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The Importance of the angiotensin type-1 receptor in the vascular response to injury

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Title:
The Importance of the angiotensin type-1 receptor in the vascular response to injury a study with autoimmunization and antisense
Alternate title:
Study with autoimmunization and antisense
Creator:
Meng, Frank H., 1968-
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English
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xiii, 111 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Angioplasty ( jstor )
Antibodies ( jstor )
Autoantibodies ( jstor )
Balloons ( jstor )
Dendrimers ( jstor )
Gene therapy ( jstor )
Messenger RNA ( jstor )
Physical trauma ( jstor )
Rats ( jstor )
Receptors ( jstor )
Angiotensin I ( mesh )
Autoimmunity ( mesh )
Carotid Artery, Common ( mesh )
Department of Physiology thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Physiology -- UF ( mesh )
Gene Expression Regulation ( mesh )
Gene Therapy ( mesh )
Muscle, Smooth, Vascular ( mesh )
Oligonucleotides, Antisense ( mesh )
Peptides ( mesh )
Rats, Sprague-Dawley ( mesh )
Receptors, Angiotensin ( mesh )
Renin-Angiotensin System ( mesh )
Research ( mesh )
Wound Healing ( mesh )
Wounds and Injuries ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1997.
Bibliography:
Bibliography: leaves 97-110.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Frank H. Meng.

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THE IMPORTANCE OF THE ANGIOTENSIN TYPE-I RECEPTOR IN THE VASCULAR RESPONSE TO INJURY: A STUDY WITH AUTOIMMUNIZATION AND ANTISENSE











By

FRANK H. MENG


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


1997



























Copyright 1997

by

Frank H. Meng




























This dissertation is dedicated to my wife, Jane, for all the love, care, support and encouragement she has given me through the years.













ACKNOWLEDGMENTS


I would like to extend my sincere thanks to the chairman of my advisory committee, Dr. M. Ian Phillips, for providing me with the great support and opportunity to learn under his supervision. His guidance and encouragement have led me through all these years in graduate school. I learned many modem techniques in his lab, and more important I have learned to think as a scientist. I also would like to thank my advisory committee members, Dr. Stephen Baker, Dr. Jeffrey Hughes, Dr. Colin Sumners and Dr. Bruce Stevens for their helpful comments and discussions on my project. I am especially grateful to Dr. Hughes who allowed me to use his lab facilities and shared his expertise with me on the Dendrimer research. I am extremely grateful to the members in Dr. Phillips' lab, Dr. Sara Galli, Birgitta, Gayle, Kevin, Leping, Harold, Robert, Jon, Bing, Tibor, Dkgmara, Jianfeng, Clare, Adrian and Dan for their help, friendship and support. Finally, I would like to extend my special thanks to my wife, Jane, for her love and understanding.












TABLE OF CONTENTS


ACKNOW LEDGM ENTS .......................................................................................... iv

LIST OF FIGURES ...................................................................................................... vii

LIST OF TABLES ......................................................................................................... ix

AFFREVATIONS ........................................................................................................... x

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

CHAPTERS
1 INTRODUCTION ............................................................................................ 1
R esteno sis ............................................................................................... 1
Pathophysiology of Restenosis ............................................................ 3
The Renin-Angiotensin System (RAS) ............................................... 4
The Renin-Angiotensin System and Restenosis ....................................... 11
Experimental M odels of Restenosis ...................................................... 15
M ethods to Inhibit Restenosis ............................................................... 16
The Antisense Technology ................................................................... 21
Summary ........................................................................................... 25

2 HYPOTHESIS AND SPECIFIC AIM S ...................................................... 26

3 M ATERIALS AND M ETHODS ................................................................ 28
Experiments on Autoimmunization .................................................... 28
Experiments on Central Ang II Inhibition ...................................... 34
Experiments on Dendrimer Delivery System ...................................... 37

4 AUTOIMMUNIZATION AGAINST ANGIOTENSIN TYPE-I RECEPTOR
PREVENTS THE NEOINTIMAL PROLIFERATION FOLLOWING
ANGIOPLASTY ........................................................................................ 42
Introduction ...................................................................................... 42
Results ............................................................................................... 45
Discussion ......................................................................................... 47











5 ANTISENSE OLIGONUCLEOTIDE TO AT, RECEPTOR mRNA INHIBITS
CENTRAL ANGIOTENSIN INDUCED THIRST AND VASOPRESSIN ......... 61
Introduction ...................................................................................... 61
R esults ............................................................................................... 63
D iscussion ......................................................................................... 71

6 DENDRIMER BASED GENE DELIVERY SYSTEM AND ITS
APPLICATION IN RESTENOSIS ............................................................... 76
Introduction ...................................................................................... 76
R esults ............................................................................................... 79
D iscussion ......................................................................................... 88

7 GENERAL CONCLUSIONS ..................................................................... 94

R E FE R E N C E S .............................................................................................................. 97

BIOGRAPHICAL SKETCH ....................................................................................... 111











LIST OF FIGURES


Figure pg

1-1. Schematic elucidation of the components and functional steps of renin angiotensin
system ......................................................................................................... 6

1-2. Schematic elucidation of the signal transduction of AT, receptor ............... 14

3-1. Schematic elucidation of the procedure of balloon catheterization on rat carotid artery.. 41 4-1. Western-blot analysis of membrane proteins from rabbit adrenal glands using
antiserum from immunized rats ....................................................................... 48

4-2. The effect of immune serum contaiHing autoantibody to the N-terminal of the
AT, protein on PKC translocation ................................................................ 49

4-3. Immunohistochemical identification of AT, receptor on the sections of rabbit
arteries using the rat AT, autoantibody ............................................................... 50

4-4. Immunohistochemical staining of sections from rat carotid arteries using a rabbit
polyclonal antibody against AT1 receptor ........................................................ 51

4-5. 121-Sar,Ile-Ang II autoradiography analysis of multiple transverse sections of
carotid arteries ............................................................................................... 52

4-6. Photomicrographs of representative histological sections from sections of rat left
common carotid arteries 2 weeks after balloon injury ..................................... 53

4-7. Bar graphs represent the ratios of intimal/medial areas in the two groups of rats
that underwent balloon catheterization .......................................................... 54

4-8. Bar graphs represent the ratios of intimal/medial areas in three groups of rats that
underwent balloon catheterization .................................................................. 55

5-1. Effect of AS-ODN for AT1 receptor mRNA on drinking to Ang II i.c.v ................... 65

5-2. Effect of AS-ODN and SC-ODN on drinking with repeated injections .................... 66

5-3. The drinking responses of rats to Ang H i.c.v ........................................................ 67


vii








5-4. Effect of repeated injection of Ang I i.c.v. on plasma AVP level ................................. 68

5-5. Effect of AS-ODN, SC-ODN or saline treatment on AVP release to Ang II i.c.v ......... 69

5-6. Effect of oligodeoxynucleotide treatment on AT, receptor binding in the
hypothalam ic block ........................................................................................ 70

6-1. Purification of FITC labeled Dendrimers (4ei Generation) .................................. 82

6-2. Gel retardation experiment on ODN-DEN complex ............................ 83

6-3. Serum elimination of generation 4, 6 and 10 dendrimer and CF .................... 84

6-4. Serum elimination of generation 6 dendrimer, free 15 mer ODN and DEN-ODN
conjugates .................................................................................................. . . 85

6-5. Tissue distribution of Generation 6 dendrimer ................................................... 86

6-6. Effect of AS-ODN for AT receptor on neointimal formation .............. 87

7-1. Summarization of physiological events following angioplasty and inhibitory
functions of autoantibody and antisense on the AT, receptor ............. 96


viii













LIST OF TABLES

Table agg



1-1. Gene Therapy for Restenosis ......................................................................... 19

1-2. Antisense Therapy for Restenosis .................................................................. 20

6-1. Pharmacokinetical Parameters of Dendrimers ............................................... 81











ABBREVIATIONS


ACE: ACSF:

Ang II: Ao: AP-I: AS-ODN: AT,: AT2: AVP: BCIP: bFGF: BSA:

CMV: CNS: DEN:

ELISA: EtOH: FITC:


Angiotensin Converting Enzyme Artificial Cerebelspinal Fluid Angiotensin II Angiotensinogen Activator protein 1 Antisense Oligodeoxynucleotides Angiotensin type-I receptor Angiotensin type-2 receptor Arginin Vasopressin 5-bromo-4-chloro-3-indolyl phosphate basic fibroblast growth factor Bovine Serum Albumin Cytomegalovirus Central Nervous System Starburst" dendrimers Enzyme Linked Immunosorbent Assay Ethanol

Fluorescein Isothiocyanate











G-protein: MAP kinase: MAP: MECATOR:



NO: PBS: PKC: RAS: SC-ODN: VSMC:


Guanine Nucleotide Binding Protein Mitogen associated protein Kianse Multiple Antigenic Peptides Multicenter European Research trial with Cilazapril after Angioplasty to prevent Transluminal Coronary Obstruction and Restenosis Nitric Oxide

Phosphate buffered Saline protein kinase C Renin angiotensin system Scrambled Oligodeoxynucleotides Vascular Smooth Muscle Cell










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

THE IMPORTANCE OF THE ANGIOTENSIN TYPE-I RECEPTOR IN THE
VASCULAR RESPONSE TO INJURY: A STUDY WITH AUTOIMMUNIZATION AND ANTISENSE By

Frank H. Meng

December, 1997


Chairperson: M. Ian Phillips, Ph.D., D.Sc. Major Department: Physiology



Arterial injury induces VSMC proliferation and migration. This leads to neointimal growth and reduction in lumenal diameter. The local RAS has been suggested to promote this process mediated by AT, receptor. To test the role of angiotensin II in a rat common carotid' artery model of restenosis, we utilized autoimmunization and antisense inhibition strategies.

In the autoimmunization study, SD rats were immunized with a synthetic peptide" corresponding to amino acid sequence 14-23 of the N-terminal the AT, receptor. The autoantibody prevented neointimal regrowth by 65% compared to sham control. The results indicate that when the AT, receptor is chronically inhibited by an autoantibody, the


xii









regrowth response to arterial injury is significantly reduced. This suggests that Ang I and AT, receptors are necessary for the growth mechanism in this model of arterial restenosis.

In the antisense inhibition study, we designed AS-ODN targeting to the AT, receptor mRNA. We tested it in a well-established model, the central Ang II induced drinking and AVP release. AS-ODN treatment significantly reduced drinking and AVP response to central Ang II injection. The results demonstrate that the antisense inhibition of brain AT, receptor gene expression decreases the Ang II induced drinking and AVP response. This indicates that antisense inhibition is capable to block AT, gene expression.

In order to facilitate uptake of AS-ODN into vessel walls, we tested the dendrimers based gene delivery system in treating restenosis. We investigated the pharmacokinetics and tissue distribution of generation 4,6 and 10 polyamidoamine Starburst� dendrimers. Our results showed dendrimer significantly increased the half life of oigonucleotide in the plasma. We complexed AS-ODN for AT, with generation six DEN and tested on a rat model of restenosis. The results showed the AT, AS-ODN delivered by DEN significantly reduced neointimal formation. We conclude that vascular RAS and AT play an obligatory role in development of restenosis. The potential of using AS-ODN as a therapeutic method still needs further investigation.


xiii












CHAPTER 1
INTRODUCTION


Restenosis



Coronary artery disease is the leading cause of death in the United States and in many other countries. The plaques deposited inside the coronary artery, narrow the blood vessel and decrease or completely block the blood supply to the heart. One of the most successful treatments of this disease is arterial angioplasty. The procedure was introduced into the clinic in 1967 by Fogarty. Since then it has become a well established and frequently performed procedure around the world. It is estimated that 350,000 procedures are carried out annually (Hillegass et al. 1994). The procedure has normally an initial success rate of opening obstructed coronary arteries of 95%. However, in spite of the fact that good symptomatic improvement occur in the majority of cases, the procedure is complicated by restenosis in 30-50% of patients, regardless of the type of angioplasty procedure used (Epstein et al. 1994). This means more than 100,000 cases of failure and hundreds of million dollars of loss each year.

It is basically accepted by researchers in the cardiovascular field that abnormal growth of the VSMC lining artery walls plays a key role in the blockage of arteries during coronary artery diseases. This abnormal growth also contributes to reblockage (restenosis) of the arteries that have been opened by balloon angioplasty or replaced in

1






2

bypass operation. The major cause of restenosis is exaggerated healing response of medical VSMC to vascular injury. Angioplasty is carried out to restore blood flows to ischemic coronary arteries. However, there has always been arterial wall injury inevitably associated with this procedure. The injury damages endothelium which normally secretes nitric oxide to prevent VSMC from growth. The injury also stimulates a variety of growth promoters for a repairing procedure (Epstein et al. 1994). Growth factors stimulate VSMC to migrate and to proliferate into lumen to form neointima, where they continue to proliferate and secrete extracellular matrix. The neointimal mass continues to expand and eventually reblocks the blood vessels (Wilcox1993). Neointimal formation is the result of cell migration, followed by cell proliferation and matrix secretion. At late stage, lumenal narrowing is due to both intimal smooth muscle proliferation and collagen and elastic deposition (Clowes et al. 1983). The problem of controlling restenosis becomes largely the problem of controlling the VSMC proliferation.

Many factors are involved in regulating of VSMC proliferation. Some researchers suggested that most endogenous vasoconstrictive substances are also growth promoters and most endogenous vasodilator substances are growth inhibitors (Dzau 1992). RAS is one of the most important systems involved in restenosis. Traditionally, the reninangiotensin system is an endocrine system which is involved in regulation of fluid homeostasis and blood pressure (Guyton 1986). The discovery of tissue RAS has led angiotensin research to a new era. Using modem technologies, researchers have found the components of RAS in various tissues, including the blood vessels (Dzau et al. 1987). Molecular cloning of the three types of angiotensin receptor subtypes (ATIA, ATIB and






3

AT2) allowed us to carry out more intensive studies on their characteristics and their physiological functions (Griendling et al. 1993). Understanding of their genetic structure also opened an avenue for gene therapy on angiotensin related diseases. For instance, antisense inhibition of RAS has drawn a lot of attention among the researchers.



Pathophysiolgy of Restenosis



Under normal conditions, VSMC are quiescent. During angioplasty, inflation of the balloon causes an increase in lumen' size. This gain in lumen size has been shown due to both loss of mass in plaque and overstretch of vessel walls (Clowes et al. 1988). Unfortunately, the loss of mass damages endothelial cells, and the overstretch of vessel walls traumatizes VSMC in the media. The direct consequence is to break the balance between the growth promoters and inhibitors. For the side of growth inhibitors, removal of endothelial cells directly causes a reduction in nitric oxide production. Nitric oxide is released, by endothelial cells in response to the increase of blood flow. The immediate effect is to produce vasodilatation and VSMC relaxation (Palmer et al. 1987). This will cause an increase in lumen size. Apparently, NO is not only a vasodilator, but also a growth inhibitor which can prevent VSMC from growing. The intact endothelial cell's layer serves as a screen to stop migration of VSMC into lumen. For the side of growth promoters, a number of growth promoters are produced. For instance, Ang II released from endothelial cells and VSMC through a paracrine mechanism begins to stimulate






4
produced. For instance, Ang II released from endothelial cells and VSMC through a paracrine mechanism begins to stimulate VSMC to migrate into lumen, leading to cell proliferation there (Dzau 1992). This proliferation is highly exaggerated, then the consequence is reblockage of lumen.

VSMC are highly proliferable cells. Cultured rat aortic smooth muscle cells have been used as an experimental model system for the studies of different growth modulators. These cells show a high tendency of proliferation even without growth factors' stimulation. The cells also have a high density of AT, receptor on their membrane surface. They are characterized by a high responsive to Ang II stimulation (Rosendorff 1996). The detailed mechanism of how Ang II is involved in restenosis will be discussed in following paragraphs.



The Renin-Angiotensin System MRAS)



The classic RAS is an endocrine system which is very important in humoral regulation of the circulation. This type of RAS exists in body fluids and has traditional characteristics of hormones. Its major component, Ang II, is one of the most powerful vasoconstrictive substances known. It is estimated that one millionth of a gram of Ang II can increase the arterial pressure of a human 50 mmHg or more (Guyton 1986). The basic function of this hormone is to cause vasoconstriction, thereby to increase total peripheral resistance and to elevate blood pressure.







The Components of the RAS

Figure 1-1 illustrates the components and functional steps by which the classic RAS helps in the regulation of blood pressure. Renin is an enzyme which is synthesized and secreted by juxtaglomerular cells of the kidneys. The function of renin is to cleave angiotensinogen to release a 10 amino acid peptide, angiotensin I. Angiotensin I has no vasoconstrictor. The active vasoconstrictor in the RAS is Ang II which is an 8 amino acid peptide converted from Ang I by ACE mostly in the endothelium of the lungs. Ang II can be inactivated and degraded by angiotensinase. The principal effects of Ang II include vasoconstriction and salt and water retention. Angiotensin Receptors

There are two major types of angiotensin receptors, AT1 and AT2. In rats the AT1 receptor is further classified into ATIA and ATIB subtypes according to their structure differences. The different Ang II binding sites were first described by their pharmacological characteristics. For example, Ang II type-i receptor specifically binds Losartan (DuP753) and Ang II type-2 receptor specifically recognizes PD123319. Recent advances in molecular cloning of the cDNAs of these receptor subtypes revealed the true structure different in their genetic level. The cDNAs of the AT receptor were first cloned from rat aortic VSMC and bovine adrenal zona glomerulosa cells (Murphy et al. 1991 and Sasaki et al. 1991). This type of AT was also recognized as ATIA. Later the other AT1 subtype, ATIB was cloned from rat adrenal (Murphy et al. 1992) gland and pituitary. Two AT subtypes can be also found in mouse genomic DNA; however there is no evidence that the divergence to ATIA and ATIB exists in humans (Smith and











Circulating RAS system


Bran -ANystem Angiotensinogen

Renin giotensino, n Il Renin Angiotensin I ACE

ACE Angiotensin 11 Angiotensin II =Agonn


Aminopeptidase A AT1 AT2


AT2 Angiotensin III


Antagonist AT2 \Aminopeptidase B (losartan) Antagonist (PD123319) Angiotensin 3-8






Figure 1-1. Schematic elucidation of the components and functional steps of renin angiotensin system.






7

Timmermans 1994). Rat ATIA is localized on chromosome 17 and ATIB on chromosome

2. In humans, chromosome 3 bears the single AT, gene.

Rat ATIA and ATB share about 96% identity in their amino acid sequences. However, they have remarkable differences in the noncoding regions which may reflect potential variety in the regulation of gene expression. They are both 395 amino acid proteins with molecular weight of 41 KDa. Hydropathy analysis of amino acid sequences suggests that AT, receptor is seven transmembrane domains, G-protein coupled receptor. Previous studies have shown that AT, receptor is responsible for most traditional Ang II functions.

When Ang II binds to its AT, receptor, the binding activates a specific protein signaling system. These signal transduction pathways include activation of phospholipase C, phospholipase D, calcium channels and other ion channels. The signal transduction pathways can be different in different tissues. In VSMC, after Ang II binds to AT,, phosphatidylinostiol bisphosphate is hydrolyzed and diacylglycerol and inositol triphosphate are increased. During the same time frame, there is a transient increase in intracellular calcium level. The immediate consequence of these intracellular signals is the activation of protein kinases, including PKC, tyrosine kinases, and a calcium-calmodulindependent protein kinase. These kinases furthei phosphorylate a number of other proteins such as MAP kinase, myosin light chain and vimentin, and these proteins mediate cellular functions of smooth muscle, such as contraction.

The cDNA of AT2 receptor subtype was cloned in 1993 from a rat pheochromocytoma cell line (PC12w) (Kawamura et al. 1993). The AT2 cDNA






8

comprises 2,868 nucleotides and encodes a 363 amino acid protein with seven putative transmembrane domains. It shares only 32-35% identical amino acid sequence with the AT, subtype, and this identity is mainly concentrated in the putative transmembrane regions. The AT2 gene has been characterized in humans (Inagami et al. 1995). This gene is located on the X chromosome in both human and rat (Hein et al. 1995). No subtype for AT2 has been reported.

AT2 receptor subtype is characterized by its specific binding to PD123319 and CGP42112A. This receptor subtype is expressed at very high levels in the developing fetus. By contrast, in the adult, its expression is restricted to the adrenal glands, uterus, ovary, heart and specialized nuclei in the brain. AT2 has also been shown to be a G proteincoupled receptor. Kang et al. (1994) demonstrated that AT2 modulated K+ current through Gi. Buisson et al. (1995) showed that AT2 mediated inhibition of T-type Ca2+ current in the NG1088-15 cell line through a pertussis-toxin insensitive, G protein. The clear physiological function of AT2 receptor has not been identified. However, the piling evidence suggested that AT2 may play a role in some processes such as cellular growth, differentiation or adhesion. Interestingly, although -AT2 disappears quickly after birth in most parts of the body, it can be re-expressed in certain pathological situations involving tissue repair, such as vascular neointima formation and wound healing (Nakajima et al. 1995). Gyurko et al first noted that the AT, receptors increase IP3 hydrolysis and the AT2 receptors decreased IP3 hydrolysis in rat skin slices (Gyurko et al. 1992). A report from Dzau's group suggested that AT2 plays an opposite role against AT, in neointima formation after angioplasty (Nakajima et al. 1995). They observed that overexpression of






9
the AT2 receptor attenuated neointimal formation in rats. Also in cultured smooth muscle cells, AT2 receptor transfection reduced proliferation and inhibited MAP kinase activity. Tanaka et al. (1995) and Yamada et al. (1996) suggested that AT2 could trigger apoptosis in rat ovary granulosa cells and PC 12w cells. They further suggested that the mechanism of AT2 induced apoptosis was mediated by the dephosphorylation of MAP-kinase. ASODN to MAP kinase phosphatase 1 inhibited the AT2 receptor-mediated MAP kinase dephosphorylation and blocked the AT2 receptor mediated apoptosis. Taken together, all these data indicate that AT2 may play an important role in developmental biology and pathophysiology.

There are several selective antagonists for AT, and AT2 receptors. Actually the initial classification of AT, and AT2 was based on their different binding characteristics to antagonists. Losartan binds to both ATIA and ATIB subtype. PD123319 is the specific antagonist for the AT2 receptor. These two are also the most frequently used antagonists for the AT, and the AT2 both in vitro and in vivo.

I

Tissue RAS

The existence of tissue RAS, independent of the circulating RAS, was first described in the early 1970s. Tissue RAS occurs in a variety of organs, such as brain, heart, blood vessels and many other organs in the body (Phillips 1987 and Dzau 1987). Modem molecular technology has helped to identify the components of RAS, such as Ao, Renin and Ang II receptors, in a large variety of tissues. These components were proposed to interact with each other by means of a paracrine and autocrine function. In






10

the paracrine mode, one cell produces Ang II and delivers it to a neighboring target cell which has receptors to bind and respond to the Ang II stimulation. The autocrine mode describes a cell which produces Ang 1I, releases it exogenously, and this feeds back via membrane receptor onto the same cell to regulate the rate of synthesis (Phillips et al. 1993).

The brain was among the first tissues that were proposed to have a tissue RAS independent of the circulating RAS. Every key component of RAS has been identified in the brain. Since the brain is protected by the blood brain barrier from circulating Ang II, an independent brain tissue RAS was suggested and thoroughly investigated by many groups (Phillips 1987). Both AT, and AT2 receptors are found in the brain. The AT, receptors are distributed in areas associated with cardiovascular effects of central Ang H, such as organum vasculosum lamina terminalis, supraoptic and paraventricular nucleus. The AT2 subtype ais located at locus coeruleus, inferior olive and mediodorsal thalamic nucleus. Its function in brain has not be clearly elucidated. Recently two groups used gene disruption technique to study the possible function of the AT2 receptor on knockout mice. Hein et al show that AT2 knockout mice develop normally, but have an impaired drinking response to water deprivation as well as a reduction in spontaneous movements. They also found that baseline blood pressure of the mutants is normal, but they show an increased vasopressor response to injection of angiotensin H (Hein et al. 1995). On the other hand, Ichiki et al. (1995) reported disruption of the mouse AT2 gene resulted in a significant increase in blood pressure and increased sensitivity to the pressor action of






11

angiotensin 11. The controversies between the results of two research groups on the AT2 knockout mice suggest that further investigation will be necessary.

There are three distinctive physiological effects of brain angiotensin receptors when Ang I is given centrally. The effects are an increase in blood pressure, AVP release and motivation to drink. The effects have been shown to be mediated exclusively by the AT, receptor, since losartan and the AT, AS-ODN blocked these responses (Hogarty et al. 1992, Meng et al. 1994).

In the vascular system, Re et al (1982) first showed that there was renin in the dog aorta. Since then every major component in RAS has been discovered, including Ao, ACE, AT, and AT2 receptors. The AT receptors are located on the membrane surface of medial VSMC and also in the nucleus (Tang et al. 1992). Renin and Ao were found to be at endothelium and media and adventitia. ACE was found in endothelium and some parts of media. AT and AT2 were both located at medial VSMC. The effects of Ang II in the vascular system are twofold. In response to Ang II, there are both short and long term effects; vasoconstriction and VSMC growth. Stimulation of the AT2 receptor may cause dephosphorylation of MAP kinase in VSMC (Nakajima et al. 1995).



The Renin-Angiotensin System and Restenosis



RAS is an endocrine system which controls body fluid and electrolyte homeostasis. Classical RAS is a blood borne, circulating hormonal system. The target organs for Ang H are the blood vessels, kidney and the adrenal cortex, in which Ang II through its type 1






12

receptor mediates vasoconstriction, decreased glomerular filtration and aldosteron secretion. The over all effects will be conservation of water, increased Na+ reabsorption and increased blood pressure.

In addition to circulating RAS, every component of RAS has been found in the vasculature and a paracrine mechanism has been proposed. The endothelial cells secrete both NO and Ang II. Through a paracrine effect, Ang II and NO are released to act on neighboring VSMC. The VSMCs secrete Ang II only. Through a paracrine effect, Ang II is released on other VSMC. Circulating Ang II may also reach the VSMC. When the layer of endothelial cells is intact, there is a balance between the growth promoting effect of Ang II and antigrowth effect of nitric oxide, and circulating Ang II probably does not reach the media directly. Damage to the endothelial cells, as for example after balloon injury, changes the balance. The growth promoting effects of Ang II become a dominating force. Interestingly, Schwartz suggested that the proliferating VSMC were actually descendants from single colonies of cells that migrated into lumen and these cells in neointima have a stronger response to Ang II than the cells in media (deBlois et al, 1996). They are regulated differently in response to systemic infusion of Ang II. Since the VSMC in neointima have also been showed to have a higher density of AT, receptor on their membrane, it is very likely that Ang II is one of the early factors involved in VSMC migration (Viswanathan 1993, 1994). The mechanism can be proposed as follows: After removal of endothelial cells by balloon injury, the damage and distention to VSMC cause an increase in Ang II production and also a upregulation of other components of RAS in the vasculature. The Ang II stimulates the cells that have a higher density of AT,




13
receptors to migrate into lumen and the cells begin to proliferate and secrete collagen and elastin. This is a wound-healing process leading to repair of the damage caused by angioplasty. Unfortunately, VSMC can not fully replace the functions of endothelial cells. Instead of NO, VSMCs secrete Ang II. So the repairing process becomes largely exaggerated.

Binding of Ang II to its AT, receptor activates a cascade of acute and delayed cellular events. Direct effects include the activation of phospholipase C and generation of metabolites that modulate calcium-sensitive protein kinase C (PKC) activity and cytoplasmic calcium concentration. Ang H binding also activates calcium channels, causing rise to calcium influx to increase VSMC contraction. The PKC mediated protein phosphorylation activates nuclear elements, with long term consequences with regard to gene expression, protein synthesis, mitogenesis and vascular hypertrophy. This long term effect of Ang II is very important to restenosis and arteriosclerosis. The mechanism which is proposed by Takeuchi et al. (1990) is as follows. Ang II binding to its AT, receptor leads to a rapid increase in c-fos and c-jun mRNAs levels. The c-fos and c-jun have been shown to 'form a heterodimeric transcriptional complex called AP-1 which is able to manipulate target gene expression. This type of Ang II stimulation can be blocked by staurosporine, a PKC inhibitor, or by salarlasin, an Ang H receptor blocker. These results indicate that Ang II induced gene expression and cell growth is partially mediated by PKC. On the other hand, calcium has been shown to be important in MAP kinase activation. MAP kinase is also one of the most important growth modulator (Fig 1-2).










'AT1


_ )No Cell
Growth


Figure 1-2. Schematic elucidation of the signal transduction of the AT1 receptor





15

Experimental Models of Restenosis



To model human responses to angioplasty restenosis and balloon catheter injury, three animal modes are frequently used. The most well developed and extensively investigated animal mode of restenosis is the rat common carotid artery. The model was introduced by Clowes et al in 1983. In their study, they proposed the VSMC migration and proliferation are the key factors for restenosis. The knowledge gained from the rat model has contributed to the understanding and interpretation of the restenosis response in humans. However, there are some disadvantages to the current rat model. There is no thrombotic component to the response to angioplasty and the rat is resistant to the dietary induction of hypercholesterolemia.

The rabbit reinjury system is another widely used animal model. Rabbits are fed a very high-cholesterol diet, and primary injury is reduced with a balloon catheter in the iliofemoral artery. Six weeks later, the same site is reinjured with a balloon catheter. This model allows some thrombosis and rabbits are easy to produce hypercholesterolemia.

Porcine models of restenosis have gained a lot of popularity since their use started in the 1970s. This model is perhaps the best model of restenosis resembling human restenosis. The porcine vascular system is very much similar to that of humans. The major disadvantage of the pig is its size. The pig is more expensive to keep and requires a larger scale in administration of drugs.






16

Methods to Inhibit Restenosis





A large number of different drugs have been tried both on human and laboratory animal models. These drugs include thrombosis inhibitors (aspirin, dipyridamole, enoxaparin, heparin, vapiprost, and warfarin), and VMSC migration inhibitors (cilazapril and heparin). However, so far none of these drugs have producted any significant results in clinical trials.

In 1989 Powell et al first reported that calizapril, an ACE inhibitor, significantly reduced restenosis in rats when it was delivered via the drinking water for two weeks before angioplasty. After this success, several other research groups demonstrated beneficial effects of ACE inhibitors on different animal models. The ATI antagonists, losartan and TCV116, were also reported to be effective (Kauffman et al. 1991 and Kawamura et al. 1993). However, problems associated with these inhibitors and antagonists (such as side effects, need for repeated ,administration and the problems associated with dose and time of dosing) have prevented these drugs from effectively treating human restenosis. For instance, although ACE inhibitors have been shown to be effective in preventing animal restenosis in the lab, the human trial by MECATOR failed to confirm any beneficial effects of ACE inhibitors. Later, Rakugi et al (1994) concluded that patients would have to be put on a much larger dose of ACE inhibitors for a longer time in order to inhibit tissue RAS. Traditionally, drugs work on the protein level. Although they can inhibit protein functions, they usually need repeat administration. Non-specific effects and protein upregulation associated with drug inhibition






17

are frequently observed. Although drug therapies have not solved the restenosis problem in the clinic, they provide us with important insights about mechanisms of this disease. For instance, ACE inhibitors work on both Ang II and bradykinin pathways. They block Ang I synthesis and also enhance NO synthesis. Studies with AT, antagonist, losartan, prove that the blockage of Ang II synthesis is the most important part of ACE inhibitor function. Since losartan and TCV116, the AT, specific antagonists inhibit restenosis just as well as ACE inhibitors. This result also confirms that the growth promoting effects of Ang II is mediated through AT1 receptor. Recently Iwai et al. (1997) showed that renin levels in injured blood vessels were increased during the first 3 days after angioplasty and that administration of quinapril significantly reduced neointimal formation. In another experiment they indicated that rat peritoneal macrophage/monocyte cells expressed renin mRNA. Macrophage/monocyte cells may be a source of tissue renin in some pathological conditions (Iwai et al. 1996). They suggested that the upregulation of renin might be the earliest event in vascular RAS activation. These data together show that RAS in vasculatqre plays an important role in restenosis. Blockage of RAS could be the potential treatment for this disease.



Gene Therapy

Viral vector mediated gene transfer holds great potential in treating restenosis (Table 1-1). There are several vectors available today. They are retroviral vectors, herpes virus vectors adenovirus vectors, and adeno-associate virus vectors. Each vector has its own characteristics. For instance, the retroviral vector will only infect dividing cells. It







18

works perfectly on cell cultures of dividing cells. However, the efficiency is fairly low when it was used on animals. The herpes virus vector is good for transfecting neuronal cells only. The adeno virus vector has been the most frequently used vector for gene delivery in the vascular system. Its advantages include high efficiency and infectivity for both replication and nonreplication cells. Studies show that the adeno virus vector is able to transfer genes into endothelial and smooth muscle cells. However, the major disadvantage is the immune reaction which it generates. Adeno-associated virus vector is a fairly new vector. It shows great potential for gene therapy. It is non-pathogenic and able to infect most cell types with high efficacy. It incorporates into a genome, so it allows stable and long lasting expression.

The two most significant experiments in gene therapy on restenosis were done by two research groups led by Nabel at the University of Michigan and Leiden at the University of Chicago. Nabel's group transfected balloon injured porcine arteries with an adeno virus vector encoding the herpes virus thymidine kinase (tk). The tk phosphorylates the nucleoside analog ganciclovir and causes DNA chain termination in the transfected cells. The intimal hyperplasia was significantly decreased after a course of ganciclovir treatment (Ohno et al. 1994). Leiden's group used a replication-defective adenovirus encoding a nonphosphorylatable Rb gene. Rb (retinoblastoma) gene is a cell cycle control gene whose nonphosphorylated form is antiproliferative. The nonphosphorylatabe Rb gene product transferred by adeno viral vector significantly blocked restenosis in a porcine femoral artery (Chang et al. 1995). The tk gene transfer method was also repeated in a rat carotid artery model of restenosis and showed results similar to Nabel's report (Guzman et







19

al. 1994). Although gene therapies have many potential advantages over drug therapies in treating restenosis, the safety has always been the biggest problem for this approach. The concern has led many researchers to look for alternatives to non-viral gene therapy methods, such as antisense-ODN inhibition.



TABLE 1-1. GENE THERAPY FOR RESTENOSIS

Vector Type Gene Animal Model References Adenoviral vector Herpesvirus Pig Ohno et al. Science thymidine kinase 1994
(tk)
Adenoviral vector Retinoblastoma (Rb) Pig Chang et al. Science 1995
Adenoviral vector Herpesvirus Rat Guzman et al.
thymidine kinase PNAS-USA 1994
(tk)
Sendai Viral Atrial Natriuretic Rat Morishita et al. J Liposome Peptide (ANP) Clin Invest 1994


Antisense Inhibition

Since restenosis is largely due to the migration and proliferation of VSMC, the antisense technology makes perfect sense for treatment of restenosis. Started in early 1980s, researchers have designed and tested a large number of AS-ODNs targeting numerous growth factors and growth regulatory elements to look for the treatment for restenosis. Simons et al (1992) reported that AS-ODN against proto-oncogene c-myb gene had antigrowth effects in rat arteries. They used Fi27 pluronic gel to facilitate delivery of AS-ODN and achieved -80% reduction in neointimal formation. Shi (1994) et al showed that proto-oncogene c-myc AS-ODN inhibited neointimal hyperplasia using a porous catheter delivery system in pigs. This catheter can infuse AS-ODN solution with





20

certain pressures into vascular wall to facilitate uptake. Bennett et al (1994) demonstrated that the c-myc antisense was also effective in reducing neointimal formation in a rat carotid artery model using F127 pluonic gel applied on adventitia. Morishita et al. (1994a) showed that AS-ODN against a cell cycle regulatory enzyme, cyclin-dependent kinase 2 kinase (cdk 2 kinase) gene was able to decrease neointimal formation. Their delivery system was sendai viral liposomes. In their pharmacokinetics study, they showed that the sendai viral liposome could help to retain AS-ODN in blood vessels for 1 week (Morishita et al. 1994b).

TABLE 1-2. ANTISENSE THERAPY FOR RESTENOSIS
Targeting Gene Delivery System Animal Model References
c-myb F,27 Pluronic Gel Rat Simons et al. Nature 1992
c-myc Transcatheter Pig Shi et al.
Circulation 1994
cdk 2 kinase Sendai Viral Rat Morishita et al.
Liposome PNAS-USA 1993
bFGF Adenoviral vector Rat Hanna et al. J. Vasc.
Surg. 1997


Another antisense approach is to package antisense sequences into expression vectors. Expression vectors will produce antisense mRNA to downregulate gene expression of sense mRNA. For instance, the antisense sequences against (basic fibroblast growth factor) bFGF packaged in an adenovirus vector was shown to be effective in reducing rat restenosis (Hanna et al. 1997). This approach allows the antisense sequence to produced continuously. Antisense inhibition has offered tremendous potential in fighting restenosis. However, the questions, such as which growth factor is the key and which type of AS-ODN is best for patients, still needs to be answered. The delivery






21

system has always become one of the most difficult hurdles in adopting antisense application.



The Antisense Technology



General

For centuries, researchers have been looking for the "magic bullet"-a drug able to reverse the illness without side effects. Since lots of diseases are caused by overproduction of certain "bad" proteins, most of the work has been focused on protein inhibitors. Recently, however, a number of researchers have turned their attention to the genetic level, the machinery which is responsible for producing the proteins. Gene therapy has evolved into a fascinating field which holds great potential to cure diseases, such as AIDS, cancers and cardiovascular diseases. The aims of gene therapy generally fall into two categories, replacement of abnormal genes with normal genes or inhibition of disease causing gene products. Antisense oligonucleotide inhibition belongs to the second category. AS-ODN are specially designed DNA or RNA fragments which are able to interfere with gene expression by binding to DNA or mRNA inside the cells. This new approach for gene manipulation was first proposed by Zamecnik and Stephenson in 1978. In their pioneer experiment for the antisense inhibition, they inhibited Rous sarcoma virus replication with a 13 mer antisense oligonucleotide (Zamecnik and Stephenson 1978). Many researchers have found success using that technique during the past 19 years. Recent discovery of naturally occurring antisense RNA suggests that prokaryotes are actually using antisense RNA in regulating their gene






22

expression (Wagner et al. 1994). It was also proposed by some researchers that, besides microbes, plant and animal cells might also use antisense strategy to control gene expression (Knee et al. 1991). In most cases, researchers use short strings of synthetic antisense nucleotides instead of a large antisense genes-although some groups are still working on that. Clinical trials are now in progress for the AS-ODNs in treating several human diseases including acute myologenous leukemia, IV infection and CMV (cytomegalovirus) infection (Anderson et al. 1996). Isis company has completed its third phase clinical trial for the ASODN treatment of CMV infection.



Design of AS-ODN

It is proposed that AS-ODN can work on any of the following processes to block the gene expression. These processes are uncoiling of DNA, transcription of DNA, export of RNA, RNA splicing, and RNA translation. The sequences of AS-ODN are short (usually 1530 mers). Since phosphodiester ODN has a fast degradation, most AS-ODN in use now is backbone modified. The two most popular modifications are methylphosphonation and phosphorothioation. The methylphosphonation was designed by Ts'o and Mller (1979). They replaced an oxygen atom in each phosphate group with a methyl group (CH3). This step helped to increase the cellular uptake and provided resistance to break down by enzymes. Phosphorothioates were introduced by Chang et al (1989). They exchanged an oxygen atom with a negatively charged sulfur atom. The phosphorothioates are water soluble and resistant to enzymes. Even now there is no standard rule in selecting target sequences. In general, researchers have found that most regions of the RNA including 5'- and 3'-untranslated, AUG






23

initiation, coding, slicing junctions and introns can be targeted. The only way to determine which sequence is most effective is through experiments. Wagner (1994) suggests that for any 20 mer phosphorothioate ODN, up to 50 sequences should be screened to find an effective AS-ODN. For 15 mer, screening six sequences is efficient to find at lease two sequences to be effective. In our laboratory, we found this is an exaggeration and we have successfully designed useable antisense ODNs by initially designing as few as three sequences.



Mechanism of Action

AS-ODN is theorized to work with at least three different mechanisms. First, ASODN can bind to DNA and form a triple helix to block DNA uncoiling and transcription to mRNA. Secondly, AS-ODN can bind to mRNA to interfere with splicing, transporting and translation into protein. Thirdly, AS-ODN can stimulate ribonuclease H (RNAse H) and destroy the DNA-mRNA hybrids. No matter -which mechanism is involved, the final result should be a reduction in the protein level, and inhibitory effects on the targeting protein related physiological effects.



Use of Controls

It is always important to use proper controls in experiments to make sure that the effects are real antisense effects. Most frequently used control sequences in antisense research, including sense (S), scrambled (SC), mismatch and inverted. It was also suggested to measure changes of other proteins with similar life cycles along with target proteins. This will show us if the AS-ODN is specifically inhibiting the target protein.








Non-Specific Effects of AS-ODN

Although antisense technology was introduced as a "magic buflet"-a new drug without side effects-the reality is most currently used AS-ODN still can not avoid producing side effects and non-antisense effects, especially when they were used in high concentration. For example, in a cell culture study, we found non-specific cell growth inhibition when the ASODN concentration exceeded 25 pM. In order to achieve specific antisense effects, the concentration used must be relative low (<10 pM).



Antisense Inhibition in RAS

Before the introduction of antisense inhibition in RAS, there were already several approaches to inhibit RAS, including ACE inhibitors, renin inhibitors and angiotensin receptor antagonists. However, these drgs are all short acting. In our lab, we began to explore the possibility of using antisense strategy for RAS inhibition in 1992. We designed a 15 mer ASODN to AT, receptor mRNA and a 18 mer AS-ODN to Ao mRNA. Our experiments clearly showed that antisense technology is effective in manipulating gene production in RAS. We found that AS-ODNs against AT, receptor significantly reduced the central Ang II induced drinking response when given intracerebraventricularly (Meng et al. 1994). The receptor binding study showed the ATI AS-ODN decreased AT, receptor protein in the hypothalamic region of rat brains. The AS-ODN targeting angiotensinogen (Ao) mRNA significantly reduced high blood pressure in SHR when it was administered centrally and peripherally (Wielbo et al. 1995, 1996). Recently we used antisense sequence packaged in an AAV vector and achieved






25

long term reduction of blood pressure in SHR (Phillips 1997). In summary, all these data suggest that AS-ODN inhibition is effective in RAS gene inhibition.







Despite the intensive investigation of the role of tissue RAS in the development of restenosis, the controversies remain. It is necessary to use new technologies and novel approaches to fiu-ther address this problem. Experimental evidence indicates strong connections of the tissue RAS and vascular diseases. In the present study, we exploited autoimmunization and antisense inhibition to investigate the role of RAS in the development of vascular response to injury restenosis. Our results confirmed the important role of Ang II and its AT, receptor. Further, we suggest that vascular Ang II is involved in the initiation of the growth response to injury. Antisense inhibition provides a useful tool to study the mechanism involved in vascular injury, and is also a potential therapeutic method for treating restenosis.












CHAPTER 2
HYPOTHESIS AND SPECIFIC AIMS



Hypothesis


There is controversy surrounding the role of RAS in restenosis. The current hypothesis is that Ang I is an initiating and critical factor in response to vascular injury. I will test this by making rats that develop their own autoimmunity to the AT, receptor to establish the importance of the AT, receptor in restenosis. Second, I will test specific antisense-ODN to AT, mRNA for inhibition of AT, receptor. Third, I will develop a novel means of delivery of AS-ODN with a dendrimer for potential therapy.



Specific Aims



Specific Aim I

I will autoimmunize animals against their AT, receptor to test specifically and chronically whether Ang II stimulation is critical for the vascular response to injury. Specific Aim 2

I will test the specific inhibitory effect of AS-ODN targeted to AT, receptor mRNA. To accomplish this goal I will use the approach of inhibiting central Ang II effects. Centrally Ang U induced drinking and AVP release will be used as indicators.






27

Specific Aim 3

I will develop and test a dendrimer based delivery system as an alternative of liposome delivery of AS-ODN in vivo. Specific Aim 4

I will inhibit arterial angioplasty induced neointimal formation by inhibition of AT, receptor using AS-ODNs delivered by dendrimers.












CHAPTER 3
MATERIALS AND METHODS



Experiments of Autoimmunization



Peptide Synthesis and Immunization

Adult male Sprague-Dawley rats (200-225 g) were acquired from Harlan (Indianapolis, In, USA). The animals were kept in individual cages in a room with a 12-hr light, 12-hr dark cycle. They were given tap water to drink and standard rat chow ad libitum. Peptide synthesis was carried out at the Protein Core, University of Florida. The peptide sequence was designed corresponding to amino acid sequence 14-23 of the first extra-cellular domain of the AT, receptor. The peptide was anchored to polylysine cores to form a multiple antigenic peptides (MAP) according to the method of Tam (1988). This design completely eliminates the conventional step of conjugation of peptides to carriers. HPLC and mass-spectrophotometer were used to check the sequence and purity of the products. For each injection, rat was given 400 pg of peptide mixed with 400 PI of Freund's adjuvant. The animals were immunized with multiple dorsal subcutaneous injections on day 1, 20 and 40.








Animal Model

The animals that produced significant titer (higher than 1000) of antibody after the second injection were used in the experimental group. Rats given the same protocol of only Freund's adjuvant injections were used in the control group. At day 45, both groups of rats were anesthetized with sodium phenobarbital (30-40 mg/kg body weight) (i.p.). Under sterile surgery conditions, a 2 French Fogarty catheter (Baxter Healthcare, Irvine, CA) was introduced into the left femoral artery and threaded through to the left common carotid artery. The balloon was inflated in the carotid artery with saline and was passed three times up and down the artery to produce deendothelializing effects (Clowes et al. 1988). After surgery, animals were returned to cages and kept for two weeks. All animals were kept according to the AALAC guidelines for animal care and the experiments were approved by the IACUC committee of the University of Florida.



Cell Culture

Male Sprague-Dawley rats (200-250 g) were acquired from Harlan. Rats were anesthetized with phentobarbital and aortas were removed. Tissue samples was transferred to 5ml dishes and were digested in 0.67 mg/ml of type II collagenase (Sigma, St. Louis, MO) at 37C for 30 min. After the digestion, adventitia was removed and tissues were incubated with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% of fetal bovine serum (EBS) overnight. The following morning, the tissues were digested again with 0.67 mg/ml of type II collagenase and 2.5 mg/ml of elastase (Sigma) for 60-90 min. The tissue were tritureated with pasture pipettes to speed up the process.






30

The digestion was stopped by diluting with DMEM with 10% FBS. Cells were spun for 5 min at 1000 rpm and plated onto culture dishes containing DMEM with 10% FBS.



Western Blotting and ELISA for the Antibody Production

Blood was taken from the tail at days 25, 45 and 60. Blood samples (500 pl) were collected in 1.5 ml centrifuge tubes and stored at room temperature for 30 min. Plasma was collected after centrifugation and kept at -200C until the day of measurement. Polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the method of Lammeli (1970). Membrane proteins extracted from rabbit adrenal glands were amended with 2x sample lysis buffer and boiled for 5 min before loading. After loading the samples on the 10%/6 pre-cast gel (BioRad, CA), the gels were electrophoresed at a constant voltage of 200 V for 45 min on a BioRad Gel Electrophoresis System. Rainbow molecular weight standard markers were used in all SDS-PAGEs. After electrophoresis, the gels were transferred to PVDF membranes (Bio-Rad) using the transfer buffer systems of Towbin et al (1979), or Szewczyk and Kozloff (1985). Transferred membranes were blocked for 1 hour in the presence of 5% bovine serum albumin (BSA) at room temperature, then the blots were probed by 1st (rat anti-AT1 autoantibody) and 2nd (Goat anti-rat IgG alkaline phosphatase, Sigma) antibodies. The antigenic bands were visualized by the incubation in substrate solution (NBT-BCIP).

Quantitation of autoantibody production was determined by ELISA. Microtiter wells were precoated with 600 ng/well of the synthetic peptide diluted in 50 mM sodium carbonate-bicarbonate buffer (pH--9.6) and incubated at 4"C overnight. Sodium






31

carbonate-bicarbonate buffer was added to the microtiter well at the same time to serve as a control. The microtiter wells were incubated at 3r*C with 5% milk in PBS for 1 hr before washing five times in washing buffer which was composed of lx PBS + 0.05% Tween-20. The serum samples containing rat autoantibody were diluted to 1:300, 1:900, 1:2700 and 1:8100, then added to the wells. After 1 hr incubation at room temperature, the wells were washed with washing buffer five times. The optical density was read from a Dynatech 600 microplate reader after the incubation with goat anti-rat IgG complexed to alkaline phosphatase (Sigma) and substrate solution.



Protein Kinase C Assay

Protein kinase C (PKC) assay was carried out by using Calbiochem nonradioactive protein kinase assay kit (Calbiochem-Novabiochem Co., San Diego, CA). Briefly, VSMC cultures were pre-treated with 100 pl of immune serum and control serum in DMEM for 60 minutes. PKC activation was achieved by adding Ang II with final concentration of 100 nM in the culture dishes. After 3 minutes incubation with Ang H (Dixon et al. 1994), incubating solution was aspirated off and cells were washed with ice cold PBS and scraped in 0.5 ml of homogenization buffer (20 mM Tris-HCI, 5 mM EDTA, 10 mM EGTA, 0.3% 3-mercaptoethanol, 1 mM PMSF, 10 mM Benzamidine). Cells were sonicated for 30 seconds on ice and centrifuge at 100,000 x g for 60 minutes at 4�C. The supernatant was used as the cytosolic fraction. The pellet was resuspended in PBS buffer, plus 0.5% Triton-X-100, for an additional 30 min. The suspension was centrifuged for another 30 min at 4�C at 100,000 x g and was used as the particulate






32

fraction. Protein concentration was determine using Lowry method (Lowry et al. 1951) and 500 Vg of protein was used in -each assay. The O.D. of each well in the assay was read on a Dynatech Immunoassay System at 492 mn.



Serum Transfusion

Blood samples from immunized and control groups were collected after rats were sacrificed. Sera were collected after spinning the samples at 1000 x g and stored at -20'C for the future transfusion study. Sprague-Dawley rats (300-325 g) were given two infusions of 0.5 ml serum one at 24 hours before surgery and one 1 hr after surgery from the femoral vein. The rats was divided into two groups (n=3 each). One group received serum samples from immunized rats, and the other group received serum from control rats. Both groups were subjected to the same procedure of angioplasty after serum transfusion. Rats were returned to their cages and kept for two weeks. At the end of the second week, rats were sacrificed and left common arteries were dissected out for the morpholQgical examination.



Autoradiography

The method of autoradiography used was described previously in detail (Ambuhl 1995). Briefly, rats were deeply anesthetized with sodium pentobarbital and perfused with 0.9% saline solution intracardially. Both carotid arteries were removed and frozen at 10�C for sectioning. The sections were cut on a cryostat (20 gm) and mounted onto gelatin-coated slides. The radio-ligand which was used in all experiments was 125I-Sarlle-







33

Ang II. Non-specific, AT,, and AT2 binding were determined in the presence of 1 jiM of Ang ]I or PD123319 or Losartan. Autoradiograms were generated by exposing the slides to X-ray films for 4 weeks. The photos were taken directly from an image analysis system (MCID, Imaging Research, Ontario, Canada).



Morphological Examinations

Two weeks after balloon catheterization, rats were deeply anesthetized with sodium pentobarbital and perfused via the heart with saline solution. The left and right common carotid arteries were dissected and fixed in 4% paraformaldehyde in PBS for 4 hours. The arteries were sliced at 20 pm thickness transversely on a cryostat machine. The tissues were stained with hematoxylin for the morphological examination. The ratios of the areas of neointima/media were measured and the data expressed as a ratio of intimal/media areas.



Immunohistochemistry

The immunohistochemical staining includes two separate experiments. In the first experiment, we applied rat autoantibody to recognize the AT, receptor on the rabbit. These aortas were from atherosclerotic rabbits in which the AT, receptor is highly upregulated in the aortas (Yang et al. 1997). The second experiment involved using a rabbit polyclonal antibody to AT, receptor to stain the carotid arteries from both immunized rats and control rats. The immunohistochemistry was carried out by using an ABC peroxidase staining kit (Pierce Chemical Company, USA). Briefly, tissues were







34

sliced into 30 gm sections on cryostat, after an overnight incubation in lx PBS at 40C overnight. After incubation with I" and 2d antibody, AT, signals were visualized by exposing sections to 3,3-diaminobenzidine.



Experiments on Central Ang II Inhibition



Animals

Adult male SHR and Sprague-Dawley (SD) rats (250-300 g) were acquired from Harlan (Indianapolis, IN. USA). The animals were kept in individual cages in a room with a 12 h light-12h dark cycle. They were given tap water to drink and standard rat chow to eat ad libitum.



Surgr

Rats were anesthetized with sodium pentobarbital (30-40 mg/kg body weight i.p) and a stainless steil, 23-gauge cannula was placed in the right lateral ventricle, using a Kopf stereotaxic instrument (stereotaxic coordinates: 1.0 mm lateral, 1.0 mm caudal to the bregma; 5.0 mm below the skull surface). The cannula was anchored with stainless steel screws in the skull and covered by dental acrylic. A steel wire obturator was placed in the cannula to maintain patency. Animals were returned to home cages to recover from surgery for 5 days. Five days after surgery a catheter filled with heparin (100 U/ml) was placed in the left common carotid artery under sodium pentobarbital (30-40 mg/kg body weight i.p.) anesthesia for blood sampling. The experiments were performed 24 hours after the carotid catheterization. The







35

obturator was removed and a 30 gauge injector, connected to a Hamilton microliter syringe (no. 700) by silastic tubing, was inserted in the guide cannula. (1) Ang H (50 ng) dissolved in 2 1 of artificial cerebrospinal fluid (ACSF) was injected. This injection was used to establish the control responses and to verify to cannula placement (a positive drinking response is a good indication that the cannula is in the ventricle). It has been shown by Hogarty et al (1992) that ACSF has no effect on drinking or AVP release when given centrally. The dose of 50 ng/Ang II was established by prior dose-response studies in this laboratory. (2) One hour after the Ang 11 injection, 50 pg antisense ODN targeted to the AT receptor, or scrambled ODN as control dissolved in 4 Wi isotonic saline was injected i.c.v. into the lateral ventricles. (3) Ang 1 (50 ng) (i.c.v) was administered 24 hours later to test the degree of inhibition by AS-ODN. (4) The experiments were performed in both Sprague-Dawley and SHR groups. In two SpragueDawley groups, the rats also received 2 i.c.v. injections at 24 h intervals with 50 pg of antisense, or scrambled ODN or 4 1 isotonic saline.



Oligodeoxynucleotides

Antisense (AS) oligodeoxynucleotides was synthesized as 15-mer to bases +63 to +77 of angiotensin II type 1 receptor mRNA. Scrambled control ODN was a 15 mer with a random sequence of the same bases. The ODN was modified by backbone phosphorothioation. ODNs were synthesized in the DNA Synthesis Laboratory, University of Florida, Gainesville, FL. The sequences were as the follows: AS: 5' TAACTGTGGCTGCAA-3', Sc 5'-AATTGGTGTGTTTCGTTC-3'.








Vasopressin Assay

Vasopressin assay was performed according to the procedure of Hogarty et al (1992). Briefly ODN treated rats were injected i.c.v with Ang ]1 (50 ng), and blood samples for the vasopressin assay in plasma were drawn at 1 min after injections. Blood samples (1-2 ml) were collected in chilled tubes containing 0.3 M ethylenediaminetetraacetic acid (EDTA) (50 pd/ml blood) from the carotid catheter. Plasma was colected after centrifugation and stored at -20�C until the day of extraction. The assay used was based on that of Raff et al (1991). Plasma AVP was measured using an antibody, raised in rabbits, against AVP. Cross-reactivity with other hormones (e.g. oxytocin. vasotocin, angiotensin I, angiotensin H) was <0.001%. AVP was extracted by absorption to bentonite with an 80% recovery. Plasma (0.5 ml) was extracted and then reconstituted by using assay buffer. Radiolabeled iodide [25I]AVP (DuPont) was used as the tracer and AVP (sigma) was used as a standard. The detection limit of the assay was 0.078 pg/tube.



Drinking

Drinking was measured for 30 min after Ang 11 injection. The water intake was read directly from scaled drinking bottles and is expressed as ml/30 min.



Radioligand Binding Assay

Sprague-Dawley rats were given three i.c.v. injections of antisense, or scrambled ODN (50 jig each injection) or 4 l isotonic saline at 24 h intervals. Rats were killed by decapitation 6 h after the third injection and the hypothalamic block including the hypothalamus, thalamus







37

and septum was dissected. Membrane proteins (100 pg) extracted from the hypothalamic block were used in the binding assays. ['51]SI-AII (0.2 nM) was used as radioligand in all experiments. The total volume for each tube is 500 pl. The incubation time was 90 min at room temperature. Ang II and losartan (IpM each tube) were used to determine specific binding and AT, receptor binding respectively.



Experiments on Dendrimer Delivery System



Dendrimer Labeling and Purification

Dendrimer generations four (Aldrich Inc.), six (Polysciences Inc.), and ten (Dendritec Inc.) were labeled -by using fluroecein isothiocyanate (FITC) (Molecular probe). The conjugation procedure was carried out in a centrifuge tube with continuous stirring for 24 hours at room temperature in a 1 to 1.1 molar excess of the isothiocyanate. G-10 Sephadex (Sigma) spin columns were used for the separation of FITC labeled dendrimer from unreacted FITC. Labeling of dendrimers was confirmed by analyzing the Den samples by thin layer chromatography (TLC) on Whatman PE Sil G/UV plates with a mobile phase of chloroform, methanol, ammonia (7:2:1).



Dendrimer and Oligonucleotide Conjugation

To determine if the dendrimers are be able to complex with oligonucleotides and form relatively stable compounds, we tested the DEN's ability to cause ODN-gel retardation on a 15% horizontal non-denaturing polyacrylamide gel system in TBE buffer.







38

FITC-labeled 15 mer antisense oligonucleotide targeting rat AT1 receptor mRNA were used in the conjugation studies with the dendrimers. The 6th generation dendrimers were complexed to the phosphorothioate antisense oligonucleotide by mixing them together in PBS buffer. The molar ratios of oligonucleotide to dendrimers were 1:1, 1:0.5, 1:0.1, 1: 0.01 and 1:0 respectively. The samples were loaded into wells after premixed with 50% glycerol. The photos were taken under UV transluminator at 360 nM.



In vivo Metabolism

Male Sprague-Dawley rats weighing 200-250 g were anesthetized with 100 mg/kg of ketamine and 2 mg/kg of xylazine intraperitoneally (i.p.). The left femoral vein was cannulated and injected with 0.5-1.0 ml of sample with contained either FITC labeled dendrimer or DEN complexed with FITC-ODN. The right femoral artery was cannulated with a PE-50 plastic catheter, and blood samples (100 pl) were collected at 1, 3, 5, 10, 15, 20, 30, 60 min after a single intravenous infusion of sample. Blood volume was replaced with same amount of physiological saline solution infused from femoral vein during the entire experiment. The blood samples were kept at room temperature for 1 hour, then centrifuged at 1000 g for 30 min. Serum (50 pl) was diluted with PBS (450 Ai). FITC signals in plasma were determined at wavelength 487 nm (ex) and 525 nm (em) by using a Perkin Elmer LS5OB Luminescence spectrophotometer.







39

Tissue Distribution of Dendrimer Based Delivery System for AS-ODN

Twenty-four hours after sample infusion, rats were sacrificed and tissues including brain, kidney, liver, skeletal muscle and blood vessels (aorta and common carotid artery) were removed. The tissues were sliced to 20 pM sections and mounted to glass slides. The FITC signals were visualized by using confocal microscopy.



Antisense Inhibition of Restenosis

Sprague Dawley rats whose body weight range from 300-375 were acquired from Harlan (Indianapolis, IN). Rats were anesthetized with sodium pentobarbital (30-40 mg/kg body weight) (i.p.) and a 2 French Fogarty catheter (Baxter Healthcare, ) was introduced from left femoral artery to the left common carotid artery. The balloon was inflated by saline and was passed three times up and down at left common carotid artery to produce deendothelializing effects. After balloon catheterization, another infusion catheter was introduced from left external carotid artery into left common carotid artery. The artery segments were tight up in order to retain the substances released from infusion catheter (Fig 3-1). Rats received 100 pg of AS-DEN (antisense), SC-DEN (scrambled) or DEN (dendrimer only) of infusions at certain pressure to facilitate uptake into vessel walls. After surgery, animals were returned to cages. Animals were scarified at different time points ranged from 7 hours to 7 days. Rats are anaesthetized and perfused with 0.9/. saline solution intracardiaclly. Both carotid arteries were removed and frozen at -100C for sectioning. The sections were cut on a cryostat (20 gm) and mounted onto gelatin-coated slides.







40



Statistics

All values are expressed as mean � S.E.M. Data were analyzed by using ANOVA or student t-test, followed by the Duncan multiple range test. In all analyses, a P value of less than

0.05 was considered significant.













Internal Carotid (Infusion Catheter)


Common Carotid

0


2F catheter









Left Femoral rRight FemoralI









Fig 3-1. Schematic elucidation of the procedure of balloon catheterization on rat carotid artery.












CHAPTER 4
AUTOIMMUNIZATION AGAINST ANGIOTENSIN TYPE-I RECEPTOR PREVENTS THE NEOINTIMAL
PROLIFERATION FOLLOWING ANGIOPLASTY Introduction



Arterial angioplasty is one of the major therapeutic methods to treat the ischemic coronary heart diseases. However, arterial injury associated with this procedure results in neointimal formation, which eventually causes restenosis in 30-50% of patients (Hillegass et al. 1994, Popma et al. 1991). The restenosis is mainly due to overgrowth of vascular smooth muscle cells (VSMC). Under normal physiological conditions these cells are nonproliferative due to suppression by endothelial cells lining the vessel lumen (Palmer et al. 1987, Kinsella et al. 1986). Arterial angioplasty destroys the endothelial cells of arteries and removes the growth inhibitory function of endothelial cells. The procedure also stimulates activation of growth factors on VSMC in the media. This causes VSMC to begin to migrate and proliferate into the lumen resulting in neointimal formation (Wilcox et al. 1993). Eventually overgrowth of neointima reduces lumenal size and blocks blood flow. Many factors have been shown to be involved in this process. Among those factors, angiotensin II has been suggested to promote restenosis (Wilcox et al. 1993, Gibbons et al. 1994, 1996, Powell et al. 1989). The vascular wall is one of the many tissues that has been proposed to have a local renin angiotensin system (RAS), independent from plasma 42






43

RAS (Dzau et al. 1987). Ang II functions through its specific receptors on the cell membrane. There are two main sub-types of Ang II receptors, AT, and AT2. The AT, is responsible for vasoconstriction and the growth effects of Ang UI on blood vessels (Wilcox et al. 1993). The AT2 subtype has been shown to have anti-growth effects (Nakajima et al. 1995). The binding of angiotensin II to AT, receptor triggers a cascade of intracellular events leading the activation of phospholipase C and generation of inositol triphosphate (IP3) and diacyiglycerol (DAG) (Griendling et al. 1994). IP3 is responsible for the increase of intracellular calcium level and DAG stimulates protein kinase C (PKC) activation. Both pathways have been shown to be important for VSMC growth (Duff et al. 1995).

The attempts of using angiotensin inhibition to treat restenosis began with the study of Powell et al in 1989. In their study, they administered ACE inhibitor, Cilazapril to the balloon injured rats and achieved 80% reduction in neointimal formation (Powell et al. 1989). In the following years several studies cases have been reported using variety of ACE inhibitors and AT, antagonists to prevent restenosis in animal models (Kauffman et al. 1991, Taguchi et al. 1993, Kino et al. 1994). However, despite the extensive study of the role of the renin-angiotensin system in development of restenosis, controversies remain. In clinical trials, we still do not have a clear picture on the importance of angiotensin in development of restenosis. The MERCATOR (Multicenter European Research trial with Cilazapril after Angioplasty to prevent Transluminal Coronary Obstruction and Restenosis) trial failed to confirm a beneficial effect of ACE inhibition in human subjects (Hermans et al. 1993). However, Yamabe et al (1995) showed that the treatments with cilazapril 7 days before angioplasty significantly reduced the rate of






44
restenosis in human. Rakugi et al (1994) later suggested in their study that higher dose of cilazapril may be need to inhibit tissue RAS. When we review the protocols used, we conclude that most of the difficulties are due to the dose and time of dosing with ACE inhibitors. Apart from the protocol decision of when to and how much to administer ACE inhibitors, there is the possible response of u preglation of receptor or inadequate reduction of Ang 1I at a critical time. It is beneficial for us to go back and carefully review the results of the animal studies. The condition that appears to have been important to treatment with ACE inhibitors prior to injury (Powell et al. 1989), whereas in the MERCATOR trials cilazapril was given after injury. Utilization of new and novel techniques will also be helpful to explore this problem.

As a useful technique to explore protein functions, autoimmunization overcomes the difficulties of drug therapies, such as problems with dose and time of dosing (Soos et al. 1995, Fu et al. 1996). Autoimmunization is chronic and complete. In the present study, we designed an experiment to induce rats to produce autoimmunity against the Nterminal of AT, receptor. The N-terminal peptide is the first extracellular loop of the 7 transmembrane receptor and was shown to be important for PKC activity induced by Ang II (Vinson et al. 1994). The immunized rats were then subjected to balloon injury of the carotid artery. We hypothesized that if Ang II is critical in the vascular response to balloon injury, blocking Ang II function with a specific autoantibody would prevent VSMCs proliferation and achieve a reduction on neointimal formation. To further test our hypothesis, we also transfused antiserum containing AT, autoantibody into normal rats






45

and performed balloon injury on these recipient rats. In both experiments we achieved a significant reduction of neointimal proliferation.



Results

Over 50% (11 out of 20 rats) of the immunized rats produced a significant amount of autoantibody in the ELISA screening (with the titers of 1000-2000) after the second injection. These animals were used as immunized group for the balloon catheterization experiments.

Western blot analysis of the protein extracts from rabbit adrenal glands using the AT, antiserum shows a single band with molecular weight of 65 kDa (Fig 4-1). This result corresponds well with the molecular weight of the mature glycosylated AT, receptor (Barker et al. 1993, Desarnaud et al. 1993). Occasionally two other minor bands with molecular weights of 43 and 55 kDa were also observed. These two bands may be the result of deglycosylation during extraction procedures. The 43 kDa is the predicted molecular weight of unglycosylated AT, protein (Murphy et al. 1991, Desarnaud et al. 1993). This result indicates that the antigen induces a specific autoantibody against the AT, receptor protein in immunized rats.

PKC assay was carried out on cultured VSMC. Ang 11 (100 nM) stimulated a rapid translocation of PKC from the cytosol to the membrane in 3 minutes (Fig.4-2). After 60 minutes incubation with immune serum, the Ang II induced PKC translocation was significantly reduced, while the incubation with control serum had little impact on PKC translocation.






46

There was staining with the autoantibody of rabbit AT, receptor. Figure 4-3 shows immunohistochemistry of staining AT, protein on rabbit aorta with antiserum from the immunized rats. The autoantibody specifically recognized AT, receptor on VSMCs in media. The result of staining for AT1 receptors in the autoimmunized rats showed the presence of autoantibody blocked exogenously applied antibody from binding. There was no staining on adventitia. Fig 4-4b shows that the carotid arteries from immunized rats could not be stained using a rabbit AT polyclonal antibody. The control rats which were injected with only Freund's adjuvant showed obvious staining to the rabbit AT, antibody (Fig. 4-4a). Autoradiographies of multiple transverse sections of carotid arteries showed that the location of AT, receptor is in the VSMCs (Fig. 4-5a). The AT, receptor binding was decreased on the sections of immunized rats (Fig. 4-5b).

The effects of AT autoantibody on restenosis are shown in Figure 4-6. Autoantibody produced significant inhibitory effect on neointimal formation. Morphological analysis of the cross sections by ratios of neointimal/medial areas shows that the-.immunized group had significantly lower neointimal growth than the control group (p<0.01)(Fig. 4-7).

In order to rule out the possibility that metabolic changes associated with autoimmunization may contribute to the growth inhibition, we also carried out the blood transfusion study. In the experiment, we transfused the sera collected from control or immunized rats to two groups of new rats at the onset of blood catheterization. The rats that received twice immune serum injection (1 ml each) showed significant lower






47

neointimal formation when compared to of those received same amount of control serum (p<0.05) (Fig. 4-8).



Discussion



In the present study, we demonstrated that the neointimal formation induced by balloon angioplasty could be inhibited by actively inducing autoantibody production against the N-terminal of the angiotensin II type-I receptor. We also showed that transfusion of antiserum during early stage of balloon injury reduces arterial regrowth. Our results support the hypothesis that Ang II is a critical growth promoter in the vascular system in response to injury. Our results further suggest that Ang II is one of the early growth factors in the process of restenosis. The present study offers the first report that an antibody against the N-terminal of the AT, protein inhibits neointimal formation.

The primary pathophysiologic mechanism in restenosis is the migration and proliferation of VSMCs in the subintimal layer (Clowes et al. 1983), where they form neointima and decrease the lumenal diameter. Arterial angioplasty removes the endothelial cells, which secrete nitric oxide and heparan sulfate proteoglycan that apparently maintain VSMCs in a quiescent stte (Palmer et.al. 1987, Kinsella et al. 1986). The distention in the procedure of angioplasty traumatizes VSMC and stimulates the production of growth promoting factors (Clowes et al. 1989). Blood vessel is one of the tissues that have been shown to have a local renin-angiotensin system (RAS) (Dzau et al. 1993). Gibbons et al (1992) show that Ang II have growth effects on VSMC and the growth actions are
















MW
(KD) 97.4 66 46 -


-65 KD


Fig 4-1. Western-blot analysis of membrane proteins from rabbit adrenal glands using antiserum from immunized rats. Membrane proteins extracted from rabbit adrenal were loaded on the 10% pre-cast gel and were electrophoresed at a constant voltage of 200 V for 45 min. The gels were transferred to PVDF membranes, then the blots were probed by 1st (rat anti-AT1 antibody) and 2nd (Goat anti-rat IgG alkaline phosphatase) antibodies. The antigenic bands were visualized by incubating in substrate solution (NBT-BCIP). There was a single band with MW of 65 kDa recognized by the rat antibody.

















100
[3- Cytosol 90 E Particulate

80

0. ., 7 0 T
0
S60

50 1I
liii I
JIM1 1 11 [E I
40 11 111

eh 3011!i I...
IIII I :: 20

10



Control Ang Il Ang H + Ang H + control serum immune serum







Fig 4-2. The effect of immune serum containing autoantibody to the N-terminal of the AT, protein on PKC translocation. Cultured VSMCs were pre-incubated with immune and control serum for 60 minutes. 100 nM of Ang I was used to induce PKC translocation. Ang H induced a rapid increase in membrane-bound (particulate) PKC activity accompanied by a reduction in cytosolic PKC level. The pre-incubation of immune serum resulted in a significant blockage of Ang U induced PKC translocation (P<0.05, n=3), while control serum had no significant impact on PKC.




















W>X


Fig 4-3. Immunohistochemical identification of AT, receptor on the sections of rabbit arteries using the rat AT, autoantibody. The immunohistochemistry was carried out by using an ABC peroxidase staining kit (Pierce Chemical Company, USA). Tissues were sliced into 30 gm sections on cryostat. After incubation with 1" (rat AT, autoantibody) and 2" (Goat anti-rat IgG) antibodies, AT, signals were visualized by exposing sections to 3,3-diaminobenzidine. The rat AT, autoantibody located most of the AT, receptor on medial smooth muscle cells.









































Fig 4-4. Immunohistochemical staining of sections from rat carotid arteries using a rabbit polyclonal antibody against AT, receptor. The immunohistochemistry was carried out by using an ABC peroxidase staining kit (Pierce Chemical Company, USA). The 1' antibody was the rabbit polyclonal antibody against AT, and the 2 * antibody is goat anti-rabbit IgG provided with kit. AT, signals were visualized by exposing sections to 3,3diaminobenzidine. A, Representative section of a carotid artery from a injured normal control rat exhibited intense staining of AT, receptor on neointimal and medial VSMCs. B. The artery of autoantibody producing rat was not be able to be stained using the same rabbit antibody and experimental protocol.











Control


Immunized


Fig 4-5. 1251I-Sar,lle-Ang II autoradiography analysis of multiple transverse sections of carotid arteries. The sections were cut on a cryostat (20 p~m) and mounted onto gelatincoated slides. The radio-ligand which was used in all experiments was 125I-SarIle-Ang II. Non-specific, AT1, and AT2 binding were determined in the presence of 1 .tM of Ang II or PD123319 or Losartan. Autoradiograms were generated by exposing the slides to X-ray films for 4 weeks. The photos were taken directly from an image analysis system (MCID, Imaging Research, Ontario, Canada). The pictures represent the specific binding of AT1 receptor. A, representative section from control rats. B, representative section from immunized rats.














Control Injured


Injured + Immunized


Fig 4-6. Photomicrographs of representative histological sections from sections of rat left common carotid arteries 2 weeks after balloon injury. Two weeks after balloon catheterization, rats were deeply anesthetized and perfused via the heart with saline solution. The left and right common carotid arteries were dissected and fixed in 4% paraformadehyde for 4 hrs. The arteries were sliced at 20 Im thickness transversely on a cryostat machine. The tissues were stained by hematoxylin for the morphological examination. AB. Uninjured. C,D. Injured. E,F. Injured and immunized.















2








0













0
Ctri hnmunized




Fig 4-7. Bar graphs represent the ratios of intimal/medial areas in the two groups of rats that underwent balloon catheterization. Values are expressed as mean � SE. Data were analyzed by Student t-test. A p value of less than 0.05 was considered significant (*), and a p value less that 0.01 was considered highly significant (**). The immunized rats (n=6) showed highly significant (p<0.01) smaller regrowth (0.49 � 0.11) than the control group (1.37+4 0. 17) (n=6).





































Control Serum control


Immune serum


Fig 4-8. Bar graphs represent the ratios of intimal/medial areas in three groups of rats that underwent balloon catheterization. Values are expressed as mean � SE. Data were analyzed by ANOVA followed by the Duncan multiple range test. A p value of less than 0.05 was considered significant (*). The rats received immunized serum (n=3) demonstrated significant less neointimal regrowth (0.78 � 0.12) than those of two control groups [(1.37 � 0.17) for the control rats (n=6), and (1.53 � 0.22) for the rats received control serum (n=3)].






56
mediated by the type I receptor (AT,). In vitro studies on VSMC cultures indicate that Ang II stimulates cellular proto-oncogenes (eg. c-fos and c-jun) that are important in the regulation of cell growth (Naftilan et al. 1989, Eyall et al. 1992). These effects can be inhibited by losartan, an AT1 specific antagonist. In in vivo studies, chronic infusion of Ang II results in vascular hypertrophy (Griffin et al. 1991). Transfection of blood vessels with ACE gene causes neointimal formation (Morishita et al. 1994). Indirect evidence for the involvement of the RAS first came from the observations that neointimal formation could be prevented by ACE inhibitor or AT antagonist administration (Powell et al. 1989, Kauffman et al. 1991, Taguchi et al. 1993, Kino et al. 1994). Interestingly, VSMCs in neointima have been shown to have stronger response to Ang U than those in media (deBlois et al. 1995). Since the VSMCs in neointima have also been shown to have higher density of AT, receptor on their membrane than those in the media, it is very likely that Ang II is one of the early factors involved in VSMC proliferation and migration (Viswanathan et al. 1992).

The method of using the synthetic peptide induced autoantibodies has been well documented in studies of autoimmune diseases (Fu et al. 1996, Soos et al. 1995). Synthetic peptides induce animals to produce antibodies that react with their cognate sequences in the native protein. We took advantage of multiple antigen peptides (MAP) in our study. MAP are highly branched polylysine molecules which have capacity to complex peptides on their surface. They do not induce immune responses themselves (Tam et al. 1996). When they complex with antigenic peptides, the conjugates are able to induce specific immune response to the peptides. Numerous reports have confirmed that






57

MAP induce specific immune responses to the antigens coupled to them without any side effects (Tam et al. 1996). The immune response alone could not be responsible for the inhibition of neointimal formation since both immunized rats and rats treated with antiserum showed the similar effect of inhibition.

The specificity of the antibody was shown by using Western blotting. A 65KDa protein was specifically selected by the autoantibody. This is the correct molecular weight of mature glycosylated AT, receptor (Desarnaud et al. 1993). The antibody positively stained AT, receptor on rabbit aortas. This is more evidence that the autoantibody is capable of binding AT, receptor in the vascular system. Further proof was shown by the carotid arteries from the immunized rats which could not be stained by a polyclonal AT, antibody raised in rabbit. The lack of staining by the rabbit antibody imply preoccupation of the AT receptor by autoantibody. Immunohistochemistry showed dense staining of AT receptor in the neointima. This result is consistent with the report from Viswanathan et al. (1992). Their study showed that neointimal VSMCs have higher density of AT receptors, using autoradiography. It is possible that the VSMCs in the neointima are regulated differently from those in media (deBlois et al. 1995). The VSMCs with higher AT, receptor number and stronger response to Ang II stimulation may first migrate into the lumen, where they form neointima by replicating themselves. The cells in neointima may be the decedents of this particular type of VSMCs that have more AT receptors. In this study, immunized rat vessels showed less receptor binding sites in autoradiography. There are three possible explanations. First, the autoantibody interfered with Ang II binding. Secondly, the density of AT receptor was decreased due to the growth






58
inhibitory effect of the autoantibody on VSMC. Third, although Vinson et al. (1995) suggested that the antibody to the N-terminal part of the AT, receptor does not interfere with Ang I binding, the autoantibody may act as a false signal of ligand binding which leads to down regulation of the receptor by negative feedback.

Ang II acts on VSMCs through the activation of phospholipase C, which catalyses the breakdown of phosphatidylinositol-4,5-bisphosphate to IP3 and DAG. The main role of DAG is to activate PKC. Activation of PKC is one of the most important events in the AT, signaling cascade. It is also suggested to be a important pathway mediating Ang II induced growth effects in VSMCs (Takeuchi et al. 1990). We chose the PKC activation as the indicator for the inhibitory effect of autoantibody on the AT, receptor into presence of Ang II. The ability of the autoantibody to interfere with physiological function of the AT, receptor was confirmed by PKC assay on cultures of VSMC. Activation of PKC in VSMCs features a rapid translocation PKC from the cytosol to the cell membrane. This event happens in 1-5 minutes (Haller et al. 1992, Dixon et al. 1994). Showing that Ang II induced IKC translocation can be blocked by the antibody on VSMC cultures, we further confirmed the capability of the autoantibody to inhibit signal transduction of the AT receptor.

The immunized rats had significantly lower neointimal growth after balloon injury. This is direct physiological evidence that the AT, antibody attenuates cell proliferation. Our result is further strengthened by showing that the serum from immunized rats is able to inhibit neointimal formation when it is transfused into normal rats. The results are critical for the specificity of the antibody effect. It excludes the possibility that a






59
nonspecific increase in immune response might be attributed to the inhibitory effects on neointimal formation. Interestingly, we achieved significantly inhibitory effects by only two infusions of antiserum. The first infusion was at 24 hours before the balloon catheterization, and the second one was on the day of surgery. This experimental protocol is different from those of Ferns et al (1991) who infused a PDGF antibody and from Lindner et al (1991) who infused an bFGF antibody. They continuously boosted their animals with antibody until the animals were sacrificed. Our result may indicate that Ang II is one of the early responding factors to the vascular injury and leads us to hypothesize that down-regulation of Ang II function at the early stage of injury is able to attenuate the initiation of restenosis.

There are limited data on time course studies either with ACE inhibitor or with AT antagonists in the animal models of restenosis. In most cases of ACE inhibitors, experimental animals were put on drugs several weeks before the angioplasty and drugs were continuously available during entire period of the development of restenosis (Powell et al. 198,9). However, losartan was tested after angioplasty (Kauffifan et al. 1991). It was reported by Prescott et al (1991), that ACE inhibitors inhibit the migration of VSMCs only, losartan, however, affect both migration and proliferation. This interesting phenomena may explain why ACE are ineffective when they are administered after angioplasty (MECATOR 1994). Vascular RAS has been proposed to one of the early factors involved in initiation of restenosis (Dzau et al. 1993), but since there are many growth factors such as PDGF, bFGF and many cell cycle genes are involved the timing of the role of Ang H is not clear. In present study we support the hypothesis by showing that






60

AT, auto antibody significantly blocked restenosis and was effective when transfused at the initiation of the response to injury.

Autoantibody inhibition is obviously not meant to be a practical approach to preventing restenosis clinically, however it provides a powerful tool to explore the role of the factors that are involved in this complicated process. The experiments have been carried out in rats. There is a debate whether that rat is a useful model for human restenosis. The debate was fueled by the failure of angiotensin converting enzyme inhibition to reduce restenosis in humans as they had done in rats. However the protocols used in patients were different from the protocol used in rats. The pig has been used as an alternative model, but treatment for restenosis has not been transferred from porcine studies to the clinic. Since the rat is available and the problems of restenosis are complex, the rodent model still offers a fruitful substrate for unraveling some those complexities. Based on this study with autoimmunity to AT, receptors. We conclude that Ang 11 and the AT, receptors are involved in the initiation of growth mechanism in response to vascular injury.












CHAPTER 5
ANTISENSE OLIGONUCLEOTIDE TO AT, RECEPTOR mRNA INHIBITS
CENTRAL ANGIOTENSIN INDUCED THIRST AND VASOPRESSIN Introduction



Central injection of angiotensin II (Ang II) elicits several distinct physiological responses including an increase in blood pressure, vasopressin release, natriuresis, salt appetite, and a motivation to drink (Phillips et al. 1987). Since the brain is protected from blood-borne Ang H by the blood-brain barrier, the existence of a brain renin-angiotensin system (RAS), independent of peripheral RAS was proposed (Ganten et al. 1983). All components of the RAS have been identified in brain (Deschepper et al. 1986, Dzau et al. 1986, Lynch et al. 1986, Phillips et al. 1985 and Unger et al. 1991). Although it is still not clear how the components interact, a paracrine action has been proposed (Phillips et al. 1991). There are at least two types of Ang H receptors which have been found in brain. AT, receptors are located in the brain regions which are involved in cardiovascular control mechanisms (Aldred et al. 1993, Phillips et al. 1985). Specifically blood pressure, drinking, and AVP release are mediated by the AT, receptor (Hogarty et al. 1992, Kirby et al. 1992, Timmermans et al. 1992). AT2 receptors are located in specific areas: such as the cerebellum, the inferior olivary nucleus, the locus coreuleus and the thalamus (Wright et al. 1994). The role of AT2 receptor still need further investigation Recently, Huang et al (1996) suggested that in rat brain neuronal cultures, MAP kinases were inhibited by the AT2 receptor stimulation being stimulated by AT, receptor 61






62
activation. Spontaneously hypertensive rats (SHR) have been proposed to have an overactive brain angiotensin system which is critical for their hypertension (Phillips et al. 1988). These rats have an elevated angiotensin receptor density in the brain, particularly in the hypothalamus and brain stem (Saavedra et al. 1992). Central injection of saralasin, an Ang II receptor antagonist decreased blood pressure in the SHR when given centrally at doses that had no effect peripherally (Phillips et al. 1977). Ang II inhibitors did not produce any change in blood pressure of normotensive controls (WKY). These observations suggest that Ang II receptors play an important role in the maintenance of hypertension in SHR. However, the data on losartan (i.c.v.) lowering hypertension in SHR are inconclusive, which may be due to different doses used. The involvement of AT, receptor specifically, has been recently revealed by antisense oligodeoxynucleotide (AS-ODN) inhibition of the translation of AT, receptor mRNA (Gyurko 1993). AS-ODNs elicit their actions by binding to the mRNA of the specific target protein and inhibiting the protein synthesis. Centrally injected AS-ODN to AT, receptor mRNA produced inhibition of high blood pressure in the SHR (Gyurko 1993). Therefore, we hypothesizd that AS-ODN to AT, receptor mRNA should inhibit effects of direct injections of Ang II. While this report was in preparation, Sakai et al showed that AS-ODN to AT, mRNA inhibited drinking to centrally injected Ang II (Sakai et al. 1994). The present study is more extensive as we investigated the effects of AS-ODN on the drinking and vasopressin response to direct i.c.v. injection of Ang U in SHR and Sprague-Dawley rats using the same AS-ODN we had used to reduce hypertension in SHR






63

Results

Dipsogenic Response

SHR

Figure 5-1 shows the effect of AS-ODN on the drinking response to Ang 11 (50 ng) in SHR (n=5). Ang 11 injection caused an immediate dipsogenesis with a water intake of 11.7 � 0.49 ml/30 min. After pretreatment with AS-ODN, the drinking response to Ang H was significantly reduced to 5.0 � 0.8 ml/30 min (P<0.05). The water intake of the control group which was treated with SC-ODN shows no significant difference with that of animals before ODN treatments.



Sprague-Dawley Rats

Figure 5-2 shows the effect of AS-ODN on the drinking response to 50 ng Ang 11 in normotensive rats (n=5). The water intake induced by Ang H injection was decreased significantly (P<0.05) from 6.46 �1.35 ml/30 min to 2.74 � 0.95 ml/30 min (after I injection of 50 pig of AS-ODN) and to 2.2 � 0.98 ml/30 min (after 2 injections of 50 pig of AS-ODN). In the repeated test, the second AS-ODN did not further reduce drinking. There is no significant difference in water intake after SC-ODN treatments as compared to Ang H alone.



Comparison of Water Intake Elicited by Central Ang H Between SHR and Sprague-Dawley Rats

Figure 5-3 shows the difference in water intake elicited by central Ang IH injection in a 30 min period of time between SHR and Sprague-Dawley groups. The water intake for SHR






64
group (n=5) was 11.7 � 0.49 ml/30 min which is significantly higher (P<0.05) than that of the Sprague-Dawley group(n=5) which was 6.46 � 1.35 ml/30 min.



Plasma Vasopressin

Vasopressin was measured in the SHR The injection of Ang H (50 ng, i.c.v.) increased the plasma AVP from 1.5 � 0.7 pg/ml to 15.4 � 0.7 pg/mi (n=5). After 24 hours, a second Ang 11 injection was administered, and plasma AVP increased to 13.66 � 0.84 pg/ml. There is no significant difference between the two injections (Fig 5-4). This result showed repeated administration of Ang I in a 24 h interval did not change the level of Ang 1 induced AVP release and indicated that the protocol of one injection of Ang II followed by a second injection of Ang U1 was valid.

Figure 5-5 shows the effects of 50 jig Ang I (i.c.v.) injection on AVP release after pretreatment with 50 p*g AS-ODN, or SC-ODN or with 4 j4 saline control. AS-ODN (n=5) decreased the plasma Ang II induced AVP significantly (6.45 � 0.54 pg/ml) (P<0.01) compared to the rats pretreated with saline-only (13.04 � 1.56 pg/mlXn=5). Compared to SCODN treatment group (9.30 � 0.99 pg/ml)(n=5), the AS-ODN pretreated rats was also significantly lower (p<0.05). However the SC-ODN treatment group had a significantly lower AVP response to Ang II than the saline control group (p<0.05).



Radioligand Binding Assay

Figure 5-6 shows that both total specific binding and AT1 receptor binding in the hypothalamic block were significantly (P<0.05) decreased in the AS-ODN treatment group













15 14 13 12 11109

8
7
6

5-


-F


-F-


I
Control


**
-F-


SC-ODN


AS-ODN


Treatment






Figure 5-1. Effect of AS-ODN for AT, receptor mRNA on drinking to Ang II i.c.v. SHR rats were administered 50 ng Ang I preceded by either saline (unshaded bar), SC-ODN (hatched bar), or AS-ODN (spotted bar). The water intake of each rat in the next 30 minutes was measured. Data are expressed as mean � SEM (n=5)(p<0.01).
















109

8

7 7





- *


3 2 1

-0i
Saline I st SC-ODN 2nd SC-ODN Saline 1st AS-ODN 2nd AS-ODN
+Ang II + Ang II +Ang II +Ang II + Ang II +Ang II Treatment




Figure 5-2. Effect of AS-ODN and SC-ODN on drinking with repeated injections. SD rats were administered 50 ng Ang I preceded by either saline (unshaded bar), I dose of SC-ODN (first hatched bar), 2 doses of SC-ODN (second hatched bar), 1 dose of AS-ODN (first spotted bar) or 2 dose of AS-ODN (second spotted bar). The water intake by each rat in the next 30 minutes was measured. Data are expressed as mean � SEM (n=5)(p<0.05).





































SD SHR


Treatment









Figure 5-3. The drinking responses of rats to Ang II i.c.v. SD (first bar) and SHR (second bar) were administered 50 ng Ang H. Water intake of each rat in the next 30 minutes was measured. Data are expressed as mean � SEM (n=5)(p<0.05).








































Control 1st Ang II 2nd Ang IH


Treatment





Figure 5-4. Effect of repeated injection of Ang i i.c.v. on plasma AVP level. Rats were administered either one dose or two doses of Ang II at 24 h intervals. The blood samples were drawn at 1 minute after Ang II injection. The plasma AVP level was measured for each rat. Dates are expressed as mean � SEM (n=5).
















161412


S 10


8 6


4


2




Saline Sc As Treatment Figure 5-5. Effect of AS-ODN, SC-ODN or saline treatment on AVP release to Ang II i.c.v. Rats were administered 50 ng Ang H preceded by either saline (unshaded bar), SC-ODN (hatched bar), or AS-ODN (solid bar). The blood samples were drawn at 1 minute after Ang H administration. The plasma AVP level was measured for each rat. Data are expressed as mean � SEM (n=5). ** p<0.01 * p<0.05
















100 -[ Saline Sc
90 * AS

80= 70
0
U
~60 .








o 30


20


10


0
TSB ATI


Figure 5-6. Effect of oligodeoxynucleotide treatment on AT, receptor binding in the hypothalamic block. TSB = Total Specific Binding AT, = Angiotensin II type 1 receptor AS = Antisense Oligodeoxynucleotides SC= Scrambled Oligodeoxynucleotides.






71

compared to the SC-ODN treatment group. The SC-ODN did not change the total specific binding and AT, binding significantly from the saline control. Values are expressed as percentage of total specific binding of the saline treated brains.



Discussion



The results show that the AS-ODN to AT, receptor mRNA inhibit the physiological effects produced by Ang 1I (i.c.v.). The data presented here extend the results of Gyurko et al (1993) which showed that central injection of AS-ODN decreased blood pressure in SHR by blocking the protein synthesis of central AT, receptor. The results confirm the finding of Sakai et al (1994) that the drinking response to Ang H (i.c.v.) can be inhibited by AS-ODN to AT, receptor mRNA, and extend the finding by showing that both drinking and AVP responses to central Ang I were inhibited by AS-ODN in SHR.

Antisense technology was developed as a tool for modulating gene expression. The studies on applying this technology to inhibit gene expression in vivo are just beginning. Wahlestedt et al (1993) injected antisense ODN targeted to NPY Y, receptor into the rat cerebral ventricles which resulted in a significant reduction in cortical Y, receptor. The report from Ogawa et al (1994) described the antisense ODN targeted to progesterone receptor mRNA lowered the receptor density and inhibited the lordosis behavior. Another successful report comes from studies of Akabayashi et al (1994) in which they inhibited neuropeptide Y synthesis and suppressed feeding behavior by using the antisense ODN to neuropetide Y mRNA. In the present study AS-ODN suppressed dipsogenesis and AVP release induced by






72

Ang II injected into brain. The working hypothesis of AS-ODN action is that AS-ODN inhibit AT, receptor expression by interfering with translation of AT, receptor mRNA (Helene et al. 1990 and Akhtar et al. 1992). Confirming this mode of action, we found decreased AT, receptor density in the hypothalamic region after AS-ODN treatment.

Interestingly the AS-ODN did not completely inhibit the drinking response. Even with a repeated dose of AS-ODN treatment the inhibition was not significantly greater than with a single injection. When comparing the percentage decrease in drinking with the SC-ODN treated group as the control, we found that the decrease in drinking with the first dose of ASODN was 52.6% and with the second dose of AS-ODN the decrease was 66.3%. This result is consistent with the report of Hogarty et al (1992) which showed that central injection of losartan, an AT, receptor antagonist reduced but did not completely block the dipsogenic response induced by Ang U (i.c.v.). This may suggest that other non-AT receptors are involved in mediation of the drinking response. Hogarty et al proposed that perhaps AT2 receptors were involved. It is also possible that other receptors not yet identified may also play a role in central Ang II induced drinking. Although non-AT receptor mediated dipsogenesis is an attractive idea, the possibility of the AT receptor accounting for all of the drinking response still exists. The autoradiographic analysis of Ang II receptor sites in the hypothalamus region indicated that AS-ODN treatment resulted in a maximum of 40% decrease in the SHR (Gyurko et al. 1994). This suggests that a higher dose of AS-ODN or repeated AS-ODN treatment should be able to block more AT, receptors thus completely inhibiting Ang II dipsogenesis. However, in our receptor binding study we used three injections of AS-ODN (50 jig/injection). With this regimen only a 40/s decease in AT receptor density in






73
hypothalamic region was seen in the Sprague-Dawley rats. This is consistent with our in-vivo studies where we showed that a second AS-ODN injection did not further decrease drinking beyond the first injection. The lack of increased effects with repeated administration of ASODN indicates that antisense does not completely inhibit the receptor protein synthesis. This may be due to limited uptake in critical cells or the AT, receptor gene responds by upregulating AT, receptor synthesis. It seems unlikely that the feedback mechanism would be fast enough or efficient enough to compensate for repeated doses of AS-ODN. If the rate of ODN uptake is the rate-limiting step, even with repeated administrations, the cells may not take up any more AS-ODN. The excess AS-ODN may be diluted in CSF and then degraded. Despite the profound decrease in dipsogenic response, the observed decrease in AT receptor binding was smaller. This may be a result of substantial decreases in AT, in discrete nuclei being masked by other areas with greater AT, receptor density in the dissected tissue block. Therefore it is conceivable that a relatively small change in receptor number causes dramatic decrease in the physiological response. An alternative explanation is that the life cycle of AT, receptor is giving a false picture. The life cycle involves internalization and recycling. During these stages the receptor is not active but detectable by the receptor binding assay. This would mask the true decrease in binding of active membrane-bound receptors by the antisense. The SC-ODN had no effect on drinking response, which showed that the action of the AS-ODN is sequencespecific.

In the response to central Ang II, SHR drank 80/. more than SD rats. These data add support to the hypothesis that SHR has overactive RAS components in the brain compared to the normotensive Sprague-Dawley rats. Sakai et al also reported that injection of AT receptor






74
AS-ODN into the third ventricle partially inhibited the drinking response to Ang I in normotensive rats (Sakai et al. 1994). They also showed that AS-ODN had no effect on central carbachol induced drinking. Their study obviated the need to include the caiiachol induced drinking, but we have addressed the question about Ang i inhibition in SHR and effects on AVP release.

The main target sources of AVP release by Ang H are SON and PVN of the hypothalamus which have high AT receptor density. Yang et al (1992) showed that Ang II depolarization of SON neurons was inhibited by losartan and Hogarty et al (1994) showed that losartan could inhibit Ang II induced AVP release. The AS-ODN treatment significantly decreased the AVP release induced by central injection of Ang II, providing further support for AS-ODN inhibition of AT, receptor expression. The SC-ODN control also slightly decreased AVP release, although the effect of AS-ODN was significantly greater. The inhibition of AVP release after SC-ODN treatment compared to saline controls is an empirical finding for which we have no explanation. In the experiment on drinking, SC-ODN had no inhibitory effect on the drinking response to central Ang I1. AVP release is related to osmolarity changes and studies suggest that AVP response to a rise in plasma osmolality is mediated by, or involves at some point, an angiotensinergic pathway in the brain (Hogarty et al. 1994, Sladek et al. 1980, Yamaguchi et al. 1982). The sensitivity of Ang II induced AVP release to AS-ODN for AT mRNA inhibition supports this view. AVP release is one of the postulated mechanisms by which SHR maintain hypertension. The present results are consistent with this concept and may be relevant to the decrease in hypertension in SHR with AS-ODN.






75
The present research is in its early stage. Fine turning of the modification of AS-ODN and improved delivery methods will ultimately allow smaller doses to be delivered to sites of action as well as enhancing or optimizing the cellular uptake efficiency. This ultimately would allow the administration of smaller doses of AS-ODN with equal or greater potency. Overall this would have fewer potential side-effects unlike some of the other chemical receptor antagonists. This is advantageous for both development as a therapeutic agent and a physiological tool. In addition, a further advantageous is the modified AS-ODN has been shown to elicit effects for extended periods of time, unlike receptor antagonists which have effects that are relatively short acting. In summary, this report demonstrates a new approach to modulate the AT, receptor gene expression in the rat brain. Our results confirm the function of the AT, receptor in controlling drinking and AVP release and also provide a potential new tool to regulate the physiological effects mediated by the AT, receptor in the brain.











CHAPTER 6
CHARICTERIZATION OF DENDRIMER BASED GENE DELIVERY SYSTEM IN VIVO AND ITS APPLICATION IN TREATING RESTENOSIS


Introduction

Restenosis is the process of reobstruction of an artery following interventional procedures such as angioplasty, atherectomy, or stenting. This disease is a multifactorial process. The migration and proliferation of VSMCs is most likely to the most consequential event (Clowes et al. 1988). Conventional drug therapeutic approaches have focused on either preventing platelet deposition, thrombus formation or inhibiting VSMCs growth. However, the problems with drug administration have prevented conventional drug therapy from achieving any clinical significance (Hermans et al. 1993). Therefore, many researchers have turned their attentions to a new approach - Gene therapy. Gene therapy is one of the fastest growing fields in the biomedical research. This emerging branch of medicine aims to correct genetic defects by transferring genetic materials into cells. One of the most dynamic research areas is antisense oligonucleotide (AS-ODN) based gene inhibition. AS-ODNs are considered a new class of therapeutic drugs that consummate their functions by binding to mRNA in a sequence-specific manner. Traditionally drugs work on the protein levels. Although they can inhibit protein functions, they usually need repeat administration. Non-specific effects and protein upregulation associated with drug inhibition are frequently observed. Antisense technology was introduced to overcome these shortcomings of traditional drugs. Many successful reports have 76






- 77

indicated that antisense approach is a very useful tool in manipulating gene products. Because of the fast metabolic degradation for natural occurring phosphodiester oligonucleotide, but the phosphorothioate prolongs the activity of antisense ODNs. At present, there are two ways to deliver AS-ODN, direct and incorporated into a delivery system. Direct administration is limited by low cellular uptake and fast elimination in vivo of AS-ODNs. Liposomes formulations, such as cationic, have the potential to enhance cellular uptake of polynucleotides into mammalian cells. However, the low efficiency andcyto-toxicity of liposomes have limited this approach in vivo.

Starburst dendrimerse are a new class of macromolecules first described by Dr. Donald A. Tomalia in the 1980s (Tomalia et al. 1985). These polyamino spherical molecules have highly branched, tree-like structures terminating in a surface of primary amines having the ability to bind anionic nucleic acids. Dendrimers are classified by the number of cascade polymer generations required. As the generation number increases there is a corresponding increased in number of primary amines and molecular weight. A newly-emerging area in dendrimer technology is the delivery of genetic material into the cell. Many in vitro reports have ascertained dendrimers are able to deliver genetic material efficiently into many cell types without damage to the organisms (Boussif et al. 1995, Haensler et al. 1993). The delivery of" AS-ODN by dendrimers as vector is being studied by other groups (Schwab et al. 1994, Poxon et al. 1996). They have been reported to have many advantages over other liposome and other particulate based delivery systems. The advantages of their products include defined polymerization reaction, reproducible product low toxicity and the ability to alter the transport and binding characteristics by changing the generation of dendrimer used. However, the in






78
w data about toxicity and metabolism need to be completely studied before dendrimers can be developed into drug delivery systems.

In the present study, we have used polyamidoamine Staburst dendrimers which were synthesized from an ethylene diamine core, resulting in a series of primary amine groups on the outermost sphere. We investigated the pharmacokinetics and tissue uptake of generation 4,6 and 10 fluorescent labeled dendrimers, free fluorescent labeled ofigonucleotides, and generation

6 DEN electrostatically complexed to fluorescent labeled ODN.

In present study, we also explored the potentiality of using dendrimer based delivery system to delivery AS-ODN to treat restenosis. The biggest problem associated with using antisense strategy to fight restenosis is achieving sufficient cellular uptake of the oligo and maintaining the antisense inhibition long enough to inhibit neointimal growth. Therefore, a suitable delivery system for AS-ODN is needed. Local delivery rather than systemic administration is a more effective way to obtain higher tissue drug levels at the site of the balloon injury. Local delivery can also minimize the potential side effects. Several local drug deliverysystems, including perfusion balloon catheters, hydrogel-coated balloon catheters, polymeric or coated stents, and many other approaches are currently under investigation. However, the low tissue uptake of the agents remains the main disadvantage of the catheter " injection systems (Femandez-Ortiz et al. 1994, Mitchel et al. 1995, Lincoffet al. 1994, Fram et al. 1994). Blood flow washes out the agents in minutes to hours. We have investigated the dendrimer as a sustained-release carnier system that could enter the vascular wall rapidly and not be washed out. We have also tested the ability of using dendrimer complexed with AS-








79

ODN to AT, receptor mRNA to decrease neointimal formation after vascular injury in the same animal model.



Results

Purification of Dendrimers

Dendrimers were labeled by a simple conjugation using fluorescein isothiocyanate and the primary amines from the dendrimer (Poxon et al. 1996). After the reaction the unreacted fluorescein needed to be isolated from the dendrimers. Spin columns filled with G-10 Sephadex were used for the purification of FITC-labeled DEN from unreacted label. Samples were run through spin columns for three times until there was no detectable free FITC signal on TLC plates. Figure 6-1 illustrates the purity of the dendrimer labeling reaction by TLC purification procedure for the 4" generation DEN. The signals of free FITC label indicated by lane 1 were gradually decreased until it could no longer be detected after the third time spin. This purification step guaranteed us for using pure FITC-labeled DEN for the rest of experiments. Similar results were obtained for the other dendrimer generations.



Dendrimer-Oligonucleotide Reaction

In order to determine the interaction of oligonucleotides and dendrimers, we performed gel retardation experiments (Fig 6-2). In this method the anionic oligonucleotides easily migrate through the gel matrix towards the cathode. As the net charge of the complex is changed due to the addition of the cationic dendrimers at first the








80

movement of the complex is slowed but with the addition of increasing. amounts of dendrimer the moment stops and is finally reversed towards the anode. Using this method we have calculated that I mole of dendrimer could react with 8 moles of oligonucleotide resulted in complete binding of the oligonucleotide. In essence the study demonstrated dendrimers could complexed with oligonucleotide and at some ratio form a positively charged complex.



Pharmacokinetics

The determination of pharmacokinetical parameters with particulate delivery systems is often a difficult process because it requires having the ability to quantitate both the drug of interest and the delivery vector. In this set of experiments we could take advantage of easy labeling methods to attach fluorescent reporters (fluorescein isothiocyanate) to both the delivery vector (DEN) and the ODN. By varying the administration of the ODN and DEN we could generate data for the ODN, free DEN, and the complex. Table 6-1 demonstrates the pharmarcokinetical parameters of different samples that we tested. These values indicated that as the dendrimer generation increases there is a corresponding increase in half life of elimination. Also from the data presented the ODN-DEN complex appears to be stable in the blood stream since there was a corresponding increase in the elimination half-life (Fig 6-3). For example, the 6'h generation of PAMAM DEN and found that it significantly (p<0.01) increased the elimination half time of 15 mer oligo (tl/2a and tl/213) from (3.45 � 0.58, 39.55 + 5.97) to (9.00 � 3.35, 395 � 53) mn (Fig 6-4).









Tissue Distribution of DEN

Tissue distribution suggested that 24 hours after infusion, FITC-Den were accumulated in kidney, liver and blood vessels (Fig 6-5). They did not cross the blood brain barrier into brain and there was not a significant amount of signal detected skeletal muscles.



Effect on Neointimal Formation

DEN-ODN (100 Lg) were delivered in situ to rat left common carotid artery. Treatment with the complexes of AS-ODN and dendrimer significantly reduced neointimal formation compared to the control. The treatments with AS-ODN alone and with SCODN complexed to dendrimer yielded no significantly changes (Fig 6-6).


TABLE 6-1. PHARMARCOKINETICAL PARAMETERS OF DENDRIMERS Generation tja/2() A, kI t 1/2P a(min) A2 k2

4 1.68 48.5 0.41 20.05 71.0 0.035

6 4.07 70.61 0.17 51.67 32.88 0.0134 101 2.93 58.8 0.236 83.66 30.9 0.008 l5merODN 3.45 18.65 0.201 39.55 8.54 0.0175 CF 2.8 43.5 0.255 19.76 43.55 0.035
















TLC demonstration of
the purification process of FITC-DEN using spin column


Lanes: 1


2 3


Fig 6-1. Purification of FITC labeled Dendrimers (4'h Generation). Dendrimers were labeled by a simple conjugation using fluorescein isothiocyanate and the primary amines from the dendrimer. Samples were run through spin columns filled with G-10 Sephadex three times until there was no detectable free FITC signal on TLC plates. The signals of free FITC label indicated by lane I were gradually decreased until it could no longer be detected after the third time spin (lane 4). Lane 1, Free FITC. Lane 2, 1" time spin. Lane 3, 2"d time spin. Lane 4, 3' time spin. Similar results were obtained for the other dendrimer generations.


4























GEL RETARDATION ASSAY FOR
DEN-ODN COMPLEX


Amount of DEN:


6 4 2 0.5 0


4- DEN-ODN 4- 15 mer ODN


Fig 6-2. Gel retardation experiment on ODN-DEN complex. The anionic oligonucleotides migrate through the gel matrix towards the cathode. As the net charge of the complex is changed due to the addition of the cationic dendrimers at first the movement of the complex is slowed but with the addition of increasing amounts of dendrimer the moment stops and is finally reversed towards the anode.











120 100-i 8060

40


20


50 60 7C


Min


Fig 6-3. Serum elimination of generation 4, 6 and 10 dendrimer and CF.


.4)
AAW AuE


G4 G6 CF GIO













120100-1 8060


40200-


10


20


I I I I I I I I . I I I I
30 40 60 60


Min

ODN

G6 DEN EDEN-ODN









Fig 6-4. Serum elimination of generation 6 dendrimer, Free 15 mer ODN and DENODN complexes.


. I I I I I ' I I I . I I I I I '


L
S..
* \,
*
* S. U *
.A ~
*


-


in


I I I











CONTROL


CONTROL


TREATED


Fig 6-5. Tissue distribution of Generation 6 dendrimer. Twenty four hours after infusion, FITC-Den were detected in liver and blood vessels. Left panel, Control. Right panel, Treated.


TREATED

















2.5



2.0



1.5
0*
.


1.0 0.5



0.0
Ctrl SC-DEN AS AS-DEN













Fig 6-6. Effect of AS-ODN for AT, receptor on neointimal formation. Balloon injured rats were treated with control (n=5), SC-DEN (n=4), AS alone (n=4) and AS-DEN (n=4). The AS-DEN treated rats showed significant decrease on the ratio of intimal/medial areas.




Full Text

PAGE 1

THE IMPORTANCE OF THE ANGIOTENSIN TYPE-1 RECEPTOR IN THE VASCULAR RESPONSE TO INJURY: A STUDY WITH AUTOIMMUNIZATION AND ANTISENSE By FRANK H. MENG 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 1997

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Copyright 1997 by Frank H. Meng

PAGE 3

This dissertation is dedicated to my wife, Jane, for all the love, care, support and encouragement she has given me through the years.

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ACKNOWLEDGMENTS I would like to extend my sincere thanks to the chairman of my advisory committee, Dr. M. Ian Phillips, for providing me with the great support and opportunity to learn under his supervision. His guidance and encouragement have led me through all these years in graduate school. I learned many modem techniques in his lab, and more important I have learned to think as a scientist. I also would like to thank my advisory committee members, Dr. Stephen Baker, Dr. Jeffrey Hughes, Dr. Colin Sumners and Dr. Bruce Stevens for their helpful comments and discussions on my project. I am especially grateful to Dr. Hughes who allowed me to use his lab facilities and shared his expertise with me on the Dendrimer research. I am extremely grateful to the members in Dr. Phillips' lab. Dr. Sara Galli, Birgitta, Gayle, Kevin, Uping, Harold, Robert, Jon, Bing, Tibor, Dkgmara, Jianfeng, Clare, Adrian and Dan for their help, friendship and support. Finally, I would like to extend my special thanks to my wife, Jane, for her love and understanding. iv

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TABLE OF CONTENTS ACKNOWLEDGMENTS LIST OF FIGURES LIST OF TABLES AFFREVATIONS ^ ABSTRACT CHAPTERS 1 INTRODUCTION j Restenosis I Pathophysiology of Restenosis 3 The ReninAngiotensin System (RAS) 4 The ReninAngiotensin System and Restenosis 1 1 ExperimentaJ Models of Restenosis 1 5 Methods to Inhibit Restenosis 15 The Antisense Technology 21 Summary ; 25 2 HYPOTHESIS AND SPECIFIC AIMS 26 3 MATERIALS AND METHODS 28 Experiments on Autoimmunization 28 Experiments on Central Ang II Inhibition 34 Experiments on Dendrimer Delivery System 37 4 AUTOIMMUNIZATION AGAINST ANGIOTENSIN TYPE-1 RECEPTOR PREVENTS THE NEOINTIMAL PROLIFERATION FOLLOWING ANGIOPLASTY 42 Introduction ^2 Results 4^ Discussion 47 V

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5 ANTISENSE OLIGONUCLEOTIDE TO ATi RECEPTOR mRNA INHIBITS CENTRAL ANGIOTENSIN INDUCED THIRST AND VASOPRESSIN 61 Introduction Results g3 Discussion 6 DENDRIMER BASED GENE DELIVERY SYSTEM AND ITS APPLICATION IN RESTENOSIS 76 Introduction Results 79 Discussion gg 7 GENERAL CONCLUSIONS 94 REFERENCES 9^ BIOGRAPHICAL SKETCH 2 1 1 vi

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LIST OF FIGURES Figure page 1-1. Schematic elucidation of the components and functional steps of renin angiotensin system g 1-2. Schematic elucidation of the signal transduction of ATi receptor 14 31. Schematic elucidation of the procedure of balloon catheterization on rat carotid artery. 41 41. Westem-blot analysis of membrane proteins from rabbit adrenal glands using antiserum from immunized rats 4g 4-2. The effect of immune serum containing autoantibody to the N-terminal of the ATi protein on PKC translocation 49 4-3. Immunohistochemical identification of ATi receptor on the sections of rabbit arteries using the rat ATi autoantibody 50 4-4. Immunohistochemical staining of sections from rat carotid arteries using a rabbit polyclonal antibody against ATi receptor 5 1 4-5. I-Sar,Ile-Ang II autoradiography analysis of multiple transverse sections of carotid arteries 52 4-6. Photomicrographs of representative histological sections from sections of rat left common carotid arteries 2 weeks after balloon injury 53 4-7. Bar graphs represent the ratios of intimal/medial areas in the two groups of rats that underwent balloon catheterization 54 48. Bar graphs represent the ratios of intimal/medial areas in three groups of rats that underwent balloon catheterization 55 51. Effect of AS-ODN for AT, receptor mRNA on drinking to Ang U i.c.v 65 5-2. Effect of AS-ODN and SC-ODN on drinking with repeated injections 66 5-3. The drinking responses of rats to Ang II i.c.v 57 VI 1

PAGE 8

5-4. Efifect of repeated injection of Ang n i.e. v. on plasma AVP level 68 5-5. Efifect of AS-ODN, SC-ODN or saline treatment on AVP release to Ang n i.c.v 69 56. Efifect of oligodeoxynucleotide treatment on AT, receptor binding in the hypothalamic block jq 61. Purification of FITC labeled Dendrimers (4'* Generation) 82 6-2. Gel retardation experiment on ODN-DEN complex 83 6-3. Serum elimination of generation 4, 6 and 10 dendrimer and CF 84 6-4. Serum elimination of generation 6 dendrimer, free 15 mer ODN and DEN-ODN conjugates 6-5. Tissue distribution of Generation 6 dendrimer 86 66. Efifect of AS-ODN for AT, receptor on neointimal formation 87 71. Summarization of physiological events following angioplasty and inhibitory functions of autoantibody and antisense on the AT, receptor 96 viii

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Table LIST OF TABLES 1 1 . Gene Therapy for Restenosis 1-2. Antisense Therapy for Restenosis 61 . Pharmacokinetical Parameters of Dendrimers Ix

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ABBREVIATIONS ACE: Angiotensin Converting Enzyme ACSF: Artificial Cerebelspinal Euid AngH: Angiotensin II Ao: Angiotensinogen AP-1: Activator protein 1 AS-ODN: Antisense Oligodeoxynucleotides AT,: Angiotensin type1 receptor AT2: Angiotensin type-2 receptor AVP: Arginin Vasopressin BCIP: 5-bromo-4-chloro-3 -indolyl phosphate bFGF: basic fibroblast growth factor BSA: Bovine Serum Albumin CMV: Cytomegalovirus CNS: Central Nen/ous System DEN: Starburst® dendrimers ELISA; Enzyme Linked Immunosorbent Assay EtOH: Ethanol FITC: Fluorescein Isothiocyanate

PAGE 11

G-protein: Guanine Nucleotide Binding Protein MAP kinase: Mitogen associated protein Kianse MAP: Multiple Antigenic Peptides MECATOR: Multicenter European Research trial with Cilazapril after Angioplasty to prevent Transluminal Coronary Obstruction and Restenosis NO: Nitric Oxide PBS: Phosphate buffered Saline PKC: protein kinase C RAS: Renin angiotensin system SC-ODN: Scrambled Oligodeoxynucleotides VSMC: Vascular Smooth Muscle Cell 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 THE IMPORTANCE OF THE ANGIOTENSIN TYPE-1 RECEPTOR IN THE VASCULAR RESPONSE TO INJURY: A STUDY WITH AUTOIMMUNIZATION AND ANTISENSE By Frank H. Meng December, 1997 Chairperson: M. Ian Phillips, Ph.D., D.Sc. Major Department: Physiology Arterial injury induces VSMC proliferation and migration. This leads to neointimal growth and reduction in lumenal diameter. The local RAS has been suggested to promote this process mediated by ATi receptor. To test the role of angiotensin II in a rat common carotid" artery model of restenosis, we utilized autoimmunization and antisense inhibition strategies. In the autoimmunization study, SD rats were immunized with a synthetic peptide' corresponding to amino acid sequence 14-23 of the N-terminal the ATi receptor. The autoantibody prevented neointimal regrowth by 65% compared to sham control. The results indicate that when the ATi receptor is chronically inhibited by an autoantibody, the xii

PAGE 13

regrowth response to arterial injury is significantly reduced. This suggests that Ang II and ATi receptors are necessary for the growth mechanism in this model of arterial restenosis. In the antisense inhibition study, we designed AS-ODN targeting to the ATi receptor mRNA. We tested it in a well-established model, the central Ang n induced drinking and AVP release. AS-ODN treatment significantly reduced drinking and AVP response to central Ang II injection. The results demonstrate that the antisense inhibition of brain ATi receptor gene expression decreases the Ang II induced drinking and AVP response. This indicates that antisense inhibition is capable to block ATi gene expression. In order to facilitate uptake of AS-ODN into vessel walls, we tested the dendrimers based gene delivery system in treating restenosis. We investigated the pharmacokinetics and tissue distribution of generation 4,6 and 10 polyamidoamine Starburst® dendrimers. Our results showed dendrimer significantly increased the half life of oligonucleotide in the plasma. We complexed AS-ODN for ATi with generation sbc DEN and tested on a rat model of restenosis. The results showed the AT, AS-ODN delivered by DEN significantly reduced neointimal formation. We conclude that vascular RAS and AT, play an obligatory role in development of restenosis. The potential of using AS-ODN as a therapeutic method still needs fiirther investigation. xiii

PAGE 14

CHAPTER 1 INTRODUCTION Restenosis Coronary artery disease is the leading cause of death in the United States and in many other countries. The plaques deposited inside the coronary artery, narrow the blood vessel and decrease or completely block the blood supply to the heart. One of the most successful treatments of this disease is arterial angioplasty. The procedure was introduced into the clinic in 1967 by Fogarty. Since then it has become a well established and frequently performed procedure around the world. It is estimated that 350,000 procedures are carried out annually (Hillegass et al. 1994). The procedure has normally an initial success rate of opening obstructed coronary arteries of 95%. However, in spite of the fact that good symptomatic improvement occur in the majority of cases, the procedure is complicated by restenosis in 30-50% of patients, regardless of the type of angioplasty procedure used (Epstein et al. 1994). This means more than 100,000 cases of failure and hundreds of million dollars of loss each year. It is basically accepted by researchers in the cardiovascular field that abnormal growth of the VSMC lining artery walls plays a key role in the blockage of arteries during coronary artery diseases. This abnormal growth also contributes to reblockage (restenosis) of the arteries that have been opened by balloon angioplasty or replaced in 1

PAGE 15

2 bypass operation. The major cause of restenosis is exaggerated healing response of medical VSMC to vascular injury. Angioplasty is carried out to restore blood flows to ischemic coronary arteries. However, there has always been arterial wall injury inevitably associated with this procedure. The injury damages endothelium which normally secretes nitric oxide to prevent VSMC from growth. The injury also stimulates a variety of growth promoters for a repairing procedure (Epstein et al.l994). Growth factors stimulate VSMC to migrate and to proliferate into lumen to form neointima, where they continue to proliferate and secrete extracellular matrix. The neointimal mass continues to expand and eventually rebloclcs the blood vessels (Wilcox 1993). Neointimal formation is the result of cell migration, followed by cell proliferation and matrix secretion. At late stage, lumenal narrowing is due to both intimal smooth muscle proliferation and collagen and elastic deposition (Clowes et al. 1983). The problem of controlling restenosis becomes largely the problem of controlling the VSMC proliferation. Many factors are involved in regulating of VSMC proliferation. Some researchers suggested that most endogenous vasoconstrictive substances are also growth promoters and most endogenous vasodilator substances are growth inhibitors (Dzau 1992). RAS is one of the most important systems involved in restenosis. Traditionally, the renin' angiotensin system is an endocrine system which is involved in regulation of fluid homeostasis and blood pressure (Guyton 1986). The discovery of tissue RAS has led angiotensin research to a new era. Using modem technologies, researchers have found the components of RAS in various tissues, including the blood vessels (Dzau et al. 1987). Molecular cloning of the three types of angiotensin receptor subtypes (ATu, ATjb and

PAGE 16

3 AT2) allowed us to carry out more intensive studies on their characteristics and their physiological functions (Griendling et al. 1993). Understanding of their genetic structure also opened an avenue for gene therapy on angiotensin related diseases. For instance, antisense inhibition of RAS has drawn a lot of attention among the researchers. Pathophysiolgy of Restenosis Under normal conditions, VSMC are quiescent. During angioplasty, inflation of the balloon causes an increase in lumen size. This gain in lumen size has been shown due to both loss of mass in plaque and overstretch of vessel walls (Clowes et al. 1988). Unfortunately, the loss of mass damages endothelial cells, and the overstretch of vessel walls traumatizes VSMC in the media. The direct consequence is to break the balance between the growth promoters and inhibitors. For the side of growth inhibitors, removal of endothelial cells directly causes a reduction in nitric oxide production. Nitric oxide is released by endothelial cells in response to the increase of blood flow. The immediate effect is to produce vasodilatation and VSMC relaxation (Palmer et al. 1987). This will cause an increase in lumen size. Apparently, NO is not only a vasodilator, but also a growth inhibitor which can prevent VSMC from growing. The intact endothelial cell's layer serves as a screen to stop migration of VSMC into lumen. For the side of growth promoters, a number of growth promoters are produced. For instance, Ang II released from endothelial cells and VSMC through a paracrine mechanism begins to stimulate

PAGE 17

4 produced. For instance, Ang II released from endothelial cells and VSMC through a paracrine mechanism begins to stimulate VSMC to migrate into lumen, leading to cell proliferation there (Dzau 1992). This proliferation is highly exaggerated, then the consequence is reblockage of lumen. VSMC are highly proliferable cells. Cultured rat aortic smooth muscle cells have been used as an experimental model system for the studies of different growth modulators. These cells show a high tendency of proliferation even without growth factors' stimulation. The cells also have a high density of ATi receptor on their membrane surface. They are characterized by a high responsive to Ang II stimulation (Rosendorff 1996). The detailed mechanism of how Ang II is involved in restenosis will be discussed in following paragraphs. The Renin-Angiotensin System (RAS) The classic RAS is an endocrine system which is very important in humoral regulation of the circulation. This type of RAS exists in body fluids and has traditional characteristics of hormones. Its major component, Ang II, is one of the most powerful vasoconstrictive substances known. It is estimated that one millionth of a gram of Ang II can increase the arterial pressure of a human 50 mmHg or more (Guyton 1986). The basic function of this hormone is to cause vasoconstriction, thereby to increase total peripheral resistance and to elevate blood pressure.

PAGE 18

5 The Components of the RAS Figure 1-1 illustrates the components and functional steps by which the classic RAS helps in the regulation of blood pressure. Renin is an enzyme which is synthesized and secreted by juxtaglomerular cells of the kidneys. The function of renin is to cleave angiotensinogen to release a 10 amino acid peptide, angiotensin I. Angiotensin I has no vasoconstrictor. The active vasoconstrictor in the RAS is Ang II which is an 8 amino acid peptide converted from Ang I by ACE mostly in the endothelium of the lungs. Ang II can be inactivated and degraded by angiotensinase. The principal effects of Ang II include vasoconstriction and salt and water retention. Angiotensin Receptors There are two major types of angiotensin receptors, ATi and AT2. In rats the ATi receptor is further classified into ATia and ATib subtypes according to their structure differences. The different Ang II binding sites were first described by their pharmacological characteristics. For example, Ang II type-1 receptor specifically binds Losartan (DuP753) and Ang II type-2 receptor specifically recognizes PD123319. Recent advances in molecular cloning of the cDNAs of these receptor subtypes revealed the true structure different in their genetic level. The cDNAs of the ATi receptor were first cloned from rat aortic VSMC and bovine adrenal zona glomerulosa cells (Murphy et al. 1991 and Sasaki et al. 1991). This type of ATi was also recognized as AT^. Later the other ATi subtype, ATm was cloned from rat adrenal (Murphy et al. 1992) gland and pituitary. Two ATi subtypes can be also found in mouse genomic DNA; however there is no evidence that the divergence to AT,a and ATib exists in humans (Smith and

PAGE 19

Angiotensinogen ^'™^5 Circulating RAS system Bra/n RAS System Renin Angiotensin I Lung y ACE Angiotensin II ngiotensinog^n J Renin! \ Aminopeptidase A y Angiotensin III . ATI ' . y Antagonist "^^^ \Aminopeptidase (losartan) Antagonist \ ^ (PD123319) . . , . o ^ ^ Angiotensin 3-8 Angiotensin II | ATI AT2 Figure 1-1. Schematic elucidation of the components and functional steps of renin angiotensin system.

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7 Timmermans 1994). Rat ATia is localized on chromosome 17 and ATib on chromosome 2. In humans, chromosome 3 bears the single ATi gene. Rat ATiA and ATib share about 96% identity in their amino acid sequences. However, they have remarkable differences in the noncoding regions which may reflect potential variety in the regulation of gene expression. They are both 395 amino acid proteins with molecular weight of 41 KDa. Hydropathy analysis of amino acid sequences suggests that ATi receptor is seven transmembrane domains, G-protein coupled receptor. Previous studies have shown that ATi receptor is responsible for most traditional Ang II functions. When Ang II binds to its ATj receptor, the binding activates a specific protein signaling system. These signal transduction pathways include activation of phospholipase C, phospholipase D, calcium channels and other ion channels. The signal transduction pathways can be different in different tissues. In VSMC, after Ang II binds to AT,, phosphatidylinostiol bisphosphate is hydrolyzed and diacylglycerol and inositol triphosphate are increased. During the same time frame, there is a transient increase in intracellular calcium level. The immediate consequence of these intracellular signals is the activation of protein kinases, including PKC, tyrosine kinases, and a calcium-calmodulindependent protein kinase. These kinases furthei phosphorylate a number of other proteins such as MAP kinase, myosin light chain and vimentin, and these proteins mediate cellular functions of smooth muscle, such as contraction. The cDNA of ATj receptor subtype was cloned in 1993 from a rat pheochromocytoma cell line (PC12w) (Kawamura et al. 1993). The AT2 cDNA

PAGE 21

8 comprises 2,868 nucleotides and encodes a 363 amino acid protein with seven putative transmembrane domains. It shares only 32-35% identical amino acid sequence with the ATi subtype, and this identity is mainly concentrated in the putative transmembrane regions. The AT2 gene has been characterized in humans (Inagami et al. 1995). This gene is located on the X chromosome in both human and rat (Hein et al. 1995). No subtype for AT2 has been reported. AT2 receptor subtype is characterized by its specific binding to PDJ23319 and CGP42n2A. This receptor subtype is expressed at very high levels in the developing fetus. By contrast, in the adult, its expression is restricted to the adrenal glands, uterus, ovary, heart and specialized nuclei in the brain. AT2 has also been shown to be a G proteincoupled receptor. Kang et al. (1994) demonstrated that AT2 modulated current through Gi. Buisson et al. (1995) showed that AT2 mediated inhibition of T-type Ca^* current in the NG 108815 cell line through a pertussis-toxin insensitive, G protein. The clear physiological fiinction of AT2 receptor has not been identified. However, the piling evidence suggested that AT2 may play a role in some processes such as cellular growth, differentiation or adhesion. Interestingly, although AT2 disappears quickly after birth in most parts of the body, it can be re-expressed in certain pathological situations involving tissue repair, such as vascular neointima formation and wound healing (Nakajima et al. 1995). Gyurko et al first noted that the AT, receptors increase IP3 hydrolysis and the AT2 receptors decreased IP3 hydrolysis in rat skin slices (Gyurko et al. 1992). A report from Dzau's group suggested that AT2 plays an opposite role against AT, in neointima formation after angioplasty (Nakajima et al. 1995). They observed that overexpression of

PAGE 22

9 the AT2 receptor attenuated neointimal formation in rats. Also in cultured smooth muscle cells, AT2 receptor transfection reduced proliferation and inhibited MAP kinase activity. Tanaka et al. (1995) and Yamada et al. (1996) suggested that AT2 could trigger apoptosis in rat ovary granulosa cells and PC12w cells. They further suggested that the mechanism of AT2 induced apoptosis was mediated by the dephosphorylation of MAP-kinase. ASODN to MAP kinase phosphatase 1 inhibited the AT2 receptor-mediated MAP kinase dephosphorylation and blocked the AT2 receptor mediated apoptosis. Taken together, all these data indicate that AT2 may play an important role in developmental biology and pathophysiology. There are several selective antagonists for ATi and AT2 receptors. Actually the initial classification of ATi and AT2 was based on their different binding characteristics to antagonists. Losartan binds to both ATia and ATib subtype. PD123319 is the specific antagonist for the AT2 receptor. These two are also the most frequently used antagonists for the ATi and the AT2 both in vitro and in vivo. Tissue RAS The existence of tissue RAS, independent of the circulating RAS, was first • described in the eariy 1970s. Tissue RAS occurs in a variety of organs, such as brain, heart, blood vessels and many other organs in the body (Phillips 1987 and Dzau 1987). Modem molecular technology has helped to identify the components of RAS, such as Ao, Renin and Ang II receptors, in a large variety of tissues. These components were proposed to interact with each other by means of a paracrine and autocrine function. In

PAGE 23

10 the paracrine mode, one cell produces Ang 11 and delivers it to a neighboring target cell which has receptors to bind and respond to the Ang n stimulation. The autocrine mode describes a cell which produces Ang 11, releases it exogenously, and this feeds back via membrane receptor onto the same cell to regulate the rate of synthesis (Phillips et al. 1993). The brain was among the first tissues that were proposed to have a tissue RAS independent of the circulating RAS. Every key component of RAS has been identified in the brain. Since the brain is protected by the blood brain barrier fi-om circulating Ang II, an independent brain tissue RAS was suggested and thoroughly investigated by many groups (Phillips 1987). Both ATi and AT2 receptors are found in the brain. The ATi receptors are distributed in areas associated with cardiovascular effects of central Ang II, such as organum vasculosum lamina terminalis, supraoptic and paraventricular nucleus. The AT2 subtype ais located at locus coeruleus, inferior olive and mediodorsal thalamic nucleus. Its function in brain has not be clearly elucidated. Recently two groups used gene disruption technique to study the possible function of the AT2 receptor on knockout mice. Hein et al show that AT2 knockout mice develop normally, but have an impaired drinking response to water deprivation as well as a reduction in spontaneous movements. • They also found that baseline blood pressure of the mutants is normal, but they show an increased vasopressor response to injection of angiotensin 11 (Hein et al. 1995). On the other hand, Ichiki et al. (1995) reported disruption of the mouse AT2 gene resulted in a significant increase in blood pressure and increased sensitivity to the pressor action of

PAGE 24

11 angiotensin H. The controversies between the results of two research groups on the AT2 knockout mice suggest that further investigation will be necessary. There are three distinctive physiological effects of brain angiotensin receptors when Ang n is given centrally. The effects are an increase in blood pressure, AVP release and motivation to drink. The effects have been shown to be mediated exclusively by the ATi receptor, since losartan and the ATi AS-ODN blocked these responses (Hogarty et al. 1992, Mengetal. 1994). In the vascular system. Re et al (1982) first showed that there was renin in the dog aorta. Since then every major component in RAS has been discovered, including Ao, ACE, ATi and AT2 receptors. The ATi receptors are located on the membrane surface of medial VSMC and also in the nucleus (Tang et al. 1992). Renin and Ao were found to be at endothelium and media and adventitia. ACE was found in endothelium and some parts of media. ATi and AT2 were both located at medial VSMC. The effects of Ang II in the vascular system are twofold. In response to Ang 11, there are both short and long term effects; vasoconstriction and VSMC growth. Stimulation of the AT2 receptor may cause dephosphorylation of MAP kinase in VSMC (Nakajima et al. 1995). The Renin-Angiotensin System and Restenosis RAS is an endocrine system which controls body fluid and electrolyte homeostasis. Classical RAS is a blood borne, circulating hormonal system. The target organs for Ang [I are the blood vessels, kidney and the adrenal cortex, in which Ang II through its type 1

PAGE 25

12 receptor mediates vasoconstriction, decreased glomerular filtration and aldosteron secretion. The over all effects will be conservation of water, increased Na* reabsorption and increased blood pressure. In addition to circulating RAS, every component of RAS has been found in the vasculature and a paracrine mechanism has been proposed. The endothelial cells secrete both NO and Ang II. Through a paracrine effect, Ang 11 and NO are released to act on neighboring VSMC. The VSMCs secrete Ang II only. Through a paracrine effect, Ang II is released on other VSMC. Circulating Ang II may also reach the VSMC. When the layer of endothelial cells is intact, there is a balance between the growth promoting effect of Ang II and antigrowth effect of nitric oxide, and circulating Ang II probably does not reach the media directly. Damage to the endothelial cells, as for example after balloon injury, changes the balance. The growth promoting effects of Ang II become a dominating force. Interestingly, Schwartz suggested that the proliferating VSMC were actually descendants from single colonies of cells that migrated into lumen and these cells in neointima have a stronger response to Ang II than the cells in media (deBlois et al, 1996). They are regulated differently in response to systemic infusion of Ang II. Since the VSMC in neointima have also been showed to have a higher density of AT| receptor on their membrane, it is very likely that Ang II is one of the early factors involved in VSMC migration (Viswanathan 1993, 1994). The mechanism can be proposed as follows: After removal of endothelial cells by balloon injury, the damage and distention to VSMC cause an increase in Ang II production and also a upregulation of other components of RAS in the vasculature. The Ang II stimulates the cells that have a higher density of AT,

PAGE 26

13 receptors to migrate into lumen and the cells begin to proliferate and secrete collagen and elastin. This is a wound-healing process leading to repair of the damage caused by angioplasty. Unfom^nately, VSMC can not fully replace the functions of endothelial ceUs. Instead of NO. VSMCs secrete Ang n. So ^ repairing process becomes largely exaggerated. Binding of Ang n to its AT, receptor activates a cascade of acute and delayed cellular events. Direct effects include the a«ivadon of phospholipase C and generation of metabolites that modulate calcium-sensitive protein kinase C (PKC) activity and cytoplasmic calcium concentration Ang n binding also activates calcium channels, causing rise to calcium influx to increase VSMC co«racti<,„. The PKC mediated protein phosphorylation activates nuclear elements, with long tem, consequences with regard to gene expression, protein synthe^s. m,togenesis and vascular hyperirophy This long tenn effect of Ang 11 is ve^, important to restenosis and aneriosclerosis. The mechanism which is proposed by Takeuchi et al. (1990) is as follows. Ang n binding to its AT, receptor leads to a rapid increase in c/os and cjun mRNAs levels. The c/os and cjun have been ahown to form a heterodimeric transcriptional complex called AP-l which is able to n-anipulate target gene expression. This type of Ang n sthnulation can be blocked by indicate that Ang „ induced gene expression and ceU growth is panially mediated by PKC. On the other hand, calcium has been shown to be important m MAP kinase activation. MAP kinase is also one of the most important growth modulator (Fig 1-2).

PAGE 27

14

PAGE 28

IS Experimental Models of Resteno58is To model human responses to angioplasty restenosis and balloon catheter injury, three animal modes are frequently used. The most well developed and extensively investigated animal mode of restenosis is the rat common carotid artery. The model was introduced by Clowes et al in 1983. In their study, they proposed the VSMC migration and proliferation are the key factors for restenosis. The knowledge gained from the rat model has contributed to the understanding and interpretation of the restenosis response in humans. However, there are some disadvantages to the current rat model. There is no thrombotic component to the response to angioplasty and the rat is resistant to the dietary induction of hypercholesterolemia. The rabbit reinjury system is another widely used animal model. Rabbits are fed a veiy high-cholesterol diet, and primary injury is reduced with a balloon catheter in the iliofemoral artery. Six weeks later, the same site is reinjured with a balloon catheter. This model allows some thrombosis and rabbits are easy to produce hypercholesterolemia. Porcine models of restenosis have gained a lot of popularity since their use started in the 1970s. This model is perhaps the best model of restenosis resembling human restenosis. The porcine vascular system is very much similar, to that of humans. The major disadvantage of the pig is its size. The pig is more expensive to keep and requires a larger scale in administration of drugs.

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16 Methods to Inhibit Restenosis Drugs A large number of different drugs have been tried both on human and laboratory animal models. These drugs include thrombosis inhibitors (aspirin, dipyridamole, enoxaparin, heparin, vapiprost, and warfarin), and VMSC migration inhibitors (cilazapril and heparin). However, so far none of these dmgs have producted any significant results in clinical trials. In 1989 Powell et al first reported that calizapril, an ACE inhibitor, significantly reduced restenosis in rats when it was delivered via the drinking water for two weeks before angioplasty. After this success, several other research groups demonstrated beneficial effects of ACE inhibitors on different animal models. The AT, antagonists, losartan and TCV„6, were also reported to be effective (Kauffinan et al. 1991 and Kawamura et al. 1993). However, problems associated with these inhibitors and antagonists (such as side effects, need for repeated .administration and the problems associated with dose and time of dosing) have prevented these drugs fi-om effectively treating human restenosis. For instance, although ACE inhibitors have been shown to be effective in preventing animal restenosis in the lab, the human ' trial by MECATOR faUed to confirm any beneficial effects of ACE inhibitors. Uter, Rakugi et al (1994) concluded that patients would have to be put on a much larger dose of ACE inhibitors for a longer time in order to inhibit tissue RAS. Traditionally, drugs work on the protein level. Although they can inhibit protein fiinctions, they usually need repeat administration. Non-specific effects and protein upregulation associated with dmg inhibition

PAGE 30

17 are frequently observed. Although drug therapies have not solved the restenosis problem in the clinic, they provide us with important insights about mechanisms of this disease. For instance, ACE inhibitors work on both Ang U and bradykinin pathways. They block Ang n synthesis and also enhance NO synthesis. Studies with AT, antagonist, losartan, prove that the blockage of Ang H synthesis is the most important part of ACE inhibitor function. Since losartan and TCV„6, the AT, specific antagonists inhibit restenosis just as well as ACE inhibitors. This result also confirms that the growth promoting effects of Ang U is mediated through AT, receptor. Recently Iwai et al. (1997) showed that renin levels in injured blood vessels were increased during the first 3 days after angioplasty and that administration of quinapril significantly reduced neointimal formation. In another experiment they indicated that rat peritoneal macrophage/monocyte cells expressed renin mRNA Macrophage/monocyte cells may be a source of tissue renin in some pathological conditions (Iwai et al. 1996). They suggested that the upregulation of renin might be the earliest event in vascular RAS activation. These data together show that RAS in vasculatnre plays an important role in restenosis. Blockage of RAS could be the potential treatment for this disease. Gene Therapy Viral vector mediated gene transfer holds great potential in treating restenosis (Table 1-1). There are several vectors available today. They are retroviral vectors, herpes virus vectors adenovims vectors, and adeno-associate vims vectors. Each vector has its own characteristics. For instance, the retroviral vector will only infect dividing cells. It

PAGE 31

18 «ve. . .e va... ^^^^^ ^ rep.oa«o„ an, „„„ep„ea.o„ ce.s. S.u.e„.ow .a. .e adeno vec.. . a5,e .0 ,e„. ..o e„do.e«. an. 3.00.. ce„. „o„ev.. .,e ^^-va„.a.e ,s .e i^nune .ac.,o„ . ..eno-a^soc,,. ... .ec.o. . --ynewveo.0. .ow. ,.a. po.e„.. ,ne .e.p, . no„,a.o.e.c a„a t " '~ ..0 a .e„o.e, . , aUowsaable and long lasting expression. .wo n,os. sig.«can. expeH™en.s in gene .e.p, on restenosis were done ^esear. gro„ps ,ed NaBe. at ..e Ut^versit, . ^.o.gan and Uiden a. -no..svectorencoding..eHe,es...s..^di„e.nase,.,TKe..p,osp..,a. ; — -e— „ in t.e t^s^ected treatment (Ohno et al 1994^ r > 994). Le,den s group used a repiieation-defeotive ade„o«™s encoding a nonpliosphorylatable Rb oen^ Bk , . u, e Rb gene. Rb (ret,noblas,o„,a) gene is a cell cycle control .e. wbose nonpbospbo^lated .o™ is antiproli^rati.e. Tbe nonpbospbo.la.abe Rb Se.producttrans.e.edb.adeno.iral.ctorsig™.antl.bloc.edrestenos,s,„aporc. — e.
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19 al. 1994). Although gene therapies have many potential advantages over d.g therapies in treating restenosis, the safety has always been the biggest problem for this approach. T1,e concern has led many researchers to look for alternatives methods, such as antisense-ODN inhibition. to non-viral gene therapy TABLE 1-1. GENE THERAPY FOR RESTENOSIS Vector Type Adenoviral vector Adenoviral vector Gene Herpesvirus thymidine kinase (tk) Retinoblastoma (Rb) Animal Model ^ig References Ohno et al. Science 1994 "pig Chang et al. Science Adenoviral vector Herpesvirus thymidine kinase (tk) ^at 1995 Guzman et al. PNAS-USA 1994 liendai Viral Liposome Atrial Natriuretic Peptide (ANP) ^at Morishita et al. J Clin Invest 1994 Antisensff Tnh.h.tjnn Sin.e restenosis is largely due to ,he n,ig™i„„ ^ p„,a.,,,,„„ ^,^3^^ -.isense .ecl^ology .akes perfect sense for ,rea..en. of „s,enosis. S.a„ed in ear, mo., researchers have designed and iesied a large ™,„ber of AS-ODNs targeting restenosis. Si„.ns e. a. (19,,) reponed that AS-ODN against prot^oncogene c^y, gene had antigrowth effects in rat arteries. Thev used F „l , iney used F,2, pluronic gel to facilitate delivety Of AS-ODNand achieved -S0% reduction in neointinta, fonnation. Shi (,,94) et ^ *owed that proto-oncogene ..... .S-OON in^hited neointi^al h« using a

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20 certain pressures into vascular wall to facilitate uptake. Bennett et al (1994) demonstrated that the cmyc antisense was also effective in reducing neointimal formation in a rat carotid arteiy model using Fu7 pluonic gel applied on adventitia. Morishita et al. (1994a) showed that AS-ODN against a cell cycle regulatory enzyme, cycUn-dependent kinase 2 kinase (cdk 2 kinase) gene was able to decrease neointimal formation. Their delivery system was sendai viral liposomes. In their pharmacokinetics study, they showed that the sendai viral liposome could help to retain AS-ODN in blood vessels for 1 week (Morishita etal. 1994b). Targeting Gene c-myb TABLE 1-2. ANTISENSE THERAPY FOR RESTFNn<;T
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21 system has always become one of the most difficult hurdles in adopting antisense application. The Antisense Technnlngy General For centuries, researchers have been looking for the "magic bullet"-a drag able to reverse tl« illness without side effeas. Since lots of diseases are caused by overproduction of oetlain "bad" proteins, most of the work has been focused on protein inhibitors. Recently, however, a number of researchers have tumed their attention to the genetic level, the n^eor which is responsible for producing the proteins. Gene therapy has evolved into a &-inau„g field which holds gtea. potent,'^ to cure diseases, such as AIDS, cancers and oardiovasaJar diseases. n,e ain. of gene therapy genendly fall into two categories, replacement of abnonnal genes with nonnal genes or inhibition of disease causing gene products. -Antisense oligonucleotide iriJbition belongs to the second category. AS^DN are specially designed DNA or RNA fragments which are able to interfere with gene expression by binding to DNA or mRNA taside the cells. TOs new approach for gene manipulation was ta proposed by Zamecnik ami Stephenson in 1978. m their pioneer experiment for the antisense inhibitior, they irf^bited Rous sarcoma vin,s replicaUon with a 13 mer antisense oUgonudeotide (Zamecnik and Stephenson ,978, Many researche,. have found success using technique during the pas. 19 years. Recent discovety of naturally occuning antisense RNA .ggests that prokatyotes .e acn,ally using antisense RNA in regulating th«r gene

PAGE 35

22 "q«ssion (Wagner et al. 1994). I, was also p^posed by so™ researchers U»t. besides microbe, plan. a^i am™, cdls ™igh. also use antisense sB^ u, con,™, gene expression (Knee e. a,. ,991). In ™os. case, researcher, use sho« srtngs of sy«he,ic an.ise„se nudeoddes instead of a large andsense ge,^a,d,ough son« g™,ps are m working on U^. CWcal Uiab are «,w in progress for U,e AS^DNs in dea&g sevenJ hun^ diseases including acute ntyologenous ,arkenja, MV inf^„ CMV (c^n»ga,o™us) infection (Anderson et al. ,996). bis company has completed its third phase dinica, tiia, for the ASODN treatment of CMV infection. Design nf AS-nnxf I. is proposed .ha. AS-ODN can wodc on any of the foBowing processes to blodc d» gene edificau„ns are me.h^phosphonatio„ and P>K.sphoroU.ioatioa The n^ylphosphonation was des,g„ed by Ts'o and Miller (1979). TH^ replaced an oxygen a.o. ^ each phosphate group with a ™eU^ group (CHJ. TOs s.ep ' helped to increase U« c^^ and provid«i resistance to break down by enzymes Phosphoro.hioa.es were introduced by Chang et a. (1989, They exchanged an oxygen atom wd, a negati vely charged ^ ato.. The phosphorothioates are water soluble and re.sta„t .oen^. ^ven now d^ere is no s^ndardn^e in selecting targe, seouenees. h, ge„e«; -earche. have fou«l d. ™os. regions of d,e RNA including y. and 3.-unti^,e, AUG \

PAGE 36

23 initiation, coding, slicing junctions and introns can be targeted. The only way to determine which sequence is most effective is through experiments. Wagner (1994) suggests that for any 20 mer phosphorothioate ODN, up to 50 sequences should be screened to find an effective AS-ODN. For 15 mer, screening six sequences is efficient to find at lease two sequences to be effective. In our laboratoiy, we found this is an exaggeration and we have successfiiUy designed useable antisense ODNs by initially designing as few as three sequences. Mechani.sm nf Artinn AS^DN is theorized to work with at least three different mechanisms. First, ASODN can bind to DNA and form a triple helix to block DNA uncoiling and transcription to mRNA. Secondly, AS-ODN can bind to mRNA to interfere with splicing, transporting and translation into protein. Thirdly, AS-ODN can stimulate ribonuclease H (RNAse H) and destroy the DNA-mRNA hybrids. No matter which mechanism is involved, the final result should be a reduction in the protein level, and inhibitory effects on the targeting protein related physiological effects. UseofCnntrnU It is always important ,o use proper comrols in experiments to make sure that the effects are real antisense effects. Most frequently used control sequences in antisense research, including sense (S), scrambled (SC), mismatch and inverted. It was also suggested to measure changes of other proteins with similar life cycles along with target prt..eins. TOs will show us if the AS.ODN is specifically inhibiting the taiget protein.

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24 Non-Specific Effects of AS-ODN Although anusense technology was introduced as a "magic bullet"-a new dnig without side effects-the reality is most currently used AS-ODN still can not avoid producing side effects and non-antisense effects, especially when they were used in high concentration. For example, in a ceU culture study, we found non-specific ceU growth inhibition when the ASODN concentration exceeded 25 ^M. In order to achieve specific antisense effects, the concentration used must be relative low (<10 ^M). Antisense Inhibition in RA
PAGE 38

25 long term reduction of blood pressure in SHR (Phillips 1997). In summary, all these data suggest that AS-ODN inhibition is eflFective in RAS gene inhibition. Summary Despite the intensive investigation of the role of tissue RAS in the development of restenosis, the controversies remain. It is necessary to use new technologies and novel approaches to further address this problem. Experimental evidence indicates strong connections of the tissue RAS and vascular diseases. In the present study, we exploited autoimmunization and antisense inhibition to investigate the role of RAS in the development of vascular response to injury restenosis. Our results confirmed the important role of Ang II and its AT, receptor. Further, we suggest that vascular Ang H is involved in the initiation of the growth response to injury. Antisense inhibition provides a useful tool to study the mechanism involved in vascular injury, and is also a potential therapeutic method for treating restenosis.

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CHAPTER 2 HYPOTHESIS AND SPECMC AIMS Hypothesis There is controversy surrounding the role of RAS in restenosis. The current hypothesis is that Ang n is an initiating and critical factor in response to vascular injury. I will test this by making rats that develop their own autoimmunity to the ATi receptor to establish the importance of the ATi receptor in restenosis. Second, I will test specific antisense-ODN to ATi mRNA for inhibition of ATi receptor. Third, I will develop a novel means of delivery of AS-ODN with a dendrimer for potential therapy. Specific Aims Specific Aim 1 I will autoimmunize animals against their ATi receptor to test specifically and chronically whether Ang II stimulation is critical for the vascular response to injury. Specific Aim 2 I will test the specific inhibitory effect of AS-ODN targeted to ATi receptor mRNA. To accomplish this goal I v^Il use the approach of inhibiting central Ang D effects. Centrally Ang n induced drinking and AVP release will be used as indicators. 26

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27 Specific Aim 3 I will develop and test a dendrimer based delivery system as an alternative of liposome delivery of AS-ODN in vivo. Specific Aim 4 I will inhibit arterial angioplasty induced neointimal formation by inhibition of ATi receptor using AS-ODNs delivered by dendrimers.

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CHAPTER 3 MATERIALS AND METHODS Experiments of Autoimmunization Peptide Synthesis and Immunization Adult male Sprague-Dawley rats (200-225 g) were acquired from Harlan (Indianapolis, In, USA). The animals were kept in individual cages in a room with a 12-hr light, 12-hr dark cycle. They were given tap water to drink and standard rat chow ad libitum. Peptide synthesis was carried out at the Protein Core, University of Florida. The peptide sequence was designed corresponding to amino acid sequence 14-23 of the first extra-cellular domain of the ATi receptor. The peptide was anchored to polylysine cores to form a multiple antigenic peptides (MAP) according to the method of Tam (1988). This design completely eliminates the conventional step of conjugation of peptides to carriers. HPLC and mass-spectrophotometer were used to check the sequence and purity of the products. For each injection, rat was given 400 ^g of peptide mixed with 400 ^1 of Freund's adjuvant. The animals were immunized with multiple dorsal subcutaneous injections on day 1, 20 and 40. 28

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29 Animal Model The animals that produced significant titer (higher than 1000) of antibody after the second injection were used in the experimental group. Rats given the same protocol of only Freund's adjuvant injections were used in the control group. At day 45, both groups of rats were anesthetized with sodium phenobarbital (30-40 mg/kg body weight) (i.p ). Under sterile surgery conditions, a 2 Trench Fogarty catheter (Baxter Healthcare, Irvine, CA) was introduced into the left femoral artery and threaded through to the left common carotid artery. The balloon was inflated in the carotid artery with saline and was passed three times up and down the artery to produce deendothelializing effects (Clowes et al. 1988). After surgery, animals were returned to cages and kept for two weeks. All animals were kept according to the AALAC guidelines for animal care and the experiments were approved by the lACUC committee of the University of Florida. Cell Culture Male Sprague-Dawley rats (200-250 g) were acquired fi-om Harlan. Rats were anesthetized with phentobarbital and aortas were removed. Tissue samples was transferred to 5 ml dishes and were digested in 0.67 mg/ml of type II collagenase (Sigma, St. Louis, MO) at ST^C for 30 min. After the digestion, adventitia was removed and tissues were incubated with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% of fetal bovine serum (FBS) overnight. The following morning, the tissues were digested again with 0.67 mg/ml of type II collagenase and 2.5 mg/ml of elastase (Sigma) for 60-90 min. The tissue were tritureated with pasture pipettes to speed up the process.

PAGE 43

30 The digestion was stopped by diluting with DMEM with 10% FBS. Cells were spun for 5 min at 1000 rpm and plated onto culture dishes containing DMEM with 10% FBS. Western Blotting and ELISA for the Antibody Production Blood was taken from the tail at days 25, 45 and 60. Blood samples (500 nl) were collected in 1.5 ml centrifuge tubes and stored at room temperature for 30 min. Plasma was collected after centrifiigation and kept at -20°C until the day of measurement. Polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the method of Lammeli (1970). Membrane proteins extracted from rabbit adrenal glands were amended with 2x sample lysis buflfer and boiled for 5 min before loading. After loading the samples on the 10% pre-cast gel (BioRad, CA), the gels were electrophoresed at a constant voltage of 200 V for 45 min on a BioRad Gel Electrophoresis System. Rainbow molecular weight standard markers were used in all SDS-PAGEs. After electrophoresis, the gels were transferred to PVDF membranes (Bio-Rad) using the transfer buffer systems of Towhin et al (1979), or Szewczyk and Kozloff (1985). Transferred membranes were blocked for 1 hour in the presence of 5% bovine serum albumin (BSA) at room temperature, then the blots were probed by 1st (rat anti-AT, autoantibody) and 2nd (Goat • anti-rat IgG alkaline phosphatase, Sigma) antibodies. The antigenic bands were visualized by the incubation in substrate solution (NBT-BCIP). Quantitation of autoantibody production was determined by ELISA. Microtiter wells were precoated with 600 ng/well of the synthetic peptide diluted in 50 mM sodium carbonate-bicarbonate buffer (pH=9.6) and incubated at 4T overnight. Sodium

PAGE 44

31 carbonate-bicarbonate buffer was added to the microtiter well at the same time to serve as a control. The microtiter wells were incubated at 3TC with 5% milk in PBS for 1 hr before washing five times in washing buffer which was composed of Ix PBS + 0.05% Tween-20. The serum samples containing rat autoantibody were diluted to 1 :300, 1 :900, 1:2700 and 1:8100, then added to the wells. After 1 hr incubation at room temperature, the wells were washed with washing buffer five times. The optical density was read fi-om a Dynatech 600 microplate reader after the incubation with goat anti-rat IgG complexed to alkaline phosphatase (Sigma) and substrate solution. Protein Kinase C Assay Protein kinase C (PKC) assay was carried out by using Calbiochem nonradioactive protein kinase assay kit (Calbiochem-Novabiochem Co., San Diego, CA). Briefly, VSMC cultures were pre-treated with 100 \il of immune serum and control serum in DMEM for 60 minutes. PKC activation was achieved by adding Ang n with final concentration of 100 nM in the culture dishes. After 3 minutes incubation with Ang II (Dkon et al. 1994), incubating solution was aspirated off and cells were washed with ice cold PBS and scraped in 0.5 ml of homogenization buffer (20 mM Tris-HCl, 5 mM • EDTA, 10 mM EGTA, 0.3% p-mercaptoethanol, 1 mM PMSF, 10 mM Benzamidine). Cells were sonicated for 30 seconds on ice and centrifiige at 100,000 x g for 60 minutes at 4°C. The supernatant was used as the cytosolic fi-action. The pellet was resuspended in PBS buffer, plus 0.5% Triton-X-100, for an additional 30 min. The suspension was centrifiiged for another 30 min at 4X at 100.000 x g and was used as the particulate

PAGE 45

32 fraction. Protein concentration was determine using Lowry method (Lowry et al. 1951) and 500 jig of protein was used in each assay. The O.D. of each well in the assay was read on a Dynatech Immunoassay System at 492 nm. Serum Tran.sfii.sinn Blood samples from immunized and control groups were collected after rats were sacrificed. Sera were collected after spinning the samples at 1000 x g and stored at -20T for the fijture transfiision study. Sprague-Dawley rats (300-325 g) were given two infiisions of 0.5 ml serum one at 24 hours before surgery and one 1 hr after surgery from the femoral vein. The rats was divided into two groups (n=3 each). One group received serum samples from immunized rats, and the other group received serum from control rats. Both groups were subjected to the same procedure of angioplasty after serum transfiision. Rats were returned to their cages and kept for two weeks. At the end of the second week, rats were sacrificed and left common arteries were dissected out for the morphological examination. Autoradio^ rflphy The method of autoradiography used was described previously in detail (Ambuhl 1995). Briefly, rats were deeply anesthetized with sodium pentobarbital and perfiised with 0.90/0 saline solution intracardially. Both carotid arteries were removed and frozen at lOX for sectioning. The sections were cut on a ciyostat (20 ,m) and mounted onto gelatin-coated slides. The radio-ligand which was used in all experiments was -I-Sar,Ile-

PAGE 46

33 Ang n. No„-spec.c, AT,. ^ AT. _ ^ ^ (MCm, Imaging Research, Ontario, Canada). Morpholoeical Fyamm^t ions Two wee^s a«. Moon ca.^e.eH.... „e. deep, .e«He.^ -u. pe„.o..«.a, .a pe*.. .ea„ ™U ,^ ~ O.O.. .eHes we. ..sec.e. 3„a fixe. . p^^^,,,^,^ , ^ The ..^es we.e s.ai„ed ,e™.o^„„ ^^^^^ on.e „e. o.„eo,„...™e.. we.e ™ea..e<. ^ ..a exp.e«e. . a ™<„ or intimal/media areas. ImmunnhUt^^hfmhtti~. we app„ea . au.oa„.o.. .o .eoo^. a.e™.,e.,e .a... . ^^^^^.^^ ^ ^ r -----rabbit polyclonal antibody to AT r... . , series from both immunized rats and control rat. ru. • — oh..tocl,e,ni.t„ was ca„,e« out by „.„g an ABC peroxidase s,ai™„g ^, Chemical Company USA, BHefl • F 'y, Briefly, tissues were

PAGE 47

34 Sliced into 30 sections on cryostat after .n « • • cryostat, after an overnight incubation in Ix PBS at 4°C overnight. After incubation with ]anH o". u . and 2 antibody. AT. signals were visualized by exposing sections to 3,3-diaminoben2idine. Animals -e ^ sp.,,,,,^ ,0) ... ,.0.300 , „ Harlan (Indianapolis, IN. USA). The h Iight-12h dark cycle. They animals were kept in individual cages in a room with a 12 libitum. were given tap water to drink and standard rat chow to eat ad Surgery and a ^ace). T1,e ca™,u,a anchored ..ai^ess «ee, sc.e.s . „e and covered by denlaJ acylic A aeel w,V, h„ y »^ ™« obmralor was placed in , he cannula ,o naintam patency Animals were rei„r„M , >, were renimed to home cases lo ra-n^,^ c. rJ recover from surgery for 5 dav — e^OOO.„.)wasp,aced.lu -^•<.a.«yundersodiu.pen.oba*i,a,(30.0„^,bod • / — sampling TT,e e ' P ) anestf,esia for blood ^Ptag. Th» «Penmen.s were perfonned 24 hours alier .h, nours after ihe carotid catheterization. The

PAGE 48

35 -.0, .po^ ^ „^ , ^ ^ indicaaon that the cannula is in the ventriclel h h. k .. ^I"*). It has been shown by Hogarty et al (1992) that ACSFhasn„e.e«o„.h«„,„.,VP,.ea.wh«„vence„.^. nwases.abashe.bypH„.os..p„^^,^,,^^^^^^^^_^^^^_^ .-P-,.sc,^,,OONaseon..o, ;"^'""'"°"^--^^------..e„..es.,).,„,o„. 0.O.V) was adn^stere. .4 hou. ,at. to test the ^ ^ ^PeH^ents we.e pe.o™e. . ^ Spta.e-Oa.ey an. SHR ^ps. . .wo Spta^ Dawley groups, the rats also received Si-, • • • ^'"^ ^ » 24 !> intervals w'th 50 ,g of ^•<«nse, or scran,bled ODN or 4 m isotonic saline. OUgodmv vniii-l,^ riH^ "•'--^«)''"«-eo«,.eswassy„thesi^„,_.„^^3,^^^^ ° ^ ' 00. was a . ^ , r: was .... b. bac^. Honda, GainesviJJe FL Tho ™' ^-"-^ weas the follows: AS 5' TAACTGTGOCTGCAA-.,Sc5.-AATTGGTOTGmCGrrc.3..

PAGE 49

Vasop.ess.assaywaspe^o™edac.^.,,,ep.ced.eofHo^ BneflyODN treated rats were injected i.c.v with Anenrsn ^ with Ang n (50 ng), and blood samples for the wa. e«^ed by ab.,,o„ ,o ben.™'. a„ 80% „ 3 '^'asma (U.5 mJ) was extracted by .sin, a.., ^^^^ . ^ > ^J'^vp (DuPont) was used as the tracer and AVP ro: w.O.O.,..be. -..ec....o.._. Drinking ^ Drinking wa.raeaa,red for 30 mk after Ang nWecUon Th J. injeaion. The water mtake was r^w directly from scaJed drinking bottles .nH ' •King bottles and is expressed as ml/30 min MeligaridBindingAssa^ Sprague-Dawley rats were given ,bre. i.c.v. injections of an.isense. or sc^nbied ODN

PAGE 50

bindmg and AT. receptor binding respectively. Dendrimer generations four (AJdrich Inc) six (Pnl m ^ • (Polysciences Inc.), and ten To determine if ,he dendrimers are be able .„ , fo™ relatively stable co ' ' '-e DEN-. ability to ea„« ODM , retardation on a IS"/ h« • . ODN-gel ~ ..te. i„ ™ buffer

PAGE 51

38 FITC-Iabeled ,5 .er antisense oligonucleotide targeting rat AT r . used in th. • ' ^ere used m the conjugation studies with the dendrimers The ^.h endnmers. The 6th generation dendrimers were complexed to the phosphorothi, PBS buffer. The molar ratios of oligonucleotide to dendrimers oate antisense oligonucleotide by mixing them together in were 1.1 i ns i n i i 0.0. an. .0 ^ _ "-^^ ' 0.. glycerol TT,, r, , 50% ^-yoero, The pho.os were .aken under W „ans,u™i„a.or a. 360 „M. ^-i^^w^Ietabolism Male Sprague-Dawley rats weighing 200 2^0 „ or.e.a„.„e.<..„^,, , . ^ ^ ~« -00 ^ *^*°''^'^-'"-P~'y(^PO. The left fe^or. vein ^ <=annulated and injected with 0 5-10 ml f , ^ ve,n was «endH„.erorDEN , ' """'"^ '^"^'^ With a r: """'^^ — ea -.^0,ao„,naftera.n„ei„.rave„o.i„
PAGE 52

39 ^i§sy£Distnbution^^ Twenty-four hours after sample infusion, rats were sacrificeH . V . . '^^'^^"^ced and tissues including bran, ladney.Lve, skeletal muscle and blood vessels raorta . vessels (aorta and common carotid arteiv) were removed. The tissues were shced to 20 ,M sections H FITC signals were ^sualized by using confocal microscopy. mg/kg body weight) fi n ) .nH , c . ) 0.p, 3 , ^^^^ ^^^^^^^ ^^^^ introduced from left femoral artery to th. i a ^^ry to the left common carotid artery Th« h.» inflated by saline and was oa.,.H.h • The balloon was was passed three times up and down af l^ft ucea from left external carotid arteiy into left The «rf. carotid artery only) of ,„fi.s,o„s a. certain pressure ,o faciiiate up,ake ,„,o , Aft«r „ "piaKe into vessel walk After surges., ani„,als were rerumed ,0 cages Anin^, -es„.u.io.„ac„ Bo. ^ ^ ^ ~ « ' Both caro„d arteries were removed and fro^e^ a, ,or r seccomng. The sections were cu. n„ , e..„nac^os,a.(ao,.)and.„„„,«,„„,„^,,,„_^

PAGE 53

40 Statistics All value, are expres^i as mean ± S.EM. Da« were analvzerf h analyzed by using ANOVA oi studentt-test,followedbytheDuncan.uItiplerangetest Inalianal p on, H ge test. In alJ analyses, a P vaJue of less than 0.05 was considered significant.

PAGE 54

41

PAGE 55

AT CHAPTER 4 -Introdiirtinn ci dj. iyy4 Popma et al IQQ]^ tu 1990, The res,e„„.i. is ™a,„,y due .o overgrowth of vascular r""^'^"'"^^^"""^----'-----ar:o r ' "^"^ — ce. o.ar..es ~ .he growth ,«,o^ — a.va.,o„ o._h ..ors o„ VSMC . the J ™ ^ .0 ".S.te an. „ . ^ ^ ^MC to «ow.M^,,_,, \ — '"-a, s^a„, Many factors have been shown to be involved in fh' has heeu suggested .0 pro . ""^^^^ -se factors, • " ^ "«9). The vascular wali is „„e of ,h Oeen proposed ,0 have a loca, • ""-f"' ™y t,ssues that has nave a local ren,„ angiotensin system fRAS) inH ^ y em (KAS), mdependent from plasma

PAGE 56

43 RAS (Dzau et al. 1987). Ang H functions through its specific receptors on the ceU membrane. There are two main sub-types of Ang H receptors, AT, and ATj. The AT, is responsible for vasoconstriction and the growth effects of Ang H on blood vessels (WUcox et al. 1993). The AT^ subtype has been shown to have anti-growth effects (Nakajima et al. 1995). The binding of angiotensin U to AT, receptor triggers a cascade of intraceUular events leading the activation of phospholipase C and generation of inositol triphosphate (IP3) and diacyiglycerol (DAG) (Griendling et al. 1994). IP3 is responsible for the increase of intracellular calcium level and DAG stimulates protein kinase C (PKC) activation. Both pathways have been shown to be important for VSMC growth (Duff et al. 1995). The attempts of using angiotensin inhibition to treat restenosis began with the study of Powell et al in 1989. In their study, they administered ACE inhibitor. Cilazapril to the balloon injured rats and achieved 8O0/0 reduction in neointimal formation (Powell et al. 1989). In the following years several studies cases have been reported using variety of ACE inhibitors and AT, antagonists to prevent restenosis in animal models (Kauffman et al. 1991, Jaguchi et al. 1993. Kino et al. 1994). However, despite the extensive study of the role of the renin-angiotensin system in development of restenosis, controversies remain. In clinical trials, we still do not have a clear picture on the importance of ' angiotensin in development of restenosis. The MERCATOR (Multicenter European Research trial with Cilazapril after Angioplasty to prevent Transluminal Coronary Obstruction and Restenosis) trial failed to confirm a beneficial effect of ACE inhibition in human subjects (Hennans et al. 1993). However, Yamabe et al (1995) showed that the treatments with cilazapril 7 days before angioplasty significantly reduced the rate of

PAGE 57

44 restenosis in human. Ralcugi et ai (1994) later suggested in their study that higher dose of cilazapril may be need to inhibit tissue RAS. When we review the protocols used, we conclude that most of the difficulties are due to the dose and time of dosing with ACE inhibitors. Apart from the protocol decision of when to and how much to administer ACE inhibitors, there is the possible response of upreglation of receptor or inadequate reduction of Ang n at a critical time. It is beneficial for us to go back and carefully review the results of the animal studies. The condition that appears to have been important to treatment with ACE inhibitors prior to injury (Powell et al. 1989), whereas in the MERCATOR trials cilazapril was given after injury. Utilization of new and novel techniques will also be helpful to explore this problem. As a useful technique to explore protein functions, autoimmunization overcomes the difficulties of drug therapies, such as problems with dose and time of dosing (Soos et al. 1995, Fu et al. 1996). Autoimmunization is chronic and complete. In the present study, we designed an experiment to induce rats to produce autoimmunity against the Nterminal>of ATi receptor. The N-terminal peptide is the first extracellular loop of the 7 transmembrane receptor and was shown to be important for PKC activity induced by Ang II (Vinson et al. 1994). The immunized rats were then subjected to balloon injury of the • carotid artery. We hypothesized that if Ang II is critical in the vascular response to balloon injury, blocicing Ang II fiinction with a specific autoantibody would prevent VSMCs proliferation and achieve a reduction on neointimal formation. To further test our hypothesis, we also transfused antiserum containing AT, autoantibody into normal rats

PAGE 58

45 and performed balloon injury on these recipient rats. In both experiments we achieved a significant reduction of neointimal proliferation. Results Over 50% (11 out of 20 rats) of the immunized rats produced a significant amount of autoantibody in the ELISA screening (with the titers of 1000-2000) after the second injection. These animals were used as immunized group for the balloon catheterization experiments. Western blot analysis of the protein extracts from rabbit adrenal glands using the ATi antiserum shows a single band with molecular weight of 65 IcDa (Fig 4-1). This result corresponds well with the molecular weight of the mature glycosylated ATi receptor (Barker et al. 1993, Desamaud et al. 1993). Occasionally two other minor bands with molecular weights of 43 and 55 IcDa were also observed. These two bands may be the result of deglycosylation during extraction procedures. The 43 kDa is the predicted molecular weight of unglycosylated ATi protein (Murphy et al. 1991, Desamaud et al. 1993). This result indicates that the antigen induces a specific autoantibody against the ATi receptor protein in immunized rats. PKC assay was carried out on cultured VSMC. Ang II (100 nM) stimulated a rapid translocation of PKC from the cytosol to the membrane in 3 minutes (Fig.4-2). After 60 minutes incubation with immune serum, the Ang II induced PKC translocation was significantly reduced, while the incubation with control serum had little impact on PKC translocation.

PAGE 59

46 There was staining with the autoantibody of rabbit ATi receptor. Figure 4-3 shows immunohistochemistry of staining AT, protein on rabbit aorta with antiserum from the immunized rats. The autoantibody specifically recognized ATj receptor on VSMCs in media. The result of staining for AT, receptors in the autoimmunized rats showed the presence of autoantibody blocked exogenously applied antibody from binding. There was no staining on adventitia. Fig 4-4b shows that the carotid arteries from immunized rats could not be stained using a rabbit AT, polyclonal antibody. The control rats which were injected with only Freund's adjuvant showed obvious staining to the rabbit AT, antibody (Fig. 4-4a). Autoradiographies of multiple transverse sections of carotid arteries showed that the location of AT, receptor is in the VSMCs (Fig. 4-5a). The AT, receptor binding was decreased on the sections of immunized rats (Fig. 4-5b). The effects of AT, autoantibody on restenosis are shown in Figure 4-6. Autoantibody produced significant inhibitory effect on neoimimal formation. Morphological analysis of the cross sections by ratios of neointimal/medial areas shows that the > immunized group had significantly lower neointimal growth than the control group (p<0.01)(Fig. 4-7). In order to rule out the possibility that metabolic changes associated with • autoimmunization may contribute to the growth inhibition, we also carried out the blood transfusion study. In the experiment, we transfused the sera collected from control or immunized rats to two groups of new rats at the onset of blood catheterization. The rats that received twice immune serum injection (1 ml each) showed significant lower

PAGE 60

47 neointimal formation when compared to of those received same amount of control serum (p<0.05) (Fig. 4-8). Discussion In the present study, we demonstrated that the neointimal formation induced by balloon angioplasty could be inhibited by actively inducing autoantibody production against the N-terminal of the angiotensin II type-I receptor. We also showed that transfusion of antiserum during early stage of balloon injury reduces arterial regrowth. Our results support the hypothesis that Ang II is a critical growth promoter in the vascular system in response to injury. Our results further suggest that Ang II is one of the early growth factors in the process of restenosis. The present study offers the first report that an antibody against the N-terminal of the AT, protein inhibits neointimal formation. The primary pathophysiologic mechanism in restenosis is the migration and proliferation of VSMCs in the subintimal layer (Clowes et al. 1983). where they form neointima and decrease the lumenal diameter. Arterial angioplasty removes the endothelial ceUs, which secrete nitric oxide and heparan sulfate proteoglycan that apparently maintain • VSMCs in a quiescent st^te (Palmer et al. 1987. Kinsella et al. 1986). The distention in the procedure of angioplasty 'traumatizes VSMC and stimulates the production of growth promoting factors (Clowes et al. 1989). Blood vessel is one of the tissues that have been shown to have a local renin-angiotensin system (RAS) (Dzau et al. 1993). Gibbons et al (1992) show that Ang II have growth effects on VSMC and the growth actions are

PAGE 61

48 Fig 4-1. Western-blot analysis of membrane proteins from rabbit adrenal glands using antiserum from immunized rats. Membrane proteins extracted from rabbit adrenal were loaded on the 10% pre-cast gel and were electrophoresed at a constant voltage of 200 V for 45 nun. The gels were transferred to PVDF membranes, then the blots were probed by 1st (rat anti-AT, antibody) and 2nd (Goat anti-rat IgG alkaline phosphatase) antibodies The antigenic bands were visualized by incubating in substrate solution (NBT-BCIP) There was a single band with MW of 65 IcDa recognized by the rat antibody

PAGE 62

49 •-> O (4-1 o o U 0. T 1 ^ Control Angll AnglHAngII + control serum immune serum Fig 4-2. The effect of immune serum containing autoantibody to the N-terminal of the AT, protein on PKC translocation. Cultured VSMCs were pre-incubated with immune and control serum for 60 minutes. 100 nM of Ang H was used to induce PKC translocation. Ang U induced a rapid increase in membrane-bound (particulate) PKC activity accompanied by a reduction in cytosoUc PKC level. The pre-incubation of immune serum resulted in a significant blockage of Ang n induced PKC translocation ^I'^u.us, n-3), while control serum had no significant impact on PKC.

PAGE 63

50 Fig 4-3. Irnmunohistochemical identification of AT, receptor on the sections of rabbit arteries using the rat AT, autoantibody. The immunohistochemistry was carried out by using an ABC peroxidase staining kit (Pierce Chemical Company, USA) Tissues were ??nd'"/^ ^"^ °" ciyostat. After incubation with l" (rat AT, autoantibody) and 2 (Goat anti-rat IgG) antibodies, AT, signals were visualized by exposing sections to 3,3-diaminoben2idine. The rat AT, autoantibody located most of the AT, receptor on medial smooth muscle cells.

PAGE 64

51 Fig 4-4. Immunohistochemical staining of sections from rat carotid arteries using a rabbit polyclonal antibody against AT, receptor. The immunohistochemistry was carried out by using an ABC peroxidase staining kit (Pierce Chemical Company, USA). The l" antibody was the rabbit polyclonal antibody against AT, and the antibody is goat anti-rabbit IgG provided with kit. AT, signals were visualized by exposing sections to 3 3diaminobenzidine. A, Representative section of a carotid artery from a injured noniial control rat exhibited intense staining of AT, receptor on neointimal and medial VSMCs B. The artery of autoantibody producing rat was not be able to be stained using the same rabbit antibody and experimental protocol.

PAGE 65

52 Control Immunized Fig 4-5. I-Sar,Ile-Ang II autoradiography analysis of multiple transverse sections of carotid arteries. The sections were cut on a cryostat (20 nm) and mounted onto gelatincoated slides. The radio-ligand which was used in all experiments was '"l-Sar,Ile-Ang II. Non-specific, ATi, and AT2 binding were determined in the presence of I of Ang II or PDi233i9 or Losartan. Autoradiograms were generated by exposing the slides to X-ray films for 4 weeks. The photos were taken directly fi-om an image analysis system (MCID, Imaging Research, Ontario, Canada). The pictures represent the specific binding of ATi' receptor. A, representative section from control rats. B, representative section fi-om immunized rats.

PAGE 66

53 Control Injured Injured + Immunized Fig 4-6. Photomicrographs of representative histological sections from sections of rat left common carotid arteries 2 weeks after balloon injury. Two weeks after balloon cathetenzation, rats were deeply anesthetized and perfused via the heart with saline solution. The left and right common carotid arteries were dissected and fixed in 4% paraformaldehyde for 4 hrs. The arteries were sliced at 20 nm thickness transversely on a cryostat machine. The tissues were stained by hematoxylin for the morphological examination. A,B. Uninjured. C,D. Injured. E.F. Injured and immunized

PAGE 67

54 2 0 Ctrl Immunized Fig 4-7. Bar graphs represent the ratios of intimal/medial areas in the two groups of rats that underwent balloon catheterization. Values are expressed as mean ± SE Data were analyzed by Student t-test. A p value of less than 0.05 was considered significant (*) and a p value less that 0.01 was considered highly significant (**). The immunized rats (n=6) showed higWy si^ficant (p<0.01) smaller regrowth (0.49 ± 0. 11) than the control group (1.37±0.17) (n=6). *^

PAGE 68

55 2 I Control Serum control Immune serum Fig 4-8. Bar graphs represent the ratios of intimal/medial areas in three groups of rats that underwent balloon catheterization. . Values are expressed as mean ± SE. Data were analyzed by ANOVA followed by the Duncan multiple range test. A p value of less than 0 05 was considered significant (*). The rats received immunized serum (n=3) demonstrated significant less neointimal regrowth (0.78 ±0.12) than those of two control groups [(1 37 ± 0.17) for the control rats (n=6), and (1.53 ± 0.22) for the rats received control serum (n=3)].

PAGE 69

56 mediated by the type I receptor (AT,). In vitro studies on VSMC cultures indicate that Ang n stimulates ceUular proto-oncogenes (eg. c-fos and o-juri) that are important in the regulation of cell growth (Naftilan et al. 1989, LyaU et al. 1992). These effects can be inhibited by losartan, an AT, specific antagonist. In in vivo studies, chronic infusion of Ang n results in vascular hypertrophy (GriflBn et al. 1991). Transfection of blood vessels with ACE gene causes neointimal formation (Morishita et al. 1994). Indirect evidence for the involvement of the RAS first came fi-om the observations that neointimal formation could be prevented by ACE inhibitor or AT, antagonist administration (Powell et al. 1989, Kauffinan et al. 1991, Taguchi et al. 1993, Kino et al. 1994). Interestingly, VSMCs in neointima have been shown to have stronger response to Ang n than those in media (deBlois et al. 1995). Since the VSMCs in neointima have also been shown to have higher density of AT, receptor on their membrane than those in the media, it is very likely that Ang n is one of the early factors involved in VSMC proliferation and migration (Viswanathanetal. 1992). The method of using the synthetic peptide induced autoantibodies has been well documented in studies of autoimmune diseases (Fu et al. 1996, Soos et al. 1995) Synthetic peptides induce animals to produce antibodies that react with their cognate • sequences in the native protein. We took advantage of multiple antigen peptides (MAP) in our study. MAP are highly branched polylysine molecules which have capacity to complex peptides on their surface. They do not induce immune responses themselves (Tam et al. 1996). When they complex with antigenic peptides, the conjugates are able to induce specific immune response to the peptides. Numerous reports have confirmed that

PAGE 70

57 MAP induce specific immune responses to the antigens coupled to them without any side effects (Tam et al. 1996). The immune response alone could not be responsible for the inhibition of neointimal formation since both immunized rats and rats treated with antiserum showed the similar effect of inhibition. The specificity of the antibody was shown by using Western blotting. A 65KDa protein was specifically selected by the autoantibody. This is the correct molecular weight of mature glycosylated AT. receptor (Desamaud et al. 1993). The antibody positively stained AT, receptor on rabbit aortas. This is more evidence that the autoantibody is capable of binding AT, receptor in the vascular system. Further proof was shown by the carotid arteries fi-om the immunized rats which could not be stained by a polyclonal AT. antibody raised in rabbit. The lack of staining by the rabbit antibody imply preoccupation of the AT, receptor by autoantibody. Immunohistochemistry showed dense staining of AT. receptor in the neointima. This result is consistent with the report fi-om Viswanathan et al. (1992). Their study showed that neoimimal VSMCs have higher density of AT, receptors using autoradiography. It is possible that the VSMCs in the neointima are regulated differently fi-om those in media (deBlois et al. 1995). The VSMCs with higher AT. receptor number and stronger response to Ang II stimulation may first migrate into • the lumen, where they form neointima by replicating themselves. The cells in neointima may be the decedents of this particular type of VSMCs that have more AT, receptors. In this study, immunized rat vessels showed less receptor binding sites in autoradiography. There are three possible explanations. First, the autoantibody interfered with Ang U binding. Secondly, the density of AT. receptor was decreased due to the growth

PAGE 71

58 inhibitory effect of the autoantibody on VSMC. Third, although Vinson et al. (1995) suggested that the antibody to the N-terminal part of the AT, receptor does not interfere with Ang n binding, the autoantibody may act as a false signal of ligand binding which leads to down regulation of the receptor by negative feedback. Ang n acts on VSMCs through the activation of phospholipase C, which catalyses the breakdown of phosphatidylinositoU.S-bisphosphate to IP3 and DAG. The main role OfDAG is to activate PKC. Activation of PKC is one of the most important events in the AT, signaling cascade. It is also suggested to be a important pathway mediating Ang n induced growth effects in VSMCs (Takeuchi et al. 1990). We chose the PKC activation as the indicator for the inhibitory effect of autoantibody on the AT, receptor into presence of Ang n. The ability of the autoantibody to interfere with physiological function of the AT, receptor was confirmed by PKC assay on cultures of VSMC. Activation of PKC in VSMCs features a rapid translocation PKC fi-om the cytosol to the cell membrane. This event happens in 1-5 minutes (Haller et al. 1992, Dbcon et al. 1994). Showing that Ang n induced ?KC translocation can be blocked by the antibody on VSMC cultures, we further confirmed the capability of the autoantibody to inhibit signal transduction of the AT, receptor. The immunized rats had sigmficanl[y lower neointimal growth after balloon injury. This is direct physiological evidence that the AT, antibody attenuates cell proliferation. Our result is further strengthened by showing that the senrm from immumzed rats is able to inhibit neointimal formation when i, is transfosed into normal rats. The results are critical for the specificity of the antibody effect. It excludes the possibility that a

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59 nonspecific increase in immune response might be attributed to the inhibitory effects on neointimal formation. Interestingly, we achieved significantly inhibitoiy effects by only two infiisions of antiserum. The first infiision was at 24 hours before the balloon catheterization, and the second one was on the day of surgery. This experimental protocol is different fi-om those of Ferns et al (1991) who infiised a PDGF antibody and from Lindner et al (1991) who infiised an bPGF antibody. They continuously boosted their animals with antibody until the animals were sacrificed. Our result may indicate that Ang n is one of the early responding factors to the vascular injury and leads us to hypothesize that down-regulation of Ang H function at the early stage of injury is able to attenuate the initiation of restenosis. There are limited data on time course studies either with ACE inhibitor or with AT, antagonists in the animal models of restenosis. In most cases of ACE inhibitors, experimental animals were put on drugs several weeks before the angioplasty and drugs were continuously available during entire period of the development of restenosis (Powell et al. 198^). However, losartan was tested after angioplasty (Kauffman et al. 1991). It was reported by Prescott et al (1991), that ACE inhibitors inhibit the migration of VSMCs only, losaitan, however, affect both migration and proUferation. This interesting phenomena may explain why ACE are ineffective when they are administered after angioplasty (MECATOR ,994). Vascular RAS has been proposed to one of the early factors involved in initiation of restenosis (Dzau et al. 1993), but since there are many growth factors such as PDGF, bPGF and many cell cycle genes are involved the timing of the role of Ang U is not clear. In present study we support the hypothesis by showing that

PAGE 73

60 AT, auto antibody significantly blocked restenosis and was effective when transfused at the initiation of the response to injury. Autoantibody inhibition is obviously not meant to be a practical approach to preventing restenosis clinically, however it provides a powerful tool to explore the role of the factors that are involved in this compUcated process. The experiments have been carried out in rats. There is a debate whether that rat is a useful model for human restenosis. The debate was fueled by the failure of angiotensin converting enzyme inhibition to reduce restenosis in humans as they had done in rats. However the protocols used in patients were different fi-om the protocol used in rats. The pig has been used as an alternative model, but treatment for restenosis has not been transferred from porcine studies to the clinic. Since the rat is available and the problems of restenosis are complex, the rodent model still offers a fruitful substrate for unraveling some those complexities. Based on this study with autoimmunity to AT. receptors. We conclude that Ang U and the AT, receptors are involved in the initiation of growth mechanism in response to vascular injury.

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CHAPTERS ANTISENSE OLIGONUCLEOTIDE TO AT. RFrpprno t>xt . CENTRAL ANCO^Sm INDUCED ^i^STZ%"^''pS" Introduction Central injection of angiotensin D (Ang n) elidt. seve,.! distinct physiologic^ -ponses includmg an increase in blood pressure, vasopressin release, natriure^s, salt appetite, and a nK,UvaUon to drink (P«ps e. al. ,987). S^ce the is protected fron, blood-bonte Ang n by the blood-brain barrier, the existence of a brain renin-an^otensin systen, (RAS), independent of peripheral RAS was proposed (Ganten e, al. 1983). All components of the RAS have been identified in brain (Deschepper et al. 1986. Dzau e, al. 1986, Lynch e. al. 1986, Phillips etal. 1985 and (Jnaeretal l9on ii.k i. •. „ unger et al. 1991). Although ,t ,s sfU not clear how the components intetaC a paracrine action has been proposed (PMips et al. ,991). T^ere are at leas, two •ypes of Attg n receptots which h.ve been found ^ b^. AT, r^o. are located a, the brain regions which are involved a, cardiovascular control mechanisms (Aldred et al ,993 P-ps e. a,. 1 985). Specifically blood pressure, drin^^ and AVP release are mediated by the AT.receptorfHogattyeta,. ,992, KiAyetal. 1992, Timme^ansetal. 1992) AT. receptors located in specific areas: .ch as the cerebellun. the Werior olivary „„cleu. the locus coreul^s and the thalamus OVri^t et al. ,994, The role of AT. ,^ptor sti. need fi,nher -estigation Recently, Huang et al (,996) .ggested that . b^ neuronal cuLres MAP I— were i„hibi,«l by the AT. receptor sttaulation being stimulated by AT, t^ptor 61

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62 activadoa Spomaneously hype,«^ve «s (SHR) have been propose to have an ove«ctive bn>in angiotensin system wl^ch is critical for their hypertension (Phillips e. al. 1988). Tl,ese «s have an elevated angiotensin receptor density in the bn^ partcularly in hypotiuian^ and bt^in stem (Saaved. e. al ,992) C^^ injection of sanlasin, an Ang n ,«ep,or antagonist decreased blood pres^ne " the SHR when given cemtally at doses that had no effect periphetany (Phillips e. al. ,977). Ang U inhibitots did no. produce any change h blood 9^. of ^.nnotensive controls (WKY). These obse^ations suggest that Ang D tecepto. Phy an Unportant role in the maintenance of hypertension ta SHR However, the data on 10^ (i cv.) lowering hypetten^on in SHR are inconolusiv, which may be due to dfferent doses us«i. V. involvement of AT, ,«:ep.or sp«fical,y, has been ,^tiy ^^aled by andsense oligodeoxy„„deotide (AS-ODN) Mbition of the fanslation of AT, receptor mRNA (03^ .993). AS-ODNs e.dt the. actions by bindhg to the mRNA of the specific tatget protein and i„h,l,i.ing the ptotein synthe^s. Cenn^y ^ ^^^^ ^ mRNA produced Mbition of high blood pressure in the SHR (Gyurko 1993). Tl^o,, we -^.hesi^ that AS^DNtoAT,receptormRNAshouldinhibi.effec.sof direct injection^^ Ang n. While this ^ was in preparation, Salcai et al showed that AS^DN to AT, mRNA inhibited drinking to centtaliy inieced Ang „ (Sa,^ « al. ,994). present stody is mote ' e-ns-ve as we .vestigat^. the effects of AS^DN on the drinldng and vasopressin ,^„se .0 dnect i cv. .Jeoion of Ang n in S«. and Sp^.Oawley tats using the same AS-ODN we had used to reduce hypertension in SHR

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63 Results DiESQgenicResEQnse SHR Figure 5., ^ws U,e effea of AS-ODN on .he drinldng respond ^ „ SHR („=5). Ang n injection an imn,edia.e dipsogenesis wid, a w«er inake of n .7 ± 0.49 mWO min. After pretreatmen. with AS-ODN. the drinking ^on^ ,„ j^^ „ „^ significant,, reduced to 5.0 . O S ntWO ntin (P<0.05). T1. water intake of the control gtoup Which was treated with SC-ODN shows no sig^ficant diflerence with that of ^ais before ODN treatments. Fi^e 5-2 show, the etfec of AS-ODN on the drinking response to 50 „g Ang n in nomtotensive ™s („=5, T.e water intake ^ced hy Ang n injection was dec.^ .^can.,y(P<005, .on, M6.,.35™«0™intoZ74 . 0.95„,.30™i„(after,, section of ^0 Mg of AS-ODN) and to 2, . OM ^0 ^ (after 2 injections of 50 pg of AS-ODN). h, U« repeated test, the second AS-ODN did not ^„her reduce drinking. TTtere is no .gniftcant dtference L, water intake after SC^DN treatments as compared to Ang n alone. Rats ^ Figure 5-3 shows the difference in water intake el I C« groups. The water intake for SHR water intake elicited by central Ang H injection 30 min period of time between SHR and Sprague-Dawley

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64 group (n=5) was 1 1.7 ± 0.49 ml/30 min which is significantly higher (P<0.05) than that of the Sprague-Dawley group(n=5) which was 6.46 ± 1.35 ml/30 min. Plasma Va.sf>p r<>«;g;n Vasopressi, wa. meamed in Ihe SHR. The injecdon of Ang B (50 ng, i.cv.) ~i plasma AVP fro™ , 5 ^ 0.7 p^,„, ,o 15.4 . 0.7 pg/™| („=5). After 24 hours, a second Ang n injection wa. administered, and plasma AVP incteased to 13.66 ± 0.84 pg/ml. There U no si^eant difference betwe«, the two injections (Fig 5^). TOs showed repeated ad™inistm,on of Ang n n, a 24 h in.e™l did «,t change the level of Ang n induced AVP release and indicated that the protocol of one i^ection of Ang n followed by a second injection of Ang n was vaUd. Figure 5-5 shows the effects of 50,„g Ang n (i.cv.) injecuon on AVP release after pretreatmen. w,dr 50 pg AS-ODN, or SC^DN or with 4 m1 saline control. AS^DN (n=5) deceased the plasma Ang n induced AVP si^canUy (6.45 * 0.54 pg/ml) (P
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65 Treatment bar), or AS^DN spo«^L^ '^^t^' ? Il"' S^-ODN (halched

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66 Treatment Figure 5-2. Effect of AS-ODN and SC-ODN on drinking with repeated injections SD rats 7^f!TTui ° P'"^^ ^'^^ ^« (""^had^i bar), 1 dose of SC-ODN (fim hatched bar), 2 doses of SC-ODN (second hatched bar). 1 dose of AS-ODN (first spotted bar) or 2 dose of AS-ODN (second spotted bar). The water intake by each rat in tJe n^ minutes was measured. Data are expressed as mean ± SEM (n=5Xp<0.05).

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67 Figure 5-3. The drinking responses of rats to Ang 0 i.c.v. SD (first bar) and SHR (second bar) were administered 50 ng Ang D. Water intake of each rat in the next 30 minutes was measured. Data are expressed as mean ± SEM (n=5)(p<0.05).

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68 Control IstAngll 2ndAngII Treatment Figure 5-4. Effect of repeated injection of Ang D i.e. v. on plasma AVP level. Rats were administered either one dose or two doses of Ang H at 24 h intervals. The blood samples were drawn at 1 minute after Ang H injection. The plasma AVP level was measured for each rat. Dates are expressed as mean ± SEM (n=5).

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69 Figure 5-5. Effect of AS-ODN, SC-ODN or saline treatment on AVP release to Ang D i.e. v. Rats were administered 50 ng Ang n preceded by either saline (unshaded bar), SC-ODN (hatched bar), or AS-ODN (solid bar). The blood samples were drawn at 1 minute after Ang n administration. The plasma AVP level was measured for each rat. Data are expressed as mean ± SEM (n=5). ** p<0.01 * p<0.05

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70 TSB ATI Figure 5-6. Effect of oligodeoxynucleotide treatment on AT, receptor binding in the hypothalamic block. TSB = Total Specific Binding AT, = Angiotensin H type 1 receptor AS Antisense Oligodeoxynucleotides SC= Scrambled Oligodeoxynucleotides.

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71 compared to the SC-ODN treatment group. The SC-ODN did not change the total specific binding and ATi binding significantly fi-om the saline control. Values are expressed as percentage of total specific binding of the saline treated brains. Discussion The results show that the AS-ODN to ATi receptor mRNA inhibit the physiological effects produced by Ang II (i.e. v.). The data presented here extend the results of Gyurko et al (1993) which showed that central injection of AS-ODN decreased blood pressure in SHR by blocking the protein synthesis of central ATi receptor. The results confirm the finding of Sakai et al (1994) that the drinking response to Ang II (i.e. v.) can be inhibited by AS-ODN to ATj receptor mRNA, and extend the finding by showing that both drinking and AVP responses to central Ang II were inhibited by AS-ODN in SHR Antisense technology was developed as a tool for modulating gene expression. The studies on applying this technology to inhibit gene expression in vivo are just beginning. Wahlestedt et al (1993) injected antisense ODN targeted to NPY Y, receptor into the rat cerebral ventricles which resulted in a significant reduction in cortical Y, receptor. The report from Ogawa et al (1994) described the antisense ODN targeted to progesterone receptor mRNA lowered the receptor density and inhibited the lordosis behavior. Another successful report comes from studies of Akabayashi et al (1994) in which they inhibited neuropeptide Y synthesis and suppressed feeding behavior by using the antisense ODN to neuropetide Y mRNA In the present study AS-ODN suppressed dipsogenesis and AVP release induced by

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72 Ang n ii^'ected into brain. The working hypothesis of AS-ODN action is that AS-ODN inhibit ATi receptor expression by interfering with translation of ATi receptor mRNA (Helene et al. 1990 and Akhtar et al. 1992). Confirming this mode of action, we found decreased ATi receptor density in the hypothalamic region after AS-ODN treatment. Interestingly the AS-ODN did not completely inhibit the drinking response. Even with a repeated dose of AS-ODN treatment the inhibition was not significantly greater than with a single injection. When comparing the percentage decrease in drinking with the SC-ODN treated group as the control, we found that the decrease in drinking with the first dose of ASODN was 52.6% and with the second dose of AS-ODN the decrease was 66.3%. This result is consistent with the report of Hogarty et al (1992) which showed that central injection of losartan, an AT, receptor antagonist reduced but did not completely block the dipsogenic response induced by Ang H (i.e. v.). This may suggest that other non-AT, receptors are involved in mediation of the drinking response. Hogarty et al proposed that perhaps AT2 receptors were involved. It is also possible that other receptors not yet identified may also play a role in central Ang H induced drinking. Although non-AT, receptor mediated dipsogenesis is an attractive idea, the possibility of the AT, receptor accounting for all of the drinking response stiU exists. The autoradiographic analysis of Ang U receptor sites in the hypothalamus region • indicated that AS-ODN treatment resulted in a maximum of 40% decrease in the SHR (Gyurico et al. 1994). This suggests that a higher dose of AS-ODN or repeated AS-ODN treatment should be able to block more AT, receptors thus completely inhibiting Ang H dipsogenesis. However, in our receptor binding study we used three injections of AS-ODN (50 ugAinjection). With this regimen only a 40% decease in AT, receptor density in

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73 hypothalamic region was seen in the Sprague-Dawley rats. This is consistent with our in-vivo studies where we showed that a second AS-ODN injection did not further decrease drinicing beyond the first injection. The lack of increased effects with repeated administration of ASODN indicates that antisense does not completely inhibit the receptor protein synthesis. This may be due to limited uptake in critical cells or the ATi receptor gene re^nds by upregulating ATi receptor synthesis. It seems unlikely that the feedback mechanism would be fast enough or eflBcient enough to compensate for repeated doses of AS-ODN. If the rate of ODN uptake is the rate-limiting step, even with repeated administrations, the cells may not take up any more AS-ODN. The excess AS-ODN may be diluted in CSF and then degraded. Despite the profound decrease in dipsogenic response, the observed decrease in ATi receptor binding was smaller. This may be a result of substantial decreases in AT| in discrete nuclei being masked by other areas with greater AT, receptor density in the dissected tissue block. Therefore it is conceivable that a relatively small change in receptor number causes dramatic decrease in the physiological response. An alternative explanation is that the life cycle of AT, receptor is giving a false picture. The life cycle involves internalization and recycling. During these stages the receptor is not active but detectable by the receptor binding assay. This would mask the taie decrease in binding of active membrane-bound receptors by the antisense. The SC-ODN had no effect on drinking response, which showed that the action of the AS-ODN is sequencespecific. In the response to central Ang II, SHR drank 80% more than SD rats. These data add support to the hypothesis that SHR has overactive RAS components in the brain compared to the normotensive Sprague-Dawley rats. Sakai et al also reported that injection of AT, receptor

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74 AS-ODN into the third ventricle partially inhibited the drinking response to Ang n in normotensive rats (Sakai et al. 1994). They also showed that AS-ODN had no effect on central carbachol induced drinking. Their study obviated the need to include the carbachol induced drinking, but we have addressed the question about Ang II inhibition in SHR and effects on AVP release. The main target sources of AVP release by Ang U are SON and PVN of the hypothalamus which have high ATi receptor density. Yang et al (1992) showed that Ang n depolarization of SON neurons was inhibited by losartan and Hogarty et al (1994) showed that losartan could inhibit Ang H induced AVP release. The AS-ODN treatment significantly decreased the AVP release induced by central injection of Ang n, providing further support for AS-ODN inhibition of AT, receptor expression. The SC-ODN control also slightly decreased AVP release, although the effect of AS-ODN was significantly greater. The inhibition of AVP release after SC-ODN treatment compared to saline controls is an empirical finding for which we have no explanation. In the experiment on drinking, SC-ODN had no inhibitory effect on the drinking response to central Ang n. AVP release is related to osmolarity changes and studies suggest that AVP response to a rise in plasma osmolality is mediated by, or involves at some point, an angiotensinergic pathway in the brain (Hogarty et al. 1994, Sladek et al. 1980, • Yamaguchi et al. 1982). The sensitivity of Ang H induced AVP release to AS-ODN for AT, mRNA inhibition supports this view. AVP release is one of the postulated mechanisms by which SHR maintain hypertension. The present results are consistent with this concept and may be relevant to the decrease in hypertension in SHR with AS-ODN.

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75 The present research is in its eariy stage. Fine turning of the modification of AS-ODN and improved delivery methods will ultimately allow smaller doses to be delivered to sites of action as well as enhancing or optimizing the cellular uptake eflBciency. This ultimately would allow the administration of smaller doses of AS-ODN with equal or greater potency. Overall this would have fewer potential side-effects unlike some of the other chemical receptor antagonists. This is advantageous for both development as a therapeutic agent and a physiological tool. In addition, a further advantageous is the modified AS-ODN has been shovwi to elicit effects for extended periods of time, unlike receptor antagonists which have effects that are relatively short acting. In summary, this report demonstrates a new approach to modulate the ATi receptor gene expression in the rat brain. Our results confirm the function of the AT| receptor in controlling drinking and AVP release and also provide a potential new tool to regulate the physiological effects mediated by the ATi receptor in the brain.

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CHAPTER 6 CHARICTERIZATION OF DENDRIMER BASED GENE DELIVERY SYSTEM IN VIVO AND ITS APPLICATION IN TREATING RESTENOSIS Introduction Restenosis is the process of reobstruction of an artery following interventional procedures such as angioplasty, atherectomy, or stenting. This disease is a multifactorial process. The migration and proliferation of VSMCs is most likely to the most consequential event (Clowes et al. 1988). Conventional drug therapeutic approaches have focused on either preventing platelet deposition, thrombus formation or inhibiting VSMCs growth. However, the problems with drug administration have prevented conventional drug therapy from achieving any clinical significance (Hennans et al. 1993). Therefore, many researchers have turned their attentions to a new approach Gene therapy. Gene therapy is one of the fastest growing fields in the biomedical research. This emerging branch of medicine aims to correct genetic defects by transferring genetic materials into cells. One of the most dynamic research areas is antisense oligonucleotide (AS-ODN) based gene inhibition. AS-ODNs are considered a new class of therapeutic drugs that consummate their fiinctions by binding to mRNA in a sequence-specific manner. Traditionally drugs work on the protein levels. Although they can inhibit protein functions, they usually need repeat administration. Non-specific effects and protein upregulation associated with dmg inhibition are frequently observed. Antisense technology was introduced to overcome these shortcomings of traditional dnigs. Many successfiil reports have 76

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77 indicated that antisense approach is a very usefiil tool in manipulating gene products. Because of the fest metabolic degradation for natural occurring phosphodiester oligonucleotide, but the phosphorothioate prolongs the activity of antisense ODNs. At present, there are two ways to deliver AS-ODN, direct and incorporated into a delivery system. Direct administration is limited by low cellular uptake and fast elimination in vivo of AS-ODNs. Liposomes formulations, such as cationic, have the potential to enhance cellular uptake of polynucleotides into mammalian cells. However, the low eflBciency and cyto-toxicity of liposomes have limited this approach in vivo. Starburst dendrimers® are a new class of macromolecules first described by Dr. Donald A. Tomalia in the 1980s (Tomalia et al. 1985). These polyamino spherical molecules have highly branched, tree-like structures terminating in a surface of primary amines having the ability to bind anionic nucleic acids. Dendrimers are classified by the number of cascade polymer generations required. As the generation number increases there is a corresponding increased in number of primary amines and molecular weight. A newly-emerging area in dendrimer technology is the delivery of genetic material into the ceU. Many in vitro reports have ascertained dendrimers are able to deliver genetic material efficiently into many ceU types without damage to the organisms (Boussifetal. 1995, Haensler et al. 1993). The delivery of • AS-ODN by dendrimers as vector is being studied by other groups (Schwab et al. 1994, Poxon et al. 1996). They have been reported to have many advantages over other liposome and other particulate based deUvery systems. The advantages of their products include defined polymerization reaction, reproducible product low toxicity and the ability to alter the transport and binding characteristics by changing the generation of dendrimer used. However, the in

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78 wvo data about toxicity and metabolism need to be completdy studied before dendrimers can be developed into drug delivery systems. In the present study, we have used polyamidoamine Starburst® dendrimers wiiich wctc synthesized from an ethylene diamine core, resulting in a series of primary amine groups on the outermost ^here. We investigated the pharmacokinetics and tissue uptake of generation 4,6 and 10 fluorescent labeled dendrimers, free fluorescent labeled oligonucleotides, and generation 6 DEN electrostatically complexed to fluorescent labeled ODN. In present study, we also explored the potentiality of using dendrimabased delivery system to delivery AS-ODN to treat restenosis. The biggest problem associated with using antisense strategy to fight restenosis is achieving suflBdent cellular uptake of the oligo and maintaining the antisense inhibition long enough to inhibit neointimal growth. Therefore, a suitable delivery system for AS-ODN is needed. Local delivery rather than systemic administration is a more effective way to obtain higher tissue drug levels at the site of the balloon injury. Local delivery can also minimize the potential side effects. Several local drug delivery^ systems, including perfusion balloon catheters, hydrogel-coated balloon catheters, polymeric or coated stents, and many other approaches are currently under investigation. However, the low tissue uptake of the agents remains the main disadvantage of the catheter " injection systems (Fernandez-Ortiz et al. 1994, Mitchd et al. 1995, Lincoff et al. 1994, Fram et al. 1994). Blood flow washes out the agents in mi^iutes to hours. We have investigated the dendrimer as a sustained-release carrier system that could enter the vascular wall rapidly and not be washed out. We have also tested the ability of using dendrimer complexed with AS-

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79 ODN to ATi receptor mRNA to decrease neointimal formation after vascular injury in the same animal model. Results Purification of Dendrimers Dendrimers were labeled by a simple conjugation using fluorescein isothiocyanate and the primary amines from the dendrimer (Poxon et al. 1996). After the reaction the unreacted fluorescein needed to be isolated from the dendrimers. Spin columns filled with G-10 Sephadex were used for the purification of FITC-labeled DEN from um-eacted label. Samples were am through spin columns for three times until there was no detectable free FITC signal on TLC plates. Figure 6-1 illustrates the purity of the dendrimer labeling reaction by TLC purification procedure for the 4* generation DEN. The signals of free FITC label indicated by lane 1 were gradually decreased until it could no longer be detected after the third time spin. This purification step guaranteed us for using pure FITC-labeled DEN for the rest of experiments. Similar results were obtained for the other dendrimer generations. Dendrimer-Oligoniirleotide Reartinn In order to determine the interaction of oligonucleotides and dendrimers, we performed gel retardation experiments (Fig 6-2). In this method the anionic oligonucleotides easily migrate through the gel matrix towards the cathode. As the net charge of the complex is changed due to the addition of the cationic dendrimers at first the

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80 movement of the complex is slowed but with the addition of increasing amounts of dendrimer the moment stops and is finally reversed towards the anode. Using this method we have calculated that 1 mole of dendrimer could react with 8 moles of oligonucleotide resulted in complete binding of the oligonucleotide. In essence the study demonstrated dendrimers could complexed with oligonucleotide and at some ratio form a positively charged complex. Pharmacnicinptirg The delenmnation of pharmacokinetical parameters with particulate delivery systems is often a difficult process because it requires having the ability to ^uantitate both the dmg of interest and the delivety vector. In this set of experiments we could take advantage of ea^ labeling methods to attach fluorescent reponers (fluorescein isothiocyanate) to both the dehvety vector (DEN) and the ODN. By vatying the administration of the ODN and DEN we could generate data for the ODN, free DEN. and the complex. Table 6-1 demonstrates the phamrarcokinetical parameters of different samples that we tested. These values indicated that as the dendrimer generation increases there is a corresponding increase in half life of elimination. Also from the data presented the ODN-DEN complex appears to be stable in the blood stream since there was a con-esponding increase in the elimination half-life (Fig 6-3). For example, the 6* generation of PAMAM DEN a,^ found that it si^cantly (p<0,0,) mcreased the elin^ation half time of f5 mer oligo (W2a and t,/2p) ftom (3.45 ± 0.58, 39.55 ± 5.97) to (9.00 1 3.35, 395 ± 53) min (Fig M).

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81 Tissue Distribution of PFN Tissue distribution suggested tliat 24 hours after infiision, FITC-Den were accumulated in kidney, liver and blood vessels (Fig 6-5). They did not cross the blood brain barrier into brain and there was not a significant amount of signal detected skeletal muscles. Effect on Neointimal Formation DEN-ODN (100 ^ig) were delivered in situ to rat left common carotid artery. Treatment with the complexes of AS-ODN and dendrimer significantly reduced neointimal formation compared to the control. The treatments with AS-ODN alone and with SCODN complexed to dendrimer yielded no significantly changes (Fig 6-6). TABLE 6-1. PHARMARCOKINETICAL PARAMETERS OF DENDRIMERS Generation tl/2a (min) A, k, tl/2P(imn) A2 k2 4* 1.68 ~48l ~OAl 20.05 71.0 0.035 6-^ 4.07 70.61 0.17 51.67 32.88 0.0134 10"" 2.93 58.8 0.236 83.66 30.9 0.008 15merODN "CF 3.45 18.65 0.201 39.55 8.54 0.0175 Ti "431 0.255 19.76 43.55 0.035

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82 TLC demonstration of the purification process of FITC-DEN using spin column Lanes: 12 3 4 Fig 6-1. Purification of FITC labeled Dendrimers (4* Generation). Dendrimers were labeled by a simple conjugation using fluorescein isothiocyanate and the primary amines from the dendrimer. Samples were run through spin columns filled with G-10 Sephadex three times until there was no detectable fi-ee FITC signal on TLC plates. The signals of fi"ee FITC label indicated by lane 1 were gradually decreased until it could no longer be detected after the third time spin (lane 4). Lane 1, Free FITC. Lane 2, l" time spin. Lane 3, 2™* time spin. Lane 4, 3* time spin. Similar results were obtained for the other dendrimer generations.

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83 GEL RETARDATION ASSAY FOR DEN-ODN COMPLEX Amount of UEN: 6 4 2 0.5 0 UKN-ODN 15 mer OUN Fig 6-2. Gel retardation experiment on ODN-DEN complex. The anionic oligonucleotides migrate through the gel matrix towards the cathode. As the net charge of the complex is changed due to the addition of the cationic dendrimers at first the movement of the complex is slowed but with the addition of increasing amounts of dendrimer the moment stops and is finally reversed towards the anode.

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84 120-1 — I — I — I — I — I — I — 1 — I — I — I — I — I — I 1 — r -I 1 1 — r o (0 c 8G 604020 0 I I I — I — I — I — I — I — I — I — I — III I — I I I 10 20 30 40 50 I I II 60 7C G4 G6 CF G10 Min Fig 6-3. Serum elimination of generation 4, 6 and 10 dendrimer and CF.

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85 120 ~ I I I "T — I — I 1 — I — I — I 1 — I — I — j — I — I — I — I — I r 100 < H 80 60 4020I I 1 I I I I — I — I 1 — I — r— ] — I — I — I — I — I — 10 20 30 40 60 Min T — I 1 1 — r — r 60 70 Fig 6-4. Serum elimination of generation 6 dendrimer, Free 15 mer ODN and DEN ODN complexes.

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Fig 6-5. Tissue distribution of Generation 6 dendrimer. Twenty four hours after infusion, FITC-Den were detected in liver and blood vessels. Left panel. Control. Right panel, Treated.

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87 "i Ctrl SC-DEN AS-DEN Fig 6-6. Effect of AS-ODN for AT rajs were ,rea.ed with control (.=5) SC^Z°^'t''T' ^T'''"" '"i"'^ -.S.OH..rea.e.ra..owe.4.„J.^J^^^^^

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88 Discussion various attempts ,„ ai.er ,he restenosis process by conventional pha™,aco,„gica, or .echa^ca. approaches (e. s.e„.i„„ Have no, ye. proven co.p,e,e success in a c,.,ca, «Hu,g. The a„,ise„se oligonuCeocide approach could open a new avenue for ,he potential. However, .here has to be a sui.ab,e deliver sys.e™ for anrisense .herapy There are normally .wo ways .„ adn.nis.er dru,s in resrenosis research. sys.en.cally and locally Conve„,„„al dr.,s are ad„..s.ered per os to be effecive sys.en,cally such as ACB .n^-bUor and AT, a„.a,onis.s. There are son,e possibili.ies .o ad„,.s.er AS-ODNs — y, or even per os, especially .0 use modified ODNs and carrier sys.e™ .0 However, i. is „ ac^owled.ed a.on. resrenosis researchers .ha. local dehve. — ons Of .herapeuric a.en.s wi.h prolonged re.e„.io. Ucal dru. delive. ac-.y. Sine .he balloon ca,he.eri..io„ ,ha. leads .0 resrenosis is done in sur,. .he aPP.:ca.io„ofAS.OO.a...s..e,sappropria.e.Curre„.,ocaldelive.™e.hods.^^^^^^

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89 ODN „ c..yt and inhibited restenosis in rats (Simons e. ai^ m2y CoaUng stents is a potentially ttsefU, tech,.,ue for achieving sustained loca, adn.,.s.ration. Hydroge, systents can enhance the uptake and ,oca, concentration of ODNs that they deliver (Shi e. al. 1994). they will not be able to increase the stability of ODNs. Cationic liposomes are the prevailingly gene deliver syste™ in antisense research Conic lipids, often with dioleoylphosphatidylethanolantine (DOPE) as an additional lipid deLvery is thought to be endocytosis (Uppalainen e. al. ,994). Catio.c lipids have been (.«6) show that addition of the liposonte/ODN co.ple.es to cultured cells results in a drantatic increase in nuclear accumulation of the fluorescent ODN. The in^bito. acti.ty Conic liposomes are nomrally ser^n. sensitive. The transfec.ion of cells needs be -edou.i„se™™fteemediaT.sse.,ne
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90 Nanoparticles which share some features of dendrimer is another promising deUveiy system for AS-ODN therapy in restenosis. Nanoparticles are 30-500 nm diameter polymeric spherical particles. There are several different types of biodegradable polymers available including biopolymers (gelatin, albumin, casein, polycyanoacralate, lectins, etc.) and synthetic polymers (polycyanoacralate, polyesters, polyanhydride, polycaprolactione, etc.). They have various drug release characteristics ranging from several hours to several months. Oligonucleotides adsorbed onto nanoparticles have been showed to have enhanced stability against nucleases and with a more ideal cellular deposition (Chavany et al. 1992, Godard et al. 1995). Chavany et al (1994) have demonstrated an increase in half life of nonoparticle-bound oligonucleotide from 2 min to over 1200 min when exposed to snake venom phosphodistrase. Wilensky et al (1995) demonstrated that arteries were able to retain radioactive latex micropraticles delivery using standard porous balloon catheters in experimental animal for up to 7 days. Through the use of these particulate systems investigators have been able to alter the pharmacokinetics profile of the entrapped ODN. Unfortunately, the particulate nature of the delivery system is a limitation. We have been working on PAMA dendrimers as an alternative deliven, system for gene therapy since 1995. Dendrimers are produced through a cascade polymerization resulting nanopatides with ending branch with a polyamino sutface. These molecules range in size from 10 to 100 mn, with each generation of the polymer adding -10 nm to the diameter of the molecule. The number of surface primary amino groups doubles with each generation, reaching 4960 for tenth generation of dendrimer (Tomalia 1995). At physiological pH (7.4) the majority of the

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91 ami„o groups are iomzed^ Thus there is a catiomc surface which can interact with anionic molecules electrostatically. The defined structure of these molecules and their large number of surface amino groups has led to dendrimers being employed as a substrate for the attachment of any bioactive molecules which are negatively charged. PAMAMA Dendrimers have been shown to complex with antibodies Studies using antibodydendrimer complex in experimental animals have documented these complexes to be nontoxic and cells specific. Denderimers have also demonstrated the ability to deliver oligonucleotides (Bidinska et al. 1996), and plasmid DNA (Kulcowska^tallo et al. 1996) to a variety of cultured cells. In these cases a new molecular idemity is formed between the opposite charged molecules. A, most ratios of dendrimer complexed to oligonucleotides the complex is soluble and not prone to aggregation. The new formed complex has been shown to be resistant to nuclease degradation and to enhance cellular uptake of ODN in tissue. Stability of oligonucleotides is a necessary requirement for the application of antisense technology to inhibit gene expression in vivo. This often precludes the use of naked phosphodiester ODNs because of the fas. degradation in sentm. Significant efforis have been made toward the development of nuclease-resistant oligonucleotides; in particular phosporo.Woa.es and methylphosphosntaes. However, these modification may increase toxicity and non-amisense effects. Dendrimers provide us with the potemiality of using phosodiester ODNs which are the natural forms of DNA. Bielinska et al (1996) showed that dendrimers could enhance both stability of uptake of phosphodiester ODNs. This may eventually allows us to use the natural form of ODNs in place of expensive and sometimes toxic modified ones. In.eres.ing,y, ,he binding of ODNs

PAGE 105

92 to dendrimer does no. interfere with the physiological effects of ODNs. It is possible that .he binding of the ODN phosphate backbone does no. change the property of bases to form hydrogen bonds wi.h the complememary sequences. Dendrimer have the ability to achieve prolonged systemic circulation effea. Conjugation of ODN to dendrimer increased the elimination half of ODN in blood. Our result show that .he phanMcoldne.ics da.a bes. fi. imo a nvo compartmen, .odel in which conuin a and p eliminadon phases. The (.l/2a and .,/2p) were generarion 4 (1.68, 20.05). gene,a.ion 6 (4.07, 51.67). and generaUon 10 (2.9. 83.66) min respecively We evalua.ed .he' 6' generation of PAMAM DEN for the dectrostatic interaaion with ODN and found that i, significantly (p
PAGE 106

93 blood ve^ls. After we appUed the ODN/de„d™,er complexes, we found ,ha, AS-ODN began .o function and achieved -50% reduction in neoL^ttaa, formation. We conclude that the dendrimer deliveo^ is capable of increasbtg stabUity, uptake and efficiency of AS-ODN. There has been no repot, on toxicity of dendrimers. ,n our study, we have no. dendri^er ,„ay no. be ™ade from biodegradable materials, repetitive administration may eventually lead to accumulation and to.ci.y. More study still need to be cabled out to ODN once so toxic effects of repeated doses are not an issue. .n summaty. our study demonstrate that dendrimers are highly efficient delivety Stability and inhibitory effect of AS-ODN.

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CHAPTER? GENERAL CONCLUSIONS The components of RAS have been located in vascular system in human, rats and other animal.. Vasoconstrictor Ang II has been shown to promote cellular hypertrophy and hyperplasia in many cell types including VSMCs. The growth functions of Ang II have been given important consequences regarding restenosis induced by angioplasty. However, previous studies which were based on ACE inhibitors and AT, antagonists failed to achieve unifonn conclusion in this issue. The resource of the controversies is the differem doses and time of dosing. Other problem such as receptor upregulation may also contribute to the failure of these drugs. This research project used two novel techniques, autoimmuni^ation and antisense inhibition to address this problem. We firs, tested the effectiveness of the AS-ODN to the AT, mRNA on a well characterized animal model in our laboratoty, centrally Ang II induced drinking and AVP release. Given 50 ^g of AS-ODN to the lateral ventricle of rats significantly inhibited drinking and AVP response to Ang 11, Our results indicate that antisense strategy can be successfully used in RAS. We exploited dendrimer based delivery system to facilitate uptake of AS-ODN into blood vessels Dendrimer sigMficantly increased uptake and .ability Of AS-ODN. AS-ODN to AT, mRNA delivered with dendrimer significantly inhibited neoinitimal formation after balloon injury. 94

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95 The other strategy we used was autoiramunization against AT, receptor protein. We immumzed rats with a peptide corresponding to the N-termi„al of AT, receptor The immunized rats and control rats then were subjected to balloon catheterization. Our results demonstrated that immumzed rats had significant less neointimal formation compared to sham control. Funher. we transfcsed amiserum into normal rats and inhibited neoimimal growth. We conclude that when Ang U and the AT, receptor were inhibited by an autoantibody to the N-terminal of the AT,, the growth response to the vascular injury was significantly inhibited. In summao(Fig. 7-1), our data with autoimmunity to the AT, receptor confirm and extend the hypothesis that vascular RAS is important for the mechanism involved in .he development of restenosis. Our study suggests that Ang I, is one of the it^tiating factors in the response to vascular injuty. The present study also demonstrates that ASODN is effective in reducing restenosis and we have developed a dendrimer delive,y system for administering AS-ODN efficiently for prolonged effects. We conclude that AS-ODN complex dendrimer deUve^ offers a potentially new therapeutic approach for restenosis and vascular response to injury.

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96 Lumen Side CZE ndotheliai^ I^ ^^"^^ve by , . / \ angioplasty (-) / NO \ (.) mRNA (-) Dendrimer AS-ODN Inhibition 1 receptor (-) Antibody Blockade

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REFERENCES Akabayash, A., Wahlestedt, C, Alexander, J.T., and Leibowitz, S.F., Specific inhibition of endogenous neuropeptide Y synthesis in arcuate nucleus by antisense oligonucleotide suppresses feeding behavior and insulin secretion. Mol. Brain Res. 1994;21:55-61 Akhtar, S., and Juliano, R.L,. Cellular uptake and intracellular fate of antisense oligonucleotide Trends Cell Biol. 1992;2:39-144 Aldred, G.P., Chai, S.Y., Song, K., Zhuo, J., MacGregor, DP. and Mendelsohn, F.A., Distribution of angiotensin II receptor subtypes in the rabbit brain Regul Pept 1993;44(2): 119-130 Ambuhl, P., Gyurko, R. and Phillips, M.I., A decrease in angiotensin receptor binding in rat brain nuclei by antisense oligonucleotides to the angiotensin AT, receptor. Regulatory Peptides 1995;59:171-182. Anderson, K.P., Fox, M.C., Brown-Driver, V., Martin, M.J., and Azad, R.F., Inhibition of human cytomegalovirus immediate-early gene expression by an antisense oligonucleotide complementary to immediate-early RNA. Antimicrob. Agents Chemother 1996 40(9)2004-11 Barth, R.F., Adams, D M., Soloway, A.H., Alam, F., and Darby, M.V., Boronated starburst dendrimer-monoclonal antibody immunoconjugates: evaluation as a potential delivery system for neutron capture therapy. Bioconjug. Chem. 1994; 5(1): 58-66 Bennett, MR., Anglin, S., McEwan, J R., Jagoe, R., Newby, A.C., and Evan, G.I., Inhibition of vascular smooth muscle cell proliferation in vitro and in vivo by c-myc antisense oligodeoxynucleotides. J. Clin. Invest. 1994; 93(2): 820-828 Berecek, K.H., and Swords, B.H., Central role for vasopressin in cardiovascular regulation and the pathogenesis of hypertension. Hypertension 1 990; 16(3):2 13-224 Bielinska, A., Kukowska-Latallo, J.F., Johnson, J., Tomalia, D.A., Baker, JR. Jr., Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers. Nucleic Acids Res 1996;24:2176-82 97

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98 Boussif, O., Lezoualch, F., Zanta, M.A., Mergn, M.D., Scherman, D., Demeneix, B., and Behr, J.P., A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. PNAS USA 1995;92:7297-7301 Buisson, B., Laflamme, L., Bottari, S.P., de-Gasparo, M., Gallo-Payet, N., Payet, M.D., A G-protein is involved in the angiotensin AT2 receptor inhibition of the T-type calcium current in non-differentiated NG108-15 cells. J. Biol. Chem. 1995; 270(4): 1670-4 Chang, E.H., Yu, Z., Shinozuka, K., Zon, G., Wilson, W.D., Strekowska, A, Comparative inhibition of ras p21 protein synthesis with phosphorus-modified antisense oligonucleotides. Anticancer Drug Des. 1 989; 4(3): .22 1 -232 Chang, M.W., Barr, E., Seltzer, J., Jiang, Y.Q., Nabel, G.J., Nabel, E.G., Parmacek, MS., and Leiden, J.M., Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science 1995; 267(5197): 518-22 Chavany, C, Le, D.T., Couvreur, P., Puisieux, F., and Helene, C, Polyalkylcyanoacrylate nanoparticles aspolymeric carriers for antisense oligonucleotides. Pharm. Res. 1992; 9:441-449 Chavany, C, Saison, B.T., Le, D.T., Puisieux, F., Couvreur, P., and Helene, C, Adsorption of oligonucleotides onto polyisohexlcyanoacrylate nanoparticles protects them against nucleases and increases their cellular uptake. Pharm. Res. 1 994; 1 1 : 1 3701 378 Clowes, A.W., Clowes, M.M., Kocher, O., Ropraz, P., Chaponnier, C, Gabbiani, G., Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin. J. Cell Biol. 1988;107(5): 1939-1945 Clowes, A.W., Reidy, M.A., and Clowes, M.M., Mechanisms of stenosis after arterial injury. Lab. Invest. 1983a; 49(2):208-215. Clowes, A.W., Reidy, M.A., Clowes, M.M., Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab. Invest 1983b 49(3)327-33 deBlois, D., Viswanathan, M., Su, J.E., Clowes, A.W., Saavedra,S.M., and Schwartz, S.M., Smooth muscle DNA replication in response to angiotensin II is regulated differently in the neointima and media at different times after balloon injury in the rat carotid artery. Role of ATi receptor expression. Arterioscler. Thromb. Vase Biol 1996 16(9): 1130-7

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99 Desamaud, F., Marie, J., Lombard, C, Larguier, R., Seyer, R., Lorca, T., Jard, S., Bonnafous, J.C., Deglycosylation and fragmentation of purified rat liver angiotensin II receptor: application to the mapping of hormone-binding domains. Biochem. J. 1993 289 (Ptl): 289-97 Deschepper, C.F., MeUon, S.H., Cumin, P., Baxter, J.D., and Ganong, W.F., Analysis by immunocytochemistry and in situ hybridization of renin and its mRNA in kidney, testis, adrend and pituitary of the rat. Proc. Natl. Acad Sci. USA 1 986;83 : 7752-7756 Dzau, V.J., Implications of local angiotensin production in cardiovascular physiology and pharmacology, ^/w. J. Cardiol. 1987; 59: 59A-65A Dzau VJ, Autocrine and paracrine mechanisms in the pathophysiology of heart failure. Am. J. Cardiol 1992;70: 4G-1 IC Dzau, V.J., Ingelfinger, J.R., Pratt, R.E., and Ellison, K.E., Identification of messenger RNA sequences in mouse and rat brains. Hypertension 1986;8:544-548 Dzau, V.J., Pratt, R., and Gibbons, G.H., Angiotensin as local modulating factor in ventricular dysftinction and failure due to coronary artery disease. Drugs 1994 47(suppl.4):l-13. Epstein, SE., Speir, E., Unger, EF., Guzman, RJ, Finkel, T., The basis of molecular strategies for treating coronary restenosis after angioplasty. J. Am. Coll. Cardiol 1994;23:1278-1288 Feigner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northop, J.P., Ringold, G.M., and Danielsen, M., Lipofection: A highly efficient, lipid-mediated DNAtransfection procedure. />A^y45 USA 1987;84:7413-7417 Fernandez-Ortiz, A., Meyer, B.J., Maihac, A., Falk, E., Badimon, L., Fallon, J.T., Fuster, v., Chesebro JH, and Badimon JJ. A new approach for local intravascular drug delivery: lontophoretic balloon. Circulation 1 994;89 : 1 5 1 81 522 Ferns, G. A., Raines, E.W., Spmgel, K.H., Motani, A.S., Reidy, M.A., and Ross, R., Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF. Science I991;253(5024):l 129-1 132. Fogarty, T.J., The balloon catheter in vascular surgery. Rev. Surg. 1967; 24(1): 9-19 Fram, D., Mitchel, and Aldin, A., Waters D, Noremberg F, McKay R. Intramural delivery of H-heparin with a new site-specific local drug delivery system: The D^ catheter J. Am Coll. Cardiol. 1 994;23 : 1 86A. Abstract.

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100 Frechet, J.M., Functional polymers and dendrimers: Reactivity, molecular architecture, and interfacial energy. Science 1994; 263(5154); 1710-5 Fu, MLX, Schulze, W., Wallukat, G., Hjalmarson, A., and Hoebeke, J., A synthetic peptide corresponding to the second extracellular loop of the human M2 Acetylcholine receptor induces pharmacological and morphological changes in cardiomyocytes by active immunization after 6 months in rabbits: Clinical Immunology and Immunopathology 1996;78(2):203-207. Furuta, H., Guo, D.F., and Inagami, T., Molecular cloning and sequencing of the gene encoding human angiotensin II type 1 receptor. Biochem. Biophys. Res. Comm. 1992; 183: 8-13 Ganten, D., Hermann, K., Unger, T., and Lang, R.E., The tissue renin-angiotensin systems: focus on brain angiotensin, adrenal gland and arterial wall. Clin. Exp. Hypertens. A. 1983;5(78): 1099-1 118 Gibbons, G.H., Dzau, V.J., Molecular therapies for vascular diseases. Science 1996; 272(5262): 689-693 Gibbons, G.H. and Dzau, V.J., The emerging concept of vascular remodeling. The New England Journal of Medicine 1994;330 (20): 143 1-1438 . Gibbons, G.H., Pratt, R.E., and Dzau, V.J., Vascular smooth muscle cell hypertrophy vs. hyperplasia. Autocrine transforming growth factor-beta 1 expression determines growth response to angiotensin II: J. Clin. Invest. 1992;90:456-461. Godard, G., Boutorine, A.S., Saison, B:E., and Helene, C, Antisense effects of cholesterol-oligodeoxynucleotide conjugates associated with poly(alkylcyanoacrylate) nanoparticles. Eur. J. Biochem. 1995;232 404-410. Griendling, KK., Murphy, TJ., Alexander, RW., Molecular biology of the reninangiotensin system. Circulation 1993 Jun;87(6): 1816-1828 Griffin, S.A., Brown, W.C., MacPherson, F., McGrath, J.C., Wilson, V.G., Korsgaard, N., Mulvany, M.J., Lever, A.F., Angiotensin II causes vascular hypertrophy in part by a nonpressor mechanism. Hypertension 1991;17:626-635. Guyton, A.C., Textbook of medical physiology. Saunder, Philadelphia, PA (1986) Guzman, L.A., Labhasetwar, V., Song, C, Jang, Y., Lincoff, A.M., Levy, R., and Topol, E.J., Local intraluminal infusion of biodegradable polymeric nanoparticles. Circulation 1996;94:1441-1448

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101 Guzman, R.J., Hirschowitz, E.A., Brody, S.L., Crystal, R.G., Epstein, S.E., and Finkel, T., In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene. Proc. Natl. Acad Sci. USA. 1994; 91(22): 10732-6 Gyurko, R., Kimura, B., Kurian, P., Crews, F.T., and Phillips, M.I., Angiotensin II receptor subtypes play opposite roles in regulating phosphatidylinositol hydrolysis in rat skin slices. Biochem. Biophys. Res. Commun. 1992; 186(1): 285-92 Gyurko, R., Wielbo, D., and Phillips, M.I., Antisense inhibition of ATi receptor mRNA and angiotensinogen mRNA in the brain of spontaneously hypertensive rats reduces hypertension of neurogenic origin. Regul. Pept. 1993 ;49: 167-174 Haensler, J., and Szoka, F.C., Polyamidoamine cascade polymers mediate efficient transfection cells in culture, C/j^/w. 1993;4:372-379 Hanna, A.K., Fox, J.C., Neschis, D.G., Saffoord, S.D., Swain, J.L., and Golden, M.A., Antisense basic fibroblast growth factor gene transfer reduces neointimal thickening after arterial injury. J. Vase. Surg, 1997; 25(2) : 320-325 Hein, L., Barsh, G.S., Pratt, R.E„ Dzau, V.J., and Kobilka, B.K., Behavioural and cardiovascular effects of disrupting the angiotensin II type-2 receptor in mice Nature 1995; 377(6551): 744-7 Hein, L., Dzau, V.J., and Barsh, G.S., Linkage mapping of the angiotensin AT2 receptor gene (Agtr2) to the mouse X chromosome. Genomics 1995; 30(2): 369-71 Helene, C, and Toulme, J.J., Specific regulation gene expression by antisense, sense and antigene nucleic acids, Biochim. Biophys. Acta. 1990;1049:99-125 Hermans, W.R., Rensing, B.J., Foley, D P., Tijssen, J.G., Rutsch, W., Emanuelsson, H Danchin, N., Wijns, W., Chappuis, F., and Serruys, P.W., Patient, lesion, and procedural variables as risk factors for luminal re-narrowing after successftil coronary angioplasty: a quantitative analysis in Cilazapril after angioplasty to prevent transluminal coronary obstruction and restenosis (MERCATOR) study group: Journal of Cardiovascular Pharmacology 1993;22(4 Suppl.): S45-57. Hillegass, W.B., Ohman, E.M., Leimberger, J.D., Califf, R.M., A meta-analysis of randomized trials of calcium antagonists to reduce restenosis after coronary ancioolastv Am. J. Cardiol. 1994; 73(12): 835-839 ^ e f j Hogarty, D.C., Speakman, E.A, Puig, V., and Phillips, M.I., The role of angiotensin, AT, and AT2 receptors in the pressor, drinking and vasopressin responses to central angiotensin Brain Res. 1992;586(2):289-294

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102 Hogarty, D.C., Tran, D.N., and Phillips, M.I., Involvement of angiotensin receptor subtypes in osmotically induced release of vasopressin. Brain Res. 1994;637:126-132 Huang, X.C., Richards, E.M., and Sumners, C, Mitogen-activated protein kinases in rat brain neuronal cultures are activated by angiotensin II type 1 receptors and inhibited by angiotensin II type 2 receptors. J. Biol. Chem. 1996; 271: 15635-15641 Ichiki, T., Labosky, P.A., Shiota, C, Okuyama, S., Imagawa, Y., Fogo, A., Niimura, F., Ichikawa, I., Hogan, B.L., Inagami, T., Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor: Nature 1995; 377(6551): 748-50 Inagami, T., Yamano, Y., Bardhan, S., Chaki, S., Guo, D.F., Ohyama, K., Kambayashi, Y., Takahashi, K., Ichiki, T., and Tsuzuki, S., Cloning, expression and regulation of angiotensin II receptors. Adv. Exp. Med. Biol. 1995; 377: 311-7 Iwai, N., and Inagami, T., Identification of two subtypes in the rat type 1 angiotensin II receptor. FEBSLett., 1992; 298: 257-260 Iwai, N., Inagami, T., Ohmichi, N., and Kinoshita, M., Renin is expressed in rat macrophage/monocyte cells. Hypertension 1996; 3: 399-403 Iwai, N., Izumi, M., Inagami, T., and Kinoshita, M., Induction of renin in medial smooth muscle cells by balloon injury. Hypertension 1997; 29: 1044-50 Kambayashi, Y., Bardhan, S., Takahashi, K., Tsuzuki, S., Inui, H., Hamakubo, T., Inagami, T., Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition, y. Biol. Chem. 1993;268(33): 24543-24546 Kang, J., Posner, P., and Sumners, C, Angiotensin II type 2 receptor stimulation of neuronal K"^ currents involves an inhibitory GTP binding protein. Am. J. Physiol 1994 267(5 Pt 1): CI 389-97 Kauffman, R.F., Bean, J.S., Zimmerman, K.M., Brown, R.F., and Steinberg, M.I., Losartan, a nonpeptide angiotensin II (Ang II) receptor antagonist, inhibits neointima formation following balloon injury to rat carotid arteries. Life Sci. 1991; 49(25): PL223-8 Kawamura, M., Terashita, Z., Okuda, H., Imura, Y., Shino, A., Nakao, M., Nishikawa, K., TCV-116, a novel angiotensin II receptor antagonist, prevents intimal thickening and impairment of vascular flinction after carotid injury in rats J. Pharmacol Exp Ther 1993; 266(3): 1664-9

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103 Kawano, Y., Yoshida, K., Matsuoka, H., and Omae, T., Chronic effects of central and systemic administration of losartan on blood pressure and baroreceptor reflex in spontaneously hypertensive rats. Am. J. Hypertens. 1994;7:536-542 Kino, H., Hama, J., Takenaka, T., Sugimura, K., Kamoi, K., Shimada, S., Yamamoto, Y., Nagata, S., Horiuchi, M., Katori, R., Effect of an angiotensin II receptor antagonist, TCV116, on rat carotid artery neointimal formation after balloon injury. Blood Press Suppl. 1994; 5: 43-8 Kinsella, M.G., and Wight, T.N., Modulation of sulfated proteoglycan synthesis by bovine aortic endothelial cells during migration. J. Cell Biol. 1986; 102(3): 679-687 Kirby, R.F., Thunhorst, R.L., and Johnson, A.K., Effects of a non-peptide angiotensin receptor antagonist on drinking and blood pressure responses to centrally administered angiotensin in the rat. Brain Res 1992;576(2):348-350 Knee, R., Murphy, P R., Regulation of gene expression by natural antisense RNA transcnpts. Neurochem. Int. 1997;31(3):379-392 Kukowska-Latallo, J.F., Bielinska, A.U., Johnson, J., Spindler, R., Tomalia, D.A,, and Baker, J.R. Jr., Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers. PNAS USA 1996; 93(10): 4897-902 Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacterophage T4. Nature 1970;227:680-685. Lappalainen, K., Urtti, A., Soderling, E., Jaaskelainen, I., Syrjanen, K., Syijanen, S., Cationic liposomes improve stability and intracellular delivery of antisense oligonucleotides into CaSki cells. Biochim. Biophys. Acta. 1994; 1 196: 201-8 Lincoff, A., Furst, J., Penn, M., Lee, P., Maclsaac, A., Chisolm, G., and Topol, E., Efficiency of solute transfer by a microporous balloon catheter in the porcine coronary model of arterial injury. J. Am. Coll. Cardiol. 1994;28: 18A. Abstract Lindner, V., and Reidy, M.A., Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor. Proc. Natl. Acad. Sci. USA 1991;88(9):3739-3743. Lyall, F., Doman, E.S., McQueen, J., Boswell, F., Kelly, M., Angiotensin II increases proto-oncogene expression and phosphoinositide turnover in vascular smooth muscle cells via the angiotensin II ATI receptor. J. Hypertens. 1992;10:1463-1469.

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104 Lyall, F., Gillespie, D., and Morton, J. J., Angiotensin II stimulates c-jun expression in cultured vascular smooth muscle cells: superinduction by emetine. Eur. J. Intern. Med . 1992;2:271-273. Lynch, K.R., Simnad, V.T., BenoAri, E.T., Maniatis, T., Zinn, K:, and Garrison, J.C., Localization of preangiotensinogen messenger RNA sequences in the rat brain. Hypertension 1986;8:540-543 Meng, H., Wielbo, D., Gyurko, R., and Phillips, M.I., Antisense oligonucleotide to ATi receptor mRNA inhibits central angiotensin induced thirst and vasopressin. Reg. Pept. 1994; 54: 543-551 Miller, P.S., Yano, J., Yano, E., Carroll, C, Jayaraman, K., Ts'o, P.O., Nonionic nucleic acid analogues. Synthesis and characterization of dideoxyribonucleoside methylphosphonates. Biochemistry 1979;18(23):5134-5143 ^ . Mitchel, J., Azrin, M., Fram, D., Schwedick, M., Alberghini, T., Waters, D., and McKay, M., Intramural deposition of urokinase at the angioplasty site: comparative efficiency of systemic and local drug delivery techniques. Circulation 1994;90(suppl I): 1-20. Abstract. Morishita, R., Gibbons, G.H., Ellison, K.E., Lee, W., Zhang, L., Yu, H., Kaneda, Y., Ogihara, T.. and Dazu, V.J., Evidence for direct local effect of angiotensin in vascular hypertrophy. In vivo gene transfer of angiotensin converting enzyme. J Clin Invest. 1994a;94(3):978-984. Morishita, R., Gibbons, G.H., Ellison, K.E., Nakajima, M., von-der-Leyen, H., Zhang, L., Kaneda, Y., Ogihara, T., and Dzau, V.J., Intimal hyperplasia after vascular injury is inhibited by antisense cdk 2 kinase oligonucleotides. J. Clin. Invest 1994b 93(4) 145864 Morishita, R., Gibbons, G.H., Kaneda, Y., Ogihara, T., and Dzau, V.J., Pharmacokinetics of antisense oligodeoxyribonucleotides (cyclin Bl and CDC 2 kinase) in the vessel wall in vivo: enhanced therapeutic utility for restenosis by HVJ-liposome delivery Gene 1994c149(1): 13-9 Murphy, T.J., Alexander, RW., Griendling, K.K., Riunge, M.S., and Bernstein, K.E., Isolation of a cDNA encoding the vascular type-1 angiotensin n receptor. Nature 1991;35 1 :233-236 Murphy, T.J., Takeuchi, K., and Alexander, R.W., Molecular cloning of AT, angiotensin receptors. Am. J. Hypertens. 1992; 5: 236S-242S Naftilan, A.J., Pratt, R.E., Eldridge, C.S., Lin, H.L., Dzau, V.J., Angiotensin II induces cfos expression in smooth muscle via transcriptional control. Hypertension. 1989,13:706-

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105 Nakajima, M., Hutchinson, H.G., Fujinaga, M., Hayashida, W., Morishita, R., Zhang, L., Horiuchi, M., Pratt, R.E., and Dzau, V.J., The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the ATI receptor: Gain-of-fiinction study using gene transfer. Proc. Natl. Acad. Sci. USA 1995;92(23): 10663-10667. Ogawa, S., Olazabal, U.E., Parhar, I.S., and Pfafl^ D.W., Effects of intrahypothalamic administration of antisense DNA for progesterone receptor mRNA on reproductive behavior and progesterone receptor inursunoreactivity in female rat. J. Neurosci. 1994;14{3Pt 2): 17661774 Ohno, T., Gordon, D., San, H., Pompili, V.J., Imperiale, M.J., Nabel, G.J., Nabel, E.G., Gene therapy for vascular smooth muscle cell proliferation after arterial injury. Science 1994; 265(5173): 781-4 Palmer, R.M.J., Ferrige, A.G., and Monc^de, S., Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524-526. Phillips, ML, Functions of brain angiotensin. Aunu. Rev. Physiol. 1987; 49: 413-435 Phillips, M.I., Levels of angiotensin and molecular biology of the tissue renin angiotensin systems. Regul. Pept. 1 993 ;43( 1-2): 1-20 Phillips, M L, Mohuczy-Dominiak, D., Coffey, M., Galli, S.M., Kimura, B., Wu, P., Zelles, T., Prolonged reduction of high blood pressure with an in vivo, nonpathogenic, adeno-associated viral vector delivery of ATl-R mRNA antisense. Hypertension 1997;29(1 Pt2):374-380 Phillips, M.I., and Kimura, B., Brain angiotensin in the developing spontaneously hypertensive rat. J. Hypertens. 1988;6(8):607-612. Phillips, M.I., Kimura, B., and Gyurko, R., Angiotensin receptor stimulation of transforming growth factor-5 in rat skin and wound healing. Angiotensin Receptor, Plenum Press, New York. (1994),377-396. Phillips, M.I., Mann, J.F., Haebara, H., Hoffinan, W.E., Dietz, R, Schilling, P., and Ganten, D., Lowering of hypertension by central saralasin in the absence of plasma renin Nature \91T 270(5636):445.447 Phillips, M.L, Shen, L., Richards, E.M., and Raizada, M.K., Immunohistochemical mapping of angiotensin ATi receptors in the brain. Regulatory Peptides 1993;44(2):95-

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106 Phillips, M.I., Speakman, E.A., and Kimura, B., Tissue renin-angiotensin systems, Im Cellular and molecular biology of the renin-angiotensin system. Raizada, M.K., Phillips, M.I., and Sumners, C, (Eds.) CRC Press, Boca Raton, FL (1993), 97-130 Phillips, M.I., and Stenstrom, B., Angiotensin II in rat brain comigrates with authentic angiotensin n in high pressure liquid chromatography. Circ. Res. 1985;56:212-219 Popma, J.J., Califf, R.M., Topol, E.J., Clinical trials of restenosis after coronary angioplasty. Circulation 1991 ;84(3): 1426-1436 PoweU, J.S., Clozel, J.P., Muller, R.K.M., Kuhn, H., Hefti, F., Hosang, M., and Baumgartner, H.R., Inhibitors of Angiotensin-Converting Enzyme prevent myointimal proliferation after vascular injury. Science 1989;245:186-188. Poxon, S.W., Mitchell, P.M., Liang, E., and Hughes, J.A., Dendrimer delivery of oligonucleotides. Drug Delivery 1996;3:255-261 . Rafl^ H., Kane, C.W., and Wood, C.E., Arginine vasopressin response to hypoxia and hypercapnia in late gestation fetal sheep. Am. J. Physiol. 1 99 1;260:R 10771081 Rakugi, H., Wang, D.S., Dzau, V.J., and Pratt, R.E., Potential importance of tissue angiotensin-converting enzyme inhibition in preventing neointima formation. Circulation 1994; 90(1): 449-55 Re, R., Fallon, J.T., Dzau, V., Ouay, S.C., and Haber, E., Renin synthesis by canine aortic smooth muscle cells in culture. Life Sci. 1982;30: 99-106 Richards, E.M., Lu, D., Zelezna, B., Phillips, M.I., Trolliet, M., Sumners, C, and Raizada, M.K., Inhibition of central angiotensin responses by angiotensin type-1 receptor antibody. Hypertensin 1993;21(6-2): 1062-1065. RosendorfF, C, The renin-angiotensin system and vascular hypertrophy. J. Am. Coll Cardiol. 1996; 28: 803-812 Saavedra, J.M., Brain and pituitary angiotensin. Eruiocrine Reviews 1992; 13(2):329-80 Sakai, R.R., He, P.F., Yang, X.D., Ma, L.Y., Guo, Y.F., Reilly, J.J., Moga, C.N., and Fluharty, S.J., Intracerebroventricular administration of AT, receptor antisense oligonucleotide inhibits the behavioral actions of angiotensin D. J. Neurochem. 1994;62:2053-2056 Sarzani, R., Brecher, P., and Chobanian, A.V., Growth factor expression in aorta of normotensive and hypertensive rats. J. Clin. Invest. 1989;83:1404-1408.

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107 Sasaki, K., Yamano, Y., Bardhan, S., Iwai, N., Murray, J.J., Hasegawa, M., Matsuda, Y., and Inagami, T., Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor. Nature 1991; 351(6323): 230-3 Schwab, G., Chavany, C, Duroux, I., Goubin, G., Lebeau, J., Helene, C, and SaisonBehmoaras, T., Antisense olgonucleotides adsorbed to polyalkylcyanoacrylate nanoparticles specifically iiihibit mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice. Proc. Natl. Acad Sci. USA 1994;9 1(22): 10460-10464 Serruys, P.W., Herrman, J.P., Simon, R., Rutsch, W., Bode, C, Laarman, G.J., van, D R., van den Bos, A., Uman, V.A., Fox, K.A., and Helvetical Investigators. A comparison of hirudin with heparin in the prevention of restenosis after coronary angioplasty. N. Engl. J. Med. 1995;333:757-763 Shi, Y., Fard, A., Galeo, A., Hutchinson, H.G., Vermani, P., Dodge, G.R., Hall, D.J., Shaheen, F., and Zalewski, A., Transcatheter delivery of c-myc antisense oligomers reduces neointimal formation in a porcine model of coronary artery balloon injury. Circulation 1994; 90(2): 944-51 Simons, M., Edelman, E.R., DeKeyser, J.L., Langer, R., and Rosenberg, R.D., Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature 1992; 359(6390): 67-70 Sladek, CD., and Joynt, R.J., Role of angiotensin in osmotic control of vasopressin release by organ-cultured rat hypothalamo-neurohypophyseal system. Endocrin. 1980;106:173-178 Smith, R.D., and Timmermans, P.B., Human angiotensin receptor subtypes. Curr. Opin. Nephrol. Hypertens. 1994; 3(1): 112-22 Soos, J.M., Hobeika, A.C., Butfiloski, E.J., Schiffenbauer, J., and Johnson, H.M., Accelerated induction of experimental allergic encephalomyelitis in PL/J mice by a nonVB8-specific superantigen. Proc. Natl. Acad. Sci. USA 1995;92:6082-6086. Szewczyk, B., and Kozloff, L.M., A method for the efficient blotting of strongly basic proteins fi-om sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose. Anal. Biochem. 1985;150(2):403-407. Taguch, J., Abe, J., Okazaki, H., Ochiai, M., Ohno, M., Takuwa, Y., and Kurokawa, K., Angiotensin converting enzyme inhibitors or DuP753 prevent neointimal formation following balloon injury with single topical or multiple systemic application. Biochem & Biophys Res Commun. 1 993 ; 1 96(2) : 969-974 .

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108 Takeuchi, K., Nakamura, N., Cook, N.S., Pratt, R.E., Dzau, V.J., Angiotensin n can regulate gene expression by the AP-1 binding sequence via a protein kinase C-dependent pathway, fi/oc/jm. Biophys. Res. Commun. 1990; 172(3): 1189-1194 Tarn, J.P., Synthetic peptide vaccine design: synthesis and properties of high-density multiple antigenic peptide system. Proc. Natl. Acad. Sci. USA 1988;85:5409-5413. Tan, J.S., Butterfiedl, D.E., Voycheck, C.L., Caldwell, K.D., and Li, J.T., Surface modification of nanoparticles by PEO/PPO block copolymers to minimize interactions with blood components and prolong blood circulation in rats. Biomaterials 1993; 14823833 Tanaka, M., Ohnishi, J., Ozawa, Y., Sugimoto, M., Usuki, S., Naruse, M., Murakami, K., and Miyazaki, H., Characterization of the AT2 receptor on rat ovarian granulosa cells. Adv-Exp. Med Biol. 1996; 396: 175-82 Tanaka, M., Ohnishi, J., Ozawa, Y., Sugimoto, M., Usuki, S., Naruse, M., Murakami, K., Miyazaki, H., Characterization of angiotensin II receptor type 2 during differentiation and apoptosis of rat ovarian cultured granulosa cells. Biochem. Biophys. Res. Commun. 1995;207(2):593-598 Tang, S.S., Rogg, H., Schumacher, R., and Dzau, V.J., Characterization of nuclear angiotensin-U-binding sites in rat liver and comparison with plasma membrane receptors. Endocrinology 1 992; 131: 374-80 Timmermans, P.B., Chiu, A.T., Herblin, W.F., Wong, PC, and Smith, R.D., Angiotensin U. receptor subtypes. Am. J. Hypertens. 1992;5(6 Pt 1):406-410 Tomalia^ D.A., Dendrimer Molecules. Scientific American 1995;62-65 Towbin, H., Staehelin, T., and Grodon, J., Electrophoretic transfer of proteins fi-om polyacrylamide gels to nitrocellulose sheets: Procedures and some applications. Proc. Natl. Acad Sci. USA 1979;76(9):4350-4354. Tsutsumi, K., and Saavedra, J.M., Quantitative autoradiography reveals different angiotensin n receptor subtypes in selected rat brain nuclei. J. Neurochem. 1991;56:384-351 Unger, T., Gohlke. P., Paul, M., and Rettig, R., Tissue renin-angiotensin systems: fact or ^ctionlJ. Cardiovas Pharmacol. 1991;18(suppl)2:S20-25. Vinson, G.P., Ho, M M., and Puddefoot, J.R., The distribution of angiotensin II type-1 receptors, and the tissue renin-angiotensin systems. Mol. Med Today 1995; 1(1): 35-39

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109 Viswanathan, M., Seltzer, A., and Saavedra, J.M., Heterogeneous expression of angiotensin II ATi receptors in neointima of rat carotid artery and aorta after balloon catheter mpry. Peptides 1994; 15(7): 1205-12 Viswanathan, M., Stromberg, C, Seltzer, A., and Saavedra, J.M., Balloon angioplasty enhances the expression of angiotensin II ATi receptors in neointima of rat aorta. J. Clin. Invest. 1992; 90(5): 1707-12 Wagner, E.G., Simons, R.W., Antisense RNA control in bacteria, phages, and plasmids. Annu. Rev. Microbiol. 1994;48:713-742 Wagner, R.W., Gene inhibition using antisense oligodeoxynucleotides. Nature 1994 24; 372(6504): 333-5 Wagner, R.W., Matteucci, M.D., Lewis, J.G., Gutierrez, A.J., Moulds, C, and Froehler, B.C., Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines. Science 1 993 ; 260(5 1 1 3): 1 5 1 0-3 Wahlestedt, C, Merlo, P.E., Koob, G.F., Yee, F., and Heilig, M., Modulation of anxiety and neuropeptide Y-Yl receptors by antisense oligodeoxynucleotides. Science 1993;259:528-531 Wielbo, D., Semia, C, Gyurko, R., and Phillips, M.I., Antisense inhibition of hypertension in the spontaneously hypertensive rat. Hypertension 1995; 25(3): 314-319 Wielbo, D., Simon, A., Phillips, M.I., and Totfolo, S., Inhibition of hypertension by peripheral administration of antisense oligodeoxynucleotides. Hypertension 1996 28(1) 147-51 Wilcox, J.N., Molecular biology: Insight into the causes and prevention of restenosis after arterial intervention. Am. J. Cardiol. 1993; 72: 88E-95E Wilensky, R.L., March, K.L., Gradus, P L, Schauwecker, D., Michaels, M B., Robinson, J., Carlson, K., and Hathaway, D R., Regional and arterial localization of radioactive microparticles after local delivery by unsupported or supported porous balloon catheters Am. Heart. J. 1995;129:852-859 Wright, J.W., and Harding, J.W., Brain angiotensin receptor subtype in the control of physiological and behavioral responses. Neuroscience and Biobehavioral Reviews 199418 2153 Yamabe, T., Imazu, M., Yamamoto, H., Ueda, H., Hattori, Y., Hayashi, Y., Sekiguchi, Y., Ito, M., and Yamkido, M., Effect of Cilazapril on vascular restenosis after percutaneous transluminal coronary angioplasty. Coronary Artery Diseases 1995 6(^7") 573-579. ' ^ ^

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110 Yamada, T., Horiuchi, M., Dzau, V.J., Angiotensin n type 2 receptor mediates programmed cell death. Proc. Natl. AcfuL Sci. USA 1996; 93(1): 156-160 Yamaguchi, K., Sakaguchi, T., and Kamoi, K., Central role of angiotensin in hyperosmolalityand hypovolemia-Induced vasopressin release in conscious rats. ActaEndocri. 1982; 101:524530 Yang, C.R., Phillips, M.I., and Renaud, L.P., Angiotensin n receptor aaivation depolarizes rat supraoptic neurons in vitro. J. Physiol. 1992;263:R1333-1338 Zamecnik, P.C., and Stephenson, M.L., Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sci. USA 1978; 75(1): 280-4 Zelezna, B., Richards, E.M., Tang, W., Lu, D., Sumners, C, and Raizada, M., Characterization of a polyclonal anti-peptide antibody to the angiotensin II type-1 (ATi) receptor. Biochem. Biophys. Res. Commun. 1992;183:781-788 Zelezna, B., Veslsky, L., Velek, J., Zicha, J., Kunes, J., Angiotensin ATi receptor blockade by specific antibody prevented two-kidney, one-clip renal hypertension in the rat. European Journal of Pharmacology. 1994;260:95-98. Zelphati, O., and Szoka,, Jr. F.C., Intracellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids. Pharm. Res. 1996;13(9): 1367-1372

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BIOGRAPHICAL SKETCH Frank Meng was bom in Beijing, 1968. His parents are both professors. He found biomedical research to be intriguing to him from a young age. He received numerous awards and recognition during his high school years in a variety of competitions in the biomedical field. This experience laid the foundation of his decision to become a researcher in this interesting field. Frank attended Beijing Medical University from 1986 to 1991, and received his Bachelor of Science degree. To seek new challenges and higher education, he moved to Gainesville and enrolled in the Ph.D program in medical sciences with a specialization in physiology at University of Florida in 1992. Frank is married to Jane and they have a lovely family. During free time, he enjoys tennis, biking, swimming, and traveling. 111

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. n M. Ian Phillips, Chair Professor of Physiology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully ^equate, in scope and quality, as a dissertation for the degree of Doctor of Philosoph Stepl^n Baker Professor of Pharmacology and Therapeutics I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy Colin Sumners Professor of Physiology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. | Bruce Stevens Professor of Physiology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fiilly adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosopl lugnes ^sistant Professor of Pharmaceutics This dissertation was submitted to the Graduate Faculty of the College of Medicine and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. /^ December, 1997 Dean, Cojlege of Medi« Dean, Graduate School