Citation
Role of Brain Soluble Epoxide Hydrolase in Cardiovascular Function

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Title:
Role of Brain Soluble Epoxide Hydrolase in Cardiovascular Function
Creator:
SELLERS, KATHLEEN WALWORTH ( Author, Primary )
Copyright Date:
2008

Subjects

Subjects / Keywords:
Baroreceptors ( jstor )
Blood pressure ( jstor )
Brain ( jstor )
Enzymes ( jstor )
Gene therapy ( jstor )
Heart rate ( jstor )
Hypertension ( jstor )
Neurons ( jstor )
Oxidases ( jstor )
Rats ( jstor )

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Kathleen Walworth Sellers. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
12/18/2004
Resource Identifier:
71424012 ( OCLC )

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Full Text












ROLE OF BRAIN SOLUBLE EPOXIDE HYDROLASE IN CARDIOVASCULAR
FUNCTION
















By

KATHLEEN WALWORTH SELLERS


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


2004

































Copyright 2004

by

Kathleen Walworth Sellers

















This work is dedicated to my family, who has taught me to embrace life with love,
perseverance, and grace.















ACKNOWLEDGMENTS

I could not have completed this work without the assistance from many colleagues

and friends. First and foremost, I would like to thank my mentor, Dr. Mohan Raizada,

who has consistently supported and encouraged me these last several years. It was by

chance that I worked in his research group before starting my graduate program, and I

could not have found a better group with which to work. Dr. Raizada's excitement and

fervor for new ideas have helped shape my appreciation for scientific research and the

pursuit of knowledge.

I also want to acknowledge all of the members of the Raizada research group, past

and present. I am fortunate to work in such a friendly and intellectually-stimulating

environment. Specifically, I want to thank Drs. Beverly Falcon and Matthew

Huentelman, the two graduate students in our research group who both helped me

tremendously. In addition, I would like to thank Drs. Carlos Diez, Cheng-wen Sun, Jorge

Vazquez, Shereeni Veerasingham and Hong Yang for all of their intellectual guidance.

I greatly acknowledge the members of my committee, Drs. Michael Katovich,

Gerard Shaw, Colin Sumners and Charles Wood. Each has provided me with unique

experience and expertise to further my research and career. In addition, I want to thank

all of my teachers throughout my education. Without a solid learning base, I would not

have reached this goal.









Finally, I want to thank all my family and friends who have supported me over the

years. It is they who lift my spirits and keep my sights set high. Each of my loved ones

has a purpose in my life for which I am eternally grateful.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF FIGURES ............................... ... ...... ... ................. .x

ABSTRACT .............. ..................... .......... .............. xii

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

Central Regulation of H hypertension .................................. .................... ............... ..
B rain stem .................................................................................. . 2
H ypothalam u s ................................................................................................ 4
B aroreceptor R eflex ......................................... ....... ........ .. ....... .. ..
M echanism s U underlying SN A ......................................... .......................... 5
R enin-angiotensin system ........................................ ......................... 5
R active oxygen species........................................... .......................... 7
Im plications ................................... ................................ ........10
Soluble Epoxide H ydrolase ................................................... ....... .. ............... 11
H history .................. ................ .............................. ................ 11
Epoxyeicosatrenoic Acids ...........................................................................12
V asodilatory properties ........................................ .......................... 12
EETs in the brain ................. .. ............. .. ... ... ..... .. ........ .... 14
A alternate actions of EETs ........................................ ........................ 14
Dihydroxyeicosatrenoic Acids ......................... .... ............... ............... 15
Cytochrom e P-450 ............. .. .... .................. ........ .. .. .......... .. 16
Epoxygenases in the brain................................ ......................... ........ 16
2 0-H E T E ...................................................................... 17
sE H and H hypertension .................................................................... ............... 17
sEH Polym orphism s .................. ............................. .. ...... .. .......... 19
R o d en t .........................................................................19
H u m an ....................................... ............... ................ 2 0
sEH polymorphisms in cardiovascular disease................. ............. .....21
sEH polymorphisms in other disorders ................................................21
Phosphatase Studies.............. .. .................. ..... .. ......22
Sum m ary ................................................. ............ .. ... ................. 23









2 INCREASED SEH EXPRESSION IN SHR VS. WKY RAT BRAIN: IN VITRO
A N D IN VIVO A N A L Y SIS ......... ......... .. .............. .. ......................................... 31

Introduction ......... ........... ..................... .... ......................... 31
M materials and M methods ........................................................................ ..................33
A n im als ...................................................... ................... 3 3
Im m unohistochem i stry .......................................................... ...............33
R eal-T im e R T -P C R ............................. ............................... ............. .... 34
W western B lot A analysis .................................................. ............................. 34
N euronal Cells in Prim ary Culture ................................................................... 35
Losartan Treatment of SHRs to Normalize Blood Pressure..............................35
Statistical A analysis ...................... .................. ................. ...........36
R e su lts .............................. ....... ............................. ........................... 3 6
Cellular Localization of sEH .......................... ..... ............... ..... .......... 36
sEH Expression in the SHR....................... ...................... 37
In vitro Validation with Neuronal Cells in Primary Culture .............................37
sEH Expression Following Blood Pressure Normalization ..............................38
D isc u ssio n .......................................... ....... .................. ................ 3 9

3 CENTRAL SEH INHIBITION INCREASES BLOOD PRESSURE AND HEART
R A T E IN T H E SH R ......... ...... ........... ................. .......................... ..................... 49

Introduction ............... ...... .. .......... ........ .... ........................ 49
M materials and M methods ........................................................................ ..................5 1
A n im als....................................................5 1
Statistical A naly sis ...................... .................. ............................5 1
IC V C an nu nation ......... .................................................. ............ .. ... .... ... ....5 1
A bdom inal A orta Cannulation ........................................ ......................... 51
Blood Pressure and Heart Rate Recordings.............................. ...............52
In vivo Drug D delivery .................. ........................... ........ ... ........ .... 52
R e su lts............................. .................... ..... ............ .. .... .... ....... ............. 5 3
Effect of sEH Inhibitor DCU on Blood Pressure and Heart Rate ......................53
W K Y rat ................................................................................... 53
SH R ................. ..... ..... ... .... ... ... ..... .. ...............53
Effect of sEH Inhibitor AUDA on Blood Pressure and Heart Rate ...................54
Dose response ................................... ........................... ..........54
W K Y rat ............................................ ........................... 55
S H R ........................................................................... 5 5
D iscu ssion ......... .................. .................................... ............................55

4 ROLE OF ROS IN ANG II-INDUCED REGULATION OF BLOOD PRESSURE.63

Intro du action ...................................... ................................................ 6 3
M eth o d s ..............................................................................6 5
A n im als................................. ...................................................... ............... 6 5
IC V C annulation ......... .................................................. .............. ... .... .... .. 65
A bdom inal A orta Cannulation ........................................ ......................... 65









Statistical A analysis ...................... ................ ................. ..... ...... 65
In v iv o D rug D eliv ery .............................................................. .....................6 5
D ipsogenic R esponse............ .... ............................................ .... ............... 66
R results ...............................................................................................66
Angiotensin II Pressor Response..........................................................................66
gp9lds-tat Prevents Ang II-Induced Blood Pressure .....................................67
W K Y rat ........................................... ........................... 67
SH R .................................................. ........ .............. 67
gp91ds-tat Prevents Ang II-Induced Drinking ............................... ...............68
D iscu ssio n ......... .................................... ............................6 9

5 MECHANISM OF CENTRAL SEH INHIBITION ON BLOOD PRESSURE
R E G U L A T IO N ......... ......................................................................... ........ .. ..... .. 77

Introduction ............. ..... ... ............................................................ 77
M eth o d s .............................................................................. 8 0
A n im als................................. ..................................................... ............... 8 0
IC V C annulation ........... .......................................................... ..... ...... ........ 80
A bdom inal A orta Cannulation ........................................ ........................80
Blood Pressure and Heart Rate Recordings.............................. ...............80
Statistical A analysis .......................... ............ ...........................80
In vivo D rug D delivery .............. ......... ........................... ............... .. ..... 80
Evaluation of Spontaneous Baroreceptor Reflex Gain.......................................80
Analysis of Heart Rate Variability .............. ............................................81
N euronal Firing R ecording............................................................. ............... 82
R esults.......................... ...... ................ ............................................82
Waveform Analysis of Baroreceptor Parameters ............................................82
Index of spontaneous baroreceptor reflex gain ...........................................82
High frequency analysis of pulse interval................... ...............................83
AUDA Regulation of Sympathetic Nerve Activity ............................................83
Increased EETs Linked to Increase in BP and HR ................... ................84
M S -P P O H ............................................................................... 8 4
11-N O D A ................................................................................. 85
Effects of sEH Inhibition Function through ROS ..............................................85
D iscu ssio n ......... .................................... ............................8 6

6 EFFECT OF SEH OVEREXPRESSION IN THE PVN OF SHR AND WKY RATS97

In tro d u ctio n .................................................................... ................ 9 7
M e th o d s .......................................................................... 1 0 2
A n im als................................. ................................................... ............... 10 2
IC V C annulation ......... .............................................................. .. .... .. .... .. 102
Abdom inal A orta Cannulation ........................................ ....... ...............102
W western B lot A analysis ........................................ ....... .......................... 102
Transformation of pCR 2.1 sEH cDNA into competent cells.................... 102
DN A m iniprep ................................................ ...... .............. ... 02
Large-scale Lentiviral Production ............................................. ...............103


viii









Infection of COS-7 cells with lenti-sEH ................................. ............... 105
Cytoplasmic Isolation for HPLC Analysis............................................105
R adiotelem etry R ecordings ........................................ .......................... 106
PVN Coordinate D eterm nation ............................................. ............... 106
Im m unohistochem istry .............................................................. ............ ..... 07
PVN Injection of lenti-sEH ...... ............................................................ 108
In vivo D rug D delivery ............................................... ............................ 108
Statistical A naly sis .......................... ...... ................ ............ .. .............. 108
R e su lts .................................. ......... .. .................................................................. 1 0 9
Infection of COS-7 cells with lenti-sEH ................................. ............... 109
Functionality of Lenti-sEH ........................................................... ............... 109
sEH Overexpression in WKY PVN .............. .............................................110
AUDA delivery .................. .................................... ..... .... ........ 110
EE T agonist delivery ............................................................ .... ........... .111
Localization of viral delivery ........... ................................. .................111
sEH Overexpression in SHR PVN ............................................ ..................111
D iscu ssion ................................................................................................ ..... 1 12

7 CONCLUSIONS AND FUTURE DIRECTIONS ................................................124

sEH Overexpression in Alternate Brain Nuclei................................ .................. 124
sE H E expression .............. ........ ................ ................ .................... 12 5
ROS-M ediated Pathway of EET Function .........................................................126
Validation of Baroreceptor Reflex Findings...................... ..... ..............126
Role of sEH in Glial and Endothelial Cells........................................ ............... 127
Role of Central Epoxygenases........................................ 128
Effect of sEH Manipulation in Female Rats..................................................... 128
Implications of Central sEH in Human Hypertension.................... ..................129

LIST OF REFEREN CES ......... ......... ..... ............... ..................................... 132

B IO G R A PH ICA L SK ETCH ......... ................. ...................................... .....................143
















LIST OF FIGURES


Figure pge

1-1 M ajor pathw ays of the RA S ..................................................... ...................25

1-2 Major pathways of ROS production and metabolism...........................................26

1-3 Scatter plot of gene expression in the SHR and WKY hypothalamus and brainstem
..................................................................................................................................2 7

1-4 Arachidonic acid metabolism........................................... 28

1-5 Tissue-specific functions of EETs............................................. ......... ............... 29

1-6 Cellular membrane actions of EETs............................................30

2-1 Cellular localization of sEH in the brain ...................................... ............... 43

2-2 sEH mRNA levels in SHR and WKY brain regions ...........................................44

2-3 sEH protein expression in SHR and WKY brain regions ....................................45

2-4 sEH protein expression in neuronal cultures .................................. .................46

2-5 Effect of chronic losartan delivery on SBP in the SHR ............ .................47

2-6 Effect of chronic losartan delivery on central sEH expression ..........................48

3-1 Effect of ICV DCU delivery on blood pressure and heart rate ...........................60

3-2 Dose response of ICV delivery of AUDA to SHR and WKY rats.........................61

3-3 Effect of central AUDA on blood pressure and heart rate ...................................62

4-1 Effect of central Ang II on blood pressure and heart rate. ....................................73

4-2 Effect of gp91ds-tat on Ang II-induced BP response in the WKY rat .................74

4-3 Effect of gp91ds-tat on Ang II-induced BP response in the SHR ...................75

4-4 Dipsogenic responses of ICV gp9lds-tat and Ang II .....................................76









5-1 Effect of central AUDA delivery on baroreceptor parameters..............................91

5-2 Effect of atenolol on AUDA-induced blood pressure............................................92

5-3 Effect of MS-PPOH on AUDA-induced blood pressure ......................................93

5-4 Effect of EET agonist on BP and HR in the WKY rat...........................................94

5-5 Effect of ROS inhibition on AUDA-induced BP and HR response in the SHR......95

5-6 Effect of AUDA and gp91ds-tat on neuronal firing rate........................................96

6-1 Lentiviral vector ............................................. .................... ......... 116

6-2 sEH protein expression in lenti-sEH-infected COS-7 cells ................................117

6-3 DHET measurements using HPLC analysis............................................... 118

6-4 Blood pressure and heart rate recordings following lenti-sEH and lenti-neo delivery
to th e W K Y rat P V N ................................................................... ..................... 119

6-5 Blood pressure and heart rate recordings following AUDA delivery in the lenti-
sEH -infected W KY rat ...................................................................... ................ 120

6-6 Blood pressure and heart rate recordings following EET agonist delivery in the
lenti-sEH -infected W K Y rat............................................................. ................ 121

6-7 Localization of lenti-sEH delivered to the PVN of WKY rats.............................122

6-8 Blood pressure and heart rate recordings following lenti-sEH and lenti-neo delivery
to the SH R PV N .................. ..................................... .. ........ .... 123















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

ROLE OF BRAIN SOLUBLE EPOXIDE HYDROLASE IN CARDIOVASCULAR
FUNCTION

By

Kathleen Walworth Sellers

December 2004

Chair: Mohan K. Raizada
Major Department: Physiology and Functional Genomics

Recent studies have linked peripheral soluble epoxide hydrolase (sEH) to

hypertensive animal models. However, the role of this enzyme in the central control of

blood pressure has not been elucidated. Thus, the objective was to investigate the

involvement of brain sEH in blood pressure control through expression studies,

pharmacological inhibition, and overexpression of sEH in the brain.

First, sEH expression in the hypothalamus and brainstem, two cardioregulatory

brain areas, was increased in the spontaneously hypertensive rat (SHR) compared to the

Wistar Kyoto (WKY) rat. Second, inhibition of the enzyme by intracerebroventricular

(ICV) delivery of pharmacological agents increased both blood pressure and heart rate in

the SHR, an effect opposite to that following intraperitoneal delivery. This concomitant

increase in blood pressure and heart rate in the SHR was accompanied by a depression of

the baroreceptor reflex gain.









The pathway by which sEH inhibition resulted in an increase in blood pressure and

heart rate functioned through an accumulation of its substrate, epoxyeicosatrienoic acid

(EET). Central delivery of the EETs resulted in an increase in pressor response in the

WKY rat. In addition, P-adrenergic receptor antagonist atenolol blocked the effect of

sEH inhibition, indicating the pathway involves sympathetic activation. Furthermore, the

effects of sEH inhibition were regulated by reactive oxygen species member NAD(P)H

oxidase. This generator of toxic free radicals also mediated the central pressor response

of angiotensin II. These observations suggest that central EETs and sEH inhibition are

involved in increasing blood pressure.

Finally, gene transfer using the lentiviral vector was used to overexpress sEH in

cardiovascular-relevant nuclei. In the WKY rat, heart rate decreased six beats per minute

following sEH overexpression and blood pressure remained unchanged. These studies,

taken together, suggest that an increased expression of sEH is a futile central nervous

system response in protection against hypertension.














CHAPTER 1
INTRODUCTION

Hypertension has long been regarded as a silent killer, a disease that presents few

symptoms but can generate devastating health problems and eventual death. Beginning

as a sustained increase in blood pressure, this disease can lead to end-organ damage

including heart failure. Cardiac output and total peripheral resistance determine blood

pressure. Each component of this equation is influenced by a number of physiological

factors in several organs including the kidney, heart and brain. For example, the heart

regulates its contractility through input from baroreceptors in the carotid sinus and aortic

arch that react to pressure changes (Zanchetti and Mancia 1991). An increased cardiac

contractility results in an increased cardiac output, thereby increasing blood pressure.

Similarly, increasing sodium intake leads to an increase in the body's fluid volume, thus

increasing cardiac output. Total peripheral resistance is ultimately controlled by the

levels of hypertrophy and constriction present in smooth muscle cells that shape how the

blood flows through the vasculature. While these factors define the major influences of

blood pressure, it is necessary to understand the mechanisms underlying these factors in

order to prevent and even reverse this disease.

Central Regulation of Hypertension

The brain continues to emerge as essential in the body's regulation of blood

pressure. For example, when an animal senses a predator, the sympathetic nervous

system increases blood pressure and activates other defense mechanisms to prepare the

animal for a fight or flight response. This response begins when the body senses danger









and transmits this input to the brain to activate the sympathetic nerves that send

projections to the brainstem and spinal column. Preganglionic neurons from these areas

project to postganglionic neurons that in turn project to the heart or blood vessels

(Dampney 1994). Two forms of preganglionic neurons are important in cardiovascular

function, sympathetic preganglionic neurons (SPN) and vagal preganglionic neurons.

Activation of SPN result in an increase in pressor response, while vagal preganglionic

neurons regulate the baroreceptor reflex response that controls heart rate and blood

pressure (Dampney 1994). Thus, signaling from these neurons regulates or controls

cardiac contraction and blood vessel constriction. This autonomic nervous system

signaling therefore regulates both blood pressure and heart rate from information initially

processed from areas in the brain.

In order to elucidate the involvement of the brain in blood pressure regulation via

sympathetic control, it is necessary to identify regions that signal to the SPN. Retrograde

tracers injected into the intermediolateral cell column (IML) have been used to map

afferent neuronal projections. Specifically, transneuronal retrograde tracers incorporating

either herpes or pseudorabies virus have been used recently to push the retrograde tracer

into the cell body, thereby allowing the identification of specific nuclei that signal to the

SPN (Strack et al. 1989). These techniques have identified nuclei in the brainstem,

hypothalamus, caudal ventrolateral pons, and the A5 noradrenergic cell group. Based on

a plethora of data underlying their influence on sympathetic activation, our research

group has chosen to focus on the brainstem and hypothalamus.

Brainstem

The brainstem functions to regulate essential physiological functions including

blood pressure, heart rate and respiration. This brain region houses several specific









nuclei involved with cardiovascular function, including the rostral ventrolateral medulla

(RVLM). The RVLM signals to the SPN, and bilateral denervation of the RVLM results

in a decrease in blood pressure (Guertzenstein and Silver 1974). The RVLM therefore

has influence over the pressor response. The excitatory response of the RVLM is

regulated by several known factors. Excitatory inputs include acetylcholine, angiotensin

II (Ang II), vasopressin, and excitatory amino acids (Andreatta et al. 1988; Dampney

1994; Gomez et al. 1993; Maeda et al. 1991; Willette et al. 1984b). Inhibitory factors

include GABA and opiate receptor activation (Kishi et al. 2002; Punnen et al. 1984;

Willette et al. 1984a).

The important transmission ability of the RVLM is highlighted in work done with

nitric oxide synthase (NOS), a known depressor. First, the inducible form of NOS,

iNOS, is higher in the RVLM of normotensive Wistar Kyoto (WKY) versus

spontaneously hypertensive rats (SHR), indicating a reason for the increased sympathetic

activity in the SHR (Chan et al. 2001). Second, injection of eNOS, the endothelial form,

increased GABA's inhibitory action on RVLM neurons (Kishi et al. 2002). Third,

injections of Ang II into the RVLM increased both sympathetic vasomotor activity as

well as blood pressure (Dampney et al. 2002; Kishi et al. 2002). Finally, gene therapy

using adenovirus encoding eNOS delivered to the RVLM resulted in a decrease in blood

pressure, heart rate and SNA in the WKY rat (Hirooka PBMP 2003). Thus, the RVLM

functions to increase SNA and hence blood pressure through its neuronal projections.

While some of these regulators have been identified, the complete mechanism of the

RVLM in blood pressure control is not yet understood. In order to develop









comprehensive treatments and preventative measures towards the development of

hypertension, it is necessary to elucidate and define this role.

Hypothalamus

The hypothalamus is known for its regulation of body homeostasis and contains

several specific nuclei that are linked to blood pressure regulation. The paraventricular

nucleus (PVN) of the hypothalamus contains both magnocellular and parvocellular

subdivisions, surrounding the third ventricle in an inverted triangular shape. The PVN

has neuronal projections to the IML in the spinal column and also receives and transmits

signals to the RVLM and the nucleus tractus solitarius (NTS) in the brainstem (de

Wardener 2001). In relation to sympathetic control, the PVN can directly innervate the

SPN or send projections to another excitatory nucleus such as the RVLM (Allen 2002).

In fact, the PVN has a large number of catecholaminergic fibers ascending from the

RVLM that release norepinephrine (de Wardener 2001; Palkovits et al. 1992).

Norepinephrine delivery to the PVN increased blood pressure by 30 mmHg, suggesting

that the PVN has direct role on the central control of blood pressure (Harland et al. 1989).

The PVN has a diverse collection of neurons with distinct projections; therefore, its

relation to SPN control is complicated. For example, electrical stimulation of the PVN

has both stimulated and depressed blood pressure (Dampney 1994). Hence, while the

PVN serves as a control unit for SPN action, its overall role is difficult to elucidate.

Research of this key hypothalamic nucleus continues to define its control in brain-

mediated blood pressure.

Baroreceptor Reflex

Apart from the influence of SPN on the pressor response, the baroreceptor reflex

response is also an important component in cardiovascular regulation. Long believed to









function only in the short-term regulation of blood pressure, recent evidence points to the

baroreceptor reflex response as an important regulator of the long-term control of blood

pressure (Malpas 2004). Baroreceptors are found in the aortic arch and the carotid sinus

where they transmit and receive cardiac impulses to the IML and finally the brain. These

impulses stimulate vagal preganglionic neurons that in turn affect heart rate, sympathetic

vasomotor activity and the rate of secretion of vasopressin (Dampney 1994; Sved et al.

2001). Thus, the baroreceptor reflex response functions to control the balance of blood

pressure and heart rate stimulation. The RVLM is a major mediator of the baroreceptor

reflex response (Dampney 1994). For example, baroreceptor activation leads to an

inhibitory effect in the RVLM (Colombari et al. 2001). The PVN has not been

extensively studied in baroreceptor activation control, but studies do suggest it modulates

this action through signaling with the NTS (Kannan and Yamashita 1985). The

baroreceptor reflex response plays an important role in cardiovascular regulation and is

influenced by brain nuclei also involved in SPN control.

Mechanisms Underlying SNA

Renin-angiotensin system

As discussed, firing of neurons from specific nuclei including the PVN and RVLM

to the IML result in an increase in SNA and hence an increase in blood pressure.

Mechanisms controlling these oscillations in firing rate involve a number of hormones,

neurotransmitters and other gene products, many still unknown. One major example of

how these factors in the brain regulate hypertension is the renin-angiotensin system

(RAS).

The brain RAS continues to emerge as a vital regulator of hypertension since its

discovery and characterization more than thirty years ago. Figure 1-1 illustrates the main









pathways and members of the RAS. Classically, angiotensinogen is made in the liver and

then cleaved by renal renin into angiotensin I. Angiotensin I is then converted to Ang II

by angiotensin I converting enzyme (ACE) produced in the lung. Ang II, an eight-

membered peptide, then binds to the Ang II type 1 receptor (ATiR), a G-protein coupled

receptor (Sasaki et al. 1991). This binding results in an increase in blood pressure

through vasoconstriction, increased sympathetic activity and fluid/salt volume regulation.

Ang II functions as both a hormone and as a neurotransmitter in the brain and is also

present in cardiovascular-relevant tissues including the kidney, heart and vasculature.

(Allen et al. 2000).

The initial understanding of the RAS has advanced and become more complex with

new findings. First, the RAS has been identified both systemically and in individual

tissues. Second, novel components have been identified that increase the complexity of

this system. For example, ACE2 converts either Angiotensin I to Angiotensin (1-9) or

Ang II to Angiotensin (1-7) (Turner et al. 2004). In most tissues, Angiotensin (1-7) acts

opposite to that of Ang II (Chappell et al. 2004). These contradictory effects are also

seen when Ang II binds to the AT2R. With each new discovery of the RAS, the pathway

is further elucidated and better understood.

Ang II is a major player in the central regulation of hypertension.

Intracerebroventricular (ICV) delivery of Ang II results in a dramatic increase in both

blood pressure and drinking (Di Nicolantonio et al. 1982). This response begins within

minutes and is only a short-term effect following bolus delivery. Ang II has also been

extensively studied in specific brain nuclei that function in blood pressure and dipsogenic

responses (Pan 2004). For example, Ang II injection to the subfornical organ results in a









profound dipsogenic response, while injection into the PVN, NTS or RVLM leads to a

dramatic increase in blood pressure (Bastos et al. 1997; Mangiapane and Simpson 1980;

Muratani et al. 1993; Tanaka et al. 2001). Taken together, central Ang II binding to the

AT1R in several brain nuclei induces a pressor response.

One ongoing area of study involves the mechanisms underlying this blood pressure

control. Ang II through AT1R stimulation is known to increase neuronal firing rate and

norepinephrine release from sympathoexcitatory neurons, therefore resulting in

sympathetic nerve activation (Sumners et al. 2002; Zimmerman et al. 1984). One

question that remains is what are the steps involved after ATiR stimulation and prior to

sympathetic nerve activation? Recent evidence points to the involvement of reactive

oxygen species (ROS) as a second messenger in the central mediation of RAS.

Reactive oxygen species

ROS has emerged as a major player in a number of disease states, including

cardiovascular disease. ROS members include superoxide (02O-), hydrogen peroxide

(H202), and the hydroxyl radicals. ROS are formed from oxygen converted to superoxide

by enzymes including NAD(P)H oxidase and xanthine oxidase (Figure 1-2). Superoxide

is converted to the hydrogen peroxide using superoxide dismutase (SOD) and then

normally converted to water via either catalase or glutathione peroxidase (Becker 2004).

This pathway is not destructive to cellular function and is the normal progression of

oxygen processing. Oxygen is processed through the mitochondria in order to provide

energy for metabolic functions. During certain disease states and times of physiological

stress, however, hydrogen peroxide can instead be converted to hydroxyl radicals that

lead to cellular injury. Thus, when the ROS outnumbers the available antioxidants,









oxidative stress results (Touyz 2004). This damaging progression is thought to be a

significant factor in a growing number of disease states.

NAD(P)H oxidase, one of the key enzymes in the generation of ROS, was

originally localized in phagocytes (Hanna et al. 2002). It is able to convert oxygen to its

superoxide form by converting NAD(P)H to NAD(P) and donating the released electron.

This conversion rendered it able to defend the host cell against microorganisms. More

recently, though, focus has shifted to the role ofNAD(P)H oxidase in other tissues. This

enzyme was found to be a major source of ROS in cardiovascular tissue, and it has also

been localized to both neurons and glia in the brain (Hanna et al. 2002; Zimmerman and

Davisson 2004). One key difference between NAD(P)H oxidase sources from

phagocytotic or non-phagocytotic cells is that the NAD(P)H oxidase in non-phagocytotic

sources is constitutively active, thus increasing the probability for oxidative stress.

NAD(P)H oxidase is a multimeric enzyme consisting of membranous subunits Gp91ds

and p22 and cytoplasmic subunits p67, p47, p40 and Rac (Hanna et al. 2002). The active

form the enzyme is initiated when p47 is phosphorylated and transports to the membrane

along with the other cytoplasmic subunits and binds with Gp91. Phosphorylation of p47

is driven by protein kinase C (Rey et al. 2001b).

Due to the importance of ROS in a wide array of systems, several inhibitors and

activators have emerged in the field of study. Tempol is a SOD mimetic used in a variety

of studies as an antioxidant, though its specificity is low. Recent studies used an

adenoviral vector to deliver SOD to the brain (Zimmerman et al. 2002). In addition to

SOD manipulation, NAD(P)H oxidase has also been used in therapeutic approaches. A

peptide that competes with the docking portion of gp91 was sequenced to block the









effects ofNAD(P)H oxidase. The tat peptide derived from HIV was included upstream

of the docking sequence in order to facilitate delivery in vivo (Rey et al. 2001b). This

peptide, gp91ds-tat, is a specific, effective blocker of NAD(P)H oxidase both in vitro and

in vivo.

Several studies have implicated ROS in the central Ang II-mediated pathway. ICV

delivery of SOD using adenovirus prevented Ang II-induced increases in blood pressure

and drinking (Zimmerman et al. 2002). In addition, the Ang II-induced decrease in heart

rate was abolished. These results were reproducible using both the mitochondrial and

cytoplasmic forms of SOD. Given the wide range of impact from ICV delivery, studies

were undertaken to elucidate the importance of specific brain nuclei in the ROS-Ang II

connection. Adenovirus encoding a dominant-negative inhibitor ofNAD(P)H oxidase

activator Racl was injected into the circumventricular organs (CVOs) (Zimmerman and

Davisson 2004). CVOs are distinct brain regions in that they lack a blood-brain barrier;

therefore, they are influenced by both the brain and the periphery and serve to pass

information across the two. RAS members are present in several CVOs, and previous

research highlighted the cardiovascular regulatory role of RAS in the CVOs (Muratani et

al. 1996). This adenoviral delivery resulted in preventing the pressor and drinking

responses of Ang II (Zimmerman and Davisson 2004). Thus, ROS involvement in the

Ang II pathway is localized to distinct neurons as well. As a complement to the in vivo

experiments, Ang II treatment of primary lamina terminalis cultures led to an increase in

superoxide, indicating the importance of Ang II on ROS production in vitro (Zimmerman

et al. 2002).









Implications

The central control of blood pressure is regulated by neurotransmitters, hormones

and genetic factors that act to control SNA. While we understand the importance of

specific nuclei in both the hypothalamus and brainstem, the mechanisms underlying this

control have not been fully elucidated. Hypertension is indeed a complex disease whose

establishment and progression cannot be pinpointed to one genetic malfunction. Thus, in

order to understand the genetic basis of hypertension, it is necessary to identify multiple

genes underlying the basis of this disease.

Our research group began the task of identifying genes novel to central blood

pressure regulation by analyzing differences in brain gene mRNA levels between the

SHR and WKY rat, using microarray technology. The SHR was chosen because of

similarities between this genetic strain and human forms of genetic hypertension

(Lindpaintner 1994; Louis and Howes 1990; Trippodo and Frohlich 1981). The

hypothesis was that genes involved in the central regulation of hypertension would have

different gene expression levels between the hypertensive and normotensive state.

However, this genetic profiling cannot in itself distinguish genes whose expression alters

as a consequence of hypertension.

In order to analyze 7000 genes and 1500 ESTs, the Affymetrix U34A Rat

GeneChip was used. Isolated mRNA from the hypothalamus and brainstem was

amplified, fluorescently-labeled, hybridized to the chip, and scanned in order to be

analyzed using GeneSpring software. Figure 1-3 highlights gene expression expressed as

log values in four SHR compared to four WKY samples. Each point represents the

average measurement for an individual gene between the rats in each group. Dotted lines

segregate genes with a two-fold expression change between SHR and WKY rats.









EPHX2, the gene that encodes the soluble epoxide hydrolase (sEH) protein, had the

highest expression discrepancy between the two models. The sEH gene, indicated by the

arrow, was significantly upregulated in the SHR and virtually undetectable in the WKY

rat. This result incited a review of sEH and how it may be related to hypertension. The

following section discusses the history, pathways and actions of sEH and how these

factors may function to regulate blood pressure.

Soluble Epoxide Hydrolase

History

The family of epoxide hydrolases (EH) includes sEH, microsomal EH (mEH),

leukotriene A4 hydrolase, and cholesterol 5,6-oxide hydrolase (Spector et al. 2004). sEH

was first identified as a cohort to mEH, a detoxifying agent to many foreign ingested

agents. The sEH gene was first cloned in 1985, and its dimer structure (two 62-kDa

subunits) was deduced in 1996 (Arand et al. 1996; Grant et al. 1993). A ubiquitous

enzyme, sEH converts arachidonic acid metabolites and linoleic acid epoxides to less

active forms through the addition of water (Zeldin 2001). Figure 1-4 outlines the

placement of sEH in the major pathways of arachidonic acid conversion.

The sEH enzyme can act on several substrates, and initial evidence suggested 1,2-

disubstituted aliphatic epoxides were the ideal substrates for this enzyme. In fact,

radiolabeled forms of this type of compound are used in activity studies of sEH (Borhan

et al. 1995). The active site of sEH has been identified by crystal structure analysis to be

a catalytic triad (Arand et al. 1996). Recently, more research has focused on the

structure-function relationship in sEH. Crystal structure analysis revealed that sEH is a

homodimer made up of two 62.5 kDa units. The two C-terminal domains interact with









each other, while the N-terminal domains have no calculated or observed interactions

with each other (Newman et al. 2003).

Epoxyeicosatrenoic Acids

The most well-studied role of sEH involves its enzymatic conversion of vasoactive

epoxyeicosatrenoic acids (EETs) to dihydroxyeicosatrenoic acids (DHETs). The EETs

are formed from arachidonic acid (AA) through the enzymatic action of cytochrome P-

450 (CYP) epoxygenases (Zeldin 2001). The EETs are divided into four different

regioisomer forms (5,6-, 8,9-, 11,12-, and 14,15-EETs) and are found in a variety of

tissues including brain, kidney, and vasculature (Fang et al. 2001; Spector et al. 2004).

Figure 1-5 outlines the main pathways and tissue locations of EET regioisomers. Most

forms of EETs promote vasodilation, while their DHET metabolites are relatively

inactive.

Vasodilatory properties

EETs in the vasculature have been implicated in blood pressure control due to their

ability to dilate arteries. While EETs are found in a variety of tissue including the brain

and kidney, plasma levels of these epoxygenase products predominate over all other

known sources (Rosolowsky and Campbell 1996). EETs are produced from arachidonic

acid in the endothelium; however, they are secreted from the cells to act on vascular

smooth muscle cells. Evidence suggest that EETs bind to and activate the G-protein

coupled receptor subunit Gs within the smooth muscle cell which then stimulates the

large conductance calcium-activated potassium (BK(Ca)) channel (Harder et al. 1995; Li

and Campbell 1997). Potassium influx into the cell results in hyperpolarization and

therefore dilation of the vascular smooth muscle cell. Thus, increasing EET plasma

levels decreases blood pressure by decreasing total peripheral resistance via vascular









smooth muscle dilation. Figure 1-6 illustrates this vasodilatory action from a cellular

level.

Evidence for this dilation is as follows. First, all regioisomer forms of EETs have

been found to activate BK(Ca) channels in vascular smooth muscle cells (Harder et al.

1995). Second, 11,12- and 14,15-EETs increase the open-state probability of Ca2+-

activated K+ channels in coronary smooth muscle cells (Campbell et al. 1996). Third, all

the regioisomers relax coronary vessels following contraction with thromboxane mimetic

U46619 (Campbell et al. 1996). Fourth, EETs can transiently increase Ca2+ release from

intracellular stores, which also functions to activate BK(Ca) channels (Li and Campbell

1997). While this pathway results in the overall hyperpolarization of the membrane, it is

not necessary for the EET-modulated activation of BK(Ca) channels. Finally, an

increased EET production is associated with hypertensive models, presumably as a

feedback mechanism that cannot overcome the increase in blood pressure (Omata et al.

1992; Pomposiello et al. 2001). Taken together, these studies provide comprehensive

evidence that EETs function to relax the vasculature and thus lead to a decrease in blood

pressure.

This evidence of membrane hyperpolarization has led to high speculation that EETs

function as the elusive endothelial-derived hyperpolarizing factor (EDHF) in the

vasculature. While enormous evidence has implicated nitric oxide as a major player in

relaxing arteries, complete abolition of relaxation cannot be achieved with inhibitors to

NO action alone. The remaining action is termed EDHF, and its identity remains

unknown. While EDHF is likely to be a combination of several factors, the

hyperpolarizing nature of EETs coupled with their high expression in the vasculature has









led to the hypothesis that EETs function as EDHF (Campbell et al. 1996; Fisslthaler et al.

1999).

EETs in the brain

EETs are expressed in astrocytes and endothelial cells in the brain, and evidence

suggests their vasodilatory roles found in the periphery are translated to cerebral blood

flow control in the brain (Medhora et al. 2001). All regioisomer forms of EETs are

expressed in the brain, though astrocytes preferentially intake 8,9-EETs over 14,15-EETs

(Shivachar et al. 1995). In addition, 8,9-EETs were not converted to DHETs in

astrocytes. Whether this result was due to sEH regulation or other conversion

mechanisms is unclear.

EETs from both astrocytes and endothelial cells dilate smooth muscle cells through

activation of the BK(Ca) channels (Gebremedhin et al. 1992). The central result of this

dilation is an increase in cerebral blood flow. While all regioisomers act to dilate the

cerebral arteries, 8,9-EETs and 11,12-EETs are the most potent forms in the brain

(Gebremedhin et al. 1992). Studies that inhibited the CYP-450 epoxygenases in the brain

and thereby prevented the formation of EETs resulted in the reduction of basal cerebral

blood flow by 30% (Alkayed et al. 1997). Thus, central EETs play an integral role in the

maintenance of cerebral blood flow.

Alternate actions of EETs

These four regioisomers of EETs exert different functions dependent on their tissue

location. Vasodilation in the vascular smooth muscle leads to a decrease in blood

pressure via the enlargement of arteries and thus an increase in blood flow, thereby

decreasing total peripheral resistance. A second organ potentially involved in the sEH

regulation of blood pressure is the kidney. Vasodilatory properties of EETs in the renal









vasculature result in changes in renal blood flow and hence changes in water and

electrolyte balance. All regioisomers modulate calcium influx in the vascular smooth

muscle, while 11,12-EET is the predominant form regulating calcium as well as other

studied functions in the endothelium (Spector et al. 2004). In the myocardium, all the

regioisomers inhibit the sodium ion channel, while only the 8,9- and 11,12-EETs activate

the K(ATP) ion channel (Lee et al. 1999; Lu et al. 2001).

EETs can also be converted to different metabolites using enzymes ranging from

COX to CYP co-oxidase (Zeldin 2001). These actions are dependent on the tissue type

and likely the availability of sEH, since treatment with sEH inhibitor N,N'-

dicyclohexyurea (DCU) augments the conversion by these alternate enzymes (Spector et

al. 2004). EETs can also be effectively stored in phospholipids if not immediately

converted by enzymes.

Dihydroxyeicosatrenoic Acids

Studies on the function of DHETs have produced inconsistent results compared to

the role of the EETs. For example, DHETs is generally less potent than the EETs.

Evidence in support of this includes decreases in Ca2+ uptake and relaxation of the

bovine coronary artery compared to the EETs (Campbell et al. 2002). However, DHETs

do activate the BK(Ca) channel of coronary artery myocytes and serve to dilate coronary

arterioles following constriction with endothelin (Spector et al. 2004). While the general

consensus is that the EETs overshadow the DHETs in their action, DHET function cannot

be discounted due to lack of sufficient studies done on these metabolites. Overall,

however, by converting EETs to DHETs, sEH acts to attenuate EET action and promotes

the relative inactivity of DHETs.









Cytochrome P-450

EETs are only one branch of products generated from arachidonic acid. Figure 1-4

illustrates an overview of the pathways stemming from arachidonic acid.

Cyclooxygenase converts AA into a variety of prostaglandins that have been well

characterized in their inflammatory activation role. Prostaglandins have also been

implicated in labor induction and metabolic regulation. The second conversion pathway

uses lipoxygenases that generate hydroxyeicosatrenoic acids (HETEs). HETEs are well

studied in their roles in vascular tissue. HETEs are known to be potent vasoconstrictors

in the kidney and vasculature.

The cyclooxygenase and lipoxygenase branches of arachidonic acid conversion are

therefore stimulating in their roles both in blood pressure control and other essential

physiological regulation. This section will focus, however, on the CYP conversion of

arachidonic acid and what is known and hypothesized about its role in the brain. 20-

HETE, an alternate product of CYP action, is discussed regarding its vasoactive effects

opposite to that of EETs.

Epoxygenases in the brain

Most isoforms are able to convert AA to all regioisomer forms of EETs. Isoforms

in the brain include CYP 2C11, 2C12, 2D18, 2D19, 2C29, 2C38, and 2C39, though more

may be identified in the future (Roman 2002). Interestingly, the studies done to localize

epoxygenase expression in the brain have pinpointed the enzymes to astrocytes. These

studies hypothesize that central epoxygenases have a role in microvascular control. In

contrast, though, CYP 2D19 has been localized to dopaminergic neurons, suggesting that

CYP members are not found exclusively in the glia (Thompson et al. 2000). More









research is needed to elucidate this pathway in conjunction with EET-mediated effects on

cardiovascular control.

20-HETE

Regioisomers of HETEs can also be generated by CYP, specifically co-hydroxylase

of the CYP 4A family. co-hydroxylase converts arachidonic acid to 19-HETE and 20-

HETE. 20-HETE acts as a vasoconstrictor in vessels ranging from the kidney to the

brain. While EETs activate BK(Ca) channels, 20-HETE inhibits these channels, thus

inducing contraction (Croft et al. 2000). Inhibiting 20-HETE results in a decrease in

blood pressure due to the decrease in total peripheral resistance. Levels of both 20-HETE

and CYP 4A are increased in the SHR (Omata et al. 1992). In addition, this

vasoconstrictor is activated by Ang II, further elucidating its role in hypertension (Croft

et al. 2000). Thus, 20-HETE functions are opposite to the vasodilator role reported for

EETs and its overexpression is linked to hypertension. In summary, these arachidonic

acid metabolites from the epoxygenase and co-hydroxylase pathways serve a variety of

functions throughout the body, and these functions are dependent on the converting

enzymes that regulate the activity of these metabolites.

sEH and Hypertension

sEH has been implicated in cardiovascular, renal, and inflammatory biology

functions. Recently, studies have linked sEH to hypertension through both a transgenic

model overexpressing the gene and sEH inhibitor studies in the SHR. sEH-null mice

were generated from offspring of chimeric mice with the disrupted sEH gene (Sinal et al.

2000b). Homologous recombination was used to disrupt the sEH gene in embryonic stem

cells. High performance liquid chromatography (HPLC) revealed that the sEH-null mice

had elevated levels of EETs and decreased levels of DHETs as compared to the wild-type









mice. Blood pressure was measured using tail-cuff and compared between groups. Male

sEH-null mice had decreased systolic blood pressure and did not have an increased blood

pressure in response to salt loading. Female sEH-null mice had no significant change in

systolic blood pressure. Thus, deletion of the sEH gene lowered systolic blood pressure

to that of the female mice.

An important subsequent finding was relative sEH expression in the SHR and

WKY rat from Charles River Laboratories. As previously mentioned, the SHR is a good

animal model to use for hypertension studies based on similarities to the human form of

the disease (Lindpaintner 1994; Louis and Howes 1990; Trippodo and Frohlich 1981).

Using an sEH-specific antibody, researchers found a significant elevation of sEH protein

in the SHR kidney and liver; in fact, it was hardly detectable in the WKY rat (Yu et al.

2000a). Given the overexpression of sEH in the hypertensive model, researchers sought

to determine whether the activity of this enzyme was linked to blood pressure.

The second major paper addressing the sEH control in blood pressure involved the

intraperitoneal (IP) delivery of sEH inhibitor N,N'-dicyclohexylurea (DCU). Treatment

with 3 mg/kg DCU resulted in a 65% decrease in 14,15-DHET and a 30% increase in

14,15-EET (Yu et al. 2000a). As a result, systolic blood pressure decreased by 22 mmHg

in the SHR and had no significant effect in the WKY rat. Thus, pharmacological

inhibition of sEH also results in a decrease of systolic blood pressure in hypertensive

male rats. In summary, both a genetic and a non-genetic method for decreasing sEH in

the periphery led to a decrease in systolic blood pressure and were accompanied by a

decrease in the conversion of EETs to DHETs.









Expression studies for sEH have also included alternate models of high blood

pressure aside from the Charles River source of SHRs (Imig et al. 2002). Peripheral or

central delivery of Ang II results in an increase in blood pressure and consequently

hypertension (Allen et al. 2000; Di Nicolantonio et al. 1982). Ang II was chronically

infused (60 ng/min) using an osmotic minipump, and blood pressure was recorded using

tail cuff (Imig et al. 2002). Following IP injection of the sEH inhibitor N-cyclohexyl-N-

dodecyl urea, systolic blood pressure decreased 30 mmHg in the Sprague-Dawley (SD)

rat. Following sEH inhibition, sEH expression increased two-fold in the kidney.

However, sEH overexpression is not present in all SHR sources. As discussed below, the

Heidelberg (Heid) SHR has a lower sEH expression compared to its normotensive

control. Thus, the importance of sEH overexpression in the peripheral tissues remains to

be established.

sEH Polymorphisms

Rodent

It is important to note that in the Ang II-infusion study, the basal sEH expression in

the SD rats was highly variable, ranging from present to not present. This variable

expression is typical of sEH in SD rats. SD rats are outbred, which increases the

probability of new gene patterns with each new breeding source. Interestingly, sEH has

known polymorphisms found in both rodent models and in humans. As previously

described, the SHR from Charles River has increased sEH expression in both kidney and

liver (Yu et al. 2000a). However, sEH expression in Heid SHR and WKY rats was

opposite-WKY rats have increased sEH expression in the liver and kidney (Fornage et

al. 2002). This finding was accompanied by the identification of a separate sEH allele in

the Heid rat source. The two alleles differ by four single nucleotide polymorphisms









(SNPs). Consequently, the polymorphism identified in the Heid source results in a

blunted sEH activity. Thus, presence of these polymorphisms and reduced activity can

explain the strain source difference in the Western blot analysis results.

Human

Polymorphisms in sEH have also been identified in humans; in fact, most of the

research involving the sEH role in the human focuses on polymorphism and its effects

ranging from sEH activity itself to coronary artery calcification. The human form of sEH

is 73% identical to the mouse form, with 100% similarity in the catalytic triad (Przybyla-

Zawislak et al. 2003). This highly conserved region underlies the importance of the sEH

active site across species. The human sEH gene has been localized to the p arm of

chromosome 8, specifically, 8p21-pl2. The sEH gene is not without differences in its

sequence, however, as highlighted by three separate studies.

Initially, seven SNPs were identified in liver samples from twenty-five human

subjects. Two of these SNPs resulted in amino acid substitutions, while the remaining

five were silent mutations (Sandberg et al. 2000). In the second study, a total of thirty-six

SNPs were identified in forty-eight members of the Japanese population. Five of these

SNPs were found in exons of the gene (Saito et al. 2001; Sato et al. 2004). A third study

resulted in the identification of six SNPs of the gene in a population size of seventy-two.

Three of these polymorphisms had an increased level of epoxide hydrolase activity while

one had decreased activity. The one with decreased activity also had significant

reduction in 14,15-EET hydrolysis. Interestingly, the incidence of sEH polymorphisms

in the sample group studies was higher in the Black population as compared to White or

Asian population (Przybyla-Zawislak et al. 2003).









sEH polymorphisms in cardiovascular disease

This variation in sEH polymorphisms as related to race and disease was further

examined in a recent correlation study. The sEH gene was mapped in 1201 African

American and 1506 White subjects, and groups were separated based on the presence or

absence of the genotypes Gln287/Gln287, Arg287/Gln287, Arg287/Arg287 (Fornage et

al. 2004). The Gln287 polymorphism has been shown to result in a decrease in sEH

activity. The presence of the Gln287 polymorphism was associated with a two-fold

increase in coronary artery calcification in an African American subset of the Black

population (Fornage et al. 2004). This finding suggests that a decrease in sEH activity

and thus an increase in EETs may lead to an increase in coronary artery calcification in

the African American population.

In a separate study involving a family afflicted with familial hypercholesterolemia,

the sEH polymorphism Glu287/Arg was identified (Sato et al. 2004). Results indicated

that patients with one Arg287 allele as well as defects in the LDL receptor gene had

elevated total cholesterol levels as well as plasma triglyceride levels. No positive

correlation was identified in subjects not having defects in the LDL receptor gene. Thus,

it is interesting that these different polymorphisms at the same locus elicit different

effects on sEH activity. More research is needed to elucidate possible mechanisms

underlying this hypothesis and other associations between sEH and cardiovascular

disease.

sEH polymorphisms in other disorders

The Arg287/Gln287 polymorphism was further studied to identify any correlation

between this sEH SNP and Parkinson's disease (Farin et al. 2001). The rationale behind

the study was that a decrease in sEH activity leads to an increase in active epoxide









intermediates, which may generate an increase in oxidative stress, leading to Parkinson's

disease. No significant correlation was measured in Parkinson's patients, indicating a

decrease in sEH activity was not related to Parkinson's disease. However, this study

cannot rule out other sEH roles that may be related to CNS disorders or general oxidative

stress in general. In addition, the general study size could mask a subset of Parkinson's

disease development that may in fact stem from sEH dysregulation. Finally, SNP allele

frequencies were measured in a subset of the Japanese population for future studies on

cancer. The sEH gene was included in this study based on its involvement in conversion

of toxins (Yoshimura et al. 2003). Thus, sEH is being examined in a variety of disorders

and diseases based on its widespread activity and implications. In addition, its ubiquitous

expression and polymorphisms resulting in changes in activity target this as a candidate

gene for regulation of a number of genetic-based problems.

Phosphatase Studies

Two recent PNAS publications focused on the role of sEH as a phosphatase

(Cronin et al. 2003; Newman et al. 2003). sEH was found to act as a phosphatase, though

without high specificity. For example, sEH was not mediated by common phosphatase

inhibitors including sodium orthovanadate (Newman et al. 2003). However, sEH did

effectively metabolize a number of hydroxy lipid phosphates. The connection of

phosphatase activity to the N-terminal domain was confirmed with both a plant form of

sEH lacking the N-terminal domain and a mutant N-terminal domain form of sEH

(Cronin et al. 2003). In both of these models, phosphatase activity of the enzyme was

eliminated. In addition, sEH inhibitors used to block epoxide conversion had no effect on

phosphatase activity. sEH function related to EET conversion is attributed to the C-

terminal domain, which is homologous to haloalkane dehydrogenase (Cronin et al. 2003).









These studies indicate that sEH may play an important physiological function through its

phosphatase activity. While evidence into this line of research is sparse, it is conceivable

that the number of polymorphisms in sEH across species and strains within species may

generate differences in phosphatase activity and thus a novel role for this enzyme.

Summary

Hypertension is a complex disease controlled by a number of organs including the

brain. While our understanding of the central regulation of blood pressure has a

substantial base involving connections between specific nuclei in the hypothalamus and

brainstem to the spinal cord, key mechanisms involved in the genetic basis of

hypertension have not been elucidated. In order to fully understand this disease in the

hopes of preventing and combating its progression, it is essential to identify genes and

their pathways underlying the dysregulation of blood pressure. Using gene profiling

involving microarray analysis of the hypothalamus and brainstem, sEH was found to be

overexpressed in the SHR compared to the WKY rat brain. sEH is an enzyme that

converts vasoactive EETs to their relatively inactive diol forms. Expressed in a variety of

tissues, sEH has been implicated in the peripheral control of hypertension due to its

increased expression in the hypertensive state and the resulting decrease in blood pressure

following IP delivery of an sEH inhibitor. In addition, sEH polymorphisms are now

being associated with cardiovascular disorders. Thus, sEH and its link to hypertension

are well-characterized in the periphery. However, little is known about the role of this

enzyme in the brain.

Given the previous studies of sEH and hypertension coupled with its dramatic

overexpression in the brain of the SHR, we hypothesized that sEH is involved in the









central regulation of hypertension. In order to address this hypothesis, our aims were as

follows:

1. Determine if central sEH expression is increased in the hypertensive state. These
studies outlined in Chapter 2 focus on the validation of gene profiling data as well
as whether this increased sEH expression is a cause or consequence of
hypertension.

2. What is the physiological effect of inhibiting sEH in the brain? Chapter 3 discusses
central sEH inhibitor studies in both SHR and WKY rats and the resulting blood
pressure, heart rate, and baroreceptor reflex changes.

3. Does the pressor action of central Ang II function through NAD(P)H oxidase? As
discussed earlier, the RAS plays a major role in the central regulation of blood
pressure. In addition, ROS continues to emerge as a necessary component of this
regulation. These studies in Chapter 4 address this question with central
administration ofNAD(P)H oxidase formation blockers and Ang II and resulting
blood pressure changes. This role of ROS in the brain forms the basis of the central
mechanism of sEH discussed in Chapter 5.

4. What is the central sEH mechanism that results in blood pressure and heart rate
changes? Chapter 5 outlines experiments incorporating EET mediators, ROS
production antagonists, and sympathetic blockers in order to elucidate the
mechanism by which central sEH inhibition increases blood pressure.

5. Determine if sEH overexpression in the brain alters blood pressure and heart rate.
These studies in Chapter 6 use the lentivirus to overexpress the sEH gene in the
PVN of SHR and WKY rats. Blood pressure and heart rate are recorded daily and
mechanisms underlying sEH control are studied using sEH inhibitors and EET
agonists.

Taken together, these aims dissect the role of sEH on the central regulation of blood

pressure. The results of these experiments invoke intriguing questions into the specific

effect of EETs in the brain and how this effect is regulated in the hypertensive and

normotensive state. Overall, these studies provide further insight into the central

regulation of blood pressure and propose a novel role of EETs in the brain.












Angiotensinogen
SRenin
Angiotensin I
I


NEP IACE2
Angiotensin 1-9
ACEE:

Angiotensin 1-7 4


ACE


Angiotensin II

2J

T AT1R AT2R

Vasoconstriction
SNA
Blood Pressure


Figure 1-1 Major pathways of the RAS











NAD(P)H-NAD(P)+ + H+
Xanthine Oxidase
CYP





SOD




Catalase Fe2+
Glutathione
peroxidase


Figure 1-2 Major pathways of ROS production and metabolism


H20+0


F-P




















41


1000 -
Unchanged
U Downregulated
.p<0.01 Upregulated

1000 10000 100000
WKY



Figure 1-3 Scatter plot of gene expression in the SHR and WKY hypothalamus and
brainstem. Each point represents the average measurement for a gene. Dotted
lines segregate genes with a two-fold expression change between SHR and
WKY samples. sEH, indicated by the black arrow, is significantly
upregulated in the SHR.















s EETs DHETs
e.-, 8,9-, G-8 11. 2-, 14.1 5-EETs) aG-. 8.9-. 11.12-. 14.1 -DIET,
CYP
I Cyclooxygenase
SidroQ a 20-HETE
1 9-HETE

,Y
Cyclooxygenase T
C daoxenase Prostaglandin H2 _- PGD/PGE/PGF

SrPmstacyclin

< Thromboxane
Lipoxygenase
SHETEs Leukotrienes


Figure 1-4 Arachidonic acid metabolism













Brain

IRelax VSMC; increase cerebral blood flow
Activate BK(Ca2+) channels
Distribution: Astrocyes > Endothelial cells > Neurons
Cerebral vessel dilation
Glutamate stimulates production



Heart/Vascuature

-- Relax VSMC
I '. Activate BK(Ca2+) channels
'ii ,Relax precontracted coronary vessels
8,9-, 11 ,12-EETs activate K(ATP) channel
Inhibit Na+channels
Found in endothelium and blood vessels
EDHF role


Kidney

Relax VSMC
Activate BiKCa2+) channels
Increase renal blood flow
EDHF role


Figure 1-5 Tissue-specific functions of EETs











Endothelial cell


VSMC


SMembrane Hyperpolarization
$ VSMC Contraction


Ca2+


Figure 1-6 Cellular membrane actions of EETs


AA- p E



EETs














CHAPTER 2
INCREASED SEH EXPRESSION IN SHR VS. WKY RAT BRAIN:
IN VITRO AND IN VIVO ANALYSIS

Introduction

The brain is an essential component of the mechanisms controlling blood pressure.

As previously discussed, specific nuclei of brain regions including the hypothalamus and

brainstem function in the central regulation of blood pressure (Dampney 1994). Given

the relative importance of the brain in establishing and maintaining hypertension coupled

with the inadequacy of our knowledge of the mechanisms underlying this influence, it is

imperative to study genes with discordant expression in the hypertensive state. By

elucidating the roles of these genes in the brain, we better understand the mechanisms

involved in the establishment of hypertension and therefore have more power with which

to prevent and even reverse this disease.

Chapter 1 discussed gene profiling findings that sEH had the most disproportionate

mRNA levels in hypothalamus-brainstem samples of the male SHR compared to the male

WKY rat. High sEH expression in the kidney also has been identified in hypertensive

animal models (Imig et al. 2002; Yu et al. 2000a). In addition, deletion of the EPHX2

gene encoding sEH decreased the systolic blood pressure of male mice (Sinal et al.

2000b). These studies raise several questions regarding sEH expression. First, given the

ubiquitous nature of this enzyme, is sEH overexpression in hypertensive models

generalized and therefore not confined to renal action? What are the sEH expression

levels in other cardiovascular-relevant tissues including the brain? Second, are









differences in brain sEH expression the result of hypertension, or are they independent of

blood pressure changes?

The increased sEH mRNA levels in the SHR brain from microarray analysis must

first be verified, given the unpredictability of microarray significance. Therefore, the

first part of Aim I is to use real-time RT-PCR and Western blot analysis to measure sEH

levels in the brain areas of SHR and WKY rats. The hypothesis is that both RNA and

protein levels of the hypothalamus and brainstem are increased in the SHR compared to

the WKY rat.

The second part of Aim II is to determine if central sEH expression is independent

of blood pressure changes. First, if sEH expression is increased in the hypertensive state,

is that increase present from birth, or do the expression levels change with development?

If the expression levels remain constant, then sEH expression cannot be a consequence of

hypertension. Conversely, if the levels change, then sEH expression may be sensitive to

changes in blood pressure. This question is important in understanding the role of sEH in

the development of hypertension. To address this issue, neuronal cultures were prepared

from one-day-old SHR and WKY rat pups. The hypothesis was that sEH overexpression

was present from birth and not an effect of high blood pressure.

The question of whether sEH expression is dependent on blood pressure changes

was next addressed in the adult SHR. In contrast to measuring sEH expression prior to

the development of hypertension, this experiment measured sEH expression following

blood pressure normalization with losartan, a potent pharmacological AT1R blocker (Carr

and Prisant 1996). We hypothesized that normalizing blood pressure in the SHR would









not affect sEH protein levels in the brain, indicating that sEH expression is not dependent

on blood pressure changes.

The following studies thus address two facets of sEH expression. First, they

determine whether the sEH data from the microarray studies do in fact translate to a

change in sEH expression. Second, they answer whether sEH expression is driven by

changes in blood pressure. Taken together, these experiments define central sEH

expression in the SHR and WKY rat.

Materials and Methods

Animals

Male SHR and WKY rats, weighing 220-250g at the beginning of the study, were

ordered from Charles River Laboratories (Wilmington, Mass). Rats were housed

individually and kept on 12:12 light:dark cycle in a climate-controlled room. Rat chow

(Harlan Tekland, Madison, WI) and water were provided ad libitum. Number of animals

per group are described in Results. All animal protocols were approved by the

Institutional Animal Care and Use Committee of the University of Florida.

Immunohistochemistry

Rats were anesthetized with inhaled isofluorane (5%) and perfused transcardially

with saline followed by 4% paraformaldehyde (PFA). SHR brains were post-fixed in

PFA and saturated in 20% sucrose solution. 10-20 [tM sections were cut using a cryostat

and mounted on poly-L-lysine-coated slides; the Rat Brian Atlas (Paxinos & Watson 4th

Ed 1998) was used as a reference. Sections were blocked in 2% bovine serum albumin

(Sigma-Aldrich, St. Louis MO) dissolved in Tris-buffered saline with Tween (TBS-T,

0.1M Tris-HC1, 0.9% NaC1, 0.1% Tween) for three hours. Sections were then incubated

with anti-sEH rabbit antibody overnight (1:500), a kind gift from Dr. Bruce Hammock,









University of California Davis, followed by anti-rabbit biotinylated (1:200) for two hours

and 10tlg/ml Avidin-FITC for one hour (Amersham Biosciences, Buckinghamshire,

England). The sections were double-stained with either neuronal-specific (NeuN, 1:10,

Amersham Biosciences) or glial-specific antibody gliall fibrillary acidic protein (GFAP),

1:10, a kind gift from Dr. Gerard Shaw, University of Florida] to detect cellular

distribution of sEH. sEH localization was detected with fluorescent microscopy.

Real-Time RT-PCR

The RNAqueous-4PCR kit was used to isolate DNA-free RNA from tissue samples

(Ambion, Austin TX). One-step real-time RT-PCR was performed on 50ng RNA per

sample. Briefly, samples were combined with sEH-specific primers and probe (900nM

forward: 5'-GATTCTCATCAAGTGGCTGAAGAC-3'; 900nM reverse: 3'-

GGACACGCCACTGGCTAAAT-5') and probe (250nM: 5'-

CCAGAACCCATCGGTGACCTCCAA-3') and TaqMan One-Step RT-PCR Master Mix

and Multiscribe as described by the company (Applied Biosystems, Foster City CA).

Primers and probe to 18S were used as an internal control. Reactions were carried out in

the ABI PRISM 7000 sequence detector, and the standard curve method was used to

calculate the threshold cycle (CT) for target amplification. The input amounts were then

calculated from the CT and standard curve and normalized using the 18S input amounts.

Western Blot Analysis

Total cell lysate (TCL) was isolated according to the company's research

applications instructions (Santa Cruz Biotechnology Inc, Santa Cruz CA). Twenty |tg

protein from either neuronal cultures or brain tissue were run on a 10% SDS-PAGE gel

and transferred onto a nitrocellulose membrane. Following three hours of blocking with









5% milk in TBS-T, the membrane was probed with the sEH rabbit-anti mouse antibody

(1:3000 in 1% milk/TBS-T) overnight. Specificity of this antibody has been previously

established (Sinal et al. 2000a; Yu et al. 2000b). The membrane was rinsed 3 times and

washed for 20 min in TBS-T and then incubated with anti-rabbit IgG HRP-conjugated

secondary antibody (1:3000) for 1 hour. Following final washes, the membrane was

incubated with Western Lightning Chemiluminescence Plus reagent for 1 min and then

exposed to film to visualize the bands (Perkin Elmer, Wellesley, MA).

Neuronal Cells in Primary Culture

Primary neuronal cultures (90% neurons, 10% glia) from the hypothalamus and

brainstem of one-day-old SHR and WKY rats were prepared as previously described

(Fleegal and Sumners 2003; Raizada et al. 1995; Sunn et al. 2002). The cells were

cultured grown twelve days in Dulbecco's Modified Eagle's Media (DMEM) + 10%

horse serum at 370C, 10% CO2 in 100 mm dishes. Three dishes were used per

experimental group.

Losartan Treatment of SHRs to Normalize Blood Pressure

Losartan (10mg/kg/day) was delivered in the drinking water to male SHR (n=6)

after determining the basal drinking rate to be 32 ml water/day. Six SHRs were kept on

regular drinking water as control. The water bottles containing losartan were covered

with aluminum foil to address the light sensitivity of the drug. Water intake and body

weight were monitored daily one week prior to and up to one month following the start of

losartan treatment. One rat in the control group was eliminated from the study due to its

inability to develop high blood pressure. Systolic blood pressure was recorded using the

tail cuff method three and four weeks after the start of losartan treatment. At the end of









the experiment, protein and mRNA was isolated from the hypothalamus, brainstem, and

cortex of the rats.

Statistical Analysis

All data are expressed as mean + standard error (SE). The number of animals or

samples in each group is described in the Results below. Comparisons between

experimental groups were analyzed using either one-way ANOVA (SPSS) or Student's T

test (Stat View). Differences between means within groups were assessed with the

Newman-Keuls analysis. Significance was set at a confidence level at or above 95% and

is denoted by in each figure.

Results

Cellular Localization of sEH

Immunohistochemical analysis revealed that sEH is localized to both neurons and

glia throughout the SHR brain (n=3). Neurons were double-stained with both NeuN (red)

and sEH antibody (green), and glial cells were double stained with both sEH antibody

(green) and GFAP (red), as shown in Figure 2-1. Co-staining was identified by the

yellow overlapping fluorescence detected using confocal microscopy. sEH was detected

in all areas of the midbrain and brainstem sectioned. Control sections treated only with

secondary and tertiary antibodies did not fluoresce at the intensity used for the confocal

analysis, thus indicating the fluorescence was due to sEH antibody rather than

background fluorescence. Fluorescence from the sEH antibody was localized to the

cytoplasm and neuronal projections of the cell. No sEH was detected in the nucleus.

This result corresponds with the nature of this protein, which is a cytoplasmic protein.

The GFAP antibody was also localized to the cytoplasm rather than the nucleus, and

overlapping fluorescence was observed in this area.









sEH Expression in the SHR

Profiling with microarray technology previously identified profoundly increased

sEH mRNA levels in the brain of the SHR compared with its WKY normotensive

control. Thus, the first objective was to determine if this result corresponded with RNA

and protein levels measured by real-time RT-PCR and Western blot analysis,

respectively. Figure 2-2 outlines the results of real-time RT-PCR using hypothalamus

and brainstem isolated from four male SHR and WKY rats. Samples within each rat

strain were combined; therefore, it is impossible to say with any certainty that there were

differences. White and black bars represent sEH mRNA levels to normalized to 18S

levels of WKY rat and SHR, respectively. sEH mRNA levels in the SHR were three-fold

higher hypothalamus and six-fold higher in the brainstem compared to WKY rats.

sEH protein expression was also increased in the SHR hypothalamus and

brainstem. Figure 2-3 depicts a representative radiograph (A) and quantitation of sEH

protein levels normalized to P-actin (n=4/group) (B). In TCL isolated from the

hypothalamus, relative sEH:P-actin protein ratios in SHR and WKY rats were 0.52+/0.14

OD/mm2 and 0.12+/-0.02 OD/mm2, respectively. In the brainstem samples, the ratio was

0.60+/-0.14 OD/mm2 in the SHR and 0.13+/-0.03 OD/mm2 in the WKY rats. sEH

protein levels were measured with Western blot analysis using the same sEH antibody

source used in the peripheral studies. White and black bars represent WKY rat and SHR,

respectively. sEH was barely detectable in WKY rats while they were dramatically

increased in the SHR.

In vitro Validation with Neuronal Cells in Primary Culture

Primary co-cultures from hypothalamus-brainstem of WKY and SHR have been

used in the past to elucidate cellular and molecular mechanisms of physiological









dysregulation in neural control of hypertension (Sun et al. 2003b; Yang et al. 2004).

These cultures were used to determine if sEH overexpression in these hypertensive

animals is present prior to the establishment of high blood pressure. Western blot

analysis demonstrated a four-fold increase in sEH protein levels in neuronal cultures of

SHR vs. WKY rats, indicating that sEH overexpression is present from birth in the SHR

(Figure 2-4). The ratio of sEH to p-actin protein was 0.34+/-0.15 OD/mm2 in the SHR

and 0.08+/-0.04 OD/mm2 in the WKY rats (n=4/group). Panel (A) is a representative

radiograph highlighting the sEH and P-actin protein bands. Panel (B) is the graphical

representation of sEH protein levels normalized to P-actin from the four sets of neuronal

cultures.

sEH Expression Following Blood Pressure Normalization

Delivery of losartan (10mg/kg/day) into the drinking water resulted in a significant

decrease in systolic blood pressure in the SHR one month following the initiation of

losartan treatment. Three weeks after the start of the study, systolic blood pressure was

33 mmHg lower in the rats treated with losartan [181.48+/-5.11 mmHg SHR (n=5),

148.40+/-1.71 mmHg SHR + losartan (n=6)]. This gap widened to a decrease in systolic

blood pressure by 38 mmHg (173.84+/-4.40 mmHg SHR, 136.28+/-2.05 mmHg SHR +

losartan) one month following the start of losartan treatment (Figure 2-5). Body weights

were not significantly different between groups.

At the end of the study, brains were dissected and protein isolated from losartan-

treated and control rats. Western blot analysis revealed no significant change in the ratio

of sEH to p-actin expression in the hypothalamus between the losartan-treated (0.530+/-

0.078 OD/mm2) and control rats (0.739+/-0.246 OD/mm2) (Figure 2-6). Similarly, the









ratio of sEH to p-actin expression was 0.863+/-0.026 OD/mm2 and 0.840+/-0.066

OD/mm2 in the brainstem of the losartan-treated and control-treated SHRs, respectively.

White and black bars represent losartan-treated and control samples, respectively. Thus,

sEH expression in the SHR was not affected by blood pressure depression using an AT1R

antagonist delivered orally.

Discussion

This study was the first to measure and compare central sEH expression between

hypertensive and normotensive animal models. Both sEH mRNA and protein levels were

elevated in the hypothalamus and brainstem of SHR compared to WKY rats. This

overexpression was independent of blood pressure changes because (i) sEH

overexpression existed in neuronal cultures from prehypertensive rats and (ii)

normalization of blood pressure with losartan did not alter sEH expression levels. These

expression studies validate the gene profiling data, are parallel to the expression findings

in peripheral tissue and provide a foundation on which to study sEH in the central control

of hypertension (Imig et al. 2002; Yu et al. 2000a).

Immunohistochemical studies established that sEH is expressed in both neurons

and glial cells of one-day-old SHR and WKY rats. In contrast, CYP and EETs have been

mainly localized to astrocytes and endothelial cells surrounding the cerebral vasculature

(Alkayed et al. 1997; Gebremedhin et al. 1992; Medhora et al. 2001; Shivachar et al.

1995). Thus, the role of sEH in the brain may involve divergent roles including

sympathetic nerve activation through neuronal pathways or control of cerebral blood

flow. The role of central sEH in relation to blood pressure likely stems from neuronal

control given two sets of data. First, sEH was overexpressed in neuronal cultures of one-









day-old SHRs. Second, sEH expression does not differ in glial cultures of SHR and

WKY rats (data not shown).

In the brain areas studied, sEH protein and mRNA levels were significantly

increased in the SHR compared to the WKY rat. This result correlates to the peripheral

studies that showed an increase in sEH expression in the kidney and liver of SHR and

Ang II-induced hypertensive models (Imig et al. 2002; Yu et al. 2000a). Thus, in all

tissue areas studied, sEH is more abundant in the Charles River SHR compared to the

WKY rat. This result therefore proves that sEH overexpression is not limited to the renal

and hepatic systems of the SHR but is in fact present in other cardiovascularly-relevant

areas.

This was the first study to measure sEH expression patterns in non-adult animal

models. sEH was highly expressed in neuronal cultures from one-day-old SHR

compared to WKY rats. This result indicates that sEH expression patterns exist from

birth and are not affected by the onset of hypertension. However, these results alone do

not prove that increased sEH expression in the brain is the cause of any change in blood

pressure. Future studies in which either central sEH is blocked during development in a

hypertensive strain or overexpressed in the brain of normotensive strain would serve to

answer this question.

The conclusion that central sEH expression is independent of systemic blood

pressure changes was also supported by the normalization of blood pressure experiment.

Chronic delivery of losartan into the drinking water had no effect on the sEH levels in

either the hypothalamus or the brainstem. This result suggests that depression of blood

pressure in the SHR does not alter sEH expression. Losartan is a potent AT1R blocker









that is often used to evaluate the role ofRAS (Carr and Prisant 1996). The decrease in

systolic blood pressure by almost 40 mmHg following its oral delivery highlights the

essential role RAS plays in blood pressure regulation. However, this decrease in blood

pressure was not accompanied by changes in sEH expression.

Several caveats regarding this experiment must be addressed. First, sEH

expression was only measured at one time point, three weeks following the start of

losartan delivery. Therefore, one cannot discount expression changes prior to or

following this time point. For example, perhaps sEH expression was affected during the

initial drop in blood pressure and then recovered by means of feedback mechanisms after

three weeks. This result would indicate that sEH expression might have a role in the

RAS regulation of blood pressure. Alternately, sEH expression may be affected after

four weeks of treatment or longer due to delayed mechanisms. If this postulation were

true, then sEH expression would be dependent on blood pressure changes.

Another possibility is that while sEH expression may not change following blood

pressure normalization, either the enzyme's activity or the affinity of the product for its

receptor may be affected. Regardless of expression levels, if the enzymatic activity

changes, then the role of sEH following blood pressure changes must be reassessed. sEH

activity is generally determined by HPLC analysis to compare the levels of EETs and

DHETs (Yu et al. 2000a). This analysis should be performed in the future in order to

confirm that sEH is independent of blood pressure changes.

Finally, further studies incorporating different means of altering blood pressure

should be performed since blood pressure normalization does not follow only one

mechanism. In this experiment, losartan was delivered via the drinking water; therefore,









the blood pressure was normalized by blocking peripheral AT1R that results in a decrease

in total peripheral resistance. While central sEH expression was not affected by this

method, it may have changed had the losartan been delivered to the brain, thus decreasing

sympathetic nerve activity. To verify that the sEH expression is independent of blood

pressure depression, other antihypertensive agents including diuretics should be

incorporated. Alternately, sEH gene regulation may be constitutively active in the SHR

and therefore other hypertensive models should be incorporated. Similarly, sEH

expression may only be activated with blood pressure stimulation rather than depression.

While we can conclude that decreasing blood pressure via oral delivery of losartan does

not alter central sEH expression, this result does not conclusively prove that central sEH

is independent of any change in blood pressure.

Collectively, these studies reveal that central sEH expression is higher in the SHR

compared to the WKY rats. In addition, these results suggest that central sEH expression

is independent of blood pressure changes. Given the dichotomy of central sEH

expression in the SHR and WKY rat, what effect, if any, do these expression changes

have on the central control of blood pressure? Does sEH overexpression in the brain

have a role in hypertension? To begin to answer these questions, Chapter 3 outlines

central sEH inhibition studies to determine whether pharmacological blockade of sEH

action in the brain results in a change in blood pressure.











NuEn Ab (Rodamine)


Neuron


sFH Ah + IFAP Ah


sEH Ab (FITC)


GFAP Ab (Rodamine)


Figure 2-1 Cellular localization of sEH in the brain. Following perfusion, SHR rat brains
sections (20[LM) from the midbrain were prepared for immunohistochemical
analysis of sEH and its localization to neurons and glial cells. sEH Antibody
is detected by green fluorescence, while the red fluorescence detects either
neurons (upper panel) or glia (lower panel). Yellow fluoresence marks the
overlap between sEH Antibody and cell type-specific Antibody. All images
were taken using confocal microscopy.


Glia


Fr-H Ah rIl-rrl










1.2

1

0.8

S0.6

0.4

0.2


-- WKY
SHR


Bninstem


Figure 2-2 sEH mRNA levels in SHR and WKY brain regions. RNA was isolated from
hypothalamus and brainstem regions of SHR and WKY rat brains
(n=4/combined in each group). Real-time RT-PCR incorporating sEH-
specific primers and probe was used to measure sEH cDNA levels in samples
from these extracted tissues. Data were normalized with primers and probe to
18S and are presented as arbitrary units (AU). White and black bars represent
WKY and SHR samples, respectively. Reprinted with permission from The
FASEB Journal.


Hqpdhdamisr










A.
62 kDa m 4- sEH

42 kDa p-actin

WKY SHR WKY SHR
Hypothalamus Brainstem
B WKY
0.8 B. SHR

S 0.6 -







Hypothalamus Brainstem


Figure 2-3 sEH protein expression in SHR and WKY brain regions. Protein was isolated
from hypothalamus and brainstem regions of SHR and WKY rat brains
(n=4/combined in each group). Western blot analysis was used to measure
sEH protein levels in from 40jtg total cell lysate. (A) Representative
radiograph of sEH and P-actin. (B) Data were normalized with P-actin.
White and black bars represent WKY and SHR samples, respectively. Data
are represented as mean+/-SEM. significantly different in SHR and WKY
rat (p<0.05). Reprinted with permission from The FASEB Journal.









A.
62 kDa dl-sEH


42 kDa


0.6 -


0.4-

0.2-

0-


*-P-actin


WKY SHR
Neurons


B.


WKY


SHR


Figure 2-4 sEH protein expression in neuronal cultures. Total cell lysate was isolated
from hypothalamus and brainstem co-cultures from one-day-old SHR and
WKY rats. (A) Representative radiograph of sEH and P-actin. (B)
Quantitation of sEH protein normalized with P-actin protein band. Data are
represented as mean+/-SEM. significantly different in SHR vs. WKY
samples (p<0.05, n=4/group). Reprinted with permission from The FASEB
Journal.


I I










190.0 -

180.0 -

170.0 -

"- 160.0 -
I
E 150.0 -

IL 140.0 -

130.0 -

120.0 -

110.0 -

100.0
SHR+Los SHR



Figure 2-5 Effect of chronic losartan delivery on SBP in the SHR. Losartan
(10mg/kg/day) was delivered orally to male SHRs. SHRs only receiving water
were used as control. SBP was recorded using tail cuff recordings. Data are
represented as mean+/-SEM. *significantly different between groups (p<0.05,
n=6)









EI LLosartan
-ontrol


Erainstem I-xthdamanijs


Figure 2-6 Effect of chronic losartan delivery on central sEH expression. Total cell
lysate was isolated from brainstem and hypothalamus, and Western blot
analysis performed as described in Methods to compare sEH protein levels.
sEH expression was normalized with P-actin and presented graphically.
White and black bars represent losartan and control treatments, respectively.
Data are represented as mean+/-SEM (p<0.05, n=6).


Q015 -
c 1-

~o5s














CHAPTER 3
CENTRAL SEH INHIBITION INCREASES BLOOD PRESSURE AND
HEART RATE IN THE SHR

Introduction

Pharmacological sEH inhibitors delivered IP to the SHR led to a decrease in blood

pressure (Yu et al. 2000a). This depressor effect is postulated to involve the

accumulation of vasodilatory EETs. What is the effect of central sEH inhibition on blood

pressure? Chapter 2 revealed an sEH overexpression in the hypothalamus and brainstem

of the SHR. These results correlated with increased renal and hepatic sEH expression in

hypertensive models (Imig et al. 2002; Yu et al. 2000a). Given the similarity of

expression patterns in the different tissues, does sEH also yield a pressor response in the

brain? Prior to embarking on these studies, it is necessary to understand the action and

limitations of the currently available sEH inhibitors.

Pharmacological agents used as sEH inhibitors act as competitive inhibitors by

forming hydrogen bonds and salt bridges with the active site of sEH. These compounds

have evolved both in their potency and versatility of use. The first round of inhibitors

were tight-binding with K(I) values in the nanomolar range; however, limitations

hampered the extended use of these compounds (Morisseau et al. 1999). First, the

compounds had a high crystal lattice energy that translated to an elevated melting point.

Second, they were not water-soluble and therefore were not easily dissolved in agents

that could be applied for use in vivo or in vitro.









The major sEH inhibitor used in published animal studies is N,N-dicyclohexylurea

(DCU), a 1,3-disubstituted urea that is formed by hydrolysis dicyclohexylcarbodiimide

(Morisseau et al. 2002). DCU specifically inhibits sEH and has no effect on mEH, CYP

or any other tested compounds related to the structure of sEH. DCU and related

compounds were determined to be effective sEH inhibitors because of hydrophobic

groups on either side of the urea (Morisseau et al. 1999). In physiological studies, DCU

was delivered to the SHR via IP injection, and blood pressure was recorded using

photoelectric tail cuff. DCU delivery decreased urinary excretion of 14,15-DHET by

40%, indicating the functionality of this inhibitor. sEH inhibition resulted in a decrease

in systolic blood pressure 12+/-2 mmHg after six hours (Yu et al. 2000a). This time

lapse of blood pressure effect may be due to the kinetics of the drug as well as its

processing by the liver. This study provided the first pharmacological evidence linking

sEH to blood pressure control.

While DCU is effective in its blockage of sEH function, it is limited by its

solubility in water. Thus, a new generation of soluble, potent sEH inhibitors was

synthesized. One main structural change underlying these improvements was

substituting a hydrophobic side group with a polar functional group arranged in an alkyl

chain (Kim et al. 2004). Adamantyl urea dodecanoic acid (AUDA) is one such second-

generation sEH inhibitor. It is disubstituted with an adamantyl group on one end and an

eleven-membered carbon chain on the other. It was formulated and characterized by

Bruce Hammock's research group at University of California, Davis. This

pharmacological inhibitor has an IC5o of 18+/-1 nM with mouse sEH (Morisseau et al.









2002). In comparison, DCU has a less potent IC5o of 81.8+/-0.7 nM with mouse sEH.

AUDA is therefore a promising candidate as an sEH inhibitor.

Overall, pharmacological sEH inhibitors have evolved in their potency and

specificity. We chose to use these inhibitors in vivo as blockers of sEH activity. The

following experiments outline physiological measurements following the central delivery

of either DCU or AUDA. Given the sEH expression patterns in the brain mimics that of

the periphery, we hypothesized that ICV delivery of sEH inhibitors would lead to a

decrease in blood pressure in the SHR.

Materials and Methods

Animals

Statistical Analysis

The above Methods are described in Chapter 2

ICV Cannulation

Rats were anaesthetized with inhaled isofluorane (3%) in all surgical procedures.

A stereotaxic frame was used to position a twenty-two-gauge guide cannula (PlasticsOne,

Roanoke VA) into the right lateral ventricle (AP: 1.3mm, ML: 1.5mm, 4.5mm below

skull) of male SHR and WKY rats. The cannula was secured to the skull with dental

resin assisted by 3 3/32 screws secured in the skull. Appropriate drugs (1-2[tl) were

injected into the right lateral ventricle of unrestrained rats using a 28 gauge internal

cannula, 4.5mm. Rats were allowed to recover for one week following all surgeries.

Abdominal Aorta Cannulation

A radiotelemetric pressure transducer (Data Sciences International, Arden Hills

MN) consisting of a fluid-filled catheter attached to a PA-C40 transmitter was implanted

into the abdominal aorta. The aorta was clamped proximally and the catheter inserted









caudal to the left renal artery and secured with medical adhesive. Prior to suturing the

abdomen, the transducer was checked for proper placement using a radio to transmit the

sound oscillations from pressure changes in the aorta. Rats were anaesthetized with

inhaled isofluorane (3%) during surgery and allowed to recover one week following the

surgical procedure.

Blood Pressure and Heart Rate Recordings

Blood pressure and heart rate were recorded using the implanted radiotelemetry

transducers. For each dataset acquisition, the rat cages were positioned on receivers that

collected signals from the implanted transducers. These signals were then transmitted to

an adapter with an ambient pressure monitor that relayed the signals to the DataQuest 3.1

Acquisition program (DataSciences International, Arden Hills MN). The rats were

allowed to acclimate to their new cage position for at least one hour before basal

measurements were recorded. During data collection, blood pressure (systolic, mean and

diastolic), heart rate and activity were recorded every minute for an average of ten

seconds at 500Hz. Data were acquired and transferred to Microsoft Excel for graphical

analysis.

In vivo Drug Delivery

sEH inhibitors were delivered via the ICV cannula into the right lateral ventricle of

unrestained rats. A 4.5 mm injector containing the inhibitor was inserted and secured

into the cannula. The injector was attached to a 5[tl Hamilton syringe via 0.38 mm

polethylene tubing 120 cm in length (Becton Dickinson, Sparks, MD). sEH inhibitors

DCU and AUDA (0.8-60ng, 2[l volume) were a kind gift from Dr. Bruce Hammock,

University of California, Davis. DCU and AUDA were dissolved in 10% DMSO/90%









artificial cerebrospinal fluid (aCSF: 133mM NaC1, 3.4mM KC1, 1.3mM CaC12, 1.2mM

MgC12, 6mM NaH2PO4, 32mM NaHCO3) and 100% aCSF, respectively. These

inhibitors as well as their vehicle controls were delivered as a single bolus to singly-

housed, unrestrained SHR and WKY rats.

Results

Effect of sEH Inhibitor DCU on Blood Pressure and Heart Rate

Figure 3-1 outlines the physiological response to sEH inhibition. Figures 3-1A,B

are representative recordings of blood pressure (A) and heart rate (B) in the SHR (red)

and the WKY rat (blue). The x-axis is time related to DCU injection in hours. Figures 3-

1C,D are bar graphs of peak blood pressure (C) and heart rate (D) responses to DCU,

expressed as mean +/- SEM.

WKY rat

In the WKY rat, heart rate increased from 342.4+/-7.6 bpm with vehicle delivery to

393.1+/-19.0 bpm following 90ng DCU (Figure 3-1D). There was no significant change

in blood pressure in the WKY rat (113.78+/-3.03 mmHg aCSF, 120.34+/-1.96 mmHg

DCU, n=4). The increase in heart rate began within one hour following injection and

normalized after two hours.

SHR

Central delivery of sEH inhibitors resulted in an increased blood pressure and heart

rate in the SHR. DCU (90ng) injected into the right lateral ventricle increased blood

pressure from 153.0+/-7.8 mmHg to 184.5+/-5.3 mmHg and increased heart rate from

339.6+/-15.3 bpm to 433.5+/-11.5 bpm (n=4) (Figure 3-1C,D). The increase in blood

pressure and heart rate initiated within one hour of injection and peaked at three to four









hours after injection. Blood pressure and heart rate were normalized within seven hours

following injection.

Effect of sEH Inhibitor AUDA on Blood Pressure and Heart Rate

The results from ICV injection with DCU indicate that heart rate is increased in

both the SHR and WKY rat following sEH inhibition. Blood pressure, however, was

increased only in the SHR. No pressor responses were detected in the WKY rat. In order

to verify these results, we used a second sEH inhibitor AUDA that later became available

from Dr. Bruce Hammock at University of California Davis. As previously discussed,

AUDA is more potent and has increased water solubility, thereby making it a better

pharmacological agent for testing the physiological effects of sEH inhibition.

Dose response

ICV administration of AUDA, a potent sEH inhibitor, caused a dose-dependent

increase in both blood pressure and heart rate in the SHR. Figure 3-2 is a graphical

representation of the change in blood pressure following delivery of either 0.8ng or 7.5ng

AUDA. Artificial cerebrospinal fluid was used as the control. Either concentration of

AUDA elicited only modest increases in blood pressure in the WKY rats [23.61+/-2.90

mmHg 0.8ng AUDA (n=5), 12.49+/-3.72 mmHg 7.5ng AUDA (n=4)]; however, the

0.8ng AUDA dose was the only delivery that yielded significance (Figure 3-2). The

alternate doses did not yield blood pressure increases that significantly differed from that

of aCSF injection (13.74+/-1.82 mmHg, n=4). In the SHR, a significant increase of 25+/-

0.9 mmHg in blood pressure was seen with as low as 0.8ng AUDA, and a dose of 7.5ng

AUDA increased blood pressure by 31.8+/-3.9ng AUDA (n=3).









WKY rat

Given this initial dose response, 15ng AUDA was used in the remaining studies in

order to measure the maximum physiological effects of sEH inhibition. Figures 3-3A,B

depict representative blood pressure (A) and heart rate (B) recordings prior to and

following ICV delivery of 15ng AUDA. Quantitation of blood pressure and heart rate

recordings are charted in Figures 3-3D,E. ICV delivery of 15ng AUDA did not

significantly change blood pressure in the WKY rat (106.52+/-2.40 mmHg basal change,

115.91+/-2.30 mmHg AUDA, n=6). Alternately, heart rate increased from 311.60+/-9.22

bpm to 358.06+/-7.73 bpm, a change of 46+/-12 bpm following AUDA injection (Figure

3-3).

SHR

Figures 3-3A,C depict representative blood pressure (A) and heart rate (C)

recordings prior to and following ICV delivery of 15ng AUDA. Quantitation of blood

pressure and heart rate recordings are charted in Figures 3-3D,E. Central delivery of

15ng AUDA resulted in a change in blood pressure from 148.04+/-1.18 mmHg to

180.48+/-0.25 mmHg, an increase of 32 mmHg (n=6). In addition, heart rate increased

from 334.44+/-2.52 bpm to 388.24+/-0.80 bpm, a change of 54 bpm (Figures 3-3). This

increase began to express after thirty minutes, reached maximal levels in three to four

hours and returned to basal levels in seven hours.

Discussion

These studies are the first to determine the physiological effects of inhibiting sEH

action in the brain. Central sEH inhibition resulted in an increase in both blood pressure

and heart rate in the already hypertensive SHR. While heart rate increased in the WKY

rat, blood pressure did not dramatically change following ICV delivery of sEH inhibitors.









These results were opposite from the hypothesized decrease in blood pressure and from

the depressor effect of IP delivery of DCU to the SHR. Given the whole-body

overexpression of sEH in different hypertensive models, why is the physiological effect

of sEH inhibition in the brain opposite from that in the periphery?

The understanding of how sEH in the periphery affects blood pressure is limited.

Explanations are based on the enzyme's conversion of vasodilatory EETs to relatively

inactive DHETs. The major function of EETs is to increase the open-state probability of

BK(Ca) channels in vascular smooth muscle cells (Harder et al. 1995; Li and Campbell

1997). sEH depletes the stores of EETs, thereby leading to vasoconstriction in the

vasculature. This hypothesis is supported by increased sEH expression in both the SHR

and Ang II-induced models of hypertension (Imig et al. 2002; Yu et al. 2000a).

Translation of this hypothesis to the central role of sEH, however, is complex. sEH may

function to deplete stores of EETs surrounding the cerebral arteries, leading to

constriction and decreased cerebral blood flow. However, while decreased cerebral

blood flow is highly implicated in stroke, its direct regulation on blood pressure is not

clear. In addition, sEH is found in neurons; therefore, its potential role in sympathetic

activation cannot be discounted. Thus, while sEH expression patterns are the same in the

brain and the periphery, the mechanism underlying sEH influence on blood pressure is

not.

One important question is whether ICV delivery of DCU and AUDA is an effective

means to inhibit sEH. DCU (3mg/kg, IP) was previously used in physiological studies

and effectively decreased 14,15-DHET urinary excretion by 65% (Yu et al. 2000a).

Thus, this pharmacological inhibitor injected IP acts on tissues to inhibit the activity of









this enzyme. However, the tissue-specific effect of this delivery was not known, nor do

we know what percentage of DCU was taken up and converted by the liver. In addition,

transfer efficiency of this drug to the brain is not known. This concern of sEH inhibition

effectiveness in these central studies is first addressed by the use of two inhibitors rather

than only one. Both have been found to inhibit sEH activity and not other members of

this family of inhibitors such as mEH. In addition, AUDA is more potent than DCU, the

inhibitor used in the previous studies. One caveat of this current study, however, was that

the sEH activity was not measured. Measurements of EETs and DHETs by HPLC

analysis must be incorporated into future studies to conclusively state that DCU and

AUDA delivery to the lateral ventricle attenuates sEH activity in the brain.

Cardiovascularly-relevant brain regions should be individually analyzed in order to

ascertain which regions ICV delivery of these pharmacological agents are targeted. Only

by meticulous analysis of sEH inhibition in specific brain nuclei can we conclude what

areas are involved in this pressor response.

One interesting observation was the discrepancy of blood pressure and heart rate

effects between the SHR and WKY rat. The lack of blood pressure response coupled

with an attenuated heart rate response in magnitude and duration in the WKY rat

compared to the SHR suggests differential central pathways between these two strains.

Given the low endogenous level of sEH in the brain of the WKY rat, it logically follows

that the sEH inhibitor would have a limited supply of enzyme on which to act compared

to the hypertensive rat. sEH inhibition in these two strains may act through the same

pathway but yield different results due to endogenous sEH expression. Alternately, since









the SHR is known for its dysregulation of blood pressure control, one of these central

pathways unique to the SHR or hypertension itself may involve sEH.

These inhibitor studies suggest that the overexpression of sEH in the SHR brain

may be a compensatory outcome of this hypertensive strain. We know that increased

sEH expression has not normalized the blood pressure in the SHR; however, inhibition of

this enzyme increases the blood pressure. Thus, without the increased sEH expression in

the brain, blood pressure in the SHR would be even higher. This idea cannot be

supported on an individual basis, given the increased sEH expression in neuronal cultures

from one-day-old SHR pups. This hypothesis instead is rooted in a proposed

evolutionary development of the SHR over generations. sEH overexpression in the brain

may have been selected for during reproduction due to increased embryo viability.

Alternately, whole-body sEH overexpression may have stemmed from random mutations

that collectively result in an increased blood pressure. Thus while the brain function of

sEH attenuates blood pressure, this beneficial effect is masked by the increased pressor

response from alternate pathways. Finally, ICV delivery of sEH inhibitors may not be a

comprehensive look at sEH in the brain. The cerebral vasculature selectively targets

certain brain regions, and injection into the right lateral ventricle will likely target areas

immediately surrounding the site of injection. It is necessary as discussed above to

measure the physiological effects of sEH inhibition in various brain regions in order to

elucidate its role in the central control of blood pressure.

The question, however, is how central sEH inhibition increases blood pressure and

heart rate. These studies conclude that pharmacological sEH inhibitors delivered to the

lateral ventricle results in an increase in blood pressure and heart rate in the SHR. It is






59


essential to elucidate the mechanisms underlying this blood pressure effect stemming

from the brain. Do these effects also include the conversion of EETs, the main substrate

identified for sEH? Or does this enzyme act through a different pathway unique to the

brain? The answers to these questions will be undertaken by experiments discussed in

the next two chapters incorporating known regulators of blood pressure control in the

brain.










A 225
2 SHR
75-M WKY







B 1 2 3








0 15 25 3 48

C l" D






SCSF Dc CSFC DCU
CS COC DCL C$F DCU



Figure 3-1 Effect of ICV DCU delivery on blood pressure and heart rate. DCU was
injected into the lateral ventricle of SHR and WKY rats. Representative tracings
of blood pressure (A) and heart rate (B) in SHR and WKY rats. Quantitation of
blood pressure (C) and heart rate (D) following DCU or aCSF injection. These
values represent the peak time of blood pressure response over a ten-minute
period. The first and second arrows represent the point of injection of aCSF or
DCU, respectively. Data are expressed mean +/-SEM. represents significance
between groups (p<0.05, n=4).






61


Uit
I 60
E *

C. 40
30

2 20


d0

WKY SHR


* CSF n 0.8ng AUDA m 7.5ng AUDA


Figure 3-2 Dose response of ICV delivery of AUDA to SHR and WKY rats. aCSF,
0.8ng and 7.5ng AUDA were delivered to SHR and WKY rats. Blood
pressure was recorded using radiotelemetry. Mean change in blood pressure
was plotted; error bars represent SEM. significantly different from aCSF
response (n=4, p<0.05).












2 "00 -
I


100 -

50 -

500
450 -

I 30-
wulf -


I 5HR
-= ..Y


5 35 85 135 185 235 285



1 46444


- I F I P I 18 P I i I
-15 35 85 135 185 235 285


930-
500 -

400-

300-


200 I I I I I 1 !1
-15 36 85 135
D. tnjnt

IE 1 ]75g 400
15050
001 2 250

WKY SHR


1835
E.


235


WKY
WKY


I I
285


r- Basal
m II~f


SHR


Figure 3-3 Effect of central AUDA on blood pressure and heart rate. 15ng AUDA
was delivered ICV, and radiotelemetry was used to record blood pressure and
heart rate. (A) MAP of SHR (red) and WKY rats (blue). (B,C) Representative
HR of WKY rats and SHRs, respectively. Quantitaton of MAP (D) and HR (E).
White and black bars represent basal and AUDA-induced levels, respectively, as
means +/- SEM. significantly different from basal (p<0.05, n=6). Reprinted
with permission from The FASEB Journal.


w,


~sjrs3"",,--J"














CHAPTER 4
ROLE OF ROS IN ANG II-INDUCED REGULATION OF BLOOD PRESSURE

Introduction

The role of the brain in hypertension remains to be fully elucidated. Chapter 1

outlined the major RAS pathways and evidence linking Ang II to the central control of

blood pressure. RAS members are localized to a number of cardiovascularly-relevant

brain areas including the PVN and SON in the hypothalamus and the RVLM and NTS in

the brainstem (Bastos et al. 1997; Mangiapane and Simpson 1980; Muratani et al. 1993;

Tanaka et al. 2001). An overactive RAS in these areas leads to an increase in blood

pressure due to sympathetic nerve activation. Thus, while the RAS was initially

identified as systemic, the brain RAS is an excellent example of cardiovascular control

localized to one organ.

The binding of Ang II to the AT1R induces the most significant pressor response of

the RAS members (Helin et al. 1997). Overwhelming electrophysiological and

pharmacological evidence indicates that the binding of Ang II to the AT1R stimulates

sympathoexcitatory neurons that in turn signal to the IML to induce blood pressure (Reid

1992). Specifically, Ang II increases neuronal firing rate by activating calcium channels

and inhibiting the delayed rectifier potassium current (Sun et al. 2003a; Zhu et al. 1997).

These changes in firing rate are prevented by losartan. Thus activation of

catecholaminergic neurons by Ang II is through its binding to the ATiR.

Although the overall pressor effect of central Ang II is established, the mechanisms

underlying this activation are complex and not well understood. An emerging set of









regulators in this pathway is the ROS. As previously discussed in Chapter 1, ROS results

in a dysregulation of a number of physiological pathways and has been linked to a

multitude of diseases including cancer. Hypertension is no exception. Striking evidence

has linked an overproduction of ROS to an increase in blood pressure Recently, studies

have focused on the role of ROS in the central control of hypertension (Zimmerman and

Davisson 2004; Zimmerman et al. 2002).

Treatment of primary cultures from the lamina terminalis with Ang II resulted in an

increase in superoxide, thereby linking Ang II action to ROS production (Zimmerman et

al. 2002). Gene therapy strategies also have defined the overall effect of ROS members

on blood pressure, though the underlying mechanisms are not understood. Ang II-

induced blood pressure was prevented in mice centrally injected with Adenovirus

containing either mitochondrial or cytoplasmic SOD (Zimmerman et al. 2002).

Adenovirus was also used to inhibit NAD(P)H oxidase activator Racl production in the

CVOs. NAD(P)H oxidase is a key enzyme involved in the formation of free radicals

from oxygen. Again, the blood pressure and dipsogenic responses to Ang II were

prevented (Zimmerman and Davisson 2004). This evidence indicates ROS production is

involved in the Ang II-induced blood pressure responses.

While these studies provide a basis of ROS-RAS interaction, it is necessary to

elucidate key ROS members and provide further insight into the mechanisms behind this

interaction. Given the emerging evidence that ROS is linked to the central action of Ang

II, do the Ang II-induced pressor and dipsogenic actions require ROS member NAD(P)H

oxidase specifically? To answer this question, we used NAD(P)H oxidase formation

blocker gp91ds-tat, a compound discussed in Chapter 1, whose high specificity









distinguishes the action ofNAD(P)H oxidase from other ROS members. This study

provides a comprehensive analysis of the overall central effect of NAD(P)H oxidase on

Ang II action. We hypothesized that pretreatment of the brain with gp91ds-tat would

prevent the ICV Ang II-induced pressor and dipsogenic responses in the SHR and WKY

rat.

Methods

Animals

ICV Cannulation

Abdominal Aorta Cannulation

Statistical Analysis

A description of the above Methods is found in Chapter 2.

In vivo Drug Delivery

All drugs were delivered via the ICV cannula into the right lateral ventricle of

unrestained rats. The drug solutions were delivered using an injector 4.5mm in length

that was secured in the ICV cannula. The injector was attached to 0.38mm polyethylene

tubing 120 cm in length (Becton Dickinson, Sparks, MD) with a 5[l Hamilton syringe

attached to the other end of the tubing. All injections were given to unrestrained, singly-

housed rats.

When testing the mechanism of Ang II requiring two drugs, the first drug was

injected one hour prior to the second, and the dummy cannula was replaced following

injection. 30ng Ang II dissolved in uCSF was delivered to the right lateral ventricle (11l

injection) using a 5[l Hamilton syringe with fitted tubing + injector. gp9lds-tat and its

scrambled peptide used as a control (200-800ng each, 21tl, dissolved in uCSF) were









synthesized by the Temple University Protein Synthesis Core facility (Philadelphia, PA)

and used to inhibit the action of NAD(P)H oxidase (Rey et al. 2001a; Rey et al. 2001b).

Dipsogenic Response

Water intake of WKY rats (n=3) following ICV delivery of Ang II was measured

using sterile graduated glass pipets fitted with metal sipper tubes. The pipets were filled

with water from the rats' sterile water bottles. The pipets replaced the water bottles thirty

minutes prior to the first injection. Water level in milliliters was recorded thirty minutes

prior to, at injection time, and thirty minutes post Ang II injection. Drinking was

calculated as the basal water level minus the water level thirty minutes following the Ang

II injection. Rats remained singly-housed and were not restrained during the experiment.

Results

Angiotensin II Pressor Response

Figure 4-1 depicts the changes in blood pressure and heart rate following ICV Ang

II delivery in the SHR and WKY rat. ICV delivery of 30ng Ang II to conscious, free-

moving WKY rats (n=5) resulted in an increase in blood pressure from 102.81+/-3.36

mmHg to 146.90+/-1.87 mmHg, a change of 44 mmHg (Figure 4-1C). Heart rate

increased 44 bpm, from 320.13+/-7.37 bpm to 364.22+/-25.39 bpm (Figure 4-1D).

Figure 4-1A shows representative blood pressure and heart rate recordings surrounding

Ang II delivery to the WKY rat. The central Ang II response began within two minutes

following injection, and maximum response occurred by ten minutes. Blood pressure and

heart rate were normalized one hour post injection.

Central delivery of Ang II (30ng) to the SHRs (n=3) also resulted in a pressor

response. Blood pressure increased from 148.61+/-1.09 mmHg to 191.36+/-6.14 mmHg

following Ang II treatment, a change of 43 mmHg (Figure 4-1C). Heart rate increased









from 310.15+/-15.39 bpm to 432.93+/-9.64 bpm, an increase of 123 bpm (Figure 4-1D).

Representative recordings of blood pressure and heart rate of the SHR are shown in

Figure 4-1B.

gp9lds-tat Prevents Ang II-Induced Blood Pressure

gp91ds-tat was delivered ICV to the lateral ventricle, and radiotelemetry was used

to measure BP and HR responses. Central administration of this NAD(P)H oxidase

formation blocker prior to Ang II delivery yielded no change in blood pressure or heart

rate in both the SHR and WKY rat. Pretreatment with gp9lds-tat one hour prior to ICV

delivery of Ang II attenuated the Ang II-induced increases in blood pressure and heart

rate in both rat strains (Figure 4-2).

WKY rat

Blood pressure and heart rate did not significantly change from basal levels

(108.86+/-1.83 mmHg and 312.62+/-5.89 bpm, respectively) thirty minutes following

ICV delivery of 800ng gp9lds-tat (106.81+/-3.72 mmHg and 329.54+/-8.11 bpm,

respectively). Central delivery of 800ng gp9lds-tat one hour prior to ICV delivery of

Ang II attenuated the Ang II-induced blood pressure by 67% in the WKY rat (146.90+/-

1.87mmHg Ang II, 123.55+/-3.75 mmHg gp91ds-tat + Ang II) (Figure 4-2C).

Pretreatment with 200ng gp91ds-tat did not significantly prevent the Ang II-induced

pressor response. In addition, 800ng gp9lds-tat did not prevent the heart rate increase

following Ang II delivery (364.22+/-25.39 bpm for Ang II, 358.84+/-24.15 bpm for

gp91ds-tat+Ang II) (Figure 4-2D).

SHR

Blood pressure increased slightly from basal levels (154.22+/-5.65 mmHg) thirty

minutes following ICV delivery of 200ng gp91ds-tat (167.58+/-3.62 mmHg). Heart rate









increased from 325.04+/-8.32 bpm to 375.02+/-13.07 bpm. Figure 4-3 outlines the blood

pressure and heart rate recordings and averages following deliveries of gp9lds-tat and

Ang II. Central delivery of 200ng gp91ds-tat one hour prior to ICV delivery of Ang II

attenuated the Ang II-induced peak blood pressure response by 67% in the SHR as well

(Ang II 191.36+/-6.14 mmHg Ang II, 168.22+/-5.10 mmHg gp9lds-tat + Ang II) (Figure

4-3C). Pretreatment with gp9lds-tat prevented the heart rate increase following Ang II

delivery by 58% (Ang II 432.93+/-9.64 bpm Ang II, 376.98+/-11.79 bpm gp9lds-tat +

Ang II) (Figure 4-3D).

Thus, pretreatment with gp91ds-tat resulted in an attenuation of blood pressure

increase induced by central Ang II delivery in both the SHR and WKY rat. In contrast, a

scrambled form of the peptide did not prevent the Ang II-induced blood pressure in a

WKY rat (135.16 mmHg Ang II, 137.26 mmHg scrambled + Ang II), indicating the

gp91ds-tat peptide was specific in its blockade of NAD(P)H oxidase.

gp9lds-tat Prevents Ang II-Induced Drinking

Next we determined if the dipsogenic responses of ICV Ang II administration

involve the ROS signaling pathway. Figure 4-4 is a graphical representation of the water

intake of the WKY rats following either Ang II treatment alone or central delivery of Ang

II following gp9lds-tat pretreatment (n=3). Delivery of Ang II into the lateral ventricle

resulted in 8.8+/-0.5 ml water intake within thirty minutes following injection. Drinking

began within two minutes of ICV delivery. Pretreatment with 800ng gp9lds-tat,

however, resulted in water intake of only 2.6+/-1.4 ml thirty minutes following Ang II

injection, thereby attenuating the Ang II dipsogenic response by 70% (Figure 4-4).









Discussion

These results support the hypothesis that brain Ang II increases blood pressure and

heart rate through a ROS-mediated pathway. Specifically, this study establishes that

blocking NAD(P)H oxidase in the brain prevents the pressor and dipsogenic actions of

Ang II. This response was observed both in the hypertensive SHR and in the

normotensive WKY rat. The scrambled form of the peptide did not affect Ang II-induced

blood pressure; therefore, gp91ds-tat was specific in its action. gp91ds-tat alone had no

effect on blood pressure or heart rate, indicating that these basal physiological

mechanisms do not involve NAD(P)H oxidase.

This work complements previous published studies involving the link between

central Ang II action and ROS in mice. Adenovirus encoding SOD delivered ICV also

prevented Ang II-induced blood pressure (Zimmerman et al. 2002). One caveat of that

study, however, was the sole use of SOD to inhibit ROS accumulation. While SOD is an

important component of ROS conversion, it does not discriminate whether Ang II acts

upstream of superoxide formation. gp9lds-tat, however, is a highly specific blocker of

NAD(P)H oxidase formation, thereby useful for analysis upstream of superoxide in Ang

II action (Rey et al. 2001b). Its use in this study therefore confirms the role of ROS,

specifically NAD(P)H oxidase, in Ang II-induced pressor and dipsogenic responses.

These studies were the first to measure the affect ofNAD(P)H oxidase blockade on

central Ang II pressor response in both hypertensive and normotensive models. The

comparison of WKY rat to SHR response is critical in understanding the role of ROS in

Ang II action. First, the blood pressure response between the two models was the same.

This result was unexpected given the characterized heightened pressor response in the

SHR (Wright et al. 1986). This discrepancy may be due to the large dose of Ang II









(30ng), yielding a potential maximum response in both strains. Pretreatment with

gp91ds-tat prevented the Ang II pressor effect by 67% in both the SHR and WKY rat.

Therefore, the influence ofNAD(P)H oxidase on the Ang II-induced blood pressure is

not dependent on the basal blood pressure. However, while the heart rate response was

attenuated 58% in the SHR with gap9lds-tat pretreatment, no change was measured in

the WKY rat. This discrepancy is likely due to the differences in Ang II-induced heart

rate between the two models. In these studies, Ang II-induced heart rate in the SHR was

three times that of the WKY rat, which was only transiently increased -bpm.

Pretreatment with gp91ds-tat in the SHR attenuated the heart rate response to the same

level following ICV Ang II delivery in the WKY rat. It is therefore likely that this 40

bpm change in heart rate is independent ofNAD(P)H oxidase signaling.

One caveat of this study is while it determined the overall central link of NAD(P)H

oxidase to Ang II, it did not extrapolate specific nuclei involved, nor did it address

cellular mechanisms underlying this central ROS-RAS control. The AT1R has been

localized to brain nuclei including the PVN, RVLM, and NTS, where activation through

Ang II binding elicits sympathetic nerve activation and therefore an increase in pressure

(Bastos et al. 1997; Mangiapane and Simpson 1980; Muratani et al. 1993; Tanaka et al.

2001). Thus, it is necessary to further characterize the role ofNAD(P)H oxidase on an

individual nuclei basis. Future studies should address this question in order to more

completely elucidate the mechanism of Ang II-ROS action.

Signaling of AT1R and NAD(P)H oxidase has begun to be elucidated. Most studies

in this regard have focused on the peripheral Ang II and ROS, but the mechanisms may

indeed apply to the brain as well. In summary, ATiR stimulation activates PKC and









therefore Rac, a subunit needed to activate NAD(P)H oxidase (Brandes 2003; Gregg et

al. 2003). Functional NAD(P)H oxidase can therefore convert oxygen to hazardous

H202, thus increasing ROS production. This increased ROS is theorized to increase

sympathetic nerve activity, though this mechanism is unknown.

The electrophysiological basis of the role of ROS in central Ang II pressor response

was recently elucidated by post-doctoral fellow Dr. Cheng-wen Sun of our research

group. Dr. Sun used both ROS inhibitors and generators in conjunction with Ang II and

measured firing rate and the delayed rectifier potassium current (IKv). Both Tempol and

gp91ds-tat attenuated the Ang II-induced neuronal firing rate by 70% and 50%,

respectively. Alternately, ROS generator Xanthine-Xanthine oxidase increased firing

rate above basal levels, indicating that ROS functions independently of extracellular Ang

II stimulation. In addition, the inhibition of IKv by Ang II was blocked by the

concomitant treatment with gp91ds-tat. These findings indicate that ROS is involved in

the Ang II-mediated increase in neuronal activity. In order to confirm these findings,

specific channel involvement and kinetics need to be examined. This study therefore

provides a cellular basis for the pressor and dipsogenic responses generated from the in

vivo studies. It also suggests the role of ROS in central control of hypertension acts

through neuronal activation.

Given that ROS continues to emerge as an important regulator in several disease

states coupled with this evidence linking NAD(P)H oxidase to Ang II function in the

central control of blood pressure, are endogenous levels of ROS activity elevated in

human hypertension? If there is an increase in ROS production, can this dysregulation be

corrected by either SOD mimetics or NAD(P)H oxidase blockers in order to diminish the









accumulation of superoxides? These questions must be addressed in future studies in the

hopes of reversing the hypertensive state.

In summary, the pressor and dipsogenic actions of central Ang II are mediated by

ROS action. Specifically, ROS member NAD(P)H oxidase was implicated in this

mechanism. Both in vivo and in vitro data support the regulation of central Ang II

pressor action by ROS. These results concur and expand on previous studies linking

ROS to Ang II in the brain. While the specific mechanism by which ROS increases

neuronal firing rate remains to be elucidated, this study provides a solid foundation on

which to study the involvement of NAD(P)H oxidase in the central control of blood

pressure.

This essential requirement of ROS in central pressor control invites thought as to

what other mechanisms ROS involved in the brain regulate blood pressure. For instance,

is ROS production necessary for the central action of sEH inhibition described in Chapter

3? Given the wide range of responsibilities of ROS, it is indeed plausible that ROS

production underlies all the major central blood pressure regulators. In theory, these

brain mechanisms contributing to blood pressure control may converge at the ROS

production level. Chapter 5 will outline and discuss data related to the mechanism

underlying blood pressure and heart rate increases following central sEH inhibition.






73


A 00 M BP
S- -HR


II I


t tl00

M 15 .5 5 15 25 35 45 55
B
300- 5 0




2 150 2 (4

100 r r r r 1 100 Basal
-15 -5 5 16 26 36 46 56
n C o i Ang II

Cr 400

.o 100

MV SMR KYH S HR

Figure 4-1 Effect of central Ang II on blood pressure and heart rate. 30ng Ang II was
delivered ICV to SHR and WKY rats. Blood pressure and heart rate were
recorded using radiotelemetry (see Methods). Bars represent peak increases
over 10 minutes. Representative recordings of WKY (A) and SHR (B). Red
and blue lines indicate HR and MAP, respectively. Arrows represent Ang II
injection. (C,D) Quantitation of MAP and HR, respectively. White and black
bars represent mean +/- SEM of basal and Ang II measurements, respectively
(p<0.05, n=5/WKY, n=3/SHR).






74



A 175
155-
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-75 -55 -35 -15 5 25 45
B
500


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1 200

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C D




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Figure 4-2 Effect of gp91ds-tat on Ang II-induced BP response in the WKY rat. 800ng
gp91ds-tat was injected ICV one hour prior to Ang II. Representative
recordings of MAP (A) and HR (B). Arrows represent point of drug delivery.
Quantitation of MAP (C) and HR (D). Bars represent means +/-SEM; *
significantly different between groups (p<0.05, n=5).




















t t


-70 -50 -30 -10


10 30


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: 150 -
S100 -
O 50
AI
0


10 30


*1


Angil


Figure 4-3 Effect of gp91ds-tat on Ang II-induced BP response in the SHR. 200ng
gp9lds-tat was injected ICV one hour prior to Ang II. Representative
recordings of MAP (A) and HR (B). Arrows represent points of drug
delivery. Quantitation of MAP (C) and HR (D). Bars represent means +/-
SEM; significantly different between groups (p<0.05, n=3).


250 -


- 200 -
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E 150-

100 -

50


500


230


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10-
8-
6-
4-
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30ng Ang II


800ng gp91ds-tat +
Angll 30 min


Figure 4-4 Dipsogenic responses of ICV gp91ds-tat and Ang II. Water intake following
Ang II injection with and without 800ng gp9lds-tat pretreatment was
measured. Bars represent water intake means +/- SEM (p<0.05, n=3).


Tt














CHAPTER 5
MECHANISM OF CENTRAL SEH INHIBITION ON
BLOOD PRESSURE REGULATION

Introduction

sEH action in the brain is emerging as critical in blood pressure regulation.

Chapter 3 established that central sEH inhibition results in an increase in blood pressure

and heart rate in the SHR model of hypertension. In addition, this inhibition yielded an

increase in heart rate in the WKY rat. Taken together, these results suggest a role for

sEH in the central control of blood pressure. Essentially, though, how does central sEH

inhibition result in an increase in blood pressure and heart rate in the SHR? This chapter

seeks to elucidate the mechanism underlying this physiological response.

The pressor effects of central sEH inhibition are opposite from the depressor

response following IP delivery of DCU to the SHR and therefore suggest an alternate

pathway of sEH action in the brain. Peripheral sEH functions as a regulator of

vasoactivity by converting vasodilatory EETs to their less active diol forms (Yu et al.

2000a). Decreased vasodilation in smooth muscle cells of the arterial walls leads to

increased probability of contractility, which results in an increase in blood pressure.

While this proposed vasoactive mechanism of sEH in the periphery coincides with its

increased expression in hypertension, the effect of central sEH inhibition likely diverges

from this pathway. The question is in what way? Is the action of sEH inhibition related

to a known pathway of central control of blood pressure? We sought to answer these









questions through a series of experiments designed to elucidate the pathway of central

sEH control of blood pressure.

The first goal is to extrapolate the overall physiological mechanism behind the

increase in blood pressure and heart rate following central sEH inhibition in the SHR. As

previously discussed, the SHR is characterized by several dysregulatory pathways. One

is the decreased baroreceptor reflex described in Chapter 1 (Hayashi et al. 1988).

Another is the increased sympathetic nerve activity that results in an increase in blood

pressure (Biaggioni 2003). Given that the SHR is predisposed to these pathways, we

hypothesized that AUDA-induced blood pressure and heart rate changes in the SHR

involved baroreceptor reflex dysregulation and sympathetic nerve activation.

The second goal is to elucidate the cellular and signaling mechanisms involved in

the AUDA-induced pressor response in the SHR. This undertaking is complex and likely

to involve years if not decades of study. However, the start of this discovery must begin

with what we know of sEH action, its conversion of EETs. Does the action of central

sEH inhibition involve the accumulation of its substrate, the EETs? Peripheral studies

have focused on this branch of the arachidonic acid pathway as the powerhouse behind

sEH cardiovascular effects. However, sEH was originally identified as a converter of

xenobiotic material foreign to the organism (Grant et al. 1993). In fact, several substrates

have already been identified for sEH in addition to its well-known role in EET

metabolism (Sinal et al. 2000b). Alternately, sEH itself may function to regulate channel

kinetics leading to changes in firing rate patterns or even regulate neurotransmitters such

as Ang II or vasopressin. Based on the primary role of sEH as an enzyme involved in the









epoxygenase branch of arachidonic acid conversion, however, we hypothesized that sEH

inhibition leads to an increase in blood pressure due to EET accumulation in the brain.

Aside from the question whether the sEH substrate EETs are involved in its central

action on blood pressure, it is important to begin to understand how this inhibition leads

to an increase in blood pressure. Is there a connection of sEH to known regulators of

central blood pressure control such as Ang II or another sympathetic activator? Chapter 2

presented evidence that sEH expression increases in the hypothalamus following ICV

delivery of Ang II. Chapter 4 further analyzed the role of brain Ang II signaling by

incorporating the NAD(P)H oxidase inhibitor gp91ds-tat. These studies linked central

Ang II action to ROS member NAD(P)H oxidase. ROS has also been linked to

epoxygenase member CYP 2C9 (Fleming et al. 2001b). Given that ROS activity in

cardiovascular control, both peripheral and central, continues to emerge as a strong

regulator, we hypothesized that the blood pressure effects stemming from sEH inhibition

were mediated by NAD(P)H oxidase.

Finally, what cell type in the brain is responsible for the pressor response following

central sEH inhibition? Previous studies have localized both CYP and EETs mainly to

astrocytes, though CYP has also been found in the cerebral vasculature as well as

dopaminergic neurons. Our studies discussed in Chapter 2 localized sEH to both neurons

and glia. Therefore all brain cell types are potential targets for the action of sEH

inhibition. However, overwhelming evidence reveals neurons as the main regulator in

the central control of blood pressure. For example, increased neuronal firing rate from

the RVLM and NTS to the IML result in an increase in sympathetic activation and hence









increased blood pressure. The following studies seek to identify key mediators and

pathways in the increase in blood pressure following sEH inhibition in the SHR brain.

Methods

Animals

ICV Cannulation

Abdominal Aorta Cannulation

Blood Pressure and Heart Rate Recordings

Statistical Analysis

The above Methods are described in Chapters 2 and 3

In vivo Drug Delivery

All drugs were delivered as bolus injections via the ICV cannula into the right

lateral ventricle of unrestained rats as described in Chapter 3. When testing the

mechanism of sEH inhibition requiring two drugs, the first drug was injected one hour

prior to the second, and the dummy cannula was replaced following injection. MS-PPOH

(680ng, 21tl, dissolved in uCSF), a kind gift from Dr. John Falck, University of Texas

Southwestern Medical Center, was used to block the formation of EETs (Brand-Schieber

et al. 2000b). 11-nonyloxy-undec-8-enoic acid (11-NODA, 100ng, ICjl, dissolved in

uCSF), an EET agonist, was also provided by Dr. Falck. gp9lds-tat and its scrambled

peptide used as a control (100ng each, 21tl, dissolved in uCSF) were synthesized by the

Temple University Protein Synthesis Core facility (Philadelphia, PA) and used to inhibit

the action of NAD(P)H oxidase (Rey et al. 2001a; Rey et al. 2001b).

Evaluation of Spontaneous Baroreceptor Reflex Gain

To evaluate baroreceptor reflex function, an index of gain was determined from

spontaneous changes in systolic blood pressure and pulse interval using a time-series









method for the rat, as described (Oosting et al. 1997; Waki et al. 2003). This method

correlates to traditional pharmacological methods for measuring the baroreceptor reflex.

However, because this method does not distinguish between an increase in blood pressure

and heart rate either by a decrease in the baroreceptor reflex or by the pharmacological

agent itself, this measurement is assumed to be an estimate of spontaneous baroreceptor

reflex gain. To calculate this index of baroreceptor reflex gain, spontaneous ramps of

systolic blood pressure changes (at least four beats) were extracted from the moving

average data. The data was collected at a frequency of 500 Hz. Measurements of

systolic blood pressure and pulse interval ramps were made at delays of three to five

beats based on previous findings (Oosting et al. 1997). Pulse interval was compared to

systolic blood pressure during these delays to calculate the spontaneous baroreceptor

reflex gain. Only positive slope values were used to avoid contaminating baroreceptor

reflex data with non-baroreceptor-mediated changes in pulse interval. This analysis was

completed by Dr. Hidefumi Waki, Department of Physiology, Bristol University,

England.

Analysis of Heart Rate Variability

Heart rate variability was analyzed using a fast Fourier transformation (FFT). For each

ten-minute recording period, the beat-to-beat pulse interval data were converted into data

points every 100 ms using a spline interpolation. Power spectral density was then

computed using FFT algorithm and averaged. It is generally accepted that the high

frequency (HF) component of heart rate variability is mediated by cardiac

parasympathetic tone (Pagani et al. 1986a; Pagani et al. 1986b). This analysis was

completed by Dr. Hidefumi Waki, Department of Physiology, Bristol University,

England.









Neuronal Firing Recording

Spontaneous action potentials were recorded with the whole-cell voltage clamp

configuration in current clamp mode. Neurons cultured from hypothalamus were bathed

in Tyrodes's solution containing (in mM): 140 NaC1, 5.4 KC1, 2.0 CaC12, 2.0 MgC12, 0.3

NaH2PO4, 10 HEPES, and 10 dextrose, pH adjust to 7.4 with NaOH. The patch

electrodes (resistances from 3-4 MOhms) were filled with an internal pipette solution

containing (in mM): 140 KC1, 4 MgCl2, 4 ATP, 0.1 guanosine 5'-triphosphate, 10

dextrose, and 10 HEPES, pH adjusted to 7.2 with KOH. The resting membrane potential

was defined as the potential within a 1 s time period during which there were no

spontaneously firing action potentials. Neuronal firing rate was measured as the number

fully developed action potentials depolarizationn beyond 0 mV) per second (Hz). This

study was completed by Dr Chen-wen Sun of our research group.

Results

Waveform Analysis of Baroreceptor Parameters

Because of the concomitant increase in blood pressure and heart rate in the SHR

following sEH inhibition, it was hypothesized that central AUDA delivery impaired the

ability of the baroreceptors to regulate physiological changes. In order to test this

hypothesis, spontaneous baroreceptor reflex gain and cardiac vagal tone were measured

using waveform analysis in response to sEH inhibition.

Index of spontaneous baroreceptor reflex gain

Analysis of waveform telemetry data revealed a 56+/-5% decrease in the estimate

of spontaneous baroreceptor reflex gain (p<0.05) following sEH inhibition in the SHR.

Specifically, the index of baroreceptor reflex gain decreased from a basal level of 0.82+/-

0.06 ms/mmHg to 0.36+/-0.04 ms/mmHg at the peak of the pressor response from sEH









inhibition (Figure 5-1A). In the WKY rat, the index ofbaroreceptor reflex gain was

1.09+/-0.11 at basal levels and 1.13+/-0.08 three to four hours after ICV delivery of 15ng

AUDA. Thus, no significant change in the baroreceptor reflex gain index was measured

in the WKY rat. In comparing basal levels of the two strains, the index of the

baroreceptor reflex gain of the WKY rat was 33% higher compared to the SHR, though

this difference was not significant.

High frequency analysis of pulse interval

Given the decrease in the index of baroreceptor reflex gain in the SHR following

central sEH inhibition, high frequency analysis of pulse interval was then analyzed to

determine if this component was also involved in this concomitant increase in blood

pressure and heart rate. This measurement correlates to the level of cardiac vagal tone.

In the SHR, this cardiac vagal tone indicator measurement decreased from 16.68+/-2.55

ms to 10.39+/-0.28 ms at the peak response of sEH inhibition (Figure 5-1B). This result

translated to a 33+/-8% decrease in heart rate analysis of pulse interval following ICV

AUDA administration in the SHR. In contrast, no significant changes were observed in

the WKY rats (13.91+/-0.84 ms basal, 13.74+/-0.95 ms 15ng AUDA). These data

demonstrate that increases in blood pressure and heart rate are consistent with a decrease

in baroreceptor reflex functions in the SHR.

AUDA Regulation of Sympathetic Nerve Activity

Regulation of sympathetic nerve activity is one of the major mediators of the

central control of blood pressure. Given the high impact of this system plus its neuronal

origin that corresponded with previous sEH expression studies, atenolol, a P31R

antagonist, was used to test whether the effects of sEH inhibition were related to

sympathetic nerve activity. Img/kg atenolol was delivered IV following 15ng AUDA









ICV delivery to two SHRs. The results from this preliminary study are shown in Figure

5-2. Atenolol delivery completely prevented the increase in blood pressure increase

following central AUDA administration (34.54+/-7.23 mmHg AUDA, 0.69+/-3.73

mmHg atenolol + AUDA, n=2). No statistical analysis could be completed due to the

low number of animals in this study. These data indicate that AUDA-induced increase in

blood pressure functions through sympathetic nerve activity.

Increased EETs Linked to Increase in BP and HR

The first step in deducing the mechanism underlying sEH inhibition-induced

blood pressure was to examine the role of EETs, the main physiological substrate of sEH.

Since sEH converts EETs to DHETs, inhibiting the enzyme results in the accumulation of

EETs. Thus, we hypothesized that EETs may be the vital in this central control of blood

pressure.

MS-PPOH

This hypothesis was tested initially with the use of MS-PPOH, an inhibitor of the

epoxygenase pathway that generates EETs and related PUFA epoxides (Brand-Schieber

et al. 2000a). Figure 5-3 outlines the pressor response to 15ng AUDA with and without

pretreatment of MS-PPOH in the SHR. Figures 5-3A,B are representative blood pressure

recordings from ICV delivery of AUDA alone (A) and with ICV delivery of MS-PPOH

one hour prior to sEH inhibition (B, n=5). Figure 5-3C is a graphical representation of

the pressor response resulting from the treatments. While central delivery of AUDA

increased blood pressure 38.99+/-4.29mmHg, pretreatment with MS-PPOH attenuated

this AUDA-induced increase in blood pressure by 65% in the SHR (14.43+/-8.04

mmHg). However, MS-PPOH alone had no effect on basal blood pressure.









11-NODA

While MS-PPOH provided the first evidence that sEH action was through EET

production, it was imperative to determine if the action was through EETs specifically or

through another product of epoxygenase. Thus, EET agonist 11-NODA (100ng) was

delivered ICV in the WKY rat to determine whether EET-specific agonist would have

any effect on blood pressure or heart rate (Figure 5-4). This central administration of an

EET agonist results in a transient increase in blood pressure (blue) and heart rate (red) in

the WKY rat (Figure 5-4, n=6). This increase was biphasic for blood pressure increases,

each increase lasting only about 10 min. The first peak was recorded just after delivery

and the second 15-30 minutes following the first. Blood pressure increased 13.2+/-2.5

mmHg in the first peak and 8.8+/-3.2 mmHg in the second peak (Figure 5-4B). Heart

rate increased 61+/-19 bpm (Figure 5-4C).

Effects of sEH Inhibition Function through ROS

To further examine the mechanism underlying this central pressor response, we

examined the role of ROS on sEH-inhibitor-mediated increase in blood pressure in the

SHR. Reasoning behind this study was two-fold. First, epoxygenases have been linked

to ROS, albeit in the periphery. In addition, Chapter 4 discussed the importance of ROS

member NAD(P)H oxidase is in the central control of hypertension. Figure 5-5 outlines

the blood pressure and heart rate data from this study (n=3). The top panels are

representative blood pressure and heart rate recordings of an SHR treated with AUDA

alone (Figures 5-5A,C) and AUDA with gp9lds-tat pretreatment (Figures 5-5B,D).

Figure 5-5E is a graphical representation of the peak blood pressure changes showing that

while ICV delivery of AUDA resulted in a 39.20+/-3.03 mmHg increase in mean arterial

pressure, pretreatment with gp91ds-tat caused the AUDA-induced blood pressure to only









increase 6.10+/-5.78 mmHg, an 85% attenuation. Figure 5-3F is a graphical

representation of the peak heart rate changes indicating that AUDA delivery resulted in a

85.66+/-4.99 bpm change, yet pretreatment with gp91ds-tat yielded an AUDA-induced

heart rate change of 19.24+/-12.48 bpm, thereby attenuating the heart rate response by

77%. In contrast, gp91ds-tat alone had no effect on blood pressure and heart rate. These

results indicate that ROS member NAD(P)H oxidase is involved in the sEH inhibitor-

induced increase in blood pressure and heart rate.

Discussion

These series of studies succeeded in beginning to elucidate the complex signaling

involved in the central sEH-mediated blood pressure control. Two important findings

can be extrapolated from these results. First, the pressor effects stemming from central

sEH inhibition are linked to the increase in its substrate, the EETs. Second, the

mechanism leading to the increase in blood pressure involves ROS, specifically

NAD(P)H oxidase. These two conclusions, taken together, form the foundation of how

sEH and EETs function in the brain to elicit blood pressure and heart rate effects.

EET studies involving formation blockers as well as agonists strongly suggest that

this central pathway involves the sEH substrate EETs. Pretreatment with MS-PPOH

prevented sEH increases in blood pressure in the SHR, though alone this compound had

no effect on blood pressure. This lack of blood pressure response may be explained by

the high sEH expression in the SHR that would prevent any basal effects from the EETs

While this result was good evidence that EETs were involved, the non-specificity of MS-

PPOH to EETs still questioned whether EETs were the main regulator. For example,

since MS-PPOH inhibits epoxygenase action, it blocks the formation of all epoxygenase

products (Brand-Schieber et al. 2000b). Therefore, EET agonists were delivered ICV to









determine whether increasing the agents themselves resulted in a change in blood

pressure. ICV delivery of the agonists resulted in a biphasic increase in blood pressure as

well as an increase in heart rate in the WKY rat, indicating these sEH substrates are

indeed the potentiators of sEH inhibitor pressor effects. The reason behind the biphasic

increase in pressure is unknown at this time, but may be related to differences in

immediate EET activity on the cerebral microcirculation and later effects involving

neuronal activation pathways in cardiovascular-relevant brain areas. Thus, the role of

central EETs appears to be distinct from that of its peripheral expression, which is

characterized by its vasodilatory properties that are associated with a decrease in blood

pressure. These results strongly indicate that the effects of central sEH inhibition involve

the accumulation of its substrate.

We further elucidated the central sEH pathway through studies involving oxidative

stress. The mechanism linking central sEH inhibition to the increase in blood pressure

and heart rate appears to function through ROS (Fleming 2001; Fleming et al. 2001a).

gp91ds-tat, an NAD(P)H oxidase blocker, was used to elucidate the ROS involvement.

Central delivery prevented AUDA-induced increase in blood pressure and heart rate in

the SHR. gp91ds-tat also prevented the increase in neuronal firing rate induced by sEH

inhibitors in vitro. As previously discussed, ROS had been linked to the epoxygenase

pathway; however, this is the first link of the NAD(P)H oxidase arm of ROS to EETs and

sEH.

One major question is what cell type is involved in stimulating blood pressure

following central sEH inhibition. sEH is localized to both neurons and glia, yet its

substrate is primarily localized to astrocytes. In order to begin to answer this question,




Full Text

PAGE 1

ROLE OF BRAIN SOLUBLE EPOXIDE HYDROLASE IN CARDIOVASCULAR FUNCTION By KATHLEEN WALWORTH SELLERS 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 2004

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Copyright 2004 by Kathleen Walworth Sellers

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This work is dedicated to my family, w ho has taught me to embrace life with love, perseverance, and grace.

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ACKNOWLEDGMENTS I could not have completed this work without the assistance from many colleagues and friends. First and foremost, I would like to thank my mentor, Dr. Mohan Raizada, who has consistently supported and encouraged me these last several years. It was by chance that I worked in his research group before starting my graduate program, and I could not have found a better group with which to work. Dr. Raizadas excitement and fervor for new ideas have helped shape my appreciation for scientific research and the pursuit of knowledge. I also want to acknowledge all of the members of the Raizada research group, past and present. I am fortunate to work in such a friendly and intellectually-stimulating environment. Specifically, I want to thank Drs. Beverly Falcon and Matthew Huentelman, the two graduate students in our research group who both helped me tremendously. In addition, I would like to thank Drs. Carlos Diez, Cheng-wen Sun, Jorge Vazquez, Shereeni Veerasingham and Hong Yang for all of their intellectual guidance. I greatly acknowledge the members of my committee, Drs. Michael Katovich, Gerard Shaw, Colin Sumners and Charles Wood. Each has provided me with unique experience and expertise to further my research and career. In addition, I want to thank all of my teachers throughout my education. Without a solid learning base, I would not have reached this goal. iv

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Finally, I want to thank all my family and friends who have supported me over the years. It is they who lift my spirits and keep my sights set high. Each of my loved ones has a purpose in my life for which I am eternally grateful. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES.............................................................................................................x ABSTRACT......................................................................................................................xii CHAPTER 1 INTRODUCTION........................................................................................................1 Central Regulation of Hypertension.............................................................................1 Brainstem...............................................................................................................2 Hypothalamus........................................................................................................4 Baroreceptor Reflex...............................................................................................4 Mechanisms Underlying SNA...............................................................................5 Renin-angiotensin system..............................................................................5 Reactive oxygen species.................................................................................7 Implications.........................................................................................................10 Soluble Epoxide Hydrolase........................................................................................11 History.................................................................................................................11 Epoxyeicosatrenoic Acids...................................................................................12 Vasodilatory properties................................................................................12 EETs in the brain..........................................................................................14 Alternate actions of EETs............................................................................14 Dihydroxyeicosatrenoic Acids.....................................................................15 Cytochrome P-450...............................................................................................16 Epoxygenases in the brain............................................................................16 20-HETE......................................................................................................17 sEH and Hypertension.........................................................................................17 sEH Polymorphisms............................................................................................19 Rodent..........................................................................................................19 Human..........................................................................................................20 sEH polymorphisms in cardiovascular disease............................................21 sEH polymorphisms in other disorders........................................................21 Phosphatase Studies.............................................................................................22 Summary.....................................................................................................................23 vi

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2 INCREASED SEH EXPRESSION IN SHR VS. WKY RAT BRAIN: IN VITRO AND IN VIVO ANALYSIS........................................................................................31 Introduction.................................................................................................................31 Materials and Methods...............................................................................................33 Animals................................................................................................................33 Immunohistochemistry........................................................................................33 Real-Time RT-PCR.............................................................................................34 Western Blot Analysis.........................................................................................34 Neuronal Cells in Primary Culture......................................................................35 Losartan Treatment of SHRs to Normalize Blood Pressure................................35 Statistical Analysis..............................................................................................36 Results.........................................................................................................................36 Cellular Localization of sEH...............................................................................36 sEH Expression in the SHR.................................................................................37 In vitro Validation with Neuronal Cells in Primary Culture...............................37 sEH Expression Following Blood Pressure Normalization.................................38 Discussion...................................................................................................................39 3 CENTRAL SEH INHIBITION INCREASES BLOOD PRESSURE AND HEART RATE IN THE SHR...................................................................................................49 Introduction.................................................................................................................49 Materials and Methods...............................................................................................51 Animals................................................................................................................51 Statistical Analysis..............................................................................................51 ICV Cannulation..................................................................................................51 Abdominal Aorta Cannulation............................................................................51 Blood Pressure and Heart Rate Recordings.........................................................52 In vivo Drug Delivery..........................................................................................52 Results.........................................................................................................................53 Effect of sEH Inhibitor DCU on Blood Pressure and Heart Rate.......................53 WKY rat.......................................................................................................53 SHR..............................................................................................................53 Effect of sEH Inhibitor AUDA on Blood Pressure and Heart Rate....................54 Dose response...............................................................................................54 WKY rat..............................................................................................................55 SHR.....................................................................................................................55 Discussion...................................................................................................................55 4 ROLE OF ROS IN ANG II-INDUCED REGULATION OF BLOOD PRESSURE.63 Introduction.................................................................................................................63 Methods......................................................................................................................65 Animals................................................................................................................65 ICV Cannulation..................................................................................................65 Abdominal Aorta Cannulation............................................................................65 vii

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Statistical Analysis..............................................................................................65 In vivo Drug Delivery.........................................................................................65 Dipsogenic Response...........................................................................................66 Results.........................................................................................................................66 Angiotensin II Pressor Response.........................................................................66 gp91ds-tat Prevents Ang II-Induced Blood Pressure..........................................67 WKY rat..............................................................................................................67 SHR..............................................................................................................67 gp91ds-tat Prevents Ang II-Induced Drinking....................................................68 Discussion...................................................................................................................69 5 MECHANISM OF CENTRAL SEH INHIBITION ON BLOOD PRESSURE REGULATION...........................................................................................................77 Introduction.................................................................................................................77 Methods......................................................................................................................80 Animals................................................................................................................80 ICV Cannulation..................................................................................................80 Abdominal Aorta Cannulation............................................................................80 Blood Pressure and Heart Rate Recordings.........................................................80 Statistical Analysis..............................................................................................80 In vivo Drug Delivery.........................................................................................80 Evaluation of Spontaneous Baroreceptor Reflex Gain........................................80 Analysis of Heart Rate Variability......................................................................81 Neuronal Firing Recording..................................................................................82 Results.........................................................................................................................82 Waveform Analysis of Baroreceptor Parameters................................................82 Index of spontaneous baroreceptor reflex gain............................................82 High frequency analysis of pulse interval....................................................83 AUDA Regulation of Sympathetic Nerve Activity.............................................83 Increased EETs Linked to Increase in BP and HR..............................................84 MS-PPOH.....................................................................................................84 11-NODA.....................................................................................................85 Effects of sEH Inhibition Function through ROS...............................................85 Discussion...................................................................................................................86 6 EFFECT OF SEH OVEREXPRESSION IN THE PVN OF SHR AND WKY RATS97 Introduction.................................................................................................................97 Methods....................................................................................................................102 Animals..............................................................................................................102 ICV Cannulation................................................................................................102 Abdominal Aorta Cannulation..........................................................................102 Western Blot Analysis.......................................................................................102 Transformation of pCR 2.1 sEH cDNA into competent cells....................102 DNA miniprep............................................................................................102 Large-scale Lentiviral Production.....................................................................103 viii

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Infection of COS-7 cells with lenti-sEH...........................................................105 Cytoplasmic Isolation for HPLC Analysis........................................................105 Radiotelemetry Recordings...............................................................................106 PVN Coordinate Determination........................................................................106 Immunohistochemistry......................................................................................107 PVN Injection of lenti-sEH...............................................................................108 In vivo Drug Delivery.......................................................................................108 Statistical Analysis............................................................................................108 Results.......................................................................................................................109 Infection of COS-7 cells with lenti-sEH...........................................................109 Functionality of Lenti-sEH................................................................................109 sEH Overexpression in WKY PVN..................................................................110 AUDA delivery..........................................................................................110 EET agonist delivery..................................................................................111 Localization of viral delivery.....................................................................111 sEH Overexpression in SHR PVN....................................................................111 Discussion.................................................................................................................112 7 CONCLUSIONS AND FUTURE DIRECTIONS...................................................124 sEH Overexpression in Alternate Brain Nuclei........................................................124 sEH Expression.........................................................................................................125 ROS-Mediated Pathway of EET Function...............................................................126 Validation of Baroreceptor Reflex Findings.............................................................126 Role of sEH in Glial and Endothelial Cells..............................................................127 Role of Central Epoxygenases..................................................................................128 Effect of sEH Manipulation in Female Rats.............................................................128 Implications of Central sEH in Human Hypertension..............................................129 LIST OF REFERENCES.................................................................................................132 BIOGRAPHICAL SKETCH...........................................................................................143 ix

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LIST OF FIGURES Figure page 1-1 Major pathways of the RAS....................................................................................25 1-2 Major pathways of ROS production and metabolism.............................................26 1-3 Scatter plot of gene expression in the SHR and WKY hypothalamus and brainstem ..................................................................................................................................27 1-4 Arachidonic acid metabolism..................................................................................28 1-5 Tissue-specific functions of EETs...........................................................................29 1-6 Cellular membrane actions of EETs........................................................................30 2-1 Cellular localization of sEH in the brain .................................................................43 2-2 sEH mRNA levels in SHR and WKY brain regions ..............................................44 2-3 sEH protein expression in SHR and WKY brain regions ......................................45 2-4 sEH protein expression in neuronal cultures ..........................................................46 2-5 Effect of chronic losartan delivery on SBP in the SHR...........................................47 2-6 Effect of chronic losartan delivery on central sEH expression ..............................48 3-1 Effect of ICV DCU delivery on blood pressure and heart rate ..............................60 3-2 Dose response of ICV delivery of AUDA to SHR and WKY rats..........................61 3-3 Effect of central AUDA on blood pressure and heart rate .....................................62 4-1 Effect of central Ang II on blood pressure and heart rate. .....................................73 4-2 Effect of gp91ds-tat on Ang II-induced BP response in the WKY rat ..................74 4-3 Effect of gp91ds-tat on Ang II-induced BP response in the SHR ........................75 4-4 Dipsogenic responses of ICV gp91ds-tat and Ang II ............................................76 x

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5-1 Effect of central AUDA delivery on baroreceptor parameters.................................91 5-2 Effect of atenolol on AUDA-induced blood pressure..............................................92 5-3 Effect of MS-PPOH on AUDA-induced blood pressure.........................................93 5-4 Effect of EET agonist on BP and HR in the WKY rat.............................................94 5-5 Effect of ROS inhibition on AUDA-induced BP and HR response in the SHR......95 5-6 Effect of AUDA and gp91ds-tat on neuronal firing rate..........................................96 6-1 Lentiviral vector.....................................................................................................116 6-2 sEH protein expression in lenti-sEH-infected COS-7 cells...................................117 6-3 DHET measurements using HPLC analysis...........................................................118 6-4 Blood pressure and heart rate recordings following lenti-sEH and lenti-neo delivery to the WKY rat PVN..............................................................................................119 6-5 Blood pressure and heart rate recordings following AUDA delivery in the lenti-sEH-infected WKY rat...........................................................................................120 6-6 Blood pressure and heart rate recordings following EET agonist delivery in the lenti-sEH-infected WKY rat...................................................................................121 6-7 Localization of lenti-sEH delivered to the PVN of WKY rats...............................122 6-8 Blood pressure and heart rate recordings following lenti-sEH and lenti-neo delivery to the SHR PVN.....................................................................................................123 xi

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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 ROLE OF BRAIN SOLUBLE EPOXIDE HYDROLASE IN CARDIOVASCULAR FUNCTION By Kathleen Walworth Sellers December 2004 Chair: Mohan K. Raizada Major Department: Physiology and Functional Genomics Recent studies have linked peripheral soluble epoxide hydrolase (sEH) to hypertensive animal models. However, the role of this enzyme in the central control of blood pressure has not been elucidated. Thus, the objective was to investigate the involvement of brain sEH in blood pressure control through expression studies, pharmacological inhibition, and overexpression of sEH in the brain. First, sEH expression in the hypothalamus and brainstem, two cardioregulatory brain areas, was increased in the spontaneously hypertensive rat (SHR) compared to the Wistar Kyoto (WKY) rat. Second, inhibition of the enzyme by intracerebroventricular (ICV) delivery of pharmacological agents increased both blood pressure and heart rate in the SHR, an effect opposite to that following intraperitoneal delivery. This concomitant increase in blood pressure and heart rate in the SHR was accompanied by a depression of the baroreceptor reflex gain. xii

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The pathway by which sEH inhibition resulted in an increase in blood pressure and heart rate functioned through an accumulation of its substrate, epoxyeicosatrienoic acid (EET). Central delivery of the EETs resulted in an increase in pressor response in the WKY rat. In addition, -adrenergic receptor antagonist atenolol blocked the effect of sEH inhibition, indicating the pathway involves sympathetic activation. Furthermore, the effects of sEH inhibition were regulated by reactive oxygen species member NAD(P)H oxidase. This generator of toxic free radicals also mediated the central pressor response of angiotensin II. These observations suggest that central EETs and sEH inhibition are involved in increasing blood pressure. Finally, gene transfer using the lentiviral vector was used to overexpress sEH in cardiovascular-relevant nuclei. In the WKY rat, heart rate decreased six beats per minute following sEH overexpression and blood pressure remained unchanged. These studies, taken together, suggest that an increased expression of sEH is a futile central nervous system response in protection against hypertension. xiii

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CHAPTER 1 INTRODUCTION Hypertension has long been regarded as a silent killer, a disease that presents few symptoms but can generate devastating health problems and eventual death. Beginning as a sustained increase in blood pressure, this disease can lead to end-organ damage including heart failure. Cardiac output and total peripheral resistance determine blood pressure. Each component of this equation is influenced by a number of physiological factors in several organs including the kidney, heart and brain. For example, the heart regulates its contractility through input from baroreceptors in the carotid sinus and aortic arch that react to pressure changes (Zanchetti and Mancia 1991). An increased cardiac contractility results in an increased cardiac output, thereby increasing blood pressure. Similarly, increasing sodium intake leads to an increase in the bodys fluid volume, thus increasing cardiac output. Total peripheral resistance is ultimately controlled by the levels of hypertrophy and constriction present in smooth muscle cells that shape how the blood flows through the vasculature. While these factors define the major influences of blood pressure, it is necessary to understand the mechanisms underlying these factors in order to prevent and even reverse this disease. Central Regulation of Hypertension The brain continues to emerge as essential in the bodys regulation of blood pressure. For example, when an animal senses a predator, the sympathetic nervous system increases blood pressure and activates other defense mechanisms to prepare the animal for a fight or flight response. This response begins when the body senses danger 1

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2 and transmits this input to the brain to activate the sympathetic nerves that send projections to the brainstem and spinal column. Preganglionic neurons from these areas project to postganglionic neurons that in turn project to the heart or blood vessels (Dampney 1994). Two forms of preganglionic neurons are important in cardiovascular function, sympathetic preganglionic neurons (SPN) and vagal preganglionic neurons. Activation of SPN result in an increase in pressor response, while vagal preganglionic neurons regulate the baroreceptor reflex response that controls heart rate and blood pressure (Dampney 1994). Thus, signaling from these neurons regulates or controls cardiac contraction and blood vessel constriction. This autonomic nervous system signaling therefore regulates both blood pressure and heart rate from information initially processed from areas in the brain. In order to elucidate the involvement of the brain in blood pressure regulation via sympathetic control, it is necessary to identify regions that signal to the SPN. Retrograde tracers injected into the intermediolateral cell column (IML) have been used to map afferent neuronal projections. Specifically, transneuronal retrograde tracers incorporating either herpes or pseudorabies virus have been used recently to push the retrograde tracer into the cell body, thereby allowing the identification of specific nuclei that signal to the SPN (Strack et al. 1989). These techniques have identified nuclei in the brainstem, hypothalamus, caudal ventrolateral pons, and the A5 noradrenergic cell group. Based on a plethora of data underlying their influence on sympathetic activation, our research group has chosen to focus on the brainstem and hypothalamus. Brainstem The brainstem functions to regulate essential physiological functions including blood pressure, heart rate and respiration. This brain region houses several specific

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3 nuclei involved with cardiovascular function, including the rostral ventrolateral medulla (RVLM). The RVLM signals to the SPN, and bilateral denervation of the RVLM results in a decrease in blood pressure (Guertzenstein and Silver 1974). The RVLM therefore has influence over the pressor response. The excitatory response of the RVLM is regulated by several known factors. Excitatory inputs include acetylcholine, angiotensin II (Ang II), vasopressin, and excitatory amino acids (Andreatta et al. 1988; Dampney 1994; Gomez et al. 1993; Maeda et al. 1991; Willette et al. 1984b). Inhibitory factors include GABA and opiate receptor activation (Kishi et al. 2002; Punnen et al. 1984; Willette et al. 1984a). The important transmission ability of the RVLM is highlighted in work done with nitric oxide synthase (NOS), a known depressor. First, the inducible form of NOS, iNOS, is higher in the RVLM of normotensive Wistar Kyoto (WKY) versus spontaneously hypertensive rats (SHR), indicating a reason for the increased sympathetic activity in the SHR (Chan et al. 2001). Second, injection of eNOS, the endothelial form, increased GABAs inhibitory action on RVLM neurons (Kishi et al. 2002). Third, injections of Ang II into the RVLM increased both sympathetic vasomotor activity as well as blood pressure (Dampney et al. 2002; Kishi et al. 2002). Finally, gene therapy using adenovirus encoding eNOS delivered to the RVLM resulted in a decrease in blood pressure, heart rate and SNA in the WKY rat (Hirooka PBMP 2003). Thus, the RVLM functions to increase SNA and hence blood pressure through its neuronal projections. While some of these regulators have been identified, the complete mechanism of the RVLM in blood pressure control is not yet understood. In order to develop

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4 comprehensive treatments and preventative measures towards the development of hypertension, it is necessary to elucidate and define this role. Hypothalamus The hypothalamus is known for its regulation of body homeostasis and contains several specific nuclei that are linked to blood pressure regulation. The paraventricular nucleus (PVN) of the hypothalamus contains both magnocellular and parvocellular subdivisions, surrounding the third ventricle in an inverted triangular shape. The PVN has neuronal projections to the IML in the spinal column and also receives and transmits signals to the RVLM and the nucleus tractus solitarius (NTS) in the brainstem (de Wardener 2001). In relation to sympathetic control, the PVN can directly innervate the SPN or send projections to another excitatory nucleus such as the RVLM (Allen 2002). In fact, the PVN has a large number of catecholaminergic fibers ascending from the RVLM that release norepinephrine (de Wardener 2001; Palkovits et al. 1992). Norepinephrine delivery to the PVN increased blood pressure by 30 mmHg, suggesting that the PVN has direct role on the central control of blood pressure (Harland et al. 1989). The PVN has a diverse collection of neurons with distinct projections; therefore, its relation to SPN control is complicated. For example, electrical stimulation of the PVN has both stimulated and depressed blood pressure (Dampney 1994). Hence, while the PVN serves as a control unit for SPN action, its overall role is difficult to elucidate. Research of this key hypothalamic nucleus continues to define its control in brain-mediated blood pressure. Baroreceptor Reflex Apart from the influence of SPN on the pressor response, the baroreceptor reflex response is also an important component in cardiovascular regulation. Long believed to

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5 function only in the short-term regulation of blood pressure, recent evidence points to the baroreceptor reflex response as an important regulator of the long-term control of blood pressure (Malpas 2004). Baroreceptors are found in the aortic arch and the carotid sinus where they transmit and receive cardiac impulses to the IML and finally the brain. These impulses stimulate vagal preganglionic neurons that in turn affect heart rate, sympathetic vasomotor activity and the rate of secretion of vasopressin (Dampney 1994; Sved et al. 2001). Thus, the baroreceptor reflex response functions to control the balance of blood pressure and heart rate stimulation. The RVLM is a major mediator of the baroreceptor reflex response (Dampney 1994). For example, baroreceptor activation leads to an inhibitory effect in the RVLM (Colombari et al. 2001). The PVN has not been extensively studied in baroreceptor activation control, but studies do suggest it modulates this action through signaling with the NTS (Kannan and Yamashita 1985). The baroreceptor reflex response plays an important role in cardiovascular regulation and is influenced by brain nuclei also involved in SPN control. Mechanisms Underlying SNA Renin-angiotensin system As discussed, firing of neurons from specific nuclei including the PVN and RVLM to the IML result in an increase in SNA and hence an increase in blood pressure. Mechanisms controlling these oscillations in firing rate involve a number of hormones, neurotransmitters and other gene products, many still unknown. One major example of how these factors in the brain regulate hypertension is the renin-angiotensin system (RAS). The brain RAS continues to emerge as a vital regulator of hypertension since its discovery and characterization more than thirty years ago. Figure 1-1 illustrates the main

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6 pathways and members of the RAS. Classically, angiotensinogen is made in the liver and then cleaved by renal renin into angiotensin I. Angiotensin I is then converted to Ang II by angiotensin I converting enzyme (ACE) produced in the lung. Ang II, an eight-membered peptide, then binds to the Ang II type 1 receptor (AT 1 R), a G-protein coupled receptor (Sasaki et al. 1991). This binding results in an increase in blood pressure through vasoconstriction, increased sympathetic activity and fluid/salt volume regulation. Ang II functions as both a hormone and as a neurotransmitter in the brain and is also present in cardiovascular-relevant tissues including the kidney, heart and vasculature. (Allen et al. 2000). The initial understanding of the RAS has advanced and become more complex with new findings. First, the RAS has been identified both systemically and in individual tissues. Second, novel components have been identified that increase the complexity of this system. For example, ACE2 converts either Angiotensin I to Angiotensin (1-9) or Ang II to Angiotensin (1-7) (Turner et al. 2004). In most tissues, Angiotensin (1-7) acts opposite to that of Ang II (Chappell et al. 2004). These contradictory effects are also seen when Ang II binds to the AT 2 R. With each new discovery of the RAS, the pathway is further elucidated and better understood. Ang II is a major player in the central regulation of hypertension. Intracerebroventricular (ICV) delivery of Ang II results in a dramatic increase in both blood pressure and drinking (Di Nicolantonio et al. 1982). This response begins within minutes and is only a short-term effect following bolus delivery. Ang II has also been extensively studied in specific brain nuclei that function in blood pressure and dipsogenic responses (Pan 2004). For example, Ang II injection to the subfornical organ results in a

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7 profound dipsogenic response, while injection into the PVN, NTS or RVLM leads to a dramatic increase in blood pressure (Bastos et al. 1997; Mangiapane and Simpson 1980; Muratani et al. 1993; Tanaka et al. 2001). Taken together, central Ang II binding to the AT 1 R in several brain nuclei induces a pressor response. One ongoing area of study involves the mechanisms underlying this blood pressure control. Ang II through AT 1 R stimulation is known to increase neuronal firing rate and norepinephrine release from sympathoexcitatory neurons, therefore resulting in sympathetic nerve activation (Sumners et al. 2002; Zimmerman et al. 1984). One question that remains is what are the steps involved after AT 1 R stimulation and prior to sympathetic nerve activation? Recent evidence points to the involvement of reactive oxygen species (ROS) as a second messenger in the central mediation of RAS. Reactive oxygen species ROS has emerged as a major player in a number of disease states, including cardiovascular disease. ROS members include superoxide (O 2 ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radicals. ROS are formed from oxygen converted to superoxide by enzymes including NAD(P)H oxidase and xanthine oxidase (Figure 1-2). Superoxide is converted to the hydrogen peroxide using superoxide dismutase (SOD) and then normally converted to water via either catalase or glutathione peroxidase (Becker 2004). This pathway is not destructive to cellular function and is the normal progression of oxygen processing. Oxygen is processed through the mitochondria in order to provide energy for metabolic functions. During certain disease states and times of physiological stress, however, hydrogen peroxide can instead be converted to hydroxyl radicals that lead to cellular injury. Thus, when the ROS outnumbers the available antioxidants,

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8 oxidative stress results (Touyz 2004). This damaging progression is thought to be a significant factor in a growing number of disease states. NAD(P)H oxidase, one of the key enzymes in the generation of ROS, was originally localized in phagocytes (Hanna et al. 2002). It is able to convert oxygen to its superoxide form by converting NAD(P)H to NAD(P) and donating the released electron. This conversion rendered it able to defend the host cell against microorganisms. More recently, though, focus has shifted to the role of NAD(P)H oxidase in other tissues. This enzyme was found to be a major source of ROS in cardiovascular tissue, and it has also been localized to both neurons and glia in the brain (Hanna et al. 2002; Zimmerman and Davisson 2004). One key difference between NAD(P)H oxidase sources from phagocytotic or non-phagocytotic cells is that the NAD(P)H oxidase in non-phagocytotic sources is constitutively active, thus increasing the probability for oxidative stress. NAD(P)H oxidase is a multimeric enzyme consisting of membranous subunits Gp91ds and p22 and cytoplasmic subunits p67, p47, p40 and Rac (Hanna et al. 2002). The active form the enzyme is initiated when p47 is phosphorylated and transports to the membrane along with the other cytoplasmic subunits and binds with Gp91. Phosphorylation of p47 is driven by protein kinase C (Rey et al. 2001b). Due to the importance of ROS in a wide array of systems, several inhibitors and activators have emerged in the field of study. Tempol is a SOD mimetic used in a variety of studies as an antioxidant, though its specificity is low. Recent studies used an adenoviral vector to deliver SOD to the brain (Zimmerman et al. 2002). In addition to SOD manipulation, NAD(P)H oxidase has also been used in therapeutic approaches. A peptide that competes with the docking portion of gp91 was sequenced to block the

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9 effects of NAD(P)H oxidase. The tat peptide derived from HIV was included upstream of the docking sequence in order to facilitate delivery in vivo (Rey et al. 2001b). This peptide, gp91ds-tat, is a specific, effective blocker of NAD(P)H oxidase both in vitro and in vivo. Several studies have implicated ROS in the central Ang II-mediated pathway. ICV delivery of SOD using adenovirus prevented Ang II-induced increases in blood pressure and drinking (Zimmerman et al. 2002). In addition, the Ang II-induced decrease in heart rate was abolished. These results were reproducible using both the mitochondrial and cytoplasmic forms of SOD. Given the wide range of impact from ICV delivery, studies were undertaken to elucidate the importance of specific brain nuclei in the ROS-Ang II connection. Adenovirus encoding a dominant-negative inhibitor of NAD(P)H oxidase activator Rac1 was injected into the circumventricular organs (CVOs) (Zimmerman and Davisson 2004). CVOs are distinct brain regions in that they lack a blood-brain barrier; therefore, they are influenced by both the brain and the periphery and serve to pass information across the two. RAS members are present in several CVOs, and previous research highlighted the cardiovascular regulatory role of RAS in the CVOs (Muratani et al. 1996). This adenoviral delivery resulted in preventing the pressor and drinking responses of Ang II (Zimmerman and Davisson 2004). Thus, ROS involvement in the Ang II pathway is localized to distinct neurons as well. As a complement to the in vivo experiments, Ang II treatment of primary lamina terminalis cultures led to an increase in superoxide, indicating the importance of Ang II on ROS production in vitro (Zimmerman et al. 2002).

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10 Implications The central control of blood pressure is regulated by neurotransmitters, hormones and genetic factors that act to control SNA. While we understand the importance of specific nuclei in both the hypothalamus and brainstem, the mechanisms underlying this control have not been fully elucidated. Hypertension is indeed a complex disease whose establishment and progression cannot be pinpointed to one genetic malfunction. Thus, in order to understand the genetic basis of hypertension, it is necessary to identify multiple genes underlying the basis of this disease. Our research group began the task of identifying genes novel to central blood pressure regulation by analyzing differences in brain gene mRNA levels between the SHR and WKY rat, using microarray technology. The SHR was chosen because of similarities between this genetic strain and human forms of genetic hypertension (Lindpaintner 1994; Louis and Howes 1990; Trippodo and Frohlich 1981). The hypothesis was that genes involved in the central regulation of hypertension would have different gene expression levels between the hypertensive and normotensive state. However, this genetic profiling cannot in itself distinguish genes whose expression alters as a consequence of hypertension. In order to analyze 7000 genes and 1500 ESTs, the Affymetrix U34A Rat GeneChip was used. Isolated mRNA from the hypothalamus and brainstem was amplified, fluorescently-labeled, hybridized to the chip, and scanned in order to be analyzed using GeneSpring software. Figure 1-3 highlights gene expression expressed as log values in four SHR compared to four WKY samples. Each point represents the average measurement for an individual gene between the rats in each group. Dotted lines segregate genes with a two-fold expression change between SHR and WKY rats.

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11 EPHX2, the gene that encodes the soluble epoxide hydrolase (sEH) protein, had the highest expression discrepancy between the two models. The sEH gene, indicated by the arrow, was significantly upregulated in the SHR and virtually undetectable in the WKY rat. This result incited a review of sEH and how it may be related to hypertension. The following section discusses the history, pathways and actions of sEH and how these factors may function to regulate blood pressure. Soluble Epoxide Hydrolase History The family of epoxide hydrolases (EH) includes sEH, microsomal EH (mEH), leukotriene A4 hydrolase, and cholesterol 5,6-oxide hydrolase (Spector et al. 2004). sEH was first identified as a cohort to mEH, a detoxifying agent to many foreign ingested agents. The sEH gene was first cloned in 1985, and its dimer structure (two 62-kDa subunits) was deduced in 1996 (Arand et al. 1996; Grant et al. 1993). A ubiquitous enzyme, sEH converts arachidonic acid metabolites and linoleic acid epoxides to less active forms through the addition of water (Zeldin 2001). Figure 1-4 outlines the placement of sEH in the major pathways of arachidonic acid conversion. The sEH enzyme can act on several substrates, and initial evidence suggested 1,2-disubstituted aliphatic epoxides were the ideal substrates for this enzyme. In fact, radiolabeled forms of this type of compound are used in activity studies of sEH (Borhan et al. 1995). The active site of sEH has been identified by crystal structure analysis to be a catalytic triad (Arand et al. 1996). Recently, more research has focused on the structure-function relationship in sEH. Crystal structure analysis revealed that sEH is a homodimer made up of two 62.5 kDa units. The two C-terminal domains interact with

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12 each other, while the N-terminal domains have no calculated or observed interactions with each other (Newman et al. 2003). Epoxyeicosatrenoic Acids The most well-studied role of sEH involves its enzymatic conversion of vasoactive epoxyeicosatrenoic acids (EETs) to dihydroxyeicosatrenoic acids (DHETs). The EETs are formed from arachidonic acid (AA) through the enzymatic action of cytochrome P-450 (CYP) epoxygenases (Zeldin 2001). The EETs are divided into four different regioisomer forms (5,6-, 8,9-, 11,12-, and 14,15-EETs) and are found in a variety of tissues including brain, kidney, and vasculature (Fang et al. 2001; Spector et al. 2004). Figure 1-5 outlines the main pathways and tissue locations of EET regioisomers. Most forms of EETs promote vasodilation, while their DHET metabolites are relatively inactive. Vasodilatory properties EETs in the vasculature have been implicated in blood pressure control due to their ability to dilate arteries. While EETs are found in a variety of tissue including the brain and kidney, plasma levels of these epoxygenase products predominate over all other known sources (Rosolowsky and Campbell 1996). EETs are produced from arachidonic acid in the endothelium; however, they are secreted from the cells to act on vascular smooth muscle cells. Evidence suggest that EETs bind to and activate the G-protein coupled receptor subunit Gs within the smooth muscle cell which then stimulates the large conductance calcium-activated potassium (BK(Ca)) channel (Harder et al. 1995; Li and Campbell 1997). Potassium influx into the cell results in hyperpolarization and therefore dilation of the vascular smooth muscle cell. Thus, increasing EET plasma levels decreases blood pressure by decreasing total peripheral resistance via vascular

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13 smooth muscle dilation. Figure 1-6 illustrates this vasodilatory action from a cellular level. Evidence for this dilation is as follows. First, all regioisomer forms of EETs have been found to activate BK(Ca) channels in vascular smooth muscle cells (Harder et al. 1995). Second, 11,12and 14,15-EETs increase the open-state probability of Ca 2+ -activated K + channels in coronary smooth muscle cells (Campbell et al. 1996). Third, all the regioisomers relax coronary vessels following contraction with thromboxane mimetic U46619 (Campbell et al. 1996). Fourth, EETs can transiently increase Ca 2+ release from intracellular stores, which also functions to activate BK(Ca) channels (Li and Campbell 1997). While this pathway results in the overall hyperpolarization of the membrane, it is not necessary for the EET-modulated activation of BK(Ca) channels. Finally, an increased EET production is associated with hypertensive models, presumably as a feedback mechanism that cannot overcome the increase in blood pressure (Omata et al. 1992; Pomposiello et al. 2001). Taken together, these studies provide comprehensive evidence that EETs function to relax the vasculature and thus lead to a decrease in blood pressure. This evidence of membrane hyperpolarization has led to high speculation that EETs function as the elusive endothelial-derived hyperpolarizing factor (EDHF) in the vasculature. While enormous evidence has implicated nitric oxide as a major player in relaxing arteries, complete abolition of relaxation cannot be achieved with inhibitors to NO action alone. The remaining action is termed EDHF, and its identity remains unknown. While EDHF is likely to be a combination of several factors, the hyperpolarizing nature of EETs coupled with their high expression in the vasculature has

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14 led to the hypothesis that EETs function as EDHF (Campbell et al. 1996; Fisslthaler et al. 1999). EETs in the brain EETs are expressed in astrocytes and endothelial cells in the brain, and evidence suggests their vasodilatory roles found in the periphery are translated to cerebral blood flow control in the brain (Medhora et al. 2001). All regioisomer forms of EETs are expressed in the brain, though astrocytes preferentially intake 8,9-EETs over 14,15-EETs (Shivachar et al. 1995). In addition, 8,9-EETs were not converted to DHETs in astrocytes. Whether this result was due to sEH regulation or other conversion mechanisms is unclear. EETs from both astrocytes and endothelial cells dilate smooth muscle cells through activation of the BK(Ca) channels (Gebremedhin et al. 1992). The central result of this dilation is an increase in cerebral blood flow. While all regioisomers act to dilate the cerebral arteries, 8,9-EETs and 11,12-EETs are the most potent forms in the brain (Gebremedhin et al. 1992). Studies that inhibited the CYP-450 epoxygenases in the brain and thereby prevented the formation of EETs resulted in the reduction of basal cerebral blood flow by 30% (Alkayed et al. 1997). Thus, central EETs play an integral role in the maintenance of cerebral blood flow. Alternate actions of EETs These four regioisomers of EETs exert different functions dependent on their tissue location. Vasodilation in the vascular smooth muscle leads to a decrease in blood pressure via the enlargement of arteries and thus an increase in blood flow, thereby decreasing total peripheral resistance. A second organ potentially involved in the sEH regulation of blood pressure is the kidney. Vasodilatory properties of EETs in the renal

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15 vasculature result in changes in renal blood flow and hence changes in water and electrolyte balance. All regioisomers modulate calcium influx in the vascular smooth muscle, while 11,12-EET is the predominant form regulating calcium as well as other studied functions in the endothelium (Spector et al. 2004). In the myocardium, all the regioisomers inhibit the sodium ion channel, while only the 8,9and 11,12-EETs activate the K(ATP) ion channel (Lee et al. 1999; Lu et al. 2001). EETs can also be converted to different metabolites using enzymes ranging from COX to CYP -oxidase (Zeldin 2001). These actions are dependent on the tissue type and likely the availability of sEH, since treatment with sEH inhibitor N,N-dicyclohexyurea (DCU) augments the conversion by these alternate enzymes (Spector et al. 2004). EETs can also be effectively stored in phospholipids if not immediately converted by enzymes. Dihydroxyeicosatrenoic Acids Studies on the function of DHETs have produced inconsistent results compared to the role of the EETs. For example, DHETs is generally less potent than the EETs. Evidence in support of this includes decreases in Ca2+ uptake and relaxation of the bovine coronary artery compared to the EETs (Campbell et al. 2002). However, DHETs do activate the BK(Ca) channel of coronary artery myocytes and serve to dilate coronary arterioles following constriction with endothelin (Spector et al. 2004). While the general consensus is that the EETs overshadow the DHETs in their action, DHET function cannot be discounted due to lack of sufficient studies done on these metabolites. Overall, however, by converting EETs to DHETs, sEH acts to attenuate EET action and promotes the relative inactivity of DHETs.

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16 Cytochrome P-450 EETs are only one branch of products generated from arachidonic acid. Figure 1-4 illustrates an overview of the pathways stemming from arachidonic acid. Cyclooxygenase converts AA into a variety of prostaglandins that have been well characterized in their inflammatory activation role. Prostaglandins have also been implicated in labor induction and metabolic regulation. The second conversion pathway uses lipoxygenases that generate hydroxyeicosatrenoic acids (HETEs). HETEs are well studied in their roles in vascular tissue. HETEs are known to be potent vasoconstrictors in the kidney and vasculature. The cyclooxygenase and lipoxygenase branches of arachidonic acid conversion are therefore stimulating in their roles both in blood pressure control and other essential physiological regulation. This section will focus, however, on the CYP conversion of arachidonic acid and what is known and hypothesized about its role in the brain. 20-HETE, an alternate product of CYP action, is discussed regarding its vasoactive effects opposite to that of EETs. Epoxygenases in the brain Most isoforms are able to convert AA to all regioisomer forms of EETs. Isoforms in the brain include CYP 2C11, 2C12, 2D18, 2D19, 2C29, 2C38, and 2C39, though more may be identified in the future (Roman 2002). Interestingly, the studies done to localize epoxygenase expression in the brain have pinpointed the enzymes to astrocytes. These studies hypothesize that central epoxygenases have a role in microvascular control. In contrast, though, CYP 2D19 has been localized to dopaminergic neurons, suggesting that CYP members are not found exclusively in the glia (Thompson et al. 2000). More

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17 research is needed to elucidate this pathway in conjunction with EET-mediated effects on cardiovascular control. 20-HETE Regioisomers of HETEs can also be generated by CYP, specifically -hydroxylase of the CYP 4A family. -hydroxylase converts arachidonic acid to 19-HETE and 20-HETE. 20-HETE acts as a vasoconstrictor in vessels ranging from the kidney to the brain. While EETs activate BK(Ca) channels, 20-HETE inhibits these channels, thus inducing contraction (Croft et al. 2000). Inhibiting 20-HETE results in a decrease in blood pressure due to the decrease in total peripheral resistance. Levels of both 20-HETE and CYP 4A are increased in the SHR (Omata et al. 1992). In addition, this vasoconstrictor is activated by Ang II, further elucidating its role in hypertension (Croft et al. 2000). Thus, 20-HETE functions are opposite to the vasodilator role reported for EETs and its overexpression is linked to hypertension. In summary, these arachidonic acid metabolites from the epoxygenase and -hydroxylase pathways serve a variety of functions throughout the body, and these functions are dependent on the converting enzymes that regulate the activity of these metabolites. sEH and Hypertension sEH has been implicated in cardiovascular, renal, and inflammatory biology functions. Recently, studies have linked sEH to hypertension through both a transgenic model overexpressing the gene and sEH inhibitor studies in the SHR. sEH-null mice were generated from offspring of chimeric mice with the disrupted sEH gene (Sinal et al. 2000b). Homologous recombination was used to disrupt the sEH gene in embryonic stem cells. High performance liquid chromatography (HPLC) revealed that the sEH-null mice had elevated levels of EETs and decreased levels of DHETs as compared to the wild-type

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18 mice. Blood pressure was measured using tail-cuff and compared between groups. Male sEH-null mice had decreased systolic blood pressure and did not have an increased blood pressure in response to salt loading. Female sEH-null mice had no significant change in systolic blood pressure. Thus, deletion of the sEH gene lowered systolic blood pressure to that of the female mice. An important subsequent finding was relative sEH expression in the SHR and WKY rat from Charles River Laboratories. As previously mentioned, the SHR is a good animal model to use for hypertension studies based on similarities to the human form of the disease (Lindpaintner 1994; Louis and Howes 1990; Trippodo and Frohlich 1981). Using an sEH-specific antibody, researchers found a significant elevation of sEH protein in the SHR kidney and liver; in fact, it was hardly detectable in the WKY rat (Yu et al. 2000a). Given the overexpression of sEH in the hypertensive model, researchers sought to determine whether the activity of this enzyme was linked to blood pressure. The second major paper addressing the sEH control in blood pressure involved the intraperitoneal (IP) delivery of sEH inhibitor N,Ndicyclohexylurea (DCU). Treatment with 3 mg/kg DCU resulted in a 65% decrease in 14,15-DHET and a 30% increase in 14,15-EET (Yu et al. 2000a). As a result, systolic blood pressure decreased by 22 mmHg in the SHR and had no significant effect in the WKY rat. Thus, pharmacological inhibition of sEH also results in a decrease of systolic blood pressure in hypertensive male rats. In summary, both a genetic and a non-genetic method for decreasing sEH in the periphery led to a decrease in systolic blood pressure and were accompanied by a decrease in the conversion of EETs to DHETs.

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19 Expression studies for sEH have also included alternate models of high blood pressure aside from the Charles River source of SHRs (Imig et al. 2002). Peripheral or central delivery of Ang II results in an increase in blood pressure and consequently hypertension (Allen et al. 2000; Di Nicolantonio et al. 1982). Ang II was chronically infused (60 ng/min) using an osmotic minipump, and blood pressure was recorded using tail cuff (Imig et al. 2002). Following IP injection of the sEH inhibitor N-cyclohexyl-N-dodecyl urea, systolic blood pressure decreased 30 mmHg in the Sprague-Dawley (SD) rat. Following sEH inhibition, sEH expression increased two-fold in the kidney. However, sEH overexpression is not present in all SHR sources. As discussed below, the Heidelberg (Heid) SHR has a lower sEH expression compared to its normotensive control. Thus, the importance of sEH overexpression in the peripheral tissues remains to be established. sEH Polymorphisms Rodent It is important to note that in the Ang II-infusion study, the basal sEH expression in the SD rats was highly variable, ranging from present to not present. This variable expression is typical of sEH in SD rats. SD rats are outbred, which increases the probability of new gene patterns with each new breeding source. Interestingly, sEH has known polymorphisms found in both rodent models and in humans. As previously described, the SHR from Charles River has increased sEH expression in both kidney and liver (Yu et al. 2000a). However, sEH expression in Heid SHR and WKY rats was oppositeWKY rats have increased sEH expression in the liver and kidney (Fornage et al. 2002). This finding was accompanied by the identification of a separate sEH allele in the Heid rat source. The two alleles differ by four single nucleotide polymorphisms

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20 (SNPs). Consequently, the polymorphism identified in the Heid source results in a blunted sEH activity. Thus, presence of these polymorphisms and reduced activity can explain the strain source difference in the Western blot analysis results. Human Polymorphisms in sEH have also been identified in humans; in fact, most of the research involving the sEH role in the human focuses on polymorphism and its effects ranging from sEH activity itself to coronary artery calcification. The human form of sEH is 73% identical to the mouse form, with 100% similarity in the catalytic triad (Przybyla-Zawislak et al. 2003). This highly conserved region underlies the importance of the sEH active site across species. The human sEH gene has been localized to the p arm of chromosome 8, specifically, 8p21-p12. The sEH gene is not without differences in its sequence, however, as highlighted by three separate studies. Initially, seven SNPs were identified in liver samples from twenty-five human subjects. Two of these SNPs resulted in amino acid substitutions, while the remaining five were silent mutations (Sandberg et al. 2000). In the second study, a total of thirty-six SNPs were identified in forty-eight members of the Japanese population. Five of these SNPs were found in exons of the gene (Saito et al. 2001; Sato et al. 2004). A third study resulted in the identification of six SNPs of the gene in a population size of seventy-two. Three of these polymorphisms had an increased level of epoxide hydrolase activity while one had decreased activity. The one with decreased activity also had significant reduction in 14,15-EET hydrolysis. Interestingly, the incidence of sEH polymorphisms in the sample group studies was higher in the Black population as compared to White or Asian population (Przybyla-Zawislak et al. 2003).

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21 sEH polymorphisms in cardiovascular disease This variation in sEH polymorphisms as related to race and disease was further examined in a recent correlation study. The sEH gene was mapped in 1201 African American and 1506 White subjects, and groups were separated based on the presence or absence of the genotypes Gln287/Gln287, Arg287/Gln287, Arg287/Arg287 (Fornage et al. 2004). The Gln287 polymorphism has been shown to result in a decrease in sEH activity. The presence of the Gln287 polymorphism was associated with a two-fold increase in coronary artery calcification in an African American subset of the Black population (Fornage et al. 2004). This finding suggests that a decrease in sEH activity and thus an increase in EETs may lead to an increase in coronary artery calcification in the African American population. In a separate study involving a family afflicted with familial hypercholesterolemia, the sEH polymorphism Glu287/Arg was identified (Sato et al. 2004). Results indicated that patients with one Arg287 allele as well as defects in the LDL receptor gene had elevated total cholesterol levels as well as plasma triglyceride levels. No positive correlation was identified in subjects not having defects in the LDL receptor gene. Thus, it is interesting that these different polymorphisms at the same locus elicit different effects on sEH activity. More research is needed to elucidate possible mechanisms underlying this hypothesis and other associations between sEH and cardiovascular disease. sEH polymorphisms in other disorders The Arg287/Gln287 polymorphism was further studied to identify any correlation between this sEH SNP and Parkinsons disease (Farin et al. 2001). The rationale behind the study was that a decrease in sEH activity leads to an increase in active epoxide

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22 intermediates, which may generate an increase in oxidative stress, leading to Parkinsons disease. No significant correlation was measured in Parkinsons patients, indicating a decrease in sEH activity was not related to Parkinsons disease. However, this study cannot rule out other sEH roles that may be related to CNS disorders or general oxidative stress in general. In addition, the general study size could mask a subset of Parkinsons disease development that may in fact stem from sEH dysregulation. Finally, SNP allele frequencies were measured in a subset of the Japanese population for future studies on cancer. The sEH gene was included in this study based on its involvement in conversion of toxins (Yoshimura et al. 2003). Thus, sEH is being examined in a variety of disorders and diseases based on its widespread activity and implications. In addition, its ubiquitous expression and polymorphisms resulting in changes in activity target this as a candidate gene for regulation of a number of genetic-based problems. Phosphatase Studies Two recent PNAS publications focused on the role of sEH as a phosphatase (Cronin et al. 2003; Newman et al. 2003). sEH was found to act as a phosphatase, though without high specificity. For example, sEH was not mediated by common phosphatase inhibitors including sodium orthovanadate (Newman et al. 2003). However, sEH did effectively metabolize a number of hydroxy lipid phosphates. The connection of phosphatase activity to the N-terminal domain was confirmed with both a plant form of sEH lacking the N-terminal domain and a mutant N-terminal domain form of sEH (Cronin et al. 2003). In both of these models, phosphatase activity of the enzyme was eliminated. In addition, sEH inhibitors used to block epoxide conversion had no effect on phosphatase activity. sEH function related to EET conversion is attributed to the C-terminal domain, which is homologous to haloalkane dehydrogenase (Cronin et al. 2003).

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23 These studies indicate that sEH may play an important physiological function through its phosphatase activity. While evidence into this line of research is sparse, it is conceivable that the number of polymorphisms in sEH across species and strains within species may generate differences in phosphatase activity and thus a novel role for this enzyme. Summary Hypertension is a complex disease controlled by a number of organs including the brain. While our understanding of the central regulation of blood pressure has a substantial base involving connections between specific nuclei in the hypothalamus and brainstem to the spinal cord, key mechanisms involved in the genetic basis of hypertension have not been elucidated. In order to fully understand this disease in the hopes of preventing and combating its progression, it is essential to identify genes and their pathways underlying the dysregulation of blood pressure. Using gene profiling involving microarray analysis of the hypothalamus and brainstem, sEH was found to be overexpressed in the SHR compared to the WKY rat brain. sEH is an enzyme that converts vasoactive EETs to their relatively inactive diol forms. Expressed in a variety of tissues, sEH has been implicated in the peripheral control of hypertension due to its increased expression in the hypertensive state and the resulting decrease in blood pressure following IP delivery of an sEH inhibitor. In addition, sEH polymorphisms are now being associated with cardiovascular disorders. Thus, sEH and its link to hypertension are well-characterized in the periphery. However, little is known about the role of this enzyme in the brain. Given the previous studies of sEH and hypertension coupled with its dramatic overexpression in the brain of the SHR, we hypothesized that sEH is involved in the

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24 central regulation of hypertension. In order to address this hypothesis, our aims were as follows: 1. Determine if central sEH expression is increased in the hypertensive state. These studies outlined in Chapter 2 focus on the validation of gene profiling data as well as whether this increased sEH expression is a cause or consequence of hypertension. 2. What is the physiological effect of inhibiting sEH in the brain? Chapter 3 discusses central sEH inhibitor studies in both SHR and WKY rats and the resulting blood pressure, heart rate, and baroreceptor reflex changes. 3. Does the pressor action of central Ang II function through NAD(P)H oxidase? As discussed earlier, the RAS plays a major role in the central regulation of blood pressure. In addition, ROS continues to emerge as a necessary component of this regulation. These studies in Chapter 4 address this question with central administration of NAD(P)H oxidase formation blockers and Ang II and resulting blood pressure changes. This role of ROS in the brain forms the basis of the central mechanism of sEH discussed in Chapter 5. 4. What is the central sEH mechanism that results in blood pressure and heart rate changes? Chapter 5 outlines experiments incorporating EET mediators, ROS production antagonists, and sympathetic blockers in order to elucidate the mechanism by which central sEH inhibition increases blood pressure. 5. Determine if sEH overexpression in the brain alters blood pressure and heart rate. These studies in Chapter 6 use the lentivirus to overexpress the sEH gene in the PVN of SHR and WKY rats. Blood pressure and heart rate are recorded daily and mechanisms underlying sEH control are studied using sEH inhibitors and EET agonists. Taken together, these aims dissect the role of sEH on the central regulation of blood pressure. The results of these experiments invoke intriguing questions into the specific effect of EETs in the brain and how this effect is regulated in the hypertensive and normotensive state. Overall, these studies provide further insight into the central regulation of blood pressure and propose a novel role of EETs in the brain.

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25 Figure 1-1 Major pathways of the RAS

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26 Figure 1-2 Major pathways of ROS production and metabolism

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27 Figure 1-3 Scatter plot of gene expression in the SHR and WKY hypothalamus and brainstem. Each point represents the average measurement for a gene. Dotted lines segregate genes with a two-fold expression change between SHR and WKY samples. sEH, indicated by the black arrow, is significantly upregulated in the SHR.

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28 Figure 1-4 Arachidonic acid metabolism

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29 Figure 1-5 Tissue-specific functions of EETs

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30 Figure 1-6 Cellular membrane actions of EETs

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CHAPTER 2 INCREASED SEH EXPRESSION IN SHR VS. WKY RAT BRAIN: IN VITRO AND IN VIVO ANALYSIS Introduction The brain is an essential component of the mechanisms controlling blood pressure. As previously discussed, specific nuclei of brain regions including the hypothalamus and brainstem function in the central regulation of blood pressure (Dampney 1994). Given the relative importance of the brain in establishing and maintaining hypertension coupled with the inadequacy of our knowledge of the mechanisms underlying this influence, it is imperative to study genes with discordant expression in the hypertensive state. By elucidating the roles of these genes in the brain, we better understand the mechanisms involved in the establishment of hypertension and therefore have more power with which to prevent and even reverse this disease. Chapter 1 discussed gene profiling findings that sEH had the most disproportionate mRNA levels in hypothalamus-brainstem samples of the male SHR compared to the male WKY rat. High sEH expression in the kidney also has been identified in hypertensive animal models (Imig et al. 2002; Yu et al. 2000a). In addition, deletion of the EPHX2 gene encoding sEH decreased the systolic blood pressure of male mice (Sinal et al. 2000b). These studies raise several questions regarding sEH expression. First, given the ubiquitous nature of this enzyme, is sEH overexpression in hypertensive models generalized and therefore not confined to renal action? What are the sEH expression levels in other cardiovascular-relevant tissues including the brain? Second, are 31

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32 differences in brain sEH expression the result of hypertension, or are they independent of blood pressure changes? The increased sEH mRNA levels in the SHR brain from microarray analysis must first be verified, given the unpredictability of microarray significance. Therefore, the first part of Aim I is to use real-time RT-PCR and Western blot analysis to measure sEH levels in the brain areas of SHR and WKY rats. The hypothesis is that both RNA and protein levels of the hypothalamus and brainstem are increased in the SHR compared to the WKY rat. The second part of Aim II is to determine if central sEH expression is independent of blood pressure changes. First, if sEH expression is increased in the hypertensive state, is that increase present from birth, or do the expression levels change with development? If the expression levels remain constant, then sEH expression cannot be a consequence of hypertension. Conversely, if the levels change, then sEH expression may be sensitive to changes in blood pressure. This question is important in understanding the role of sEH in the development of hypertension. To address this issue, neuronal cultures were prepared from one-day-old SHR and WKY rat pups. The hypothesis was that sEH overexpression was present from birth and not an effect of high blood pressure. The question of whether sEH expression is dependent on blood pressure changes was next addressed in the adult SHR. In contrast to measuring sEH expression prior to the development of hypertension, this experiment measured sEH expression following blood pressure normalization with losartan, a potent pharmacological AT 1 R blocker (Carr and Prisant 1996). We hypothesized that normalizing blood pressure in the SHR would

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33 not affect sEH protein levels in the brain, indicating that sEH expression is not dependent on blood pressure changes. The following studies thus address two facets of sEH expression. First, they determine whether the sEH data from the microarray studies do in fact translate to a change in sEH expression. Second, they answer whether sEH expression is driven by changes in blood pressure. Taken together, these experiments define central sEH expression in the SHR and WKY rat. Materials and Methods Animals Male SHR and WKY rats, weighing 220-250g at the beginning of the study, were ordered from Charles River Laboratories (Wilmington, Mass). Rats were housed individually and kept on 12:12 light:dark cycle in a climate-controlled room. Rat chow (Harlan Tekland, Madison, WI) and water were provided ad libitum. Number of animals per group are described in Results. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Florida. Immunohistochemistry Rats were anesthetized with inhaled isofluorane (5%) and perfused transcardially with saline followed by 4% paraformaldehyde (PFA). SHR brains were post-fixed in PFA and saturated in 20% sucrose solution. 10-20 M sections were cut using a cryostat and mounted on poly-L-lysine-coated slides; the Rat Brian Atlas (Paxinos & Watson 4 th Ed 1998) was used as a reference. Sections were blocked in 2% bovine serum albumin (Sigma-Aldrich, St. Louis MO) dissolved in Tris-buffered saline with Tween (TBS-T, 0.1M Tris-HCl, 0.9% NaCl, 0.1% Tween) for three hours. Sections were then incubated with anti-sEH rabbit antibody overnight (1:500), a kind gift from Dr. Bruce Hammock,

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34 University of California Davis, followed by anti-rabbit biotinylated (1:200) for two hours and 10g/ml Avidin-FITC for one hour (Amersham Biosciences, Buckinghamshire, England). The sections were double-stained with either neuronal-specific (NeuN, 1:10, Amersham Biosciences) or glial-specific antibody [glial fibrillary acidic protein (GFAP), 1:10, a kind gift from Dr. Gerard Shaw, University of Florida] to detect cellular distribution of sEH. sEH localization was detected with fluorescent microscopy. Real-Time RT-PCR The RNAqueous-4PCR kit was used to isolate DNA-free RNA from tissue samples (Ambion, Austin TX). One-step real-time RT-PCR was performed on 50ng RNA per sample. Briefly, samples were combined with sEH-specific primers and probe (900nM forward: 5-GATTCTCATCAAGTGGCTGAAGAC-3; 900nM reverse: 3-GGACACGCCACTGGCTAAAT-5) and probe (250nM: 5-CCAGAACCCATCGGTGACCTCCAA-3) and TaqMan One-Step RT-PCR Master Mix and Multiscribe as described by the company (Applied Biosystems, Foster City CA). Primers and probe to 18S were used as an internal control. Reactions were carried out in the ABI PRISM 7000 sequence detector, and the standard curve method was used to calculate the threshold cycle (C T ) for target amplification. The input amounts were then calculated from the C T and standard curve and normalized using the 18S input amounts. Western Blot Analysis Total cell lysate (TCL) was isolated according to the companys research applications instructions (Santa Cruz Biotechnology Inc, Santa Cruz CA). Twenty g protein from either neuronal cultures or brain tissue were run on a 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane. Following three hours of blocking with

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35 5% milk in TBS-T, the membrane was probed with the sEH rabbit-anti mouse antibody (1:3000 in 1% milk/TBS-T) overnight. Specificity of this antibody has been previously established (Sinal et al. 2000a; Yu et al. 2000b). The membrane was rinsed 3 times and washed for 20 min in TBS-T and then incubated with anti-rabbit IgG HRP-conjugated secondary antibody (1:3000) for 1 hour. Following final washes, the membrane was incubated with Western Lightning Chemiluminescence Plus reagent for 1 min and then exposed to film to visualize the bands (Perkin Elmer, Wellesley, MA). Neuronal Cells in Primary Culture Primary neuronal cultures (90% neurons, 10% glia) from the hypothalamus and brainstem of one-day-old SHR and WKY rats were prepared as previously described (Fleegal and Sumners 2003; Raizada et al. 1995; Sunn et al. 2002). The cells were cultured grown twelve days in Dulbeccos Modified Eagles Media (DMEM) + 10% horse serum at 37C, 10% CO 2 in 100 mm dishes. Three dishes were used per experimental group. Losartan Treatment of SHRs to Normalize Blood Pressure Losartan (10mg/kg/day) was delivered in the drinking water to male SHR (n=6) after determining the basal drinking rate to be 32 ml water/day. Six SHRs were kept on regular drinking water as control. The water bottles containing losartan were covered with aluminum foil to address the light sensitivity of the drug. Water intake and body weight were monitored daily one week prior to and up to one month following the start of losartan treatment. One rat in the control group was eliminated from the study due to its inability to develop high blood pressure. Systolic blood pressure was recorded using the tail cuff method three and four weeks after the start of losartan treatment. At the end of

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36 the experiment, protein and mRNA was isolated from the hypothalamus, brainstem, and cortex of the rats. Statistical Analysis All data are expressed as mean standard error (SE). The number of animals or samples in each group is described in the Results below. Comparisons between experimental groups were analyzed using either one-way ANOVA (SPSS) or Students T test (Stat View). Differences between means within groups were assessed with the Newman-Keuls analysis. Significance was set at a confidence level at or above 95% and is denoted by in each figure. Results Cellular Localization of sEH Immunohistochemical analysis revealed that sEH is localized to both neurons and glia throughout the SHR brain (n=3). Neurons were double-stained with both NeuN (red) and sEH antibody (green), and glial cells were double stained with both sEH antibody (green) and GFAP (red), as shown in Figure 2-1. Co-staining was identified by the yellow overlapping fluorescence detected using confocal microscopy. sEH was detected in all areas of the midbrain and brainstem sectioned. Control sections treated only with secondary and tertiary antibodies did not fluoresce at the intensity used for the confocal analysis, thus indicating the fluorescence was due to sEH antibody rather than background fluorescence. Fluorescence from the sEH antibody was localized to the cytoplasm and neuronal projections of the cell. No sEH was detected in the nucleus. This result corresponds with the nature of this protein, which is a cytoplasmic protein. The GFAP antibody was also localized to the cytoplasm rather than the nucleus, and overlapping fluorescence was observed in this area.

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37 sEH Expression in the SHR Profiling with microarray technology previously identified profoundly increased sEH mRNA levels in the brain of the SHR compared with its WKY normotensive control. Thus, the first objective was to determine if this result corresponded with RNA and protein levels measured by real-time RT-PCR and Western blot analysis, respectively. Figure 2-2 outlines the results of real-time RT-PCR using hypothalamus and brainstem isolated from four male SHR and WKY rats. Samples within each rat strain were combined; therefore, it is impossible to say with any certainty that there were differences. White and black bars represent sEH mRNA levels to normalized to 18S levels of WKY rat and SHR, respectively. sEH mRNA levels in the SHR were three-fold higher hypothalamus and six-fold higher in the brainstem compared to WKY rats. sEH protein expression was also increased in the SHR hypothalamus and brainstem. Figure 2-3 depicts a representative radiograph (A) and quantitation of sEH protein levels normalized to -actin (n=4/group) (B). In TCL isolated from the hypothalamus, relative sEH:-actin protein ratios in SHR and WKY rats were 0.52+/0.14 OD/mm 2 and 0.12+/-0.02 OD/mm 2 respectively. In the brainstem samples, the ratio was 0.60+/-0.14 OD/mm 2 in the SHR and 0.13+/-0.03 OD/mm 2 in the WKY rats. sEH protein levels were measured with Western blot analysis using the same sEH antibody source used in the peripheral studies. White and black bars represent WKY rat and SHR, respectively. sEH was barely detectable in WKY rats while they were dramatically increased in the SHR. In vitro Validation with Neuronal Cells in Primary Culture Primary co-cultures from hypothalamus-brainstem of WKY and SHR have been used in the past to elucidate cellular and molecular mechanisms of physiological

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38 dysregulation in neural control of hypertension (Sun et al. 2003b; Yang et al. 2004). These cultures were used to determine if sEH overexpression in these hypertensive animals is present prior to the establishment of high blood pressure. Western blot analysis demonstrated a four-fold increase in sEH protein levels in neuronal cultures of SHR vs. WKY rats, indicating that sEH overexpression is present from birth in the SHR (Figure 2-4). The ratio of sEH to -actin protein was 0.34+/-0.15 OD/mm 2 in the SHR and 0.08+/-0.04 OD/mm 2 in the WKY rats (n=4/group). Panel (A) is a representative radiograph highlighting the sEH and -actin protein bands. Panel (B) is the graphical representation of sEH protein levels normalized to -actin from the four sets of neuronal cultures. sEH Expression Following Blood Pressure Normalization Delivery of losartan (10mg/kg/day) into the drinking water resulted in a significant decrease in systolic blood pressure in the SHR one month following the initiation of losartan treatment. Three weeks after the start of the study, systolic blood pressure was 33 mmHg lower in the rats treated with losartan [181.48+/-5.11 mmHg SHR (n=5), 148.40+/-1.71 mmHg SHR + losartan (n=6)]. This gap widened to a decrease in systolic blood pressure by 38 mmHg (173.84+/-4.40 mmHg SHR, 136.28+/-2.05 mmHg SHR + losartan) one month following the start of losartan treatment (Figure 2-5). Body weights were not significantly different between groups. At the end of the study, brains were dissected and protein isolated from losartan-treated and control rats. Western blot analysis revealed no significant change in the ratio of sEH to -actin expression in the hypothalamus between the losartan-treated (0.530+/-0.078 OD/mm 2 ) and control rats (0.739+/-0.246 OD/mm 2 ) (Figure 2-6). Similarly, the

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39 ratio of sEH to -actin expression was 0.863+/-0.026 OD/mm 2 and 0.840+/-0.066 OD/mm 2 in the brainstem of the losartan-treated and control-treated SHRs, respectively. White and black bars represent losartan-treated and control samples, respectively. Thus, sEH expression in the SHR was not affected by blood pressure depression using an AT 1 R antagonist delivered orally. Discussion This study was the first to measure and compare central sEH expression between hypertensive and normotensive animal models. Both sEH mRNA and protein levels were elevated in the hypothalamus and brainstem of SHR compared to WKY rats. This overexpression was independent of blood pressure changes because (i) sEH overexpression existed in neuronal cultures from prehypertensive rats and (ii) normalization of blood pressure with losartan did not alter sEH expression levels. These expression studies validate the gene profiling data, are parallel to the expression findings in peripheral tissue and provide a foundation on which to study sEH in the central control of hypertension (Imig et al. 2002; Yu et al. 2000a). Immunohistochemical studies established that sEH is expressed in both neurons and glial cells of one-day-old SHR and WKY rats. In contrast, CYP and EETs have been mainly localized to astrocytes and endothelial cells surrounding the cerebral vasculature (Alkayed et al. 1997; Gebremedhin et al. 1992; Medhora et al. 2001; Shivachar et al. 1995). Thus, the role of sEH in the brain may involve divergent roles including sympathetic nerve activation through neuronal pathways or control of cerebral blood flow. The role of central sEH in relation to blood pressure likely stems from neuronal control given two sets of data. First, sEH was overexpressed in neuronal cultures of one

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40 day-old SHRs. Second, sEH expression does not differ in glial cultures of SHR and WKY rats (data not shown). In the brain areas studied, sEH protein and mRNA levels were significantly increased in the SHR compared to the WKY rat. This result correlates to the peripheral studies that showed an increase in sEH expression in the kidney and liver of SHR and Ang II-induced hypertensive models (Imig et al. 2002; Yu et al. 2000a). Thus, in all tissue areas studied, sEH is more abundant in the Charles River SHR compared to the WKY rat. This result therefore proves that sEH overexpression is not limited to the renal and hepatic systems of the SHR but is in fact present in other cardiovascularly-relevant areas. This was the first study to measure sEH expression patterns in non-adult animal models. sEH was highly expressed in neuronal cultures from one-day-old SHR compared to WKY rats. This result indicates that sEH expression patterns exist from birth and are not affected by the onset of hypertension. However, these results alone do not prove that increased sEH expression in the brain is the cause of any change in blood pressure. Future studies in which either central sEH is blocked during development in a hypertensive strain or overexpressed in the brain of normotensive strain would serve to answer this question. The conclusion that central sEH expression is independent of systemic blood pressure changes was also supported by the normalization of blood pressure experiment. Chronic delivery of losartan into the drinking water had no effect on the sEH levels in either the hypothalamus or the brainstem. This result suggests that depression of blood pressure in the SHR does not alter sEH expression. Losartan is a potent AT 1 R blocker

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41 that is often used to evaluate the role of RAS (Carr and Prisant 1996). The decrease in systolic blood pressure by almost 40 mmHg following its oral delivery highlights the essential role RAS plays in blood pressure regulation. However, this decrease in blood pressure was not accompanied by changes in sEH expression. Several caveats regarding this experiment must be addressed. First, sEH expression was only measured at one time point, three weeks following the start of losartan delivery. Therefore, one cannot discount expression changes prior to or following this time point. For example, perhaps sEH expression was affected during the initial drop in blood pressure and then recovered by means of feedback mechanisms after three weeks. This result would indicate that sEH expression might have a role in the RAS regulation of blood pressure. Alternately, sEH expression may be affected after four weeks of treatment or longer due to delayed mechanisms. If this postulation were true, then sEH expression would be dependent on blood pressure changes. Another possibility is that while sEH expression may not change following blood pressure normalization, either the enzymes activity or the affinity of the product for its receptor may be affected. Regardless of expression levels, if the enzymatic activity changes, then the role of sEH following blood pressure changes must be reassessed. sEH activity is generally determined by HPLC analysis to compare the levels of EETs and DHETs (Yu et al. 2000a). This analysis should be performed in the future in order to confirm that sEH is independent of blood pressure changes. Finally, further studies incorporating different means of altering blood pressure should be performed since blood pressure normalization does not follow only one mechanism. In this experiment, losartan was delivered via the drinking water; therefore,

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42 the blood pressure was normalized by blocking peripheral AT 1 R that results in a decrease in total peripheral resistance. While central sEH expression was not affected by this method, it may have changed had the losartan been delivered to the brain, thus decreasing sympathetic nerve activity. To verify that the sEH expression is independent of blood pressure depression, other antihypertensive agents including diuretics should be incorporated. Alternately, sEH gene regulation may be constitutively active in the SHR and therefore other hypertensive models should be incorporated. Similarly, sEH expression may only be activated with blood pressure stimulation rather than depression. While we can conclude that decreasing blood pressure via oral delivery of losartan does not alter central sEH expression, this result does not conclusively prove that central sEH is independent of any change in blood pressure. Collectively, these studies reveal that central sEH expression is higher in the SHR compared to the WKY rats. In addition, these results suggest that central sEH expression is independent of blood pressure changes. Given the dichotomy of central sEH expression in the SHR and WKY rat, what effect, if any, do these expression changes have on the central control of blood pressure? Does sEH overexpression in the brain have a role in hypertension? To begin to answer these questions, Chapter 3 outlines central sEH inhibition studies to determine whether pharmacological blockade of sEH action in the brain results in a change in blood pressure.

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43 Figure 2-1 Cellular localization of sEH in the brain. Following perfusion, SHR rat brains sections (20M) from the midbrain were prepared for immunohistochemical analysis of sEH and its localization to neurons and glial cells. sEH Antibody is detected by green fluorescence, while the red fluorescence detects either neurons (upper panel) or glia (lower panel). Yellow fluoresence marks the overlap between sEH Antibody and cell type-specific Antibody. All images were taken using confocal microscopy.

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44 Figure 2-2 sEH mRNA levels in SHR and WKY brain regions. RNA was isolated from hypothalamus and brainstem regions of SHR and WKY rat brains (n=4/combined in each group). Real-time RT-PCR incorporating sEH-specific primers and probe was used to measure sEH cDNA levels in samples from these extracted tissues. Data were normalized with primers and probe to 18S and are presented as arbitrary units (AU). White and black bars represent WKY and SHR samples, respectively. Reprinted with permission from The FASEB Journal.

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45 Figure 2-3 sEH protein expression in SHR and WKY brain regions. Protein was isolated from hypothalamus and brainstem regions of SHR and WKY rat brains (n=4/combined in each group). Western blot analysis was used to measure sEH protein levels in from 40g total cell lysate. (A) Representative radiograph of sEH and -actin. (B) Data were normalized with -actin. White and black bars represent WKY and SHR samples, respectively. Data are represented as mean+/-SEM. significantly different in SHR and WKY rat (p<0.05). Reprinted with permission from The FASEB Journal.

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46 Figure 2-4 sEH protein expression in neuronal cultures. Total cell lysate was isolated from hypothalamus and brainstem co-cultures from one-day-old SHR and WKY rats. (A) Representative radiograph of sEH and -actin. (B) Quantitation of sEH protein normalized with -actin protein band. Data are represented as mean+/-SEM. significantly different in SHR vs. WKY samples (p<0.05, n=4/group). Reprinted with permission from The FASEB Journal.

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47 Figure 2-5 Effect of chronic losartan delivery on SBP in the SHR. Losartan (10mg/kg/day) was delivered orally to male SHRs. SHRs only receiving water were used as control. SBP was recorded using tail cuff recordings. Data are represented as mean+/-SEM. *significantly different between groups (p<0.05, n=6)

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48 Figure 2-6 Effect of chronic losartan delivery on central sEH expression. Total cell lysate was isolated from brainstem and hypothalamus, and Western blot analysis performed as described in Methods to compare sEH protein levels. sEH expression was normalized with -actin and presented graphically. White and black bars represent losartan and control treatments, respectively. Data are represented as mean+/-SEM (p<0.05, n=6).

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CHAPTER 3 CENTRAL SEH INHIBITION INCREASES BLOOD PRESSURE AND HEART RATE IN THE SHR Introduction Pharmacological sEH inhibitors delivered IP to the SHR led to a decrease in blood pressure (Yu et al. 2000a). This depressor effect is postulated to involve the accumulation of vasodilatory EETs. What is the effect of central sEH inhibition on blood pressure? Chapter 2 revealed an sEH overexpression in the hypothalamus and brainstem of the SHR. These results correlated with increased renal and hepatic sEH expression in hypertensive models (Imig et al. 2002; Yu et al. 2000a). Given the similarity of expression patterns in the different tissues, does sEH also yield a pressor response in the brain? Prior to embarking on these studies, it is necessary to understand the action and limitations of the currently available sEH inhibitors. Pharmacological agents used as sEH inhibitors act as competitive inhibitors by forming hydrogen bonds and salt bridges with the active site of sEH. These compounds have evolved both in their potency and versatility of use. The first round of inhibitors were tight-binding with K(I) values in the nanomolar range; however, limitations hampered the extended use of these compounds (Morisseau et al. 1999). First, the compounds had a high crystal lattice energy that translated to an elevated melting point. Second, they were not water-soluble and therefore were not easily dissolved in agents that could be applied for use in vivo or in vitro. 49

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50 The major sEH inhibitor used in published animal studies is N,N-dicyclohexylurea (DCU), a 1,3-disubstituted urea that is formed by hydrolysis dicyclohexylcarbodiimide (Morisseau et al. 2002). DCU specifically inhibits sEH and has no effect on mEH, CYP or any other tested compounds related to the structure of sEH. DCU and related compounds were determined to be effective sEH inhibitors because of hydrophobic groups on either side of the urea (Morisseau et al. 1999). In physiological studies, DCU was delivered to the SHR via IP injection, and blood pressure was recorded using photoelectric tail cuff. DCU delivery decreased urinary excretion of 14,15-DHET by 40%, indicating the functionality of this inhibitor. sEH inhibition resulted in a decrease in systolic blood pressure 12+/-2 mmHg after six hours (Yu et al. 2000a). This time lapse of blood pressure effect may be due to the kinetics of the drug as well as its processing by the liver. This study provided the first pharmacological evidence linking sEH to blood pressure control. While DCU is effective in its blockage of sEH function, it is limited by its solubility in water. Thus, a new generation of soluble, potent sEH inhibitors was synthesized. One main structural change underlying these improvements was substituting a hydrophobic side group with a polar functional group arranged in an alkyl chain (Kim et al. 2004). Adamantyl urea dodecanoic acid (AUDA) is one such second-generation sEH inhibitor. It is disubstituted with an adamantyl group on one end and an eleven-membered carbon chain on the other. It was formulated and characterized by Bruce Hammocks research group at University of California, Davis. This pharmacological inhibitor has an IC 50 of 18+/-1 nM with mouse sEH (Morisseau et al.

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51 2002). In comparison, DCU has a less potent IC 50 of 81.8+/-0.7 nM with mouse sEH. AUDA is therefore a promising candidate as an sEH inhibitor. Overall, pharmacological sEH inhibitors have evolved in their potency and specificity. We chose to use these inhibitors in vivo as blockers of sEH activity. The following experiments outline physiological measurements following the central delivery of either DCU or AUDA. Given the sEH expression patterns in the brain mimics that of the periphery, we hypothesized that ICV delivery of sEH inhibitors would lead to a decrease in blood pressure in the SHR. Materials and Methods Animals Statistical Analysis The above Methods are described in Chapter 2 ICV Cannulation Rats were anaesthetized with inhaled isofluorane (3%) in all surgical procedures. A stereotaxic frame was used to position a twenty-two-gauge guide cannula (PlasticsOne, Roanoke VA) into the right lateral ventricle (AP: 1.3mm, ML: 1.5mm, 4.5mm below skull) of male SHR and WKY rats. The cannula was secured to the skull with dental resin assisted by 3 3/32 screws secured in the skull. Appropriate drugs (1-2l) were injected into the right lateral ventricle of unrestrained rats using a 28 gauge internal cannula, 4.5mm. Rats were allowed to recover for one week following all surgeries. Abdominal Aorta Cannulation A radiotelemetric pressure transducer (Data Sciences International, Arden Hills MN) consisting of a fluid-filled catheter attached to a PA-C40 transmitter was implanted into the abdominal aorta. The aorta was clamped proximally and the catheter inserted

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52 caudal to the left renal artery and secured with medical adhesive. Prior to suturing the abdomen, the transducer was checked for proper placement using a radio to transmit the sound oscillations from pressure changes in the aorta. Rats were anaesthetized with inhaled isofluorane (3%) during surgery and allowed to recover one week following the surgical procedure. Blood Pressure and Heart Rate Recordings Blood pressure and heart rate were recorded using the implanted radiotelemetry transducers. For each dataset acquisition, the rat cages were positioned on receivers that collected signals from the implanted transducers. These signals were then transmitted to an adapter with an ambient pressure monitor that relayed the signals to the DataQuest 3.1 Acquisition program (DataSciences International, Arden Hills MN). The rats were allowed to acclimate to their new cage position for at least one hour before basal measurements were recorded. During data collection, blood pressure (systolic, mean and diastolic), heart rate and activity were recorded every minute for an average of ten seconds at 500Hz. Data were acquired and transferred to Microsoft Excel for graphical analysis. In vivo Drug Delivery sEH inhibitors were delivered via the ICV cannula into the right lateral ventricle of unrestained rats. A 4.5 mm injector containing the inhibitor was inserted and secured into the cannula. The injector was attached to a 5l Hamilton syringe via 0.38 mm polethylene tubing 120 cm in length (Becton Dickinson Sparks, MD). sEH inhibitors DCU and AUDA (0.8-60ng, 2l volume) were a kind gift from Dr. Bruce Hammock, University of California, Davis. DCU and AUDA were dissolved in 10% DMSO/90%

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53 artificial cerebrospinal fluid (aCSF: 133mM NaCl, 3.4mM KCl, 1.3mM CaCl 2 1.2mM MgCl 2 6mM NaH 2 PO 4 32mM NaHCO 3 ) and 100% aCSF, respectively. These inhibitors as well as their vehicle controls were delivered as a single bolus to singly-housed, unrestrained SHR and WKY rats. Results Effect of sEH Inhibitor DCU on Blood Pressure and Heart Rate Figure 3-1 outlines the physiological response to sEH inhibition. Figures 3-1A,B are representative recordings of blood pressure (A) and heart rate (B) in the SHR (red) and the WKY rat (blue). The x-axis is time related to DCU injection in hours. Figures 3-1C,D are bar graphs of peak blood pressure (C) and heart rate (D) responses to DCU, expressed as mean +/SEM. WKY rat In the WKY rat, heart rate increased from 342.4+/-7.6 bpm with vehicle delivery to 393.1+/-19.0 bpm following 90ng DCU (Figure 3-1D). There was no significant change in blood pressure in the WKY rat (113.78+/-3.03 mmHg CSF, 120.34+/-1.96 mmHg DCU, n=4). The increase in heart rate began within one hour following injection and normalized after two hours. SHR Central delivery of sEH inhibitors resulted in an increased blood pressure and heart rate in the SHR. DCU (90ng) injected into the right lateral ventricle increased blood pressure from 153.0+/-7.8 mmHg to 184.5+/-5.3 mmHg and increased heart rate from 339.6+/-15.3 bpm to 433.5+/-11.5 bpm (n=4) (Figure 3-1C,D). The increase in blood pressure and heart rate initiated within one hour of injection and peaked at three to four

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54 hours after injection. Blood pressure and heart rate were normalized within seven hours following injection. Effect of sEH Inhibitor AUDA on Blood Pressure and Heart Rate The results from ICV injection with DCU indicate that heart rate is increased in both the SHR and WKY rat following sEH inhibition. Blood pressure, however, was increased only in the SHR. No pressor responses were detected in the WKY rat. In order to verify these results, we used a second sEH inhibitor AUDA that later became available from Dr. Bruce Hammock at University of California Davis. As previously discussed, AUDA is more potent and has increased water solubility, thereby making it a better pharmacological agent for testing the physiological effects of sEH inhibition. Dose response ICV administration of AUDA, a potent sEH inhibitor, caused a dose-dependent increase in both blood pressure and heart rate in the SHR. Figure 3-2 is a graphical representation of the change in blood pressure following delivery of either 0.8ng or 7.5ng AUDA. Artificial cerebrospinal fluid was used as the control. Either concentration of AUDA elicited only modest increases in blood pressure in the WKY rats [23.61+/-2.90 mmHg 0.8ng AUDA (n=5), 12.49+/-3.72 mmHg 7.5ng AUDA (n=4)]; however, the 0.8ng AUDA dose was the only delivery that yielded significance (Figure 3-2). The alternate doses did not yield blood pressure increases that significantly differed from that of aCSF injection (13.74+/-1.82 mmHg, n=4). In the SHR, a significant increase of 25+/-0.9 mmHg in blood pressure was seen with as low as 0.8ng AUDA, and a dose of 7.5ng AUDA increased blood pressure by 31.8+/-3.9ng AUDA (n=3).

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55 WKY rat Given this initial dose response, 15ng AUDA was used in the remaining studies in order to measure the maximum physiological effects of sEH inhibition. Figures 3-3A,B depict representative blood pressure (A) and heart rate (B) recordings prior to and following ICV delivery of 15ng AUDA. Quantitation of blood pressure and heart rate recordings are charted in Figures 3-3D,E. ICV delivery of 15ng AUDA did not significantly change blood pressure in the WKY rat (106.52+/-2.40 mmHg basal change, 115.91+/-2.30 mmHg AUDA, n=6). Alternately, heart rate increased from 311.60+/-9.22 bpm to 358.06+/-7.73 bpm, a change of 46+/-12 bpm following AUDA injection (Figure 3-3). SHR Figures 3-3A,C depict representative blood pressure (A) and heart rate (C) recordings prior to and following ICV delivery of 15ng AUDA. Quantitation of blood pressure and heart rate recordings are charted in Figures 3-3D,E. Central delivery of 15ng AUDA resulted in a change in blood pressure from 148.04+/-1.18 mmHg to 180.48+/-0.25 mmHg, an increase of 32 mmHg (n=6). In addition, heart rate increased from 334.44+/-2.52 bpm to 388.24+/-0.80 bpm, a change of 54 bpm (Figures 3-3). This increase began to express after thirty minutes, reached maximal levels in three to four hours and returned to basal levels in seven hours. Discussion These studies are the first to determine the physiological effects of inhibiting sEH action in the brain. Central sEH inhibition resulted in an increase in both blood pressure and heart rate in the already hypertensive SHR. While heart rate increased in the WKY rat, blood pressure did not dramatically change following ICV delivery of sEH inhibitors.

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56 These results were opposite from the hypothesized decrease in blood pressure and from the depressor effect of IP delivery of DCU to the SHR. Given the whole-body overexpression of sEH in different hypertensive models, why is the physiological effect of sEH inhibition in the brain opposite from that in the periphery? The understanding of how sEH in the periphery affects blood pressure is limited. Explanations are based on the enzymes conversion of vasodilatory EETs to relatively inactive DHETs. The major function of EETs is to increase the open-state probability of BK(Ca) channels in vascular smooth muscle cells (Harder et al. 1995; Li and Campbell 1997). sEH depletes the stores of EETs, thereby leading to vasoconstriction in the vasculature. This hypothesis is supported by increased sEH expression in both the SHR and Ang II-induced models of hypertension (Imig et al. 2002; Yu et al. 2000a). Translation of this hypothesis to the central role of sEH, however, is complex. sEH may function to deplete stores of EETs surrounding the cerebral arteries, leading to constriction and decreased cerebral blood flow. However, while decreased cerebral blood flow is highly implicated in stroke, its direct regulation on blood pressure is not clear. In addition, sEH is found in neurons; therefore, its potential role in sympathetic activation cannot be discounted. Thus, while sEH expression patterns are the same in the brain and the periphery, the mechanism underlying sEH influence on blood pressure is not. One important question is whether ICV delivery of DCU and AUDA is an effective means to inhibit sEH. DCU (3mg/kg, IP) was previously used in physiological studies and effectively decreased 14,15-DHET urinary excretion by 65% (Yu et al. 2000a). Thus, this pharmacological inhibitor injected IP acts on tissues to inhibit the activity of

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57 this enzyme. However, the tissue-specific effect of this delivery was not known, nor do we know what percentage of DCU was taken up and converted by the liver. In addition, transfer efficiency of this drug to the brain is not known. This concern of sEH inhibition effectiveness in these central studies is first addressed by the use of two inhibitors rather than only one. Both have been found to inhibit sEH activity and not other members of this family of inhibitors such as mEH. In addition, AUDA is more potent than DCU, the inhibitor used in the previous studies. One caveat of this current study, however, was that the sEH activity was not measured. Measurements of EETs and DHETs by HPLC analysis must be incorporated into future studies to conclusively state that DCU and AUDA delivery to the lateral ventricle attenuates sEH activity in the brain. Cardiovascularly-relevant brain regions should be individually analyzed in order to ascertain which regions ICV delivery of these pharmacological agents are targeted. Only by meticulous analysis of sEH inhibition in specific brain nuclei can we conclude what areas are involved in this pressor response. One interesting observation was the discrepancy of blood pressure and heart rate effects between the SHR and WKY rat. The lack of blood pressure response coupled with an attenuated heart rate response in magnitude and duration in the WKY rat compared to the SHR suggests differential central pathways between these two strains. Given the low endogenous level of sEH in the brain of the WKY rat, it logically follows that the sEH inhibitor would have a limited supply of enzyme on which to act compared to the hypertensive rat. sEH inhibition in these two strains may act through the same pathway but yield different results due to endogenous sEH expression. Alternately, since

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58 the SHR is known for its dysregulation of blood pressure control, one of these central pathways unique to the SHR or hypertension itself may involve sEH. These inhibitor studies suggest that the overexpression of sEH in the SHR brain may be a compensatory outcome of this hypertensive strain. We know that increased sEH expression has not normalized the blood pressure in the SHR; however, inhibition of this enzyme increases the blood pressure. Thus, without the increased sEH expression in the brain, blood pressure in the SHR would be even higher. This idea cannot be supported on an individual basis, given the increased sEH expression in neuronal cultures from one-day-old SHR pups. This hypothesis instead is rooted in a proposed evolutionary development of the SHR over generations. sEH overexpression in the brain may have been selected for during reproduction due to increased embryo viability. Alternately, whole-body sEH overexpression may have stemmed from random mutations that collectively result in an increased blood pressure. Thus while the brain function of sEH attenuates blood pressure, this beneficial effect is masked by the increased pressor response from alternate pathways. Finally, ICV delivery of sEH inhibitors may not be a comprehensive look at sEH in the brain. The cerebral vasculature selectively targets certain brain regions, and injection into the right lateral ventricle will likely target areas immediately surrounding the site of injection. It is necessary as discussed above to measure the physiological effects of sEH inhibition in various brain regions in order to elucidate its role in the central control of blood pressure. The question, however, is how central sEH inhibition increases blood pressure and heart rate. These studies conclude that pharmacological sEH inhibitors delivered to the lateral ventricle results in an increase in blood pressure and heart rate in the SHR. It is

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59 essential to elucidate the mechanisms underlying this blood pressure effect stemming from the brain. Do these effects also include the conversion of EETs, the main substrate identified for sEH? Or does this enzyme act through a different pathway unique to the brain? The answers to these questions will be undertaken by experiments discussed in the next two chapters incorporating known regulators of blood pressure control in the brain.

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60 Figure 3-1 Effect of ICV DCU delivery on blood pressure and heart rate. DCU was injected into the lateral ventricle of SHR and WKY rats. Representative tracings of blood pressure (A) and heart rate (B) in SHR and WKY rats. Quantitation of blood pressure (C) and heart rate (D) following DCU or aCSF injection. These values represent the peak time of blood pressure response over a ten-minute period. The first and second arrows represent the point of injection of aCSF or DCU, respectively. Data are expressed mean +/-SEM. represents significance between groups (p<0.05, n=4). 1

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61 Figure 3-2 Dose response of ICV delivery of AUDA to SHR and WKY rats. aCSF, 0.8ng and 7.5ng AUDA were delivered to SHR and WKY rats. Blood pressure was recorded using radiotelemetry. Mean change in blood pressure was plotted; error bars represent SEM. significantly different from aCSF response (n=4, p<0.05).

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62 Figure 3-3 Effect of central AUDA on blood pressure and heart rate. 15ng AUDA was delivered ICV, and radiotelemetry was used to record blood pressure and heart rate. (A) MAP of SHR (red) and WKY rats (blue). (B,C) Representative HR of WKY rats and SHRs, respectively. Quantitaton of MAP (D) and HR (E). White and black bars represent basal and AUDA-induced levels, respectively, as means +/SEM. significantly different from basal (p<0.05, n=6). Reprinted with permission from The FASEB Journal.

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CHAPTER 4 ROLE OF ROS IN ANG II-INDUCED REGULATION OF BLOOD PRESSURE Introduction The role of the brain in hypertension remains to be fully elucidated. Chapter 1 outlined the major RAS pathways and evidence linking Ang II to the central control of blood pressure. RAS members are localized to a number of cardiovascularly-relevant brain areas including the PVN and SON in the hypothalamus and the RVLM and NTS in the brainstem (Bastos et al. 1997; Mangiapane and Simpson 1980; Muratani et al. 1993; Tanaka et al. 2001). An overactive RAS in these areas leads to an increase in blood pressure due to sympathetic nerve activation. Thus, while the RAS was initially identified as systemic, the brain RAS is an excellent example of cardiovascular control localized to one organ. The binding of Ang II to the AT 1 R induces the most significant pressor response of the RAS members (Helin et al. 1997). Overwhelming electrophysiological and pharmacological evidence indicates that the binding of Ang II to the AT 1 R stimulates sympathoexcitatory neurons that in turn signal to the IML to induce blood pressure (Reid 1992). Specifically, Ang II increases neuronal firing rate by activating calcium channels and inhibiting the delayed rectifier potassium current (Sun et al. 2003a; Zhu et al. 1997). These changes in firing rate are prevented by losartan. Thus activation of catecholaminergic neurons by Ang II is through its binding to the AT 1 R. Although the overall pressor effect of central Ang II is established, the mechanisms underlying this activation are complex and not well understood. An emerging set of 63

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64 regulators in this pathway is the ROS. As previously discussed in Chapter 1, ROS results in a dysregulation of a number of physiological pathways and has been linked to a multitude of diseases including cancer. Hypertension is no exception. Striking evidence has linked an overproduction of ROS to an increase in blood pressure Recently, studies have focused on the role of ROS in the central control of hypertension (Zimmerman and Davisson 2004; Zimmerman et al. 2002). Treatment of primary cultures from the lamina terminalis with Ang II resulted in an increase in superoxide, thereby linking Ang II action to ROS production (Zimmerman et al. 2002). Gene therapy strategies also have defined the overall effect of ROS members on blood pressure, though the underlying mechanisms are not understood. Ang II-induced blood pressure was prevented in mice centrally injected with Adenovirus containing either mitochondrial or cytoplasmic SOD (Zimmerman et al. 2002). Adenovirus was also used to inhibit NAD(P)H oxidase activator Rac1 production in the CVOs. NAD(P)H oxidase is a key enzyme involved in the formation of free radicals from oxygen. Again, the blood pressure and dipsogenic responses to Ang II were prevented (Zimmerman and Davisson 2004). This evidence indicates ROS production is involved in the Ang II-induced blood pressure responses. While these studies provide a basis of ROS-RAS interaction, it is necessary to elucidate key ROS members and provide further insight into the mechanisms behind this interaction. Given the emerging evidence that ROS is linked to the central action of Ang II, do the Ang II-induced pressor and dipsogenic actions require ROS member NAD(P)H oxidase specifically? To answer this question, we used NAD(P)H oxidase formation blocker gp91ds-tat, a compound discussed in Chapter 1, whose high specificity

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65 distinguishes the action of NAD(P)H oxidase from other ROS members. This study provides a comprehensive analysis of the overall central effect of NAD(P)H oxidase on Ang II action. We hypothesized that pretreatment of the brain with gp91ds-tat would prevent the ICV Ang II-induced pressor and dipsogenic responses in the SHR and WKY rat. Methods Animals ICV Cannulation Abdominal Aorta Cannulation Statistical Analysis A description of the above Methods is found in Chapter 2. In vivo Drug Delivery All drugs were delivered via the ICV cannula into the right lateral ventricle of unrestained rats. The drug solutions were delivered using an injector 4.5mm in length that was secured in the ICV cannula. The injector was attached to 0.38mm polyethylene tubing 120 cm in length (Becton Dickinson, Sparks, MD) with a 5l Hamilton syringe attached to the other end of the tubing. All injections were given to unrestrained, singly-housed rats. When testing the mechanism of Ang II requiring two drugs, the first drug was injected one hour prior to the second, and the dummy cannula was replaced following injection. 30ng Ang II dissolved in CSF was delivered to the right lateral ventricle (1l injection) using a 5l Hamilton syringe with fitted tubing + injector. gp91ds-tat and its scrambled peptide used as a control (200-800ng each, 2l, dissolved in CSF) were

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66 synthesized by the Temple University Protein Synthesis Core facility (Philadelphia, PA) and used to inhibit the action of NAD(P)H oxidase (Rey et al. 2001a; Rey et al. 2001b). Dipsogenic Response Water intake of WKY rats (n=3) following ICV delivery of Ang II was measured using sterile graduated glass pipets fitted with metal sipper tubes. The pipets were filled with water from the rats sterile water bottles. The pipets replaced the water bottles thirty minutes prior to the first injection. Water level in milliliters was recorded thirty minutes prior to, at injection time, and thirty minutes post Ang II injection. Drinking was calculated as the basal water level minus the water level thirty minutes following the Ang II injection. Rats remained singly-housed and were not restrained during the experiment. Results Angiotensin II Pressor Response Figure 4-1 depicts the changes in blood pressure and heart rate following ICV Ang II delivery in the SHR and WKY rat. ICV delivery of 30ng Ang II to conscious, free-moving WKY rats (n=5) resulted in an increase in blood pressure from 102.81+/-3.36 mmHg to 146.90+/-1.87 mmHg, a change of 44 mmHg (Figure 4-1C). Heart rate increased 44 bpm, from 320.13+/-7.37 bpm to 364.22+/-25.39 bpm (Figure 4-1D). Figure 4-1A shows representative blood pressure and heart rate recordings surrounding Ang II delivery to the WKY rat. The central Ang II response began within two minutes following injection, and maximum response occurred by ten minutes. Blood pressure and heart rate were normalized one hour post injection. Central delivery of Ang II (30ng) to the SHRs (n=3) also resulted in a pressor response. Blood pressure increased from 148.61+/-1.09 mmHg to 191.36+/-6.14 mmHg following Ang II treatment, a change of 43 mmHg (Figure 4-1C). Heart rate increased

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67 from 310.15+/-15.39 bpm to 432.93+/-9.64 bpm, an increase of 123 bpm (Figure 4-1D). Representative recordings of blood pressure and heart rate of the SHR are shown in Figure 4-1B. gp91ds-tat Prevents Ang II-Induced Blood Pressure gp91ds-tat was delivered ICV to the lateral ventricle, and radiotelemetry was used to measure BP and HR responses. Central administration of this NAD(P)H oxidase formation blocker prior to Ang II delivery yielded no change in blood pressure or heart rate in both the SHR and WKY rat. Pretreatment with gp91ds-tat one hour prior to ICV delivery of Ang II attenuated the Ang II-induced increases in blood pressure and heart rate in both rat strains (Figure 4-2). WKY rat Blood pressure and heart rate did not significantly change from basal levels (108.86+/-1.83 mmHg and 312.62+/-5.89 bpm, respectively) thirty minutes following ICV delivery of 800ng gp91ds-tat (106.81+/-3.72 mmHg and 329.54+/-8.11 bpm, respectively). Central delivery of 800ng gp91ds-tat one hour prior to ICV delivery of Ang II attenuated the Ang II-induced blood pressure by 67% in the WKY rat (146.90+/-1.87mmHg Ang II, 123.55+/-3.75 mmHg gp91ds-tat + Ang II) (Figure 4-2C). Pretreatment with 200ng gp91ds-tat did not significantly prevent the Ang II-induced pressor response. In addition, 800ng gp91ds-tat did not prevent the heart rate increase following Ang II delivery (364.22+/-25.39 bpm for Ang II, 358.84+/-24.15 bpm for gp91ds-tat+Ang II) (Figure 4-2D). SHR Blood pressure increased slightly from basal levels (154.22+/-5.65 mmHg) thirty minutes following ICV delivery of 200ng gp91ds-tat (167.58+/-3.62 mmHg). Heart rate

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68 increased from 325.04+/-8.32 bpm to 375.02+/-13.07 bpm. Figure 4-3 outlines the blood pressure and heart rate recordings and averages following deliveries of gp91ds-tat and Ang II. Central delivery of 200ng gp91ds-tat one hour prior to ICV delivery of Ang II attenuated the Ang II-induced peak blood pressure response by 67% in the SHR as well (Ang II 191.36+/-6.14 mmHg Ang II, 168.22+/-5.10 mmHg gp91ds-tat + Ang II) (Figure 4-3C). Pretreatment with gp91ds-tat prevented the heart rate increase following Ang II delivery by 58% (Ang II 432.93+/-9.64 bpm Ang II, 376.98+/-11.79 bpm gp91ds-tat + Ang II) (Figure 4-3D). Thus, pretreatment with gp91ds-tat resulted in an attenuation of blood pressure increase induced by central Ang II delivery in both the SHR and WKY rat. In contrast, a scrambled form of the peptide did not prevent the Ang II-induced blood pressure in a WKY rat (135.16 mmHg Ang II, 137.26 mmHg scrambled + Ang II), indicating the gp91ds-tat peptide was specific in its blockade of NAD(P)H oxidase. gp91ds-tat Prevents Ang II-Induced Drinking Next we determined if the dipsogenic responses of ICV Ang II administration involve the ROS signaling pathway. Figure 4-4 is a graphical representation of the water intake of the WKY rats following either Ang II treatment alone or central delivery of Ang II following gp91ds-tat pretreatment (n=3). Delivery of Ang II into the lateral ventricle resulted in 8.8+/-0.5 ml water intake within thirty minutes following injection. Drinking began within two minutes of ICV delivery. Pretreatment with 800ng gp91ds-tat, however, resulted in water intake of only 2.6+/-1.4 ml thirty minutes following Ang II injection, thereby attenuating the Ang II dipsogenic response by 70% (Figure 4-4).

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69 Discussion These results support the hypothesis that brain Ang II increases blood pressure and heart rate through a ROS-mediated pathway. Specifically, this study establishes that blocking NAD(P)H oxidase in the brain prevents the pressor and dipsogenic actions of Ang II. This response was observed both in the hypertensive SHR and in the normotensive WKY rat. The scrambled form of the peptide did not affect Ang II-induced blood pressure; therefore, gp91ds-tat was specific in its action. gp91ds-tat alone had no effect on blood pressure or heart rate, indicating that these basal physiological mechanisms do not involve NAD(P)H oxidase. This work complements previous published studies involving the link between central Ang II action and ROS in mice. Adenovirus encoding SOD delivered ICV also prevented Ang II-induced blood pressure (Zimmerman et al. 2002). One caveat of that study, however, was the sole use of SOD to inhibit ROS accumulation. While SOD is an important component of ROS conversion, it does not discriminate whether Ang II acts upstream of superoxide formation. gp91ds-tat, however, is a highly specific blocker of NAD(P)H oxidase formation, thereby useful for analysis upstream of superoxide in Ang II action (Rey et al. 2001b). Its use in this study therefore confirms the role of ROS, specifically NAD(P)H oxidase, in Ang II-induced pressor and dipsogenic responses. These studies were the first to measure the affect of NAD(P)H oxidase blockade on central Ang II pressor response in both hypertensive and normotensive models. The comparison of WKY rat to SHR response is critical in understanding the role of ROS in Ang II action. First, the blood pressure response between the two models was the same. This result was unexpected given the characterized heightened pressor response in the SHR (Wright et al. 1986). This discrepancy may be due to the large dose of Ang II

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70 (30ng), yielding a potential maximum response in both strains. Pretreatment with gp91ds-tat prevented the Ang II pressor effect by 67% in both the SHR and WKY rat. Therefore, the influence of NAD(P)H oxidase on the Ang II-induced blood pressure is not dependent on the basal blood pressure. However, while the heart rate response was attenuated 58% in the SHR with gap91ds-tat pretreatment, no change was measured in the WKY rat. This discrepancy is likely due to the differences in Ang II-induced heart rate between the two models. In these studies, Ang II-induced heart rate in the SHR was three times that of the WKY rat, which was only transiently increased bpm. Pretreatment with gp91ds-tat in the SHR attenuated the heart rate response to the same level following ICV Ang II delivery in the WKY rat. It is therefore likely that this 40 bpm change in heart rate is independent of NAD(P)H oxidase signaling. One caveat of this study is while it determined the overall central link of NAD(P)H oxidase to Ang II, it did not extrapolate specific nuclei involved, nor did it address cellular mechanisms underlying this central ROS-RAS control. The AT 1 R has been localized to brain nuclei including the PVN, RVLM, and NTS, where activation through Ang II binding elicits sympathetic nerve activation and therefore an increase in pressure (Bastos et al. 1997; Mangiapane and Simpson 1980; Muratani et al. 1993; Tanaka et al. 2001). Thus, it is necessary to further characterize the role of NAD(P)H oxidase on an individual nuclei basis. Future studies should address this question in order to more completely elucidate the mechanism of Ang II-ROS action. Signaling of AT 1 R and NAD(P)H oxidase has begun to be elucidated. Most studies in this regard have focused on the peripheral Ang II and ROS, but the mechanisms may indeed apply to the brain as well. In summary, AT 1 R stimulation activates PKC and

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71 therefore Rac, a subunit needed to activate NAD(P)H oxidase (Brandes 2003; Gregg et al. 2003). Functional NAD(P)H oxidase can therefore convert oxygen to hazardous H 2 O 2 thus increasing ROS production. This increased ROS is theorized to increase sympathetic nerve activity, though this mechanism is unknown. The electrophysiological basis of the role of ROS in central Ang II pressor response was recently elucidated by post-doctoral fellow Dr. Cheng-wen Sun of our research group. Dr. Sun used both ROS inhibitors and generators in conjunction with Ang II and measured firing rate and the delayed rectifier potassium current (I Kv ). Both Tempol and gp91ds-tat attenuated the Ang II-induced neuronal firing rate by 70% and 50%, respectively. Alternately, ROS generator Xanthine-Xanthine oxidase increased firing rate above basal levels, indicating that ROS functions independently of extracellular Ang II stimulation. In addition, the inhibition of I Kv by Ang II was blocked by the concomitant treatment with gp91ds-tat. These findings indicate that ROS is involved in the Ang II-mediated increase in neuronal activity. In order to confirm these findings, specific channel involvement and kinetics need to be examined. This study therefore provides a cellular basis for the pressor and dipsogenic responses generated from the in vivo studies. It also suggests the role of ROS in central control of hypertension acts through neuronal activation. Given that ROS continues to emerge as an important regulator in several disease states coupled with this evidence linking NAD(P)H oxidase to Ang II function in the central control of blood pressure, are endogenous levels of ROS activity elevated in human hypertension? If there is an increase in ROS production, can this dysregulation be corrected by either SOD mimetics or NAD(P)H oxidase blockers in order to diminish the

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72 accumulation of superoxides? These questions must be addressed in future studies in the hopes of reversing the hypertensive state. In summary, the pressor and dipsogenic actions of central Ang II are mediated by ROS action. Specifically, ROS member NAD(P)H oxidase was implicated in this mechanism. Both in vivo and in vitro data support the regulation of central Ang II pressor action by ROS. These results concur and expand on previous studies linking ROS to Ang II in the brain. While the specific mechanism by which ROS increases neuronal firing rate remains to be elucidated, this study provides a solid foundation on which to study the involvement of NAD(P)H oxidase in the central control of blood pressure. This essential requirement of ROS in central pressor control invites thought as to what other mechanisms ROS involved in the brain regulate blood pressure. For instance, is ROS production necessary for the central action of sEH inhibition described in Chapter 3? Given the wide range of responsibilities of ROS, it is indeed plausible that ROS production underlies all the major central blood pressure regulators. In theory, these brain mechanisms contributing to blood pressure control may converge at the ROS production level. Chapter 5 will outline and discuss data related to the mechanism underlying blood pressure and heart rate increases following central sEH inhibition.

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73 Figure 4-1 Effect of central Ang II on blood pressure and heart rate. 30ng Ang II was delivered ICV to SHR and WKY rats. Blood pressure and heart rate were recorded using radiotelemetry (see Methods). Bars represent peak increases over 10 minutes. Representative recordings of WKY (A) and SHR (B). Red and blue lines indicate HR and MAP, respectively. Arrows represent Ang II injection. (C,D) Quantitation of MAP and HR, respectively. White and black bars represent mean +/SEM of basal and Ang II measurements, respectively (p<0.05, n=5/WKY, n=3/SHR).

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74 Figure 4-2 Effect of gp91ds-tat on Ang II-induced BP response in the WKY rat. 800ng gp91ds-tat was injected ICV one hour prior to Ang II. Representative recordings of MAP (A) and HR (B). Arrows represent point of drug delivery. Quantitation of MAP (C) and HR (D). Bars represent means +/-SEM; significantly different between groups (p<0.05, n=5).

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75 Figure 4-3 Effect of gp91ds-tat on Ang II-induced BP response in the SHR. 200ng gp91ds-tat was injected ICV one hour prior to Ang II. Representative recordings of MAP (A) and HR (B). Arrows represent points of drug delivery. Quantitation of MAP (C) and HR (D). Bars represent means +/-SEM; significantly different between groups (p<0.05, n=3).

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76 Figure 4-4 Dipsogenic responses of ICV gp91ds-tat and Ang II. Water intake following Ang II injection with and without 800ng gp91ds-tat pretreatment was measured. Bars represent water intake means +/SEM (p<0.05, n=3).

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CHAPTER 5 MECHANISM OF CENTRAL SEH INHIBITION ON BLOOD PRESSURE REGULATION Introduction sEH action in the brain is emerging as critical in blood pressure regulation. Chapter 3 established that central sEH inhibition results in an increase in blood pressure and heart rate in the SHR model of hypertension. In addition, this inhibition yielded an increase in heart rate in the WKY rat. Taken together, these results suggest a role for sEH in the central control of blood pressure. Essentially, though, how does central sEH inhibition result in an increase in blood pressure and heart rate in the SHR? This chapter seeks to elucidate the mechanism underlying this physiological response. The pressor effects of central sEH inhibition are opposite from the depressor response following IP delivery of DCU to the SHR and therefore suggest an alternate pathway of sEH action in the brain. Peripheral sEH functions as a regulator of vasoactivity by converting vasodilatory EETs to their less active diol forms (Yu et al. 2000a). Decreased vasodilation in smooth muscle cells of the arterial walls leads to increased probability of contractility, which results in an increase in blood pressure. While this proposed vasoactive mechanism of sEH in the periphery coincides with its increased expression in hypertension, the effect of central sEH inhibition likely diverges from this pathway. The question is in what way? Is the action of sEH inhibition related to a known pathway of central control of blood pressure? We sought to answer these 77

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78 questions through a series of experiments designed to elucidate the pathway of central sEH control of blood pressure. The first goal is to extrapolate the overall physiological mechanism behind the increase in blood pressure and heart rate following central sEH inhibition in the SHR. As previously discussed, the SHR is characterized by several dysregulatory pathways. One is the decreased baroreceptor reflex described in Chapter 1 (Hayashi et al. 1988). Another is the increased sympathetic nerve activity that results in an increase in blood pressure (Biaggioni 2003). Given that the SHR is predisposed to these pathways, we hypothesized that AUDA-induced blood pressure and heart rate changes in the SHR involved baroreceptor reflex dysregulation and sympathetic nerve activation. The second goal is to elucidate the cellular and signaling mechanisms involved in the AUDA-induced pressor response in the SHR. This undertaking is complex and likely to involve years if not decades of study. However, the start of this discovery must begin with what we know of sEH action, its conversion of EETs. Does the action of central sEH inhibition involve the accumulation of its substrate, the EETs? Peripheral studies have focused on this branch of the arachidonic acid pathway as the powerhouse behind sEH cardiovascular effects. However, sEH was originally identified as a converter of xenobiotic material foreign to the organism (Grant et al. 1993). In fact, several substrates have already been identified for sEH in addition to its well-known role in EET metabolism (Sinal et al. 2000b). Alternately, sEH itself may function to regulate channel kinetics leading to changes in firing rate patterns or even regulate neurotransmitters such as Ang II or vasopressin. Based on the primary role of sEH as an enzyme involved in the

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79 epoxygenase branch of arachidonic acid conversion, however, we hypothesized that sEH inhibition leads to an increase in blood pressure due to EET accumulation in the brain. Aside from the question whether the sEH substrate EETs are involved in its central action on blood pressure, it is important to begin to understand how this inhibition leads to an increase in blood pressure. Is there a connection of sEH to known regulators of central blood pressure control such as Ang II or another sympathetic activator? Chapter 2 presented evidence that sEH expression increases in the hypothalamus following ICV delivery of Ang II. Chapter 4 further analyzed the role of brain Ang II signaling by incorporating the NAD(P)H oxidase inhibitor gp91ds-tat. These studies linked central Ang II action to ROS member NAD(P)H oxidase. ROS has also been linked to epoxygenase member CYP 2C9 (Fleming et al. 2001b). Given that ROS activity in cardiovascular control, both peripheral and central, continues to emerge as a strong regulator, we hypothesized that the blood pressure effects stemming from sEH inhibition were mediated by NAD(P)H oxidase. Finally, what cell type in the brain is responsible for the pressor response following central sEH inhibition? Previous studies have localized both CYP and EETs mainly to astrocytes, though CYP has also been found in the cerebral vasculature as well as dopaminergic neurons. Our studies discussed in Chapter 2 localized sEH to both neurons and glia. Therefore all brain cell types are potential targets for the action of sEH inhibition. However, overwhelming evidence reveals neurons as the main regulator in the central control of blood pressure. For example, increased neuronal firing rate from the RVLM and NTS to the IML result in an increase in sympathetic activation and hence

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80 increased blood pressure. The following studies seek to identify key mediators and pathways in the increase in blood pressure following sEH inhibition in the SHR brain. Methods Animals ICV Cannulation Abdominal Aorta Cannulation Blood Pressure and Heart Rate Recordings Statistical Analysis The above Methods are described in Chapters 2 and 3 In vivo Drug Delivery All drugs were delivered as bolus injections via the ICV cannula into the right lateral ventricle of unrestained rats as described in Chapter 3. When testing the mechanism of sEH inhibition requiring two drugs, the first drug was injected one hour prior to the second, and the dummy cannula was replaced following injection. MS-PPOH (680ng, 2l, dissolved in CSF), a kind gift from Dr. John Falck, University of Texas Southwestern Medical Center, was used to block the formation of EETs (Brand-Schieber et al. 2000b). 11-nonyloxy-undec-8-enoic acid (11-NODA, 100ng, 1l, dissolved in CSF), an EET agonist, was also provided by Dr. Falck. gp91ds-tat and its scrambled peptide used as a control (100ng each, 2l, dissolved in CSF) were synthesized by the Temple University Protein Synthesis Core facility (Philadelphia, PA) and used to inhibit the action of NAD(P)H oxidase (Rey et al. 2001a; Rey et al. 2001b). Evaluation of Spontaneous Baroreceptor Reflex Gain To evaluate baroreceptor reflex function, an index of gain was determined from spontaneous changes in systolic blood pressure and pulse interval using a time-series

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81 method for the rat, as described (Oosting et al. 1997; Waki et al. 2003). This method correlates to traditional pharmacological methods for measuring the baroreceptor reflex. However, because this method does not distinguish between an increase in blood pressure and heart rate either by a decrease in the baroreceptor reflex or by the pharmacological agent itself, this measurement is assumed to be an estimate of spontaneous baroreceptor reflex gain. To calculate this index of baroreceptor reflex gain, spontaneous ramps of systolic blood pressure changes (at least four beats) were extracted from the moving average data. The data was collected at a frequency of 500 Hz. Measurements of systolic blood pressure and pulse interval ramps were made at delays of three to five beats based on previous findings (Oosting et al. 1997). Pulse interval was compared to systolic blood pressure during these delays to calculate the spontaneous baroreceptor reflex gain. Only positive slope values were used to avoid contaminating baroreceptor reflex data with non-baroreceptor-mediated changes in pulse interval. This analysis was completed by Dr. Hidefumi Waki, Department of Physiology, Bristol University, England. Analysis of Heart Rate Variability Heart rate variability was analyzed using a fast Fourier transformation (FFT). For each ten-minute recording period, the beat-to-beat pulse interval data were converted into data points every 100 ms using a spline interpolation. Power spectral density was then computed using FFT algorithm and averaged. It is generally accepted that the high frequency (HF) component of heart rate variability is mediated by cardiac parasympathetic tone (Pagani et al. 1986a; Pagani et al. 1986b). This analysis was completed by Dr. Hidefumi Waki, Department of Physiology, Bristol University, England.

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82 Neuronal Firing Recording Spontaneous action potentials were recorded with the whole-cell voltage clamp configuration in current clamp mode. Neurons cultured from hypothalamus were bathed in Tyrodess solution containing (in mM): 140 NaCl, 5.4 KCl, 2.0 CaCl 2 2.0 MgCl 2 0.3 NaH 2 PO 4 10 HEPES, and 10 dextrose, pH adjust to 7.4 with NaOH. The patch electrodes (resistances from 3-4 MOhms) were filled with an internal pipette solution containing (in mM): 140 KCl, 4 MgCl 2 4 ATP, 0.1 guanosine 5-triphosphate, 10 dextrose, and 10 HEPES, pH adjusted to 7.2 with KOH. The resting membrane potential was defined as the potential within a 1 s time period during which there were no spontaneously firing action potentials. Neuronal firing rate was measured as the number fully developed action potentials (depolarization beyond 0 mV) per second (Hz). This study was completed by Dr Chen-wen Sun of our research group. Results Waveform Analysis of Baroreceptor Parameters Because of the concomitant increase in blood pressure and heart rate in the SHR following sEH inhibition, it was hypothesized that central AUDA delivery impaired the ability of the baroreceptors to regulate physiological changes. In order to test this hypothesis, spontaneous baroreceptor reflex gain and cardiac vagal tone were measured using waveform analysis in response to sEH inhibition. Index of spontaneous baroreceptor reflex gain Analysis of waveform telemetry data revealed a 56+/-5% decrease in the estimate of spontaneous baroreceptor reflex gain (p<0.05) following sEH inhibition in the SHR. Specifically, the index of baroreceptor reflex gain decreased from a basal level of 0.82+/-0.06 ms/mmHg to 0.36+/-0.04 ms/mmHg at the peak of the pressor response from sEH

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83 inhibition (Figure 5-1A). In the WKY rat, the index of baroreceptor reflex gain was 1.09+/-0.11 at basal levels and 1.13+/-0.08 three to four hours after ICV delivery of 15ng AUDA. Thus, no significant change in the baroreceptor reflex gain index was measured in the WKY rat. In comparing basal levels of the two strains, the index of the baroreceptor reflex gain of the WKY rat was 33% higher compared to the SHR, though this difference was not significant. High frequency analysis of pulse interval Given the decrease in the index of baroreceptor reflex gain in the SHR following central sEH inhibition, high frequency analysis of pulse interval was then analyzed to determine if this component was also involved in this concomitant increase in blood pressure and heart rate. This measurement correlates to the level of cardiac vagal tone. In the SHR, this cardiac vagal tone indicator measurement decreased from 16.68+/-2.55 ms to 10.39+/-0.28 ms at the peak response of sEH inhibition (Figure 5-1B). This result translated to a 33+/-8% decrease in heart rate analysis of pulse interval following ICV AUDA administration in the SHR. In contrast, no significant changes were observed in the WKY rats (13.91+/-0.84 ms basal, 13.74+/-0.95 ms 15ng AUDA). These data demonstrate that increases in blood pressure and heart rate are consistent with a decrease in baroreceptor reflex functions in the SHR. AUDA Regulation of Sympathetic Nerve Activity Regulation of sympathetic nerve activity is one of the major mediators of the central control of blood pressure. Given the high impact of this system plus its neuronal origin that corresponded with previous sEH expression studies, atenolol, a 1R antagonist, was used to test whether the effects of sEH inhibition were related to sympathetic nerve activity. 1mg/kg atenolol was delivered IV following 15ng AUDA

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84 ICV delivery to two SHRs. The results from this preliminary study are shown in Figure 5-2. Atenolol delivery completely prevented the increase in blood pressure increase following central AUDA administration (34.54+/-7.23 mmHg AUDA, 0.69+/-3.73 mmHg atenolol + AUDA, n=2). No statistical analysis could be completed due to the low number of animals in this study. These data indicate that AUDA-induced increase in blood pressure functions through sympathetic nerve activity. Increased EETs Linked to Increase in BP and HR The first step in deducing the mechanism underlying sEH inhibition-induced blood pressure was to examine the role of EETs, the main physiological substrate of sEH. Since sEH converts EETs to DHETs, inhibiting the enzyme results in the accumulation of EETs. Thus, we hypothesized that EETs may be the vital in this central control of blood pressure. MS-PPOH This hypothesis was tested initially with the use of MS-PPOH, an inhibitor of the epoxygenase pathway that generates EETs and related PUFA epoxides (Brand-Schieber et al. 2000a). Figure 5-3 outlines the pressor response to 15ng AUDA with and without pretreatment of MS-PPOH in the SHR. Figures 5-3A,B are representative blood pressure recordings from ICV delivery of AUDA alone (A) and with ICV delivery of MS-PPOH one hour prior to sEH inhibition (B, n=5). Figure 5-3C is a graphical representation of the pressor response resulting from the treatments. While central delivery of AUDA increased blood pressure 38.99+/-4.29mmHg, pretreatment with MS-PPOH attenuated this AUDA-induced increase in blood pressure by 65% in the SHR (14.43+/-8.04 mmHg). However, MS-PPOH alone had no effect on basal blood pressure.

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85 11-NODA While MS-PPOH provided the first evidence that sEH action was through EET production, it was imperative to determine if the action was through EETs specifically or through another product of epoxygenase. Thus, EET agonist 11-NODA (100ng) was delivered ICV in the WKY rat to determine whether EET-specific agonist would have any effect on blood pressure or heart rate (Figure 5-4). This central administration of an EET agonist results in a transient increase in blood pressure (blue) and heart rate (red) in the WKY rat (Figure 5-4, n=6). This increase was biphasic for blood pressure increases, each increase lasting only about 10 min. The first peak was recorded just after delivery and the second 15-30 minutes following the first. Blood pressure increased 13.2+/-2.5 mmHg in the first peak and 8.8+/-3.2 mmHg in the second peak (Figure 5-4B). Heart rate increased 61+/-19 bpm (Figure 5-4C). Effects of sEH Inhibition Function through ROS To further examine the mechanism underlying this central pressor response, we examined the role of ROS on sEH-inhibitor-mediated increase in blood pressure in the SHR. Reasoning behind this study was two-fold. First, epoxygenases have been linked to ROS, albeit in the periphery. In addition, Chapter 4 discussed the importance of ROS member NAD(P)H oxidase is in the central control of hypertension. Figure 5-5 outlines the blood pressure and heart rate data from this study (n=3). The top panels are representative blood pressure and heart rate recordings of an SHR treated with AUDA alone (Figures 5-5A,C) and AUDA with gp91ds-tat pretreatment (Figures 5-5B,D). Figure 5-5E is a graphical representation of the peak blood pressure changes showing that while ICV delivery of AUDA resulted in a 39.20+/-3.03 mmHg increase in mean arterial pressure, pretreatment with gp91ds-tat caused the AUDA-induced blood pressure to only

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86 increase 6.10+/-5.78 mmHg, an 85% attenuation. Figure 5-3F is a graphical representation of the peak heart rate changes indicating that AUDA delivery resulted in a 85.66+/-4.99 bpm change, yet pretreatment with gp91ds-tat yielded an AUDA-induced heart rate change of 19.24+/-12.48 bpm, thereby attenuating the heart rate response by 77%. In contrast, gp91ds-tat alone had no effect on blood pressure and heart rate. These results indicate that ROS member NAD(P)H oxidase is involved in the sEH inhibitor-induced increase in blood pressure and heart rate. Discussion These series of studies succeeded in beginning to elucidate the complex signaling involved in the central sEH-mediated blood pressure control. Two important findings can be extrapolated from these results. First, the pressor effects stemming from central sEH inhibition are linked to the increase in its substrate, the EETs. Second, the mechanism leading to the increase in blood pressure involves ROS, specifically NAD(P)H oxidase. These two conclusions, taken together, form the foundation of how sEH and EETs function in the brain to elicit blood pressure and heart rate effects. EET studies involving formation blockers as well as agonists strongly suggest that this central pathway involves the sEH substrate EETs. Pretreatment with MS-PPOH prevented sEH increases in blood pressure in the SHR, though alone this compound had no effect on blood pressure. This lack of blood pressure response may be explained by the high sEH expression in the SHR that would prevent any basal effects from the EETs While this result was good evidence that EETs were involved, the non-specificity of MS-PPOH to EETs still questioned whether EETs were the main regulator. For example, since MS-PPOH inhibits epoxygenase action, it blocks the formation of all epoxygenase products (Brand-Schieber et al. 2000b). Therefore, EET agonists were delivered ICV to

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87 determine whether increasing the agents themselves resulted in a change in blood pressure. ICV delivery of the agonists resulted in a biphasic increase in blood pressure as well as an increase in heart rate in the WKY rat, indicating these sEH substrates are indeed the potentiators of sEH inhibitor pressor effects. The reason behind the biphasic increase in pressure is unknown at this time, but may be related to differences in immediate EET activity on the cerebral microcirculation and later effects involving neuronal activation pathways in cardiovascular-relevant brain areas. Thus, the role of central EETs appears to be distinct from that of its peripheral expression, which is characterized by its vasodilatory properties that are associated with a decrease in blood pressure. These results strongly indicate that the effects of central sEH inhibition involve the accumulation of its substrate. We further elucidated the central sEH pathway through studies involving oxidative stress. The mechanism linking central sEH inhibition to the increase in blood pressure and heart rate appears to function through ROS (Fleming 2001; Fleming et al. 2001a). gp91ds-tat, an NAD(P)H oxidase blocker, was used to elucidate the ROS involvement. Central delivery prevented AUDA-induced increase in blood pressure and heart rate in the SHR. gp91ds-tat also prevented the increase in neuronal firing rate induced by sEH inhibitors in vitro. As previously discussed, ROS had been linked to the epoxygenase pathway; however, this is the first link of the NAD(P)H oxidase arm of ROS to EETs and sEH. One major question is what cell type is involved in stimulating blood pressure following central sEH inhibition. sEH is localized to both neurons and glia, yet its substrate is primarily localized to astrocytes. In order to begin to answer this question,

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88 Electrophysiological studies were performed by post-doctoral fellow Dr. Cheng-wen Sun. The top two panels of Figure 5-6A are representative neuronal firing rate recordings with and without AUDA delivered to the culture media. Delivery of 0.4mg/ml AUDA resulted in a two-fold increase in neuronal firing rate in the SHR neurons (Figure 5-6B). Next, gp91ds-tat was added to the cultures in conjunction with AUDA to determine if the role of ROS observed in vivo is related to neuronal firing rate regulation. Figure 5-6A shows representative tracings of firing rate following AUDA treatment, gp91ds-tat + AUDA or AUDA + scrambled tat peptide. Figure 5-6B is a graphical representation of neuronal firing of these different treatments. AUDA-induced increase was attenuated 75% by pretreatment with 5M gp91ds-ta. Taken together, these observations provide cellular insight into the effects of sEH and NAD(P)H oxidase on neuronal activity in the SHR. These electrophysiological studies therefore supported the role of neurons in the central pressor action of sEH. The increased neuronal firing rate with AUDA treatment coupled with its attenuation in the presence of gp91ds-tat indicates effects from sEH inhibition are through neuronal targeting. However, these studies do not discount the role of glial or endothelial cells in sEH central control (Medhora et al. 2001). These cultures are 80% neurons; therefore, the other 20% of cells may be essential in providing EETs for sEH action. In order to resolve this issue, sEH and EETs in the glia should be blocked to determine if sEH inhibition still results in a pressor response. This targeted disruption can be accomplished with the use of GFAP promoters coupled with either an antisense or ribonuclease interference strategy in the brain.

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89 We propose that an increase in EETs by inhibition of sEH leads to an increase in ROS in the brain and hence an increase in blood pressure. An alternate possibility is that ROS acts upstream and actually stimulates EETs which therefore leads to an increase in blood pressure. This possibility cannot be excluded since the experiments presented here showed only a relationship between sEH inhibition and ROS. In order to resolve this issue, gp91ds-tat should be delivered prior to EET agonist delivery. If pretreatment with ROS inhibitors prevents the actions of the EET agonists, then ROS most likely acts downstream of EETs in the central control of hypertension. If, however, EET agonists still exert a pressor effect with effective ROS inhibition, then EETs are likely to act downstream of ROS, and the proposed mechanism needs to be readjusted. At this point, we hypothesize that ROS acts downstream of EETs given the direct involvement of brain ROS to blood pressure control that has been elucidated over the past decade. Because ROS seems to be a common denominator in several systems regulating hypertension, it is likely that several factors including members of the epoxygenase pathway function to regulate the production of ROS in the brain. The first two studies in this series addressed the overall mechanism underlying blood pressure effects in the SHR following sEH inhibition. Waveform analysis revealed that the baroreceptor reflex was decreased in the SHR, providing an explanation for the concomitant increase in blood pressure and heart rate. The SHR is known to be less efficient with its control of heart rate regulation, and this result further recognizes its dysfunction. The WKY rat had no change in baroreceptor reflex, thus explaining why blood pressure is increased only in the SHR.

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90 The second finding related to the overall mechanism of sEH inhibition was that atenolol blocked the effects of AUDA, suggesting involvement of sympathetic nerve activating. While this study cannot be statistically analyzed since only two rats were involved in the study, it provides a starting base on which to study sympathetic activation in future studies. Sympathetic activation is a major regulator of the brains control of blood pressure and is involved in the majority of these central pathways including the RAS (Dampney 1994; Di Nicolantonio et al. 1982). Given the activation of sympathetic nerve activity by dopaminergic neurons, this study points to neurons as the mediators of central sEH inhibition action. Taken together, these studies provide a critical look into the function and mechanism of central sEH. However, discrepancies between expression data and inhibitor data invite a more comprehensive analysis of sEH in specific brain regions. In order to fully elucidate the role of sEH in the central control of hypertension, it is important to determine what effect overexpressing the gene will have on blood pressure and heart rate. Chapter 6 discusses studies on gene therapy utilizing a viral vector to deliver sEH to cardiovascularly-relevant nuclei in the brain of hypertensive and normotensive rats.

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91 Figure 5-1 Effect of central AUDA delivery on baroreceptor parameters. 15ng AUDA was injected into the lateral ventricle of SHR and WKY rats. (A) Baroreceptor reflex gain was measured as described in Methods and is expressed as ms/mmHg. (B) HF analysis of PI, bars represent mean +/SEM (see Methods). significantly different between basal and AUDA treatments (p<0.05, n=6). Reprinted with permission from The FASEB Journal.

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92 Figure 5-2 Effect of atenolol on AUDA-induced blood pressure. 1mg/kg atenolol was delivered IV to the SHR following ICV delivery of 15ng AUDA. Blood pressure was recorded using radiotelemetry. Bars represent changes in mean arterial pressure in mmHg (n=2).

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93 Figure 5-3 Effect of MS-PPOH on AUDA-induced blood pressure. SHRs were pretreated with ICV MS-PPOH one hour prior to AUDA injection. BP was monitored using radiotelemetry. (A) Representative MAP recording following AUDA injection, (B) Representative MAP recording following MS-PPOH + AUDA injections, x-axis is time in minutes. Arrows represent point of injection. (C) Quantitation of blood pressure data, mean +/SEM. significantly different between groups (p<0.05, n=5). Reprinted with permission from The FASEB Journal.

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94 Figure 5-4 Effect of EET agonist on BP and HR in the WKY rat. 11-NODA was injected into the lateral ventricle, and BP and HR was recorded using radiotelemetry (see Methods). Representative recordings of BP and HR (A); x-axis represents time pre and post-injection. (B,C) Quantitation of BP and HR in the WKY rats, mean +/SEM. significantly different between treatments (p<0.05, n=6).

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95 Figure 5-5 Effect of ROS inhibition on AUDA-induced BP and HR response in the SHR. NAD(P)H oxidase activity blocker gp91ds-tat was injected ICV one hour prior to AUDA delivery. Representative recordings of BP (A,B) and HR (C,D). (A,C) AUDA treatment alone. (B,D) gp91ds-tat + AUDA treatment. Arrows represent points of injection. (E,F) Quantitation of BP and HR as mean +/-SEM. significantly different between groups (p<0.05, n=3). Reprinted with permission from The FASEB Journal.

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96 Figure 5-6 Effect of AUDA and gp91ds-tat on neuronal firing rate. (A) Action potential recordings of neurons +/treatment. (B) Quantitation of firing rate, mean+/-SEM. significantly different from control. significantly different from AUDA (p<0.05). This figure was used with permission from Dr. Chengwen Sun, University of Florida and reprinted with permission from The FASEB Journal.

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CHAPTER 6 EFFECT OF SEH OVEREXPRESSION IN THE PVN OF SHR AND WKY RATS Introduction Central sEH inhibition resulted in an increase in blood pressure and heart rate in the SHR, an effect opposite from that in the periphery. Additional sEH inhibitor studies indicate that central pressor action of EETs involves ROS production and sympathetic nerve activation, thereby providing a novel role for EETs in the brain. Thus far, these studies into the central role of sEH on cardiovascular function have utilized pharmacological agents delivered to the lateral ventricle to inhibit the action of sEH. These results, however, cannot distinguish the role of central sEH in distinct nuclei. This chapter examines the effect of sEH overexpression in the PVN on blood pressure and heart rate in the SHR and WKY rat. Chapter 1 discussed the complex pathways involved in the central control of blood pressure. Specific brain areas elicit different effects; for example, activation of the RVLM and CVLM yields a pressor and depressor effect, respectively, yet both nuclei are located in the brainstem (Guertzenstein and Silver 1974). Similarly, the effects of the sEH inhibitors injected into the lateral ventricle are a culmination of the responses of each brain nucleus exposed to DCU and AUDA. In order to comprehensively understand the role of sEH in the brain, it is imperative to examine its blood pressure effects when localized to distinct cardiovasculary-relevant brain nuclei. The PVN, located in the hypothalamus, is one such nucleus whose neuronal activation stimulates sympathetic nerve activity and affects blood pressure. While the 97

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98 complete mechanism of this activation is not fully understood, we know that it involves neuronal projections from the PVN to the IML in the spinal column either directly or via the RVLM in the brainstem (de Wardener 2001; Strack et al. 1989). Stimulation of the PVN produces a pressor response, while denervation of this nucleus depresses blood pressure (Dampney 1994; Harland et al. 1989). Thus, the PVN provides an intriguing start point to elucidate the role of sEH in specific brain nuclei. Given the increased expression of sEH in hypertensive models, it is logical to measure the physiological effects of overexpression in normotensive models as well as further overexpression in hypertensive models. Overexpression of a gene can be accomplished by several methods, including using gene therapy to deliver the gene of interest. Both viral and nonviral vectors can be used to accomplish this gene therapy, each with its own advantages and disadvantages. Naked DNA, a nonviral vector, is noted for its safety; however, it has low efficiency due to its low rate of transduction and limited expression time (Wivel and Wilson 1998). Similarly, liposomes are safe due to its lack of pathogenicity, but its expression is also low because it does not integrate into the genome (Aoki et al. 1997; Morishita et al. 2000; Raizada et al. 2000). Both nonviral methods have been used for gene transfer, though low efficiency has limited their success in trials. Viral vector-mediated delivery is an appealing choice for gene transfer because of their natural ability to efficiently infect different cell types. Theoretically, they are a useful alternative in in vivo studies. Viruses are extremely diverse, each with different advantages and disadvantages based on efficiency, limitations to transduction of non-dividing cells, as well as gene insert size limitations. Therefore, when choosing a gene

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99 delivery vector, it is essential to consider the target cell type, timeframe of genetic action, and gene size. Many clinical trials involving gene therapy have used the adenovirus, a family of viruses that includes members that causes the common cold. Adenovirus is an attractive choice because (i) large quantities can be produced with high efficiency; (ii) it is able to infect nondividing cells; and (iii) it results in long-term expression of the gene (Lu et al. 1998; Raizada et al. 2000). In addition, it has a wide tissue distribution and effectively diffuses from the site of injection. However, two recent clinical trials involving adenovirus resulted in adverse effects and even one death, highlighting the potential dangers of both the immunological response as well as an unknown site of integration (Amalfitano and Parks 2002; Reid et al. 2002). The adeno-associated virus, in contrast, is not immunogenic and integrates into a particular chromosome. Unfortunately, the virus has a small capacity for the gene insert and is difficult to produce on a large scale (Flotte and Carter 1995). The final major class of viral vectors is the retrovirus, a RNA virus that reverse transcribes into DNA. The retrovirus can hold a large gene insert and is able to be produced on a large scale but has an unknown site of integration (Raizada et al. 2000; Raizada et al. 1999). A subset of the retrovirus known as the lentivirus was chosen as the viral vector for the sEH overexpression studies. The lentiviral vector is a modified form of the human immunodeficiency virus type 1 (HIV-1). While the wild-type virus encodes nine genes, the genes not essential to its vector function are eliminated in this modified virus, thereby only housing four of the original nine genes. These remaining genes are known as cis-acting sequences because they are only recognized by viral proteins (Buchschacher and Wong-Staal 2000). Because safety needs be ensured with this type of virus, viral production is accomplished

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100 only when the genes, individually prepared, are combined. Due to the removal of half of the viral genome, the virus only replicates one time and therefore does not infect again, further ensuring its safety. Lentivirus was selected as the viral vector of choice for five reasons. First, it is able to infect nondividing cells, a requirement when studying neurons (Naldini et al. 1996; Verma and Somia 1997). Second, the vector can house a large transgene up to 18 kb. While sEH is only ~1.7 kb, future studies may include additional regulatory genes that can easily be cloned into the vector. Third, the vector allows long-term expression of the sEH gene, thereby determining a true progression of sEH action. Fourth, brain injections of lentivirus revealed it did not diffuse widely from the point of injection, making it an attractive candidate for targeting specific brain nuclei. Finally, the large-scale production of the lentiviral vector was optimized in our laboratory (Coleman et al. 2003), and this vector has been used and characterized in other cardiovascular studies by members of this research group (Metcalfe et al. 2004). Thus, the lentiviral vector was ideal for the overexpression of the sEH gene. The lentiviral vector used in these experiments is outlined in Figure 6-1. It, along with all retroviruses, encodes three common genes, gag, pol, and env (Coleman et al. 2003). Within the virus, gag encodes the core proteins; pol encodes the replication enzymes, and env encodes the envelope glycoprotein. Gag and Pol proteins are initially fused together and then cleaved to yield individual functional proteins including the viral body (from Gag), reverse transcriptase and other proteases (from Pol). The translated envelope proteins function to target specific cell types based on the env gene. As shown in Figure 6-1, this lentiviral vector contains vesicular stomatitis virus G-protein (VSV-G),

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101 which functions both to infect a wide array of cells as well as to aid in the production of large-scale high-titer virus (Naldini et al. 1996; Yee et al. 1994). The long terminal repeat (LTR) sites flank the genes transcribed following viral infection. The lentiviral vector contains several genes that distinguish it from other retroviruses, including rev and tat. The Rev protein functions in concert with the Rev-responsive element to guide the movement of the viral RNA to the cytoplasm from the nucleus (Lesnik et al. 2002). The Tat protein functions in increasing efficient gene production (Roebuck and Saifuddin 1999). This protein has been isolated and used in other studies as a stand-alone efficiency regulator in various transduction pathways. For example, the NAD(P)H oxidase blocker used in Chapters 4 and 5 was fused to tat in order to increase efficiency. The lentiviral vector contains the human elongation factor 1 (EF1) promoter to efficiently induce gene delivery (Zaiss et al. 2002). In summary, this lentiviral vector can easily incorporate a gene of interest and provides a safe, effective, long-term expression of the gene. The lentiviral vector is ideal for delivering the sEH gene to specific brain nuclei. The following studies outline the construction and testing of the lentiviral vector encoding the sEH gene (lenti-sEH) followed by its delivery in vivo. Blood pressure and heart rate were then measured in SHR and WKY rats overexpressing the sEH gene in the PVN.

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102 Methods Animals ICV Cannulation Abdominal Aorta Cannulation Western Blot Analysis The above Methods are described in Chapters 2 and 3 Transformation of pCR 2.1 sEH cDNA into competent cells The mouse sEH cDNA cloned into the pCR 2.1-TOPO plasmid (Invitrogen, Carlsbad, CA) was a kind gift from Dr. Curt Sigmund at the University of Iowa. The plasmid contained resistance genes to both Kanamycin and Ampicillin. The plasmid was transformed into the competent cell line TOP10 (Invitrogen, Carlsbad, CA) in order to amplify the plasmid. TOP10 cells (1x10 9 cfu/g tube) were thawed on ice. The pCR 2.1 sEH cDNA plasmid (20l) was added to the cells, and the tube was incubated on ice for 30 min. The cells were then heat-shocked at 42C for 90 seconds and then placed on ice for 2 minutes. SOC (800l) was then added, and the cells were agitated at 225 rpm at 37C for one hour. The transformation was then plated on LB+Kanamycin plates overnight at 37C. Resulting colonies were grown up in 2 ml superbroth+Kanamycin at 37C, 225 rpm. The media was cloudy with the bacterial growth, and the DNA was then extracted using DNA miniprep. DNA miniprep The bacteria were pelleted at maximum speed in the microcentrifuge for 15 seconds and resuspended in 100 l GTE solution containing 100g/ml RNase. NaOH/SDS (200l) solution was added, vortexed, and left at room temperature for 2 minutes. Cold potassium-acetate solution (150l) was added and inverted. The solution was centrifuged

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103 at maximum speed for 15 minutes, 4C. The supernatant was added to 800l 95% EtOH, and the DNA was pelleted for 10 minutes, 4C. The DNA was air-dried and suspended in Tris-EDTA buffer, pH 7.5. Large-scale Lentiviral Production Transfection Cultured human embryonic kidney (HEK293FT, Invitrogen) cells were split into 27 T-75 flasks coated with poly-d-lysine, 1x10^7 cells/flask. Once the cells were approximantely 90% confluent, they were transfected with the lenti-sEH or lenti-neo complex that was comprised of the following per flask: Serum-free media 400l pHP 7.10g pTYF-sEH or pTYF-neo 3.50g VSV-G 2.80g Tat 0.60g Superfect 28l 10% fetal bovine serum (FBS) media 5.50ml The above reagents (minus the 10% FBS media) were mixed in a polystyrene tube to prevent any DNA from adhering to the walls and incubated at room temperature for 10 minutes. The mixture was then added to the 10% FBS media (Invitrogen), and incubated at 37C for 5 minutes. The transfection media was then used to replace the old media, and the flasks were incubated at 37C for six hours. The media was then replaced with fresh 5.5 ml 10% FBS media and incubated again for 24 hours. First virus collection

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104 Thirty hours following transfection, the media was collected into four 50ml conical tubes and centrifuged at 2000xg for 10 minutes. Fresh media was added to the flasks. The media was then filtered through a PES 45m filter (Nalge Nunc, Rochester NY) to further remove debris. The filtrate was stored on ice overnight to be processed with the second collection. The next morning, the filtrate was added to two Centricon-80 ultrafiltration columns (Millipore, Billerica MA) and spun at 2000xg for one hour. This concentrate was collected by inverting it into the collection cup and spinning again at 990xg for 2 minutes at 4C. Second virus collection Forty-eight hours following transfection, the media was collected into 4 50ml conical tubes and centrifuged at 2000xg for 10 minutes. The media was then filtered through a PES 45m (Nalge Nunc) filter to further remove debris. The concentrate from the first collection was used to spike the second collection, and the two were mixed. Aliquots (30 ml) were added to four chilled SW28 rotor tubes containing 220l 60% iodixanol (Optiprep, Axis-Shield, UK). The collection was centrifuged at 20,000xg for 2.5 hours at 4C using the SW-28 swinging-bucket rotor (Beckman Coulter, Fullerton CA). Following centrifugation, the top media layer was removed to the same height distance as the iodixanol height. The contents in the four tubes were then mixed and combined into 3 ml conical tubes (Beckman Coulter). The volume was brought up to 3.5ml with CSF. The virus was then centrifuged at 8100xg for 48 hours at 4C using a SW-50.1 swinging-bucket rotor (Beckman Coulter). The supernatant was then removed and the pellet was resuspended in 20l CSF. The virus was aliquoted (1-2l each per tube) and stored at -80C.

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105 Infection of COS-7 cells with lenti-sEH To determine whether the transfection was successful, COS-7 cells derived from African green monkey kidney (a kind gift from Dr. Peter Sayeski, University of Florida) were infected with the viral preparations, and the cells were analyzed for sEH expression. COS-7 cells were chosen as the in vitro vehicle because of their very low endogenous levels of sEH protein. COS-7 cells were plated at 1x10 5 cells/well in six-well plates. Cells were infected with either lenti-sEH, lenti-neo (control), or lenti-GFP (visual control). Virus was delivered at a multiplicity of infection (MOI) of either 1 or 10. For cells infected with virus with an MOI of 1, 10l unconcentrated virus (1x10 Tu/ml) was added to each well; 100l unconcentrated virus was added to achieve an MOI equal to ten. Twelve wells were used for each treatment of lenti-sEH or lenti-neo in order to extract substantial amounts of protein for Western blot analysis of sEH protein (see Chapter 2 for protocol). In contrast, four wells were used for each treatment condition of lenti-GFP since these cells were used for fluorescence detection and not for protein extraction. Cytoplasmic Isolation for HPLC Analysis COS-7 cells in six-well plates were washed twice with phosphate-buffered saline (PBS ), removed from the well with rubber policemen and pelleted at 1500 rpm for 15 min. PBS (100l) + protease inhibitor solution was added to each tube and the contents homogenized. The samples were centrifuged at 100,000xg for 60 min and the supernatant collected for DHET analysis by HPLC as previously described (Brand-Schieber et al. 2000b).

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106 Radiotelemetry Recordings Blood pressure and heart rate were recorded using the implanted radiotelemetry transducers. For each dataset acquisition, the rat cages were positioned on receivers that collected signals from the implanted transducers. These signals were then transmitted to an adapter with an ambient pressure monitor that relayed the signals to the DataQuest 3.1 Acquisition program (DataSciences International, Arden Hills MN). The rats were allowed to acclimate to their new position for at least one hour before basal measurements were recorded. For basal measurements, blood pressure and heart rate were recorded for (four) days prior to virus injection. Daily recordings were taken between 3 and 4pm and averaged for analysis. During data collection, blood pressure (systolic, mean and diastolic), heart rate and activity were recorded every minute for an average of ten seconds at 500Hz. Data was acquiesced and transferred to Microsoft Excel for graphical analysis. PVN Coordinate Determination Coordinates to inject into the PVN were determined for both SHR and WKY rats weighing 250-270g. Initial coordinates were taken from studies done by Drs. Beverly Falcon (AP: -1.6mm, ML: +/-0.5mm, 7.3mm from dura) and Jorge Vazquez (AP: -1.3mm, ML: +/-0.5mm, 8.3 from skull) in our research group using SHRs weighing 180-220g and >250g, respectively (unpublished data). SHR and WKY rats were injected at the higher weight in the current study in order to complete the abdominal aorta cannulation approximately ten days prior to the PVN injection. This time frame allowed for a one-week recovery time from surgery as well as basal blood pressure and heart rate measurements. To determine the coordinates, 500nl dye was injected bilaterally using a 5l Hamilton syringe mounted on the stereotaxic frame. The dye was delivered over 8

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107 10 minutes, and ten minutes elapsed following the injection before the syringe was removed. The rats were sacrificed and the brain sectioned at 20m using a cryostat. Dye location was determined by comparing the stained localization to nuclei markers from the Rat Brain Atlas (Paxinos & Watson 4 th Ed 1998). Coordinates were adjusted accordingly and the process repeated until reproducible injections into the PVN were generated with dye. These coordinates were then used for subsequent studies using the viral delivery of the sEH gene. Immunohistochemistry Rats were anesthetized with inhaled isofluorane (5%) and perfused transcardially with saline followed by 4% paraformaldehyde (PFA). SHR brains were post-fixed in PFA and saturated in 20% sucrose solution. 20 M sections were cut using a cryostat and mounted on poly-L-lysine-coated slides; the Rat Brian Atlas (Paxinos & Watson 4 th Ed 1998) was used as a reference. Slides were rinsed with TBS and then washed in 0.3% hydrogen peroxide/methanol for twenty minutes to block endogenous peroxides. Sections were blocked with 2% BSA in TBS-T for three hours. Sections were incubated overnight with anti-sEH rabbit antibody (1:500) in 1% BSA, a kind gift from Dr. Bruce Hammock, University of California Davis, followed by anti-rabbit biotinylated (1:200) for two hours and streptavidin-HRP (1:100) for one hour (Amersham Biosciences, Buckinghamshire, England). sEH protein was visualized using 3,3'-diaminobenzidine (DAB) substrate in peroxide solution (Fisher Scientific, Fairlawn NJ). Between each incubation, the sections were washed three times in TBS, five minutes each. Following DAB treatment for one minute, the sections were rinsed in increasing concentrations of

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108 ethanol and finally xylene before a coverslip was mounted using Permount (Fisher Scientific). PVN Injection of lenti-sEH Male SHR and WKY rats, 250-270g (Charles River Laboratories), were used in bilateral PVN injections. Pre-determined PVN coordinates were AP: -1.3mm, ML: +/-0.5mm, 7.9mm from dura for SHR and AP: -1.2mm, ML: +/-0.5mm, 7.8mm from dura for WKY rats. Ten days following abdominal aorta cannulation, rats were injected bilaterally with 5x10 5 TU of lentivirus per injection. Half of the rats received lenti-neo (n=4/WKY, n=5/SHR) as control and half received lenti-sEH (n=4/WKY, n=4/SHR). The right lateral ventricle of the rats was cannulated for future drug delivery, and the rats were allowed to recover for one week. At the end of the study, the brain was prepared for immunohistochemical analysis of sEH in order to determine injection position and sEH localization following viral delivery (see Chapter 2 Methods for protocol). In vivo Drug Delivery All drugs were delivered via the ICV cannula into the right lateral ventricle of unrestained rats. sEH inhibitor AUDA (15ng, 2l volume) dissolved in CSF was used to compare the effect of central sEH inhibition between the rats injected with control and sEH virus. 11-nonyloxy-undec-8-enoic acid (11-NODA, 100ng, 1l, dissolved in CSF), an EET agonist, was provided by Dr. Falck. Statistical Analysis All data are expressed as mean standard error of the mean (SEM). The number of animals or samples in each group is described in the Results below. The effects of lenti-sEH delivery in the PVN were analyzed using two-way repeated measures ANOVA

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109 (SigmaStat, kindly provided by Dr. Charles Wood, University of Florida). The two factors individually analyzed in one dimension were time and group. In the remaining experiments, comparisons between experimental groups were analyzed using either one-way ANOVA (SPSS) or Students T test (Stat View). Differences between means within groups were assessed with the Newman-Keuls analysis. Significance was set at a confidence level at or above 95% and is denoted by in each figure. Results Infection of COS-7 cells with lenti-sEH Three days after infection of COS-7 cells with lenti-GFP, green fluorescence was detected, indicating a successful transfection and subsequent infection. Given the positive result of the GFP infection, protein was isolated from cells containing either sEH or control virus. Western blot analysis revealed sEH expression in cells infected with the lenti-sEH virus and no detectable sEH expression in cells infected with lenti-neo (Figure 6-2). Functionality of Lenti-sEH While sEH overexpression was clearly detected using Western blot analysis, it was imperative to verify the functionality of this enzyme. Cytoplasmic fractions were isolated from lenti-sEH or lenti-neo-infected COS-7 cells to measure DHET levels, the product of EETs under sEH regulation. HPLC analysis by Dr. Michal Scwartzman, New York Medical College, revealed 11,12-DHET levels increased from 0.03 nmol/mg to 3.26 nmol/mg and 14,15-DHET levels increased from 0.41 nmol/mg to 4.95 nmol/mg in the lenti-sEH (red) compared to lenti-neo-infected cells (blue, Figure 6-3). This analysis was only done in one group of COS-7 cell cultures and therefore cannot be analyzed for

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110 statistical significance. However, the substantial increase in DHETs in lenti-sEH-infected cells indicates that lenti-sEH infection results in sEH functionality in vitro. sEH Overexpression in WKY PVN WKY rats (250-270g) were injected with either lenti-sEH (red) or lenti-neo (blue) into the PVN. Blood pressure (Figure 6-4A) and heart rate (Figure 6-4B) were recorded daily from 3-4pm prior to and following brain injections. Two-way repeated measures ANOVA was used to analyze blood pressure and heart rate. Figure 6-4 shows that blood pressure was not significantly different between the two groups. From two to forty days post injection, rats injected with lenti-neo and lenti-sEH had average blood pressures of 105.6 mmHg and 102.2 mmHg, respectively, with a standard error of least square means of 1.7 mmHg. In the same time period, rats injected with lenti-neo and lenti-sEH had average heart rates of 317.3 bpm and 311.9 bpm, respectively, with a standard error of least squares means of 2.2 mmHg. The test for normality failed for these data sets. However, heart rate was 6 bpm lower in lenti-sEH compared to lenti-neo-injected animals (310.7 bpm versus 304.3 bpm, respectively, LS means = 1.7bpm) from twenty to forty days post injection. Collectively in both groups, blood pressure increased and heart rate decreased following viral delivery. AUDA delivery 15ng ICV AUDA delivery resulted in similar increases in heart rate in both the lenti-sEH and lenti-neo-injected groups (Figure 6-5). Heart rate increased from 321.69+/-8.93 bpm to 414.52+/-9.39 bpm in the lenti-sEH rats and from 323.33+/-8.21 bpm to 435.87+/-9.72 bpm in the lenti-neo-infected rats.

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111 EET agonist delivery 100ng 11-NODA was delivered ICV, and blood pressure and heart rate were recorded using radiotelemetry. In contrast to the biphasic peak from 11-NODA delivery in control rats, lenti-sEH-injected rats had no second pressor peak (Figure 6-6). For the first peak, blood pressure peaked at 118.85+/-2.70 mmHg in lenti-sEH rats (n=4) and 121.41+/-2.3 mmHg in lenti-neo rats(n=3). For the second peak, blood pressure peaked at 114.44+/-5.78 mmHg in lenti-neo rats and returned to basal levels (100.23+/-3.29 mmHg) in the lenti-sEH rats. Heart rate peaked at 379.99+/-5.98 bpm in lenti-sEH rats and 392.91+/-3.76 bpm in lenti-neo rats. Thus, the first pressor peak as well as the heart rate were not significantly different between the two groups. Localization of viral delivery Immunohistochemistry using the sEH antibody and visualized with DAB substrate was used to detect viral delivery of lenti-sEH in 10m brain slices. Figure 6-7 shows images from the PVN region of the four WKY rats injected with lenti-sEH and their relative positions from Rat Brain Atlas images (Paxinos & Watson, 4 th Ed 1998). sEH expression was localized bilaterally to the PVN in all rats, as detected by antibody staining as indicated by the circular region of brown color. The images were captured under visual field with 10x power and photographed with a Nikon Coolpix 990 digital camera mounted on the Leica microscope. sEH Overexpression in SHR PVN SHRs (250-270g) were injected with either lenti-sEH or lenti-neo into the PVN. Blood pressure and heart rate were recorded daily from 3-4pm prior to and following brain injections. Two-way repeated measures ANOVA was used to analyze blood pressure and heart rate. Figure 6-8 shows that neither blood pressure nor heart rate was

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112 significantly different between the two groups. From two to fourteen days post injection, rats injected with lenti-neo and lenti-sEH had average blood pressures of 146.40+/-4.27 mmHg and 146.78+/-4.77 mmHg, respectively. In the same time period, rats injected with lenti-neo and lenti-sEH had average heart rates of 340.30+/-7.33 bpm and 346.99+/-8.19 bpm, respectively. The test for normality failed for these data sets. Collectively in both groups, blood pressure and heart rate increased following viral delivery. Discussion Overexpression of sEH using the lentiviral vector has proven to be successful both in vitro and in vivo. COS-7 cells infected with lenti-sEH had dramatic overexpression of sEH as demonstrated by Western blot analysis. Thus, this gene therapy method was able to successfully infect the cells and lead to a translation of the gene insert into its respective protein. More importantly, the functionality of this protein was verified through HPLC analysis of COS-7 cells infected with lenti-sEH. The lentiviral vector used to deliver sEH is indeed functional and successfully infects cells in vitro. The pertinent question, however, was how does this sEH overexpression in cardiovascular-relevant brain nuclei affect blood pressure and heart rate? sEH gene therapy to the WKY rat resulted in only a 6 bpm decrease in heart rate twenty to forty days post injection, as compared to delivery with lenti-neo. In the SHR, no changes in blood pressure or heart rate were detected following lenti-sEH delivery up to two weeks post-injection. Changes in SHR blood pressure and heart rate will continue to be monitored and compared to the WKY rat. At this point, however, the sEH overexpression in the PVN exerts minimal control in the central control of blood pressure.

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113 It is important to discuss why the lenti-sEH-infected WKY rats had no change in blood pressure and only a small decrease in heart rate. Several possibilities can be addressed. First, the gene delivery coordinates to individual rats may not have targeted the PVN. However, immunohistochemistry of sEH in brain sections of the WKY rats following the study shows the sEH expression in the PVN of the four rats infected with lenti-sEH. Given the importance of correct localization, it is essential in all gene therapy studies to verify injection site, including at the end of the current SHR PVN study. Second, the blood pressure and heart rate may have already been at the minimum physiological level. Since the WKY rats are normotensive, they have little to no cardiovascular problems, including those involving increased heart rate. Because there is no endogenous elevated heart rate to combat, increasing sEH may not be able to lower heart rate further than basal levels. Another possibility that may work in conjunction with the second point is that WKY rats do not have the same pathway invoked in the SHR through which EETs function. We know that ROS, found to be associated with the actions of sEH inhibition, is elevated in the SHR. In the WKY rat, however, EETs may not be at the same level to induce ROS, or ROS members may not be as widely available. Thus, increasing sEH, which decreases EETs, would not affect pathophysiologies associated with ROS. One piece of evidence against this suggestion, however, is that heart rate does increase in the WKY rat with central sEH inhibition, albeit not as dramatically as in the SHR. Therefore, this pathway must be intact to some degree in order to allow for the increase in heart rate. sEH overexpression in the PVN had no effect of blood pressure or heart rate in the SHR. These results suggest several underlying possibilities. First, the high endogenous

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114 sEH levels in the SHR may be in excess of the EETs, indicating that EETs are the rate-limiting step in this conversion. Thus overexpressing the protein would not affect physiology since the SHR would already be converting the maximum level of EETs in the PVN. Second, sEH itself may not be essential in central blood pressure regulation. While the inhibitor studies clearly identified the epoxygenase branch of arachidonic acid conversion in blood pressure changes, these effects may be due to CYP function or EETs. While sEH is definitely a strong regulator of EET availability, perhaps alternate pathways mask its influence. In addition, because the sEH activity was not measured in vivo following sEH inhibition, we cannot conclusively state that ICV delivery of sEH inhibitors solely targeted sEH. Activity of sEH using HPLC analysis of DHETs should be incorporated into future gene therapy studies. To address these first two explanations, EETs should be targeted to the PVN, either by overexpression or depletion. This analysis of substrate function would determine the importance of the PVN in epoxygenase-mediated blood pressure. Third, sEH may not act through the PVN to influence blood pressure and heart rate. Though the PVN is an important regulator of cardiovascular function, it is in the company with several other brain nuclei that must be examined for sEH function. Indeed, Dr. Julian Paton, our collaborator in Bristol, England, has recently used lenti-sEH to overexpress the gene in the RVLM. Surprisingly, blood pressure increased in the SHR from 136 mmHg to 151 mmHg two weeks following viral delivery, an unexpected result. This finding corresponds with the action of sEH in the peripheral vasculature but disputes the pressor effect of ICV delivery of the sEH inhibitors. This discrepancy in the brain is likely due to differences between targeting specific nuclei and diverse brain area delivery

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115 with ICV injection. In order to resolve this contradiction, AUDA should be delivered to specific brain nuclei including the RVLM to further elucidate the role of sEH in different brain areas. These different results following gene therapy to the PVN and RVLM, though, support the hypothesis that sEH has diverse functions in different brain nuclei. One question that remains unanswered is the pattern of heart rate and blood pressure changes following viral injections in specific brain nuclei. In both control and sEH viral injections into the WKY PVN, heart rate decreased and blood pressure increased. In the SHR, both factors increased following viral delivery. The trend seen in both viral treatments indicates that the changes in physiological measurements are due to the viral injections or the injection protocol itself. This was the first study to inject lentivirus into the brain and measure blood pressure and heart rate daily using radiotelemetry; therefore, whether this physiological result is common for the lentiviral injection is not currently known. If this pressor response is indeed reproducible in other gene therapy studies, then it is necessary to determine the cause prior to considering this strategy in a therapeutic manner. Differences between strains may be due to an intrinsic dampened baroreceptor reflex in the SHR. More studies are needed to define this pressor response following central lentiviral delivery in each strain. While sEH overexpression in the PVN of SHR and WKY rats had little effect on the cardiovascular parameters measured, this work provides a solid basis for future analysis of sEH function in different brain nuclei. The functionality of the lentivirus coupled with its high titer and ability to incorporate specific promoters makes it ideal for studying gene function in the brain. Using this approach, the central role of sEH on cardiovascular function can be mapped clearly in different models of hypertension.

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116 Figure 6-1 Lentiviral vector

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117 Figure 6-2 sEH protein expression in lenti-sEH-infected COS-7 cells. COS-7 cells were infected with 1x10 5 or 1x10 6 TU lenti-sEH or lenti-neo. After 3 days, protein was extracted from the cells (see Methods), and Western blot analysis was performed to detect sEH protein levels. Representative radiograph is shown above. Bands represent sEH Ab detection, with the TU listed below each band. The first two bands are infected with lenti-sEH, and the last two are infected with lenti-neo.

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118 Figure 6-3 DHET measurements using HPLC analysis. DHET levels were measured in COS-7 cells infected with either lenti-sEH or lenti-neo. DHET regioisomers were measured as described in Methods by Dr. Michal Schwartzman, New York Medical College.

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119 Figure 6-4 Blood pressure and heart rate recordings following lenti-sEH and lenti-neo delivery to the WKY rat PVN. Blood pressure and heart rate were recorded using radiotelemetry prior to and following PVN bilateral injections of the 5x10 5 TU lentivirus. X-axis represents days post injection. (A) Mean arterial pressure; (B) Heart rate. Data are mean +/SEM (p<0.05, n=4).

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120 Figure 6-5 Heart rate recordings following AUDA delivery in the lenti-sEH-infected WKY rat. Heart rate was recorded using radiotelemetry prior to and following 15ng AUDA delivery ICV (n=4 lenti-sEH, n=3 lenti-neo). The graph is quantitation of heart rate changes. Data are mean +/SEM, p<0.05.

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121 Figure 6-6 Blood pressure and heart rate recordings following EET agonist delivery in the lenti-sEH-infected WKY rat. Blood pressure and heart rate were recorded prior to and following 11-NODA delivery ICV (n=4 lenti-sEH, n=3 lenti-neo). X-axis represents time, and arrows represent point of drug delivery. (A) Representative blood pressure recording; (B) Representative heart rate recording; (C) quantitation of blood pressure changes. Black and gray bars represent first and second blood pressure peaks, respectively. Data are mean +/SEM, p<0.05.

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122 Figure 6-7 Localization of lenti-sEH delivered to the PVN of WKY rats

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123 Figure 6-8 Blood pressure and heart rate recordings following lenti-sEH (n=4) and lenti-neo (n=5) delivery to the SHR PVN. Blood pressure and heart rate were recorded using radiotelemetry prior to and following PVN bilateral injection of 5x10 5 TU lentivirus. X-axis represents days post-injection. (A) Mean arterial pressure; (B) Heart rate. Data are mean +/SEM, p<0.05

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CHAPTER 7 CONCLUSIONS AND FUTURE DIRECTIONS What began as a single gene identified from a microarray experiment emerged as an in-depth study of the cardiovascular effects of sEH in the brain. While sEH expression in the SHR brain is elevated, central sEH inhibition resulted in further increases in blood pressure and heart rate through the accumulation of EETs. This pressor effect is mediated by central ROS and functions through sympathetic nerve activation (Figure 7-1). Gene therapy was used to elucidate brain nuclei-specific effects of sEH overexpression. Lentiviral-mediated delivery of the sEH gene to the PVN had no effect on either blood pressure or heart rate in the SHR and only a minimal decrease in heart rate in the WKY rat. Collectively, these studies provide compelling evidence that sEH is a novel regulator of the central control of hypertension, though its effects may not be mediated through the PVN. These results stimulate a number of questions and plans for future analysis of central sEH function, as discussed in the following sections. sEH Overexpression in Alternate Brain Nuclei Mapping the mechanism of central sEH and EET regulation of blood pressure will be an extensive project. Initial gene therapy studies found that the PVN has minimal influence in the central role of sEH. However, the same experimental methods applied to the RVLM resulted in an increase in blood pressure in the SHR, as deduced by Dr. Julian Patons research group at the University of Bristol, England. These findings are the foundation for future brain-nuclei directed studies of sEH. Other cardiovascularly-relevant nuclei that should be targeted include the NTS and the SON. Effects of this 124

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125 overexpression should be compared between nuclei as well as between hypertensive and normotensive strains. In addition, it is critical to deliver sEH inhibitors and EET agonists directly to these nuclei via cannulae in order to extrapolate their specific effects. Given the wide array of neuronal projections between these areas, we hypothesize that sEH overexpression will have diverse effects in different areas, following the same results already observed in the PVN and RVLM. It will be interesting to study further any nuclei whose sEH overexpression yields a decrease in blood pressure or heart rate, thus supporting the increased pressor response resulting from ICV delivery of sEH inhibitors. The lentiviral vector is ideal for nuclei-specific delivery given its minimal distribution following central injection as well as the ease of incorporating promoters to target sEH to specific cell types. sEH Expression sEH expression is higher in the SHR brain compared to its normotensive control. This result correlates to sEH overexpression in the kidney and liver, indicating a whole-body induction of this enzyme in the SHR (Sinal et al. 2000b; Yu et al. 2000a). However, prior to definitely linking the central sEH to hypertension, it is necessary to determine the expression levels in other models of hypertension. Does sEH expression play a role in the induction or progression of hypertentension, or is it unique to the SHR? Peripheral studies have indicated an increase in sEH protein levels in the Ang II infusion model (Imig et al. 2002). However, these protein levels were highly variable between rats and therefore do not provide conclusive evidence for the link of sEH to hypertension in general. Future studies into central sEH expression should include the Ang II infusion model as well as strains outside RAS manipulation including Dahl salt sensitive rats and the two kidney, one clip model of hypertension. These models incorporate

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126 pharmacological, dietary, and surgical manipulation methods that will further elucidate the role of this enzyme. ROS-Mediated Pathway of EET Function sEH inhibition-induced effects were attenuated by blocking the formation of either EETs or ROS. From these results, we postulate that EETs increase the production of ROS; however, it is possible that ROS itself is upstream of EET function and controls the regulation of EETs. Inhibiting ROS and then adding EET agonists to the brain should address this question. If the ROS inhibition blocks the effects of EET activation, then ROS is downstream of EET action. If, however, EET agonists still result in increasing blood pressure and heart rate, then ROS is likely to be upstream of the EETs, indicating that EETs cause a pressor response through an alternate pathway than hypothesized. More focus should then turn to whether ROS effects in other tissues function though EET accumulation. This is an important next question that will provide further insight into the pathway of sEH regulation of blood pressure and heart rate control in the brain. Validation of Baroreceptor Reflex Findings Waveform analysis of AUDA delivery to the brain indicated a decrease in the spontaneous baroreceptor reflex gain in the SHR. This analysis employed the use of a time-series extrapolation from spontaneous changes in heart rat that was developed in 1997 (Oosting et al. 1997). While the methods are attractive in their elimination of response to pharmacological agents, debate exits as to their accuracy. Thus, in order to verify that concomitant increases in blood pressure and heart rate in the SHR are due to a dampening of the baroreceptor reflex gain, vasoactive agents such as phenylephrine (pressor) or nitroprusside (depressor) should be injected and the reflex measured as in traditional methods.

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127 Role of sEH in Glial and Endothelial Cells Conflict exists over the cell-specific actions of sEH and EETs. Previous studies, both peripheral and central, highlight the production of EETs in endothelial cells and its vasodilatory action in vascular smooth muscle cells (Harder et al. 1995; Li and Campbell 1997). Indeed, even in the brain, EETs are localized to endothelial and glial cells, though one study did locate CYP 2D19 in dopaminergic neurons (Medhora et al. 2001; Thompson et al. 2000). However, our studies found that sEH inhibition increased neuronal firing rate in cultures from one-day-old SHR pups. In addition, while the EF1 promoter used in lenti-sEH overexpression infects most cells, it seems to preferentially target neurons when delivered to the brain (Coleman et al. 2003). While our results suggest sEH is involved in neuronal activation, we cannot discount the role of other cell types in the brain. In order to determine the cell type-specific effects involved with sEH and EET action, it is necessary to incorporate GFAP promoters to target glial cells. Overexpression of sEH or blockade of EETs specifically in the glia will further answer this question of what cell types are involved in sEH action. The pathway may involve multiple cell types as does EET action in the vasculature. For example, EETs produced in the glia may transport to the neurons where they are either converted by sEH or elicit neuronal activation through a ROS-dependent pathway. Alternately, ICV delivery of sEH inhibitors may function to decrease EET stores in the cerebral vasculature and lead to changes in cerebral blood flow. Signaling from these changes to the overall

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128 vasculature may elicit pressor effects, though possible mechanisms behind this postulation are not known. Role of Central Epoxygenases We propose that sEH inhibition elicits its pressor effects through accumulation of the EETs. However, while EET formation blocker and EET agonist data support this idea, the role of epoxygenases the central control of blood pressure also must be determined. Epoxygenases are a subset of CYP that function to convert arachidonic acid to EETs. Given the connection of ROS to epoxygenases in the coronary artery and the role of ROS in central sEH-mediated physiological effects, these enzymes should not be overlooked as mediators in the regulation of blood pressure. Perhaps their expression or activity is altered in the hypertensive state. Many epoxygenase isoforms exist, though relatively few have been identified in the brain. This discrepancy may be due to enzyme specificity or to the lack of sufficient analysis in the brain. Collectively, inhibiting central epoxygenases may have attractive therapeutic potential. If EETs do indeed function in certain brain areas to increase blood pressure in the hypertensive state, then blocking their formation may prevent this progression. While MS-PPOH did not have any depressor effects on basal blood pressure, this result stemmed from ICV delivery of the drug. Direct inhibition of epoxygenase in brain areas may have beneficial effects on blood pressure and heart rate in hypertensive subjects. Effect of sEH Manipulation in Female Rats All the current studies used male rats to evaluate the effects of central sEH. However, it is essential to determine the effect of brain sEH on the blood pressure and heart rate in females as well. The only study incorporating females was the generation of transgenic mice lacking the sEH gene (Sinal et al. 2000b). In these animals, no change in

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129 physiological parameters was observed in the females, whose blood pressure was lower than that of the males. In essence, the effect of sEH knockout in the males was to feminize their systolic blood pressure. However, these studies do not address brain-specific sEH actions and their gender differences. Does central sEH inhibition in female SHRs also result in an increase in blood pressure and heart rate? If not, can these differences be explained by hormonal regulation? Do females have similar endogenous sEH expression levels compared to the males? The importance of these studies is two-part. First, female responses are not homogeneous to those of males, so the role of an enzyme studied in one gender cannot be automatically applied to another. In order to comprehensively study sEH, it must be analyzed in both males and females. Second, potential differences in central sEH effects between genders would further elucidate mechanisms underlying the control of this enzyme. This comparison between genders may reveal sEH and EET pathways not otherwise discerned by the study of only males. Implications of Central sEH in Human Hypertension The pinnacle question is what role, if any, sEH has in the human control of hypertension. As previously discussed, sEH polymorphisms have been identified in humans (Przybyla-Zawislak et al. 2003; Saito et al. 2001; Sandberg et al. 2000; Sato et al. 2004). Polymorphisms localized to members of the African American community correlated with an increase in coronary artery calcification (Fornage et al. 2004). Little research has been done in the brain, except one study that found no correlation of sEH polymorphisms to the incidence of Parkinsons disease (Farin et al. 2001). Given the increase in blood pressure and heart rate following sEH inhibition it is important to examine potential similarities between these models and the human state of hypertension.

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130 If sEH is indeed linked to the hypertensive state, then pharmacological agents already used in animal research may be modified for effective use in clinical trials. This finding may be a novel way to treat hypertension or tachycardia in a subset of the population that has sEH SNPs and thus an underactive function of the enzyme. One question that must be addressed, however, is what effect sEH agonists would have on the periphery, given the opposite effects of sEH compared to the brain. Our studies have set the basis for sEH overexpression in the brain, but it remains to be seen whether this overexpression not directed at the brain would have adverse side effects. The products of the epoxygenase pathway of arachidonic acid in the brain have been shown, for the first time, to elicit blood pressure and heart rate responses in the hypertensive rat. This mechanism involves sympathetic activation as well as ROS production, two established mediators of pressor response. These studies could not have been completed without sEH reagents including antibodies, inhibitors, and substrate agonists that were kindly provided by multiple researchers. Future studies will undertake linking and establishing brain-specific sEH effects, determining the roles of alternate members of the epoxygenase pathway, and translating these findings to polymorphisms identified in humans. Together, this work will continue to elucidate the role of central sEH in blood pressure regulation.

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131 Figure 7-1 Proposed mechanism of brain sEH function in the central control of blood pressure

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BIOGRAPHICAL SKETCH Kathleen Sellers was born February 3 rd 1978, in Savannah, Georgia. During her primary and secondary educational training, she lived in Gainesville, Florida; Nashville, Tennessee; and Daytona Beach, Florida. Her first taste for research came in 1995 when she attended the Student Science Training Program at the University of Florida. There, she worked on a project entitled Effect of Melatonin on Progesterone Secretion in Hysterectomized Pony Mares. Kathleen earned her bachelors degree in science from Winthrop University in Rock Hill, South Carolina. While attending the university, she worked with both Dr. Dwight Dimaculangan on genetic impulses underlying blackworm regeneration and Dr. Patricia Bossart-Whitacker on pneumocystis carinii cloning. In Summer 2000, Kathleen worked in Dr. Mohan Raizadas laboratory before starting the Interdisciplinary Program in Biomedical Sciences at the University of Florida. She joined the Department of Physiology and Functional Genomics in May 2001 where she continued to work under the guidance of Dr. Raizada to examine to the central role of soluble epoxide hydrolase on blood pressure regulation. She will earn her doctorate in December 2004. 143