementation can have a significant effect on th e development of hypertension in SHR over time. However, one very important outcome that remains to be de termined is whether MIF supplementation in PVN neurons of SHR and the resulting reduction in blood pressure are acco mpanied by a reduction in oxidative stress in this tissue. Si nce it has been demonstrated in several instances that ROS can affect neuronal function by modulating neuronal firing,71 another important outcome to measure in the context of MIF supplementation in SHR PVN would be sympathetic drive. Previous experiments published by our laboratory78 should be repeated and these important parameters determined to further complete the picture of how a lack of MIF contributes to the neuropathogenesis of hypertension.

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89 BIOGRAPHICAL SKETCH Rachael Harrison was born in 1980 in Jacksonvill e, FL. She has resided in Florida all of her life and has wanted to becom e a scientist si nce she was a very small child. Rachaels career aspirations ranged from marine biology to being a science teacher through her junior high school and high school years. Rachael began to think seri ously about a career in biomedical research in her later high school years, and pursued a degr ee in molecular biology an d microbiology (with an emphasis on human health and disease) from the college of Health and Public Affairs at the University of Central Florida, where she gradua ted with honors and went on to graduate school at the University of Florida, College of Medi cine. Rachael joined the Physiology and Functional Genomics department under the supervision of Dr. Colin Sumners in fall 2004, two years after she entered graduate school in 2002. In Dr. Sumners laboratory, her re search has focused on redox biology and neurogenic hypertension. She will receive her doctora te in August of 2008 and go on to a career in cancer research at th e Sunnybrook Health Sciences Center in Toronto, Ontario.