ANTISENSE INHIBITION OF BRAIN ANGIOTENSIN IN RAT MODEL
OF GENETIC HYPERTENSION
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
I dedicate this work
to my wife and my parents
To M.Ian Phillips I owe a tremendous debt of gratitude for his continuous support
throughout my training. He is not only an excellent scientist, but also a philosopher and
mentor, whose friendship I value greatly. Of the Phillips Laboratory, I thank Sara Galli,
Birgitta, Gayle, Leping, Tibor, Phillip, and my fellow students, Bing, Hongbin and Dan, for
their friendship and support. A friend and scientific fellow, Jon Bui, is also recognized for the
endless discussions on scientific and other matters. I am grateful to the faculty of the
Department of Physiology for giving me the opportunity to train in this highly competitive
atmosphere. I am particularly grateful to the members of my committee: Drs. Mohan Raizada,
Colin Sumners, Nick Muzyczka and Fulton Crews.
TABLE OF CONTENTS
ACKNOWLEDGMENTS ....................................... iii
AB STRA CT ................................................ v
1 INTRODUCTION .................................... 1
2 HYPOTHESIS AND SPECIFIC AIMS ...................... 4
H ypothesis ...................................... 4
Specific Aims ..................................... 4
3 REVIEW OF THE LITERATURE ......................... 6
Blood Pressure Regulation ........................... 6
The Renin-Angiotensin System ....................... 9
The Antisense Paradigm ............................. 27
Gene Therapy with Antisense Expression Vector .......... 41
Summary of the Literature ........................... 47
4 MATERIALS AND METHODS ........................... 49
Oligonucleotide Experiments ......................... 49
Expression Vector Experiments ...................... 54
5 RESULTS ............................................ 58
The Plan of the Study .............................. 58
Antisense Oligonucleotide Experiments in Vivo ........... 59
Antisense Oligonucleotide Experiments in Vitro ........... 73
Antisense Expression Vector Experiments ............... 79
Summary of the Results ............................. 90
6 CONCLUSIONS ....................................... 91
Antisense Oligonucleotide Design ..................... 91
Lowering Blood Pressure with Antisense Oligonucleotides... 92
Intracellular Mechanism of Action ...................... 101
Inhibition with Antisense Expression Vector ............. 104
7 IMPLICATIONS OF THE STUDY ......................... 109
REFERENCES ............................................... 113
BIOGRAPHICAL SKETCH ................................... 131
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor
ANTISENSE INHIBITION OF BRAIN ANGIOTENSIN IN RAT MODEL
OF GENETIC HYPERTENSION
Chairman: M. Ian Phillips
Major Department: Physiology
A novel method of inhibition of the synthesis of specific proteins, antisense inhibition,
was used to decrease the activity of the brain renin-angiotensin system (RAS). Since
overactivity of the brain RAS has been implicated in the development of essential hypertension,
the hypothesis was tested that attenuation of angiotensin type-1 receptor protein synthesis with
antisense DNA in the brain of spontaneously hypertensive rats can decrease high blood
pressure. Antisense oligonucleotides and antisense expression vectors were developed. The
ability of antisense oligonucleotides to lower blood pressure was tested in vivo, when antisense
oligonucleotides were injected intracerebroventricularly in spontaneously hypertensive rats. A
long-lasting, significant drop in blood pressure showed that antisense inhibition is applicable to
physiological processes. The functional downregulation of brain angiotensin receptors was
demonstrated by an attenuated drinking response to angiotensin A, whereas the decrease in
receptor number was shown by receptor binding and autoradiography. The cellular uptake was
followed using fluorescent labeled oligonucleotides, and the mechanism of action was
investigated with northern blots. In vitro experiments established that the antisense expression
vectors are capable of decreasing the synthesis of their target proteins. More experiments are
needed to develop an effective method for antisense delivery to the brain.
Angiotensin II (Angll) is one of the major physiological regulators of blood pressure.
Circulating AngI is generated in the blood from angiotensinogen through a cascade of
proteolytic events by the renin-angiotensin system (RAS). The RAS was initially described
only in the peripheral circulation, but neurochemical and functional data points to the existence
of an independent RAS in the brain as well as in other tissues (1,2). All components of the
RAS have been shown to be produced in the brain, and distinct functions have been linked to
brain AngIl: stimulation of vasopressin release, stimulation of the sympathetic nervous system
and induction of drinking behavior are the best recognized among them (3).
The brain RAS is implicated in the development of essential hypertension (4,5).
Increased levels of AnglI and AnglI receptors in the brain of spontaneously hypertensive rats
(SHRs) suggest an overactive brain RAS in the SHR (6,7,8,9). Inhibition of AngII in the brain
by a peptide antagonist saralasin decreases blood pressure in SHR (4). The angiotensin-
converting enzyme inhibitor captopril prevents the development of hypertension in SHR (5).
The choice ofantisense inhibition for further dissection of the brain RAS was appealing
primarily because of the specificity and the versatility of the approach and also because of its
potential clinical applicability. The unique specificity of antisense inhibition is due to base to
base matching of the oligonucleotide (ODN) to the target mRNA, allowing for differentiation
between e.g. subtypes of receptors with known nucleotide sequence. The versatility is intrinsic
to antisense technology: antisense oligonucleotides can be constructed to any part of any
mRNA with known nucleotide sequence. As with any new approach of course, there are yet
unknown factors that might influence the applicability: these include cellular uptake and
mechanism of action (10). The application of antisense ODNs as clinical drugs is particularly
appealing not only because it practically takes out the "guess-work" from drug design, but it
also uses natural components, nucleotides, as building blocks, therefore the chance of a toxic
reaction is decreased. In addition, antisense sequences can be cloned into expression vectors
which carry the potential for long-term depression of specific genes.
Blood pressure regulation is one of the fundamental physiological tasks in man. It is a
fine balance between providing the necessary blood perfusion to particular organs and
preventing the heart and blood vessels from undue strain. It has to ensure not only a
continuous delivery of blood but it has to accommodate to changing needs of all the organs
under different conditions.
Its importance is well reflected by epidemiological data: even slight elevations in blood
pressure shorten life expectancy, and there is an inverse linear relationship between systolic as
well as diastolic blood pressure and lifespan (11).
According to its importance, blood pressure is regulated by multiple organs including
the heart, blood vessels, kidney and the nervous system. While classic physiology describes the
hemodynamic, hormonal and neural regulation of blood pressure on the whole animal and
single organ level, our understanding of the molecular and cellular aspects of hemodynamic
control is still at its infancy.
The deficiency of classic techniques in answering some fundamental questions is
demonstrated by the fact that while some 100 years of hypertension research enriched us with a
formidable knowledge, the origin of 95% of the hypertensive cases is still unaccounted for, and
there is an ongoing debate whether researchers should look at the kidneys, the blood vessels or
the nervous system for the culprit.
The advent of genetics and molecular biology permits us to investigate a basic aspect of
this complex disease: these new techniques investigate the roles of individual genes and their
protein products in biological processes, thus narrowing the gap between physiology and its
underpinning, biochemistry. While there is no guarantee that molecular biological
investigations will answer all the questions about hypertension, its use will undoubtedly bring
us closer to understanding this and possibly other complex diseases. In this project, we
attempted to apply one of the new molecular biological tools, antisense molecules to
investigate the role of the brain renin-angiotensin system in hypertension.
HYPOTHESIS AND SPECIFIC AIMS
We hypothesize that antisense oligonucleotides injected into the brains of
spontaneously hypertensive rats can decrease the synthesis of angiotensin type-1 (AT-1)
receptor protein and decrease blood pressure. We also hypothesize that an expression vector
expressing antisense RNA delivered to the brain can decrease AT-1 synthesis and consequently
decrease high blood pressure.
Specific Aim 1
Antisense oligonucleotides will be designed based on available information on the base
sequence of cloned AT-1 receptor cDNA. The ability of AT-1 antisense oligonucleotides to
modulate physiological responses, such as blood pressure and drinking in spontaneously
hypertensive rats will be tested.
Specific Aim 2
The effect of antisense oligonucleotides on AT-1 receptor levels in rat brain following
intracerebroventricular injection will be tested using radioligand binding on brain slices or brain
Specific Aim 3
The intracellular mechanism of action of antisense oligonucleotides will be investigated.
The oligonucleotides will be tested along with their controls in cultured cells for their
efficiency in inhibiting AT-1 receptor protein synthesis. The cellular uptake of fluorescent
labeled oligonucleotides will be followed with confocal microscopy, and the fate of the target
mRNA will be investigated with northern blot.
Specific Aim 4
The possibility of long-term delivery of antisense molecules will be explored with and
adeno-associated virus (AAV)-derived antisense expression vector. A portion of the AT-1
receptor cDNA will be cloned into the expression vector in the antisense direction. This
recombinant molecule will be tested in vitro for its ability to inhibit AT-1 receptor synthesis,
and in vivo whether it can influence hypertension when injected in the brains of SHRs.
REVIEW OF THE LITERATURE
Blood Pressure Regulation
Our current understanding of blood pressure regulation states that two main factors
determine blood pressure: cardiac output and vascular resistance (12). Cardiac output in a
healthy heart is determined by the venous return, which, in the long run, is a function of the
circulating blood volume. Thus, the more blood is circulating, or the higher is the vascular
resistance, the higher is the blood pressure.
Regulation of Blood Volume
Blood volume is mainly regulated by the kidneys as well as by the brain. The
juxtaglomerular cells of the kidney release renin when the blood pressure decreases in the
afferent arteriole or the sodium concentration is reduced in the tubular fluid. Renin cleaves the
decapeptide angiotensin I from angiotensinogen in the blood, which is converted to angiotensin
II (AngDI) by angiotensin converting enzyme (ACE) in the lungs.
Circulating AnglI regulates blood volume by stimulating aldosterone secretion in the
zona glomerulosa of the adrenal cortex, which stimulates sodium reabsorption in the distal
tubule. Sodium retention by the kidney results in more efficient water reabsorption. Thus a
decrease in blood volume elicits a cascade of events which improves water conservation.
The brain, on the other hand, has its own effector to restore fluid homeostasis.
Vasopressin, also known as antidiuretic hormone, is synthesized by neurons in the supraoptic
and the paraventricular nuclei of the hypothalamus, and released from the posterior pituitary in
response to increased plasma osmolarity as well as decreased blood pressure. Slight increases
in plasma vasopressin vastly improves water permeability of the collecting ducts of the kidney,
thus promoting water conservation.
Although it does not directly influence the total blood volume, the heart determines the
blood volume on the arterial side of the circulation, thus playing an important role in
determining the blood pressure. Its role can be demonstrated upon syncope or heart stop,
when the blood pressure decreases to the mean arterial filling pressure (about 7 mmHg), which
is the sum of the effects of blood volume and vascular tension only, without heart function.
Regulation of Peripheral Resistance
Regulation of the vascular resistance involves some of the hormones responsible for
fluid homeostasis, and also other humoral and neural actions. AngI and vasopressin, discussed
in the earlier paragraph as water-conserving hormones, are also the two most potent
vasoconstrictors of the body. AngI binds to its type-1 receptors on the vascular smooth
muscle cells and increases intracellular calcium, which is a signal for contraction. Vasopressin,
upon binding to its V-1 receptors on the vascular smooth muscle cell, elicits vasoconstriction
with a similar mechanism.
Many other hormones can elicit vasoconstriction including catecholamines, endothelin
and neuropeptide Y, whereas some are potent vasodilators such as nitric oxide or ANP.
The brain exerts control over vascular tone as well as cardiac output via the
sympathetic and parasympathetic nervous system. The sympathetic postganglionic neurons
innervate the blood vessels, heart and adrenal medulla. They release norepinephrine and elicit
vasoconstriction in different vascular beds, increase venous return by contracting large veins,
increase heart rate and the release of epinephrine from the adrenal medulla.
The sympathetic preganglionic neurons are found in the intermediolateral column of the
thoracic and upper lumbar spinal chord, and provide tonic stimulation of the peripheral
sympathetic system. Loss of this tonic activity results in collapse of blood pressure. The origin
of tonic excitatory activity are the adrenaline-synthesizing cells in the Cl area of the rostral
ventrolateral reticular nucleus of the medulla oblongata (RVM), the integrative center of
regulation of circulation. Cl cells fire in phase with cardiac systole because baroreflex inhibits
Baroreflex originates from stretch-receptors in the carotid artery and the aorta, and
travels through the IX. and the X. cranial nerves to the nucleus tractus solitarii (NTS).
Increased blood pressure activates the stretch-receptors which results in inhibition of the RVM
vasomotor cells by GABA-erg neurons from the NTS. Inhibition of RVM in turn causes
vasodilation by decreasing sympathetic activity. Baroreflex activation also causes a decrease in
heart rate by activating cardiovagal efferents.
Diencephalic and Telencephalic Control of Blood Pressure
There are supramedullary pathways that are shown to exert profound effects on blood
pressure regulation. The specific nuclei involved in these networks are found in all levels of the
brain: the parabrachial nuclei of the pons, the periaqueductal gray of the midbrain,
hypothalamic nuclei including the paraventricular, supraoptic, lateral and anterior nucleus, the
central and medial amygdaloid nucleus and areas of the limbic cortex including the cingulate,
parahippocampal, insular and orbitofrontal cortices (14). The functions of these areas are only
partially understood, but they are thought to play a role in integrating cardiovascular functions
with other physiological functions including stress reaction, emotion and behavior, and
malfunction of some of these brain areas are implicated in the development of essential
It is apparent from this brief overview of blood pressure regulation that blood pressure,
a vital body function, is secured with several feed-back loops. These regulatory mechanisms
are apparently built onto each other, and on the top of this regulatory hierarchy is the brain.
The Renin-Angiotensin System
The best recognized function of the renin-angiotensin system is to directly or indirectly
preserve fluid homeostasis and normal blood pressure. The renin-angiotensin system (RAS)
was originally described in the peripheral circulation (15). According to the classical
description, the only endogenous ligand for angiotensin receptors was the octapeptide
angiotensin II (AngIl), which is derived from its prohormone angiotensinogen through
generation of angiotensin I by renin and consequent conversion of angiotensin I to angiotensin
II by angiotensin converting enzyme (ACE). This of course still holds true with the addition
that some other derivatives of the octapeptide such as AngII (Ang 2-8) and AnglI(3-8) have
since been proposed to be endogenous ligands, and alternative routes for synthesis of AngIl
from angiotensinogen have been postulated (16,17). In the classical view, the prohormone
angiotensinogen is synthesized and secreted constitutively by the liver into the circulation,
where some of it is transformed to AngI by renin in the kidney and further cleaved by ACE
mainly in the lungs to form AngII. Now we know that there are several other RASs located in
tissues rather than in the circulating blood and they act independently of each other (18). The
primary sites of action for the circulating AngIl are the blood vessels, kidney and the adrenal
cortex, where AT-1 receptors mediate the well known effects of AngI: vasoconstriction in the
blood vessels, decreased glomerular filtration in the kidney and aldosteron secretion of the
glomerulosa cells of the adrenal gland (19). All three actions are aimed at conserving body
fluids and elevating blood pressure. Accordingly, the rate limiting step in AngII synthesis is the
conversion of angiotensinogen to AngI by renin, which is released by juxtaglomerular cells of
the kidney in response of decreased fluid and/or Na+ load.
The function of the local tissue RASs are considerably less well understood. What
northern blots and in situ hybridizations tell us is that components of RAS are not only found
but also synthesized locally in tissues (18). It is also suggestive for the existence of the
independent tissue RAS's that these tissues, the brain, kidney, heart, blood vessel wall, and
testis are capable of maintaining local AngII concentrations significantly different from that of
the plasma. Evidence accumulates that the locally produced AnglI in these tissues acts in a
paracrine and/or autocrine fashion serving physiological and perhaps developmental regulatory
Classification of angiotensin 1 receptors
Angiotensin II binding sites were characterized first in 1974 (20) in adrenal cortex and
soon after in blood vessels and brain (21). Although isolation and chemical characterization of
the receptor protein proved to be difficult due to the instability of the receptor protein,
pharmacological data suggested that there is probably more than one type of AnglI receptors.
For example, the peptidic AngII analogue des-Asp-AngII proved to be equipotential with
AngII in increasing serum aldosterone, but increased blood pressure 10 times less efficiently
(22). Receptor binding assays with radiolabeled AngII suggested the presence of a high affinity
and a low affinity AngII binding site in rat hepatic membranes, and the GTP analogue GppNHp
was found to influence receptor binding only in the high-affinity site (23,24). Dithiothreitol, a
sulfhydryl-reducing agent cleaving covalent bonds between cystein amino acids, reduced AnglI
binding only on the high-affinity site suggesting structural differences between the two AngII
binding sites (25). Different second messenger systems seemed to be activated by AnglI
depending on tissue localization: in the kidney AngII was found to activate phospholipase C in
glomerular mesangial cells, whereas in tubular epithelial cells it appeared to be coupled to
adenylate cyclase (26).
Defining the two major AnglI receptor subtype was greatly facilitated by the
development of the new, nonpeptidic AngIl antagonists. The first of these, losartan (formerly
DuP753), displaced AngI completely in rabbit adrenal cortex, liver and vascular smooth
muscle and displaced AngII partially in rat adrenal cortex, adrenal medulla and uterus. AngII
binding in human uterus and PC12W cells was not influenced by losartan. The losartan-
sensitive binding sites were sensitive to dithiothreitol and also to nonhydrolyzable GTP
analogues such as GppNHp and GTP-gamma-S. The losartan-insensitive AngII binding site in
rabbit uterus was shown to be sensitive to another type of nonpeptidic AngII antagonist,
PD123319. The PD123319-sensitive site was not sensitive to the nonhydrolyzable GTP
analogue 5'-guanylyl-imidodiphosphate, and was dissimilar to the losartan-sensitive site in that
it did not influence inositol phosphate metabolism, vascular contractile activity or blood
pressure regulation (27).
Together, these two drugs serve as basis for classification of AngI receptors: the
accepted nomenclature calls AT-1 the losartan-sensitive binding site, whereas PD123319-
sensitivity defines the AT-2 subtype (28).
Recent advances in molecular cloning of the cDNAs and consequently the whole genes
for angiotensin receptors not only validated the classification of AngI receptors based on their
pharmacological properties, but also expanded the number of the different receptor subtypes.
Clones of the AT-1 receptor cDNA from rat vascular smooth muscle (29) and adrenal gland
(30) was found to be different, and screening rat genomic library showed that there is two
similar but distinct genes for AT-1 receptor in rat, which were later termed AT-la for the
vascular type and AT-lb for the adrenal type. The presence of two AT-1 genes has been also
shown in mice but not in humans (31).
Pharmacological data suggested AT-2 receptor heterogeneity in the brain and in
neuroblastoma cell line NIE-115. AT-2 receptors located in the ventral and mediodorsal
thalamic nuclei, medial geniculate nuclei and in the locus ceruleus of the rat brain are sensitive
to guanine nucleotides, pertussis toxin and dithiothreitol and named AT-2a, whereas AT-2
receptors in the inferior olive and in the hypoglossal nucleus are not sensitive to any of these
reagents and suggested to be classified as AT-2b (32). In N1E-1 15 cells two population of
AT-2 receptors were distinguished based on their sensitivity to PD123319 and dithiothreitol
Molecular structure of the AT-1 receptor
Regardless of species or subtype, the open reading frame of all AT-1 receptor genes
code for a protein of 359 amino acids constituting a 41 kDa protein, which is in good
agreement with the molecular weight of the deglycosylated form of AT-1 receptor protein
purified from adrenal gland (34). Hydropathy analysis of the amino acid sequence showing
seven groups of non-hydrophilic amino acids suggest that the AT-1 receptor protein belongs to
the superfamily of G-protein coupled receptors possessing seven membrane-spanning regions.
All four extracellular loops connecting the transmembrane regions have a cysteine
residue capable of forming disulfide bridges. It is suggested that the cysteines on the second
and the third loop form one disulfide bridge, whereas the first and the fourth loop form the
other creating a well-like structure transversing the cell membrane (35). Disruption of these
disulfide bridges with sulfhydryl reagents such as dithiothreitol would destroy this tertiary
structure and this is the probable explanation for the effect of these reagents on decreasing
binding affinity to AnglI.
Cloning AT-1 cDNA into expression vectors and expressing it in COS-7 cells which
normally do not possess AnglI receptors has shown that upon Angnl stimulation these
recombinant receptors are capable of activating inositol trisphosphate (IP3) and causing
transient elevation of intracellular Ca', as well as stimulating phospholipase C activation (29).
Transfection of CHO cells showed that the cloned AT-1 is capable of inhibiting cAMP
production and that the IP3 stimulation is mediated by Gq protein, whereas the cAMP inhibition
is mediated by ,,. These observations confirmed that the cloned AT-1 cDNA codes for a
functional receptor and it is similar in its actions to the naturally occurring one.
Further dissection of the functional domains of the AT-1 receptor required molecular
techniques such as site-directed mutagenesis. Mutating individual amino acids revealed that
Lys-199 of the fourth transmembrane domain is critical for ligand binding: it was proposed that
the negatively charged charboxyl-terminus of AngI lands on the positively charged amino
group of this lysine. Mutating of any of the cysteine residues reduced AngI binding
drastically, whereas eliminating the three Asn residuals which are potential sites for N-
glycosylation had no effect on receptor binding (36). It appears that intracellular domains
responsible for G-protein binding are found on the second intracellular loop and on the
intracellular carboxyl terminal of the protein: mutation of any of these areas prevented the
characteristic shift from high-affinity to low affinity state upon exposure to non-hydrolizable
GTP analogues such as GTP-y-S. This is in apparent contrast with the adrenergic receptors
where a large third cytoplasmic loop is thought to interact with G-proteins.
Regulation of AT-1 gene expression
The gene encoding the AT-1 receptor is comprised of at least five exons and four
introns in rodents as well as in humans. The whole gene spans a region over 55 kilobases. The
first four exons are short sequences and are subject to alternative splicing: whereas the fifth
exon is always present in the mRNA, the first, second, third and/or the fourth might be missing.
The entire coding region (1077 bp) is found on the fifth exon. Whereas there is two AT-1
gene in rats and mice, called AT-la and AT-lb, coding for two proteins about 95% similar but
differing significantly in their untranslated regions, there is only one AT-1 gene is found in
southern blots of human DNA. The 5' flanking region of the human gene contains two putative
TATA boxes, two CAAT boxes, two overlapping SPI sequences, a GC box and a cAMP-
responsive element (CRE) (37).
The rat AT-la gene promoter region located upstream of the first exon also includes
several consensus elements: two TATA boxes (a third can be found 5' to the fourth exon),
several transcription factor binding sites including two AP-1 site capable of binding early gene
products, an SP-1 site which can interact with RNA polymerase I, several glucocorticoid-
responsive elements (GRE) and a CRE site (35). The presence of the GREs gives explanation
for the stimulation of AT-1 mRNA in response to aldosterone and dexamethasone, and the
CRE is thought to be responsible for AT-1 downregulation by AngII (38).
AngII downregulates AT-1 mRNA rapidly: AngIl treatment causes 80%/ loss of AT-1
mRNA in 3 hours in adrenocortical carcinoma cells. A similar effect can be achieved by the
combination of calcium ionophore and phorbol ester, suggesting the involvement of protein
kinase C in the downregulation of AT-1 mRNA (39). Protein kinase C in turn, is thought to
act on the AP-1 and AP-2 sites of the promoter region of the AT-1 gene (40).
Downregulation of AT-1 mRNA by AngII was also observed in vascular smooth muscle cells
and mesangial cells (41,42). In vivo studies, however, suggest that AngII also can upregulate
its receptor expression: AngII infusion for two weeks increased AT-1 mRNA in the adrenal
gland, whereas bilateral nephrectomy decreased it (43).
The life-cycle of the AT-1 receptor
The AT-1 receptor, like many other receptors, undergo cellular internalization upon
ligand binding. Electronmicroscopic studies utilizing colloidal gold-labeled Angl showed that
the ligand bound first diffusely to the cell surface. In the following minutes, however, receptor-
ligand complexes aggregated into groups and internalized forming small membrane vesicles
called coated pits (44). The receptor-ligand complexes are thought to dissociate in lysosomes,
where AngI is degraded. The fate of the receptor molecule is uncertain: a fraction of the
receptors is recycled to the cell surface, whereas most of it degrades in lysosomes (45). In
vascular smooth muscle, there is significant perinuclear accumulation of radiolabeled AngII
following intravenous injection suggesting direct nuclear action of AngII-receptor complex
Binding of an antagonist to the AT-1 receptor generally does not result in
internalization. Whether internalization is actually necessary for activating the appropriate
second messenger systems remains to be seen.
Intracellular messenger systems coupled to AT-1 receptors
The AT-1 receptors, based on their predicted molecular structure, belong to the
superfamily of the G-protein coupled receptors. Experimental evidence also shows the active
involvement of at least two types of G proteins- Gq and G,- in intracellular signaling. Gq has
been shown to mediate AT-1 stimulation on phospholipase C, whereas i is thought to mediate
AT-1 inhibition of adenyl cyclase. Stimulation of phospholipase C by AngII in several tissues
results in hydrolysis of phosphatidyl-inositol-bisphosphate, a minor membrane protein into
inositol-(1,4,5)trisphosphate (IP3) and diacylglycerol. IP3 then binds to its intracellular receptor
and releases Ca' into the cytosol. These events are observed within 1-2 minutes after AT-1
receptor stimulation (46,47,48). A slower component of the stimulation can be observed when
diacylglycerol, along with cytoplasmic Ca+, activates protein kinase C. Increased Ca++ also
contributes to the activation of calcium-calmodulin dependent protein kinase. Protein kinase C
activation leads to the cell-specific response by phosphorylation of MAP kinase, tyrosine
kinases and myosin light chain (49). Vasoconstriction by blood vessel smooth muscle cells or
aldosterone secretion by glomerulosa cells in the adrenal are examples of cellular responses
mediated by these intracellular mechanisms.
Coupling AT-1 to the inhibitory G, protein results in inhibition of the action of
adenylate cyclase in hepatocytes, bovine adrenal cells and heart myocytes (50-52). Inhibition
of cAMP production antagonizes actions of protein kinase A such as sodium transport
inhibition in proximal tubule epithelium. In rat brain homogenates, however, stimulation of
adenylate cyclase was detected (53).
Angi is also known for inducing Ca' influx from the extracellular fluid. AngU can
operate ligand-gated channels or modify the action of voltage-gated T or L type Ca++-channels
either through direct intramembrane action or by activating intracellular protein kinase C
Besides phospholipase C, AngII can activate other phospholipases through the AT-1
receptor such as phospholipase D and phospholipase A2. Phospholipase D hydrolizes
phosphatidylcholine to phosphatidic acid and choline upon stimulation with AngII in vascular
smooth muscle cells. The proposed second messenger is the phoshatidic acid, whereas the
choline is released into the extracellular medium. Activation of phospholipase A2 in proximal
tubule cells and vascular smooth muscle cells by AngII results in the release of arachidonic
acids from phospholipids. Arachidonic acid is the precursor for prostaglandin and leukotriene
synthesis. Indeed, AngI has been shown to induce prostaglandin synthesis in vascular smooth
muscle cells (50).
Genetics of the Renin-Angiotensin System and Hypertension
Studies of monozygotic and heterozygotic twins indicate that the development of
hypertension is due to inherited as well as environmental factors (56). The success of animal
breeding experiments in producing inbred hypertensive rats (SHR) is also an evidence for a
strong genetic component in the etiology of hypertension.
Blood pressure is a quantitative trait which shows continuous variations from low to
high values in human populations. Quantitative traits are generally thought to be polygenic,
that is, a product of the effect of several genes.
Genetic linkage studies searching for chromosomal loci cosegregating with high blood
pressure in animal models and in humans indicated several candidate genes that might cause
hypertension. Most persistently, molecular variants of angiotensinogen are shown to be linked
to the hypertensive phenotype. M235T, a variant of the angiotensinogen gene coding for
threonine instead of methionine at position 235 (which is outside the renin cleavage site) has
been shown to be present significantly more often in hypertensive subjects than in
normotensives, and plasma angiotensinogen levels were also significantly higher in female
M235T-positive subjects. An other molecular variant, T174M was also associated with
essential hypertension (57).
The linkage of T235M variant to hypertension was confirmed in a different population
in Japan (58), but not in the United Kingdom (59), although these latter investigators also
found significant linkage between angiotensinogen locus and hypertension.
The same molecular variant of angiotensinogen gene was also found to be associated
with the development of preeclampsia, a severe form of pregnancy-induced hypertension (60).
Transgenic experiments also indicate the possible role of angiotensinogen in the
development of hypertension. Transgenic mice TGM(rAOGEN)123 carrying a copy of the rat
angiotensinogen gene expressed rat angiotensinogen in the liver and the brain and developed
hypertension (61). Duplication of one allele of the angiotensinogen gene in mice resulted in
elevated blood pressure, while disruption of one allele caused hypotension (62).
Other components of the renin-angiotensin system were also investigated as candidate
genes for hypertension. Investigations of the potential involvement of the renin gene were
inconclusive (63). Linkage analysis of the rat genome strongly suggested the pathogenic role
of ACE (64,65), but cloning expression and functional analysis showed only few differences in
base sequence and no apparent difference in enzyme function between SPSHR and WKY
A deletion-insertion polymorphism in the ACE gene, however, appeared to be
associated with heart disease. The homozygous deletion (DD) genotype was found to be in
excess in middle-aged men with left ventricular hypertrophy (67).
The Brain Renin-Angiotensin System
It was suggested as early as in 1961 that AngIl might have physiological effects on the
brain (68). The possibility that AngII is synthesized in the brain was suggested in 1971, and
conclusive evidence for the presence of AngII in neurons was provided by Ganten, Fuxe and
Phillips showing renin- and AngI-immunoreactivity in neurons in the brain (69,70). Further
immunocytochemical studies revealed that AngII is found in the brain in neurosecretory
vesicles, which is suggestive for synthesis of AngII by these neurons (71). Physiological data
showed that acute or chronic changes in circulating AngII levels failed to change brain AngI
levels, which points to the existence of an independently regulated brain RAS (72). In vitro
experiments showed that radiolabeled amino acids are incorporated into AngIl by cultured
primary rat brain cells which is a direct evidence for the ability of brain tissue to synthesize
Components of the brain RAS: angiotensinogen
Even though there is little doubt that AngII is synthesized in the brain, it is not filly
understood where and how the synthesis occur. The prohormone angiotensinogen is a
relatively abundant protein of the brain extracellular fluid. Angiotensinogen secretion has been
shown from brain slices to the fluid media (74). Angiotensinogen mRNA is widely distributed
in the brain; highest levels were measured in the hypothalamus (75,76). Angiotensinogen
mRNA is found to be colocalized with glial fibrillary acid protein (GFAP)-staining cells,
suggesting that glial cells are responsible for the production of brain angiotensinogen (77). In
contrast, immunocytochemical data shows angiotensinogen-like immunoreactivity in neurons
as well as in glia (78). Angiotensinogen secretion is thought to be constitutive rather than
regulated, based on observations showing that cells themselves contain little angiotensinogen,
and depolarizing agents such as K' do not evoke increase in cerebrospinal angiotensinogen
levels (79). It seems, therefore, that most of the angiotensinogen in the brain is synthesized by
glial cells of the hypothalamus, and it is constitutively secreted into the extracellular fluid.
The only substrate for the aspartyl protease renin is angiotensinogen. Other enzymes
such as cathepsin D can also cleave angiotensinogen, but whether this actually happens under
physiological circumstances is not known (80). The presence of renin in brain tissue has been
shown with functional assays showing renin-like activity as well as immunohistochemically.
Renin-like activity has been found ubiquitously but in a low level in the brain, with the highest
levels in the brainstem and in circumventricular organs such as the pituitary and the pineal gland
(79). Immunocytochemical assays using anti-salivary and anti-kidney renin antibodies showed
highest staining in the supraoptic and paraventricular nucleus (81,82). Others found more
widespread immunoreactivity covering most parts of the brain (83). Renin mRNA has been
found in the cortex and in low amounts in the brainstem, but not in the hypothalamus (84).
Brain renin appears to be regulated independently from plasma-renin: infusion of renin or renin
antibody into the peripheral circulation failed to produce significant elevation or decrease in
brain renin-like activity (85). Sodium depletion, which increases plasma renin activity,
decreased brain renin activity (86).
Cultured primary brain cells also produce immunochemically detectable renin, showing
the capacity of brain cells for endogenous renin synthesis. Brain renin appears to be in
synaptosomal preparations, and depolarization of cortical cells results in renin release (87).
These data suggest that renin-like enzyme activity and immunoreactivity is present in the
mammalian brain, even though the two do not necessarily overlap. The overall renin levels are
low: most of it is found on the cortex and it appears to be intracellular.
Angiotensin converting enzyme (ACE)
The enzymatic properties of brain ACE closely resembles to that of the lung and
kidney ACE: all of them are peptidylpeptide carboxyhydrolases anchored to the outside of the
cell membrane. Besides AngI, ACE also cleaves bradykinin, luteinizing hormone releasing
hormone, enkephalins and substance P (88). ACE is found mostly in the choroid plexus,
subfornical organ, area postrema, and, in lesser amounts, in the substantial nigra, locus ceruleus
and the hypothalamus (89). There is also soluble ACE in the cerebrospinal fluid.
AngII immunoreactivity in the brain has been demonstrated in the hypothalamus,
medulla oblongata and limbic system. AngIl has been found in synaptic nerve terminals in the
median eminence, paraventricular nucleus, supraoptic nucleus and subfomrnical organ. The
caudate-putamen and the sympathetic lateral column also contains immunoreactive AnglI (69).
Biochemical identification of brain AngLI using HPLC showed that brain AnglI is identical to
its circulating counterpart (90).
Several pathways have been identified having AngII-containing neurons. AngI-
containing fibers from the subfomical organ and organum vasculosum lamina terminalis
(OVLT) converge at the median preoptic nucleus and send fibers to either to the brainstem
through the medial periventricular hypothalamus or to the paraventricular and supraoptic
nucleus from where angiotensinerg fibers go to the neurohypophysis (91). A separate
pathway responsible for the drinking response (see below) has been identified starting from the
OVLT through the median preoptic area projecting to the midbrain.
In summary, while acknowledging that our knowledge is far from complete about how
AnglI is generated in the brain, the current theory states that angiotensinogen, synthesized and
secreted constitutively into the extracellular space by glial cells is cleaved by renin, which is
secreted from nerve endings upon depolarization and appears to be the rate limiting factor.
The generated AngI is then converted to AnglI by either membrane bound or soluble ACE.
AngII then can be taken up by neurons and utilized as a peptide neurotransmitter or it can act
extracellularly as neuromodulator.
Angiotensin Receptors of the Brain
Angiotensin receptors are fairly widespread in the mammalian brain (92). They are
found in circumventricular organs where they are accessible to circulating AnglI as well as in
areas protected by the blood-brain barrier. The method of choice for identifying angiotensin
receptors in the brain is autoradiography, where slide-mounted brain slices are incubated with
radiolabeled AngI analogues, and the slices then are exposed to x-ray film. Such experiments
yielded precise maps of angiotensin receptor localizations in the brain.
The two major receptor subtypes--AT-1 and AT-2--which can be differentiated in
autoradiographic studies by using subtype-specific receptor antagonists show different spatial
and temporal (developmental) distribution (93). There are areas which contain exclusively AT-
1 receptors: the piriform cortex, organum vasculosum lamina terminalis, median preoptic
nucleus, supraoptic and suprachiasmatic nucleus, subfomrnical organ, basolateral amygdaloid
nucleus, paraventricular nucleus, lateral olfactory tract, dentate gyms, subiculum, area
postrema, nucleus of the solitary tract and choroid plexus. Other areas contain only AT-2
receptors: the lateral septal nucleus, ventral and mediodorsal thalamic nucleus, parasolitary,
medial amygdaloid and medial geniculate nucleus, locus coeruleus and inferior olive. Some
areas contain both AT-1 and AT-2 receptors within the same nucleus: in the superior
colliculus, cingulate and cerebellar cortex (94).
During fetal development, angiotensin receptors first appear in the last third of the
pregnancy in the rat brain. The angiotensin receptor number progressively increases up until
the first two weeks after birth, when the receptor density reaches an approximately ten times of
the adult level. The majority of these receptors are type-2, such as the ones in the inferior
olive, paratrigeminal nucleus, hypoglossal nucleus and in the meninges. There are some areas
in the fetal brain with AT-1 receptors, such as the nucleus of the solitary tract and the choroid
plexus. As the rats reach young adulthood, there is no major change in the pattern of AT-1
receptor expression, whereas a drastic decrease can be detected in the AT-2 receptors (95). In
the lateral septal nucleus, ventral and mediodorsal thalamic nuclei, locus ceruleus, sensory
trigeminal nucleus, parasolitary nucleus, inferior olive, medial amygdaloid nucleus and medial
geniculate nucleus the AT-2 binding significantly decreases, whereas in other areas such as the
cerebellar cortex, anterior pretectal nucleus, nucleus of the optic tract, ventral tegmental area,
posterodorsal tegmental, hypoglossal nucleus, central medial and paracentral thalamic nuclei,
laterodorsal thalamic nucleus and oculomotor nucleus the AnglI binding disappears.
Effects of Central Angiotensin on Brain Receptors
Three major effects of Angi on brain angiotensin receptors can be observed most
consistently: pressor response, drinking behavior and vasopressin release from the posterior
pituitary (1). Even though these are distinct reactions, they are related to each other and all of
them result in restoration of body fluid homeostasis. A fourth, less obvious effect serving the
same goal is the increase in salt appetite (96). AnglI injected into the brain also can elicit
oxytocin release form the posterior and luteinizing hormone and ACTH release from the
anterior pituitary. AngI may also influence higher brain functions such as memory (97,98).
The most readily observable reaction to i.c.v. AngII is the drinking response (99). Fifty
ng AngI injected into the lateral brain ventricle induces drinking behavior in 1-2 minutes, and
the animals will drink 5-15 mnil water in 30 minutes (100). The main brain areas involved in
mediating the drinking response are localized in the anterior hypothalamus and include the
SFO, the AV3V region including the organum vasculosum lamina terminalis (OVLT) and the
median preoptic nucleus (MnPO). It is generally thought that the SFO, as a circumventricular
organ, is responsible for drinking reaction induced by circulating AngII, whereas the AV3V
region transmits central AngII signals (101). Preventing AngIl to reach the AV3V region
blocked central AngI effects (102). Applying AngII to the OVLT resulted in neuronal
excitation which can be blocked by specific antagonists (103). On the other hand, lesioning
the SFO attenuated the central effects of intravenously injected AngII (104).
It is believed that there are two drinking pathways, both of them originating in the
AV3V region: one passes through the periventricular zone of the hypothalamus and projects to
the brainstem, while the other sends fibers to the PVN and SON and to the posterior pituitary
(16). As to how these neurons bring about the motor and cognitive functions required for the
act of drinking is less well understood.
The other well known reaction to i.c.v. AngII is increased blood pressure. Low doses
of AngII will result in pronounced and prolonged increase in blood pressure when given i.c.v.
(68). The main coordinating center in the brain for mediating this pressor response is thought
to be the PVN. The PVN receives efferents from the SFO, which is shown to mediate the
effects of circulating AngII of central blood pressure regulation, and the OVLT, which is a
major site of action for brain-borne AngII (105,101). The PVN has large amounts of AngII
immunoreactivity as well as one of the highest concentrations of AngII receptors. The PVN,
among other efferents, projects to the posterior pituitary, NTS and the intermediolateral horn
of the spinal cord. It is thought that brain AngII elevates blood pressure by increasing
vasopressin release from the posterior pituitary, stimulating the sympathetic nervous system
and decreasing vagal input, including baroreflex input (1). The involvement of the sympathetic
nervous system is suggested by results showing that sympathetic blockade can diminish pressor
response to AnglI (106,107). Norepinephrine release was found to be increased in the
hypothalamus in response to i.c.v. AngI administration (108,109). The possible involvement
of the baroreflex modulation is supported by the notion that some of the AT-1 receptors in the
NTS are located on nerve endings of vagal afferents (110,111).
The third major effect of central AngI involves the posterior pituitary. AngI
receptors in the supraoptic nucleus (SO) and in the PVN can stimulate vasopessin as well as
oxytocin secretion from the posterior pituitary (112,113). AVP in turn, has immediate
(vasoconstriction) and prolonged antidiureticc action) effect on blood pressure. Inhibition of
AVP's action by V1 AVP antagonists or hypophysectomy decreases, but does not eliminate the
pressor effect of i.c.v. AngII (114,115). Pressor response to i.c.v. AngHI is also attenuated in a
genetic model of diabetes insipidus, the Brattleboro rat (116).
Besides these better known actions, AngHI has been proposed to influence other
neurosecretory cells. Lesioning dopaminerg nigrostriatal pathways inhibited AngI induced
drinking (117). The opiate-antagonist naloxone was also shown to inhibit both pressor and
drinking response to AngII (118,119). The involvement of central inhibitory GABA-erg
neurons was proposed when it was shown that GABA injections into brain ventricles inhibited
the pressor response and the release of vasopressin (120). Whether these neurons are
interneurons, modulatory neurons or parallel pathways to the AngI-neurons, is not clear. The
final efferents of the brain RAS on blood pressure are thought to be sympathetic motor fibers
and vasopressin secretion. Indeed, hypophysectomy and pharmacological blockade of alpha-1
adrenoceptor abolished the pressor response to centrally injected AngI (121).
The Antisense Paradigm
Antisense inhibition of gene expression was first proposed by Zamecnik et al. in 1978,
who inhibited Rous sarcoma virus replication with a 13mer antisense oligonucleotide (122).
Consequently it was discovered that prokaryotes use endogenous antisense RNA molecules
(123). These natural antisense molecules are produced most frequently by transcribing parts of
the gene to be regulated, but from the non-template strand under a promoter directed opposite
to the "sense" promoter. The endogenous antisense RNAs influence gene expression, plasmid
replication and transposition by binding to mRNA coding for essential replication proteins, or
by binding to a primer RNA required for DNA replication (124). It has been shown that
eukaryotic cells also synthesize antisense RNAs (125). It was, however, not until the advent of
the automatic DNA synthesizers that custom-made oligonucleotides became widely available,
and functional testing of antisense oligonucleotides targeted to different proteins began.
Antisense inhibition is an extremely attractive investigative and pharmacological
approach, since it offers base-to-base specificity to the target protein and versatility comparable
to the complexity of the genetic code itself It has become clear, however, that there are a
number of issues to be considered before using antisense in any experimental or clinical setting.
These include (1) selection of target sequence; (2) stability of antisense oligos in body fluids;
(3) the mechanism of cellular uptake; (4) possible intracellular sites of action and (5)
effectiveness, specificity and toxicity.
Selection of the Target Sequence
An antisense oligodeoxynucleotide is a short (15-30 nucleotides long), synthetic, single
stranded DNA molecule, which is complementary in sequence to the messenger RNA (mRNA)
of the target protein. When the antisense DNA hybridizes with the mRNA, it can inhibit the
synthesis of the target protein. Thus in principle, the concept is very simple. Designing an
effective antisense oligonucleotide, however, is not simple. The ambiguity in antisense
oligonucleotide design stems from incomplete knowledge about the mechanism of action of
such molecules, as well as from conflicting experimental results.
The original concept of antisense inhibition assumes that antisense molecules bind to
the mRNA of the target protein in the cytoplasm, and prevent either ribosomal assembly or
read-through (124). Most oligonucleotides therefore are targeted to the initiation codon AUG,
or part of the coding region downstream from it. When designing such an antisense
oligonucleotide, one has to consider two antagonistic factors: the affinity of oligonucleotide to
its target sequence, which is dependent on the number and composition of complementary
bases, and the availability of the target sequence, which is dependent on the folding of the
mRNA molecule (126).
There have been attempts to correlate oligonucleotide efficiency and these two factors:
affinity and availability. There is a general agreement that increasing affinity of antisense
oligonucleotides positively influences effectiveness, but results are conflicting on the
importance of the secondary structure of the target molecule. Several reports suggested that
antisense oligonucleotides targeted to different regions of the RNA have unequal efficiencies
(127,128). These differences may be related to the predicted secondary structure of the target
mRNA (129-131), but other experimenters did not find significant differences in antisense
efficiency between oligonucleotides targeting different segments of the RNA (126,132,133). It
would seem logical that the folding of the mRNA influences target sequence availability, but
one also has to consider that the stretches of the RNA double helices which are responsible for
the secondary structure of the mRNA incorporate a weaker G-U base pairing besides A-U and
G-C and are generally short and rarely perfect. Therefore an ODN that has only strong
(Watson-Crick) base pairing with 100% complementarity will form the more
thermodynamically favorable structure with its target RNA (134). If this is the case, the
antisense DNA could resolve the RNA secondary structure, and hybridize with the targeted
Besides inhibition of translation, other possible antisense mechanisms of action have
been proposed. Based on studies of cellular uptake of labeled oligonucleotides, the picture
emerged that most antisense oligonucleotides quickly migrate to the cell nucleus, suggesting an
intranuclear site of action (135). This suggests a more direct action on gene expression:
antisense molecules might inhibit pre-RNA splicing, transport of mRNA from the nucleus to
the cytoplasm, or bind to the DNA inhibiting transcription. Accordingly, effective antisense
oligonucleotides were designed targeting exon-intron splicing sites (136) or the major groove
of the DNA (137).
The experimental evidence accumulated in the past few years of antisense research
suggest that a large number of antisense oligonucleotides targeted to different regions of a
variety of mRNAs were successful in downregulating their target proteins, although the
number of failed attempts can not be gauged. It is, however, hard to recognize a general
pattern by which one can design an effective antisense oligonucleotide to a particular target.
The three regions that are repeatedly suggested to be better targets are the 5' cap region, the
AUG translation initiation codon and the 3' untranslated region of the mRNA (126,138,139).
Since most mRNAs have an AUG initiator codon site, targeting 12-15 of the neighboring bases
should produce inhibitory oligos. Unfortunately, many mRNAs in vertebrates have the Kozak
consensus sequence around the AUG start codon, and there is a danger of creating nonspecific
oligos by taking this approach. In any case, all designed oligos must be checked with the
GeneBank for existing sequences to avoid homology with other mRNAs.
A possible reason why there is no general rule of thumb for antisense oligonucleotide
design is that different mechanisms of action are present in different experimental situations
(131). If this is the case, the safest way for finding the optimal target sequence is through
testing a series of oligonucleotides. Common rules for antisense action may exist, but factors
like differential cellular uptake and discharge of different oligonucleotides or intracellular
feedback mechanisms on mRNA or protein production make them difficult to identify. Further
systematic investigation is needed to elucidate the molecular mechanisms of antisense inhibition
in order to be able to design antisense molecules with predictable results.
Stability of Oligonucleotides
Oligonucleotides in their natural form as phosphodiesters are subject to rapid
degradation in the blood, intracellular fluid or cerebrospinal fluid by exo- and endonucleases.
The half-life of phosphodiester oligonucleotides is in the range of minutes in blood and tissue
culture media. The half-life of oligonucleotides is .somewhat longer in cerebrospinal fluid, and
there are reports of detecting intact oligonucleotides 24 hours after injection into the cerebral
ventricles (140). Several chemical modifications have been proposed to prolong the half-life of
oligonucleotides in biological fluids while retaining their activity and specificity. The majority
of these modifications are backbone-modifications, which effect the phosphodiester bond
Probably the most widely used modified oligonucleotides are phosphorothioates, where
one of the oxygen atoms in the phosphodiester bond between nucleosides is replaced with a
sulfur atom. These phosphorothioate oligonucleotides have greater stability in biological fluids
than normal oligos. The half-life of a 15-mer phosphorothioate oligo is 9 hours in human
serum, 14 hours in tissue culture media with 10% fetal bovine serum and 19 hours in
cerebrospinal fluid (141). Phosphorothioate oligos can be synthesized with automated DNA
synthesizers, but because these machines replace either of the oxygen atoms in a given
phosphodiester bond, the resulting product is a 2-diastereoisomer in the case of an n-mer
instead of a uniform compound, which may complicate not only experimental data
interpretation but also pharmacological production of a given phosphorothioate antisense
oligonucleotide (142). By replacing both oxygen in the phosphodiester bond to sulfur one
would expect even greater resistance to nucleases, and also the problem of diastereoisomerism
could be eliminated. However, this type of oligonucleotides, the phosphorodithioates is hard to
synthesize, and even though they show high resistance to endonucleases, they have been
studied much less than monothioates (143).
Phosphorothioate oligonucleotides retain the negative charge of each nucleotide which
is characteristic to phosphodiester oligonucleotides, which might explain the similar behavior of
phosphorothioate oligos and phosphodiester oligos in cellular uptake and possibly intracellular
Replacing one of the oxygens in the phosphodiester bond with a methyl group results
in two degrees of magnitude of increase of nuclease resistance. Single base modification can
even protect the neighboring phosphodiester bonds. In serum containing tissue culture media
where 3'-exonuclease activity is the most prominent, methylphosphate modification of the 3'
nucleotide can increase the half-life of an oligonucleotide from 2 hours to 10 hours (144).
One or more of the oxygen atoms can be replaced with a variety of other compounds
such as alkyl phosphotriester, phosphoramidate or boranophosphate which all expand the half-
life of oligonucleotides in ex-vivo experiments, but only limited data is available on their
biological activity. Other modifications aiming at protecting oligonucleotides from enzymatic
digestion include 2'-sugar modifications (145), Y'-cholesterol modifications (146) and peptide
nucleic adcids (147).
Cellular Uptake of Oligonucleotides
In order to hybridize with the target mRNA, antisense oligonucleotides have to cross
the cell membrane. Investigators used two major methods for detecting intracellular
oligonucleotides: radiolabeling and fluorescent labeling. Fluorescent labeling offers convenient
detecting methods with fluorescent or confocal microscopes, whereas radioactive tags offer
labeling without changing the chemical configuration of the molecule. One of the first
comprehensive studies dissecting oligonucleotide uptake by cultured cells utilized fluorescent
(acridine)-labeled oligonucleotides (148). This group detected saturable uptake of
oligonucleotides plateauing within 50 hours with 11% of the administered oligonucleotides
taken up by the cells. The uptake was faster for shorter oligonucleotides than for longer ones.
Decreasing the temperature prevented the oligonucleotide uptake almost completely, indicating
an active uptake mechanism. Any sequence or size of unlabeled oligonucleotide competed
with the labeled oligonucleotide for uptake, even DNA as small as a single nucleotide or as big
as yeast DNA. Free nucleosides however, were not competitive, showing that the phosphate
group is essential for this uptake mechanism. An 80 kDa oligonucleotide binding protein was
isolated using oligo(dT) cellulose beads and this protein has given rise the theory of the
receptor mediated uptake of oligonucleotides.
Others used 35S labeled oligonucleotides to study their uptake and intracellular
localization without altering the chemical structure (149). They described oligonucleotide
uptake which was concentration-dependent, reached a plateau in 16 hours and resulted in an
intracellular oligonucleotide concentration 100 times of that of the tissue culture medium
indicating an active uptake mechanism. An efflux mechanism has also been described
indicating temperature-dependent secretion of the oligonucleotides from the cells to the
Separation of the nuclei from the rest of the cells indicated that only about 20% of the
cell-associated oligonucleotides was found in the nucleus, which was indicative for a
predominantly cytoplasmic uptake. Other groups, however, describe fast translocation of the
oligonucleotides from the cytoplasm to the nucleus which was found to be independent of the
ATP pool or temperature, suggesting diffusion through nuclear pores (150,151).
While some of the data might seem to be controversial, most of the differences are
probably due to different cell types and different oligonucleotides used. Indeed, quite dramatic
differences in oligonucleotide uptake rate has been shown between different cell lines (149). It
is generally believed that most types of oligonucleotides enter the cell by adsorptive or fluid
phase endocytosis, but the existence of the oligonucleotide receptor has to be proven by
purification of the protein.
Mechanism of Action
Antisense oligonucleotides have several potential sites of action, and different types of
oligonucleotides might not utilize the same one. According to the most obvious theory
antisense oligonucleotides inhibit translation by hybridizing to their respective mRNA thus
preventing either ribosomal assembly or ribosomal sliding along the mRNA. This kind of
action would assume that antisense oligonucleotides are acting in the cytosol and they do not
affect measurable mRNA levels. Indeed, there are several articles reporting antisense effects
without detectable change in target mRNA levels (152).
In cases where decreased mRNA levels are detected, the most likely explanation is the
digestion of the RNA portion of the mRNA-antisense DNA hybrid by RNase H. RNase H is
found in the cytoplasm as well as in the nucleus, and it is normally involved in DNA
duplication. In cell-free systems the role of RNase H is clearly demonstrated where the cleaved
RNA products were detected as smaller fragments, and in the case of chicken lysozyme, even
truncated polypeptides were detected (153). In cells, however, detection of the RNA
fragments appears to be difficult probably because the accelerated degradation of the truncated
mRNA fragments. Indeed, mRNA stripped from its cap sequence or poly-A tail is subject to
fast degradation. RNase H is thought to be activated by not only phosphodiester but also
phosphorothioated oligonucleotides, whereas oligonucleotides with modifications that do not
maintain the negative charge of the natural form will not activate RNase (153). Activation of
RNase H also seems to be advantageous because the enzyme leaves the antisense
oligonucleotide intact, so it is free to hybridize with another mRNA, making the reaction
catalytic rather than stochiometric.
In several cases however, the antisense oligonucleotides seem to be taken up by the
nucleus, suggesting other possible mechanisms of action. Antisense DNA can hybridize to its
target mRNA or pre-mRNA in the nucleus, forming a partially double-stranded structure which
would inhibit its transport from the nucleus to the cytoplasm, thus preventing transcription.
Antisense oligonucleotides targeted to intron-exon junction sites can prevent proper splicing,
and consequently the maturation of the transcript.
There is also a theoretical possibility of the antisense oligonucleotides' binding to the
DNA by triple helix formation or by binding to the locally opened loop created by RNA
polymerase (154). These appear to be possible only in special cases, e.g. triple helix formation
by antisense oligonucleotide binding in the major groove of the DNA can only happen if the
target sequence contains only purines on one strand and pirimidines on the other. Initial hopes
that triple helix formation by oligonucleotides or peptide nucleic acids can provide long-term
inhibition of transcription have been faded when it become clear that even covalent
modifications or DNA cleavage is corrected in the cell by various repair mechanisms. It is also
not clear how antisense oligonucleotides can have access to DNA in the chromatin.
Pharmacology ofAntisense Oligonucleotides
Antisense inhibition can be considered pharmacologically a drug-receptor interaction
where the oligonucleotide is the drug and the target sequence is the receptor. In order to
binding occur between the two, a minimum level of affinity is required which is provided by
hydrogen bonding between the Watson-Crick base pairs and base stacking in the double helix
which is formed. In order to achieve pharmacological activity, a minimum number of 12-14
bases can provide the minimum level of affinity (155).
The specificity of the antisense oligonucleotide-target sequence interaction is provided
by the Watson-Crick base pairing. Considering that roughly 1% of the human genome
contains coding regions, an oligonucleotide of 11-15 nucleotides long is specific enough
statistically to be complementary to a single sequence (156). Increasing the length of the
antisense should result in higher level of specificity, but there is an upper limit of about 30
nucleotides above which oligonucleotides are not taken up effectively by cells. Instead of
increasing length, testing the antisense sequence against genetic databases for possible cross-
reaction with other than the target sequence is advisable.
Sequence-specificity can be ascertained experimentally by using control
oligonucleotides such as sense, which is identical to the target sequence, scrambled, which has
the same base composition as the antisense but in a scrambled order and mismatch, which is
similar to the antisense but contains 3-4 mismatches while maintaining the same base
composition. Naturally, the more the control sequence resembles the antisense, the more
stringent control it will provide.
Antisense oligonucleotides can inhibit protein synthesis in cultured cells in pM doses.
This inhibition, however, never results in a total knockout of the target protein, but rather in a
partial decrease. Increasing the dose generally. does not result in complete inhibition, instead,
nonspecific effects might take place. Indeed, the therapeutical window for antisense
oligonucleotides is rather narrow: when testing for the optimal dose, small increments in the
low pM range should be used (140). This limited ability for protein synthesis inhibition is
certainly disadvantageous when targeting disease genes that need to be shut down completely,
but might be advantageous in e.g. neuropharmacology where modulation rather than
eradication of gene function is needed.
Phosphodiester oligonucleotides are degraded to their naturally occurring nucleotide
building blocks relatively quickly, therefore no toxic reaction is expected from even high doses.
Studies on phosphorothioated oligonucleotides in rats show that following intravenous
injection phosphorothioated oligonucleotides are taken up from the plasma mainly by the liver,
fat and muscle tissues. Phosphorothioate oligonucleotides are excreted virtually completely
through the urine in 3 days mainly in their original form. An apparent mild increase in plasma
LDH, and to a lesser extent in AST and ALT indicated a possible transient liver-toxicity (157).
The exceptionally long presence of the phosphorothioated oligonucleotide in the
organism is promising from a pharmacological point of view because sufficient tissue levels
could be maintained with infrequent administration. The fact that there is hardly any
metabolism of the compound is important because there is a theoretical possibility that these
unnatural nucleotides can be built into DNA of dividing cells possibly causing mutations.
In Vivo Applications of Antisense Oligonucleotides
Neuroendocrin functions such as vasopressin secretion has been successfully
suppressed with antisense oligonucleotides: injection of vasopressin antisense into the lateral
brain ventricle of the rat resulted in reduced vasopressin immunoreactivity in the hypothalamic
nuclei and in temporary diabetes insipidus which lasted up to 9 hours (158). In oxytocin
antisense treated rats where oligonucleotides were infused into the supraoptic nucleus of the
lactating rat there was a decrease in the number of milk ejection reflexes, and consequently,
there was significantly less weight gain observed in the litter (159).
Opioid receptors are also susceptible to antisense treatment. When injected
intrathecally in mice, the delta opioid receptor antisense has been shown to significantly inhibit
the antinociceptive action of deltorphin, a selective delta opioid agonist, whereas it was
ineffective in altering the pain response to g or K opioid agonists (160).
Antisense oligonucleotides have proven to be valuable research tools in identifying
functions of proteins. With no classical receptor antagonist available, Wahlestedt et al. used
antisense oligos in establishing the role of neuropeptide Y (NPY) in anxiety.
Intracerebroventricular injections of an NPY-1 antisense resulted in decreased density of NPY-
1 receptors in the cortex, with consequent change in behavior (161).
The role of kinesins in axonal transport was demonstrated in the optic nerve: when
antisense oligonucleotides to kinesin heavy chain were injected into the vitreous of rabbits,
retinal kinesin synthesis decreased, and to a similar extent, rapid axonal transport of proteins
was inhibited (162).
Animal behavior can be changed by interfering the synthesis of specific proteins in the
brain. Suppression of the synthesis of adrenocorticotropin (ACTH) and beta-endorphin in the
hypothalamic arcuate nucleus of the rat substantially decreased the grooming behavior normally
observed in a novel environment (163).
Reproductive behavior such as lordosis in female rats can be changed by inhibiting
progesterone receptor synthesis in the hypothalamus with antisense injections into the third
brain ventricle, or directly into the ventromedial hypothalamic nucleus (164-166). Inhibition of
two different forms of GABA synthetic enzyme GAD65 and GAD67 both led to a decrease in
female rat receptivity when injected into the medial hypothalamus and the midbrain central
gray, but with different time courses: GAD67 took effect within 24 hours and lasted for 4 days,
whereas GAD65 acted with a 48 hours delay and lasted 5 days (167). These differences might
be explained with differing turnover rates of the targeted proteins and the oligonucleotides.
Inhibition of catecholamine synthesis in the ventral tegmental area of the brain by
antisense oligonucleotides targeted to tyrosine hydroxylase mRNA led to reduction in operant
behavior, and this effect was observable for several days after the injection (168).
Dopamine receptors, which are known to induce rotational behavior in rats lesioned in
the corpus striatum, can be inhibited subtype-specifically with antisense. D2 dopamine receptor
antisense inhibited rotational behavior induced by intracerebroventricular injection of the D2
specific agonist quinpirole, whereas D, antisense was not effective (169). Conversely, D,
antisense inhibited behavioral responses elicited by the D, agonist SKF38393, and the D2
antisense proved to be ineffective (170).
Inhibition of the immediate-early genes with antisense oligonucleotides has become a
popular tool for investigating specific cellular responses requiring gene expression.
Intrahippocampal injection of c-jun antisense oligonucleotides impaired learning and memory
formation in rats (171). Superfusion of c-fos antisense onto rat spinal cord was shown to
suppress heat-induced c-Fos protein expression (172). Inhibition of c-fos expression in the rat
nucleus accumbens blocked cocaine-induced locomotor stimulation (173). On the other hand,
inhibition of c-fos expression unilaterally in the neostriatum lead to induction of ipsilateral
rotational behavior after d-amphetamine administration (174).
Important pathophysiological events such as arterial restenosis after balloon
cathetherization can be effectively influenced with antisense oligonucleotides. Inhibition of two
cell cycle regulatory enzymes, cyclin-dependent kinase 2 kinase and cell division cycle 2 kinase
with antisense oligonucleotides resulted in a nearly complete abolition of neointima formation
which is thought to be responsible for arterial restenosis (175). Application of c-myc antisense
oligonucleotides in apluronic gel to the outside of balloon-cathether injured blood vessel
resulted in 75% decrease in c-myc expression and significantly reduced neointima formation
(176). This result also suggests the remarkable penetrating ability of the oligonucleotides by
which they reached the neointima of the rat carotid from the outside of the vessel.
The outcome of cerebral infarction can be influenced with antisense oligonucleotides.
Synthesis of NMDA receptors, which are thought to be responsible for neuronal death
following cerebral vascular occlusion, can be inhibited by antisense treatment. Such an
inhibition reduces the volume of the infarction produced experimentally by the occlusion of the
middle cerebral artery (152).
Inhibition of tumor growth is one of the major fields of research in antisense
development. Indeed, the first in vivo application of antisense inhibition was designed to
downregulate N-myc and consequently inhibit neuroectodermal tumor growth. Antisense
oligonucleotides were delivered in an osmotic pump to the vicinity if the xenographic tumor,
and loss ofN-myc protein along with 50% loss in tumor mass was observed (177).
Gene Therapy with Antisense Expression Vector
Why do we Need Gene Therapy?
The answer in short is, that because contemporary research shows that a score of
diseases are caused by malfunctioning genes. Some of these faulty genes are already identified:
the mutated chloride-channel gene CFTR for cystic fibrosis, or BRCA1 for hereditary breast
cancer, and there are certainly a lot more to come. Curing these diseases will require the
correction or replacement of the disease-genes with the correct ones.
Not only inherited diseases are caused by genetic disorders, however, most cancers
now are viewed as acquired genetic diseases where the irregular expression of a cell regulatory
protein causes uncontrolled cell division.
Viral diseases are clearly caused by genes: viruses can be viewed as genetic
misinformation packaged into a tiny protein capsid. Some of the foreign viral genes can
integrate into the host cell's chromosome and cause disease in various ways, including cell
death or malignant transformation.
There are other conditions where there is no mutation, foreign DNA invasion or
expression of normally silent genes, but there is malfunction in the degree of the expression of
normal genes. It would be difficult to estimate the number of diseases which can be included
into this group, but experimental evidence suggests that hypertension is one of them.
What also links these disorders together is that currently there is no cure for them, only
treatment of the symptoms or in some instances, not even that. Gene therapy aims at
eliminating the cause of these diseases by correcting the genetic malfunctions.
The ideal solution for genetic disorders would be the replacement of the faulty gene
segment with a normal one by homologous recombination in all or at least in a clinically
significant fraction of the affected cells. Unfortunately, the frequency of homologous
recombination upon introduction of the correct gene is very low. It seems, however, that it
might be not all that critical introducing the correct gene into the exact same location where the
faulty gene is; and suppression of an overactive gene can be achieved form another locus on the
genome without excising it, with e.g. an antisense gene.
As it turns out, some of the best agents to date in the fight against genetic malfunctions
are the same viruses that cause some of these disorders; only the correct genes have to be put
into them. Viruses evolved for the specific task of delivering DNA (or RNA) into cells, and
researchers take advantage of this ability by using viruses for delivering recombinant genes into
There are several viruses which have been tested for gene delivery, and each has its
advantages, but none of them fits perfectly to the description of the "ideal viral vector". To be
the perfect vector, a virus should fulfill all of the following criteria:
-it should be safe. This means that it cannot be a virus known to cause disease, or it
has to be re-engineered to be harmless. It cannot elicit immune response, and it cannot
integrate into the genome randomly carrying the risk of disrupting other cellular genes. It also
has to be replication-deficient for the prevention of the spread of it to unwanted tissues or
-it should be efficient. The virus has to infect the target cells with high frequency in
order to achieve biological effect.
-it should be easy to manipulate. The virus should to be able to accommodate the gene
of interest, along with its regulatory sequences (promoter), and the recombinant DNA has to
be packaged with high efficiency into the viral capsid proteins.
Viruses Adopted for Gene Therapy
The most popular viral vectors in the past have been retroviruses. These have been
used primarily because of their high efficiency in delivering genes to cells (178). They proved
to be effective in cell culture systems, but they are notorious for their random integration into
the genome which raises concerns about their safety when used in vivo. Retroviruses also
require cell division for chromosomal integration, which makes them limited mostly to tumor
Adenovirus vectors have been tested successfully in their natural host cells, the
respiratory endothelium, as well as other tissues such as muscle and brain (179-181). Most
adenovirus vectors in their current form are episomal, that is they do not integrate into the host
DNA. They provide high levels of expression for a significant amount of time, but the
episomal DNA will invariably turn inactive. Repeated infections, on the other hand, result in
inflammatory response with consequent tissue damage.
Herpesvirus vectors hold the promise for delivering genes into the nervous system.
They have the ability to spread from one neuron to the other, raising the possibility of infecting
cells of the central nervous system from peripheral injections. Controlling of this spread,
however, proved to be difficult, and continuous expression of the inserted gene is hindered by
latency periods intrinsic to herpesviruses.
Adeno-associated virus (AAV), has been gaining attention because its safety and
efficiency (182). It has been successfully used for delivering antisense RNA against alpha-
globin (183) and HIV LTR (long terminal repeat) (184), and it is our vector of choice for
delivering antisense RNA to the AT-I receptor.
AAV is a parvovirus, discovered as contamination of adenoviral stocks. It is a
ubiquitous virus (antibodies present in 85% of US human population), which has not been
linked to any disease. It is also classified as dependovirus, because its replication is absolutely
dependent on the presence of a helper-virus, which can be adenovirus or herpes virus. Five
serotypes have been isolated, of which AAV-2 is the best characterized. AAV has a single-
stranded linear DNA which is encapsidated into capsid proteins VP1, VP2 and VP3 to form an
icosohedral virion of 20-24 nm in diameter.
The AAV DNA is approximately 4.7 kilobases long. It contains two open reading
frames and it is flanked by two terminal repeats. Each terminal repeat can form a T-shaped
hairpin structure. These terminal repeats are the only essential components of the AAV for
chromosomal integration. They also serve as origins of replication. There are two major genes
in the AAV genome: rep and cap. rep codes for proteins responsible for viral replication,
whereas cap codes for capsid proteins VP1-3. Three promoters have been identified and
named p5, p19 and p40, according to their map position. Transcription from p5 and p19 result
in production of rep proteins, and transcription from p40 produces the capsid proteins (185).
The life cycle of adeno-associated virus
Upon infection of a human cell, AAV integrates to the q arm of chromosome 19 (186).
For chromosomal integration the only essential component are the terminal repeats. The viral
components responsible for site-specific integration are yet to be identified. With no helper
virus present, AAV infection remains latent indefinitely. Upon superinfection of the cell with
helper virus the AAV genome is excised, replicated, packaged into virions and released to the
Adeno-associated virus as expression vector
There are several factors that prompted researchers to study the possibility of using
AAV as an expression vector. One is that the requirements for delivering a gene to the host
chromosome is surprisingly little: the presence of the terminal repeats- which are each only 3%
of the AAV genome- is sufficient for chromosomal integration. No viral proteins required,
and the cellular proteins that are needed for integration are most likely ubiquitous cellular
The second characteristics that makes AAV a good vector candidate is its relative
complicated rescue mechanism. As opposed to integration, productive infection requires
several factors including superinfection with a second virus as well as transcription of viral
proteins, which gives several opportunities for preventing the spread of recombinant AAV
vectors to non-target areas.
Another reason why AAV is a premier candidate for gene therapy is its exceptionally
broad host range. It infects all mammalian tissues tested apparently with the only exception of
vascular endothel cells (187).
Preparation of recombinant AAVs is relatively easy: 94% of the genome can be
replaced for the gene(s) of interest. There are, however, disadvantages to such a small virus.
Recombinant DNA larger than the wild-type AAV is not encapsidated effectively, so the 4.4
kilobases available space has to accommodate the promoter, coding sequence, polyA tail and
possibly a marker protein gene. Other difficulty is the preparation of the viral stocks: addition
ofadenovirus as helpervirus to packaging cells contaminates AAV stocks. It is also a matter of
concern that the site-specific integration of AAV into the ql3.4-ter of chromosome 19 appears
to be lost when cells are infected with recombinant AAV.
In summary, AAV appears to be one of the best candidates for gene delivery. Most of
the aforementioned problems appear to be technical and should be solved satisfactorily in the
Expression of Antisense Sequences with Vectors
Long-term suppression of synthesis of individual proteins is desirable in conditions
where overexpression of endogenous genes or expression of foreign genes is thought to be the
cause of the disease. Inhibition of synthesis of certain proteins for extended periods of time is
also useful when studying protein function, e.g. in development.
Antisense expression vectors produce antisense RNA, which can hybridize with the
target mRNA and prevent its translation by either promoting its degradation, preventing its
transport from the nucleus to the cytoplasm or interfering with ribosomal function.
AAV-derived expression vectors have been successfully used to inhibit alpha-globin
expression in an erythroleukemia cell line where a 1.0 kb fragment of the alpha-globin gene was
inserted in the antisense direction under either thymidine kinase, SV40 or the alpha-globin
promoter. 91% inhibition in alpha-globin mRNA production was achieved with the alpha-
globin's own promoter, whereas the other two promoters proved to be less efficient (183).
A much shorter antisense sequence (64 base pairs long) targeting the human
immunodeficiency virus (HIV) long terminal repeat was expressed with an AAV vector using
the SV40 promoter in 293 cells. Greater than 99% reduction in infectious HIV-1 production
and the absence of cellular toxicity was observed for over a period of 20 days (184).
Recombinant AAV in the Brain
An AAV expression vector has been successfully tested in the central nervous system.
A recombinant AAV virus expressing tyrosine hydroxylase significantly raised dopamine levels
in the striatum of monkey brains. The hormonal change was sufficient to produce long-lasting
behavioral changes (188).
Summary of the Literature
Despite the immense knowledge accumulated over decades in circulation research, the
cause of essential hypertension is still unknown. The prevailing view today is that blood
pressure is determined by both genetic and environmental factors. Moreover, the inheritable
component of hypertension is thought to be the sum of the effects of several genes. Most of
the candidate genes for hypertension are members of the renin-angiotensin system.
The role of the brain RAS in the development of the hypertension has been indicated by
large volume of experimental evidence. The recent development of subtype-specific AngII
receptor antagonists, however, failed to support unequivocally the pivotal role of brain AngII
Emerging new molecular techniques such as antisense allow us to approach these
questions from a molecular genetic point of view. Besides using antisense as a research tool,
evaluating it as a potential clinical agent is equally important.
MATERIALS AND METHODS
Adult male spontaneously hypertensive rats (SHR) and Sprague-Dawley (SD) rats
(250-300g) were acquired from Harlan. The animals were kept in individual cages in a room
with 12 hours light-12 hours dark cycle. They were given tap water to drink and standard rat
chow to eat ad libitum. For intracerebroventricular cannulation, each animal was anesthetized
with sodium pentobarbital (65 mg/kg body weight) intraperitoneally, and a stainless steel (23-
gauge) cannula was placed in the right lateral brain ventricle, using a Kopf stereotaxic
instrument (coordinates: 1.0 mm lateral, 1.0 nmmn caudal to the bregma; 5.0 numm below the skull
surface). The animals were allowed to recover for 5 days before further surgical procedures or
blood pressure readings were carried out.
Blood Pressure Measurements
Blood pressure and heart rate were monitored by a Statham pressure transducer via a
heparinized (100 U/ml) catheter placed in the left common carotid artery. Animals were
allowed to recover from carotid catheterization for 24 hours before experimentation. All
experimental protocols were approved by the Animal Care Committee of the University of
Florida, Gainesville, FL.
Antisense (AS), sense (S) and scrambled (SC) oligonucleotides were synthesized as
15-mer phosphorothioated oligonucleotides on an Applied Biosystems 394 DNA/RNA
Synthesizer at the DNA Synthesis Core Facility at the University of Florida. AS
oligonucleotides were complementary to bases +63 to +77 of the AT-la receptor mRNA (29).
S oligonucleotides were identical to this target sequence, whereas SCR oligonucleotides had
the same base composition as AS but in a scrambled order. Prior to synthesis, oligonucleotide
sequences were tested for sequence-homology against all available DNA sequences in the
GeneBank. The purity of oligonucleotides was routinely tested with capillary electrophoresis.
Oligonucleotides were dissolved in 0.9% saline solution, aliquotted and stored on -20 C.
SHRs were treated with i.c.v. injections of 50 pg/4gl antisense, scrambled
oligonucleotide or saline 24 hours before the drinking tests. Through the same i.c.v. cannula,
50 ng AngII (acetate salt, Sigma) dissolved in 2 pgl artificial cerebrospinal fluid was injected in
conscious, freely moving rats. The rats were presented with scaled drinking bottles, and the
amount of consumed water was recorded over a 30 minutes period.
24 hours after oligonucleotide treatment the rats were sacrificed, the brains were
removed and frozen on dry ice. Cryostat-sections were cut at 20 gm thickness on -20 C, and
thaw-mounted onto gelatin-coated slides. The slides were preincubated in assay buffer (150
mM NaCl, 50 mM sodium phosphate, 1 mM EDTA, 0.1 mM bacitradcin, pH 7.4) for 30
minutes and then incubated in the same buffer with the addition of 500 pM 12I-Sar-Ile-Angll
for 2 hours. Sections were incubated with the radioligand in the presence or absence of 1 pM
AngII to determine nonspecific and total receptor binding. The sections were washed in assay
buffer and in distilled water, and dried under cool air. Autoradiograms were generated by
apposition of the slide-mounted tissue sections with X-ray film (Hyperfilm-3H, Amersham) for
3 days. Densitometric analysis of the autoradiograms was carried out with a video-based
computerized image analysis system (MCID, Imaging Research, Ontario, Canada).
Receptor Binding Assay on Hypothalamic Cell Membranes
Under metophane anesthesia, rats were decapitated and the brains were dissected. The
hypothalamic block, including the hypothalamus, septum and thalamus was homogenized in 4
ml ice cold TE buffer (50 mM Tris, ImM EDTA, 150 mM NaCI, pH 7.2) for 10 s using a
Tekmar SDT 1810 homogenizer and centrifuged at 2,000 g for 10 minutes. The pellet was
discarded and the supernatant was re-centrifuged at 50,000 g on 4 C for 30 minutes. The
resulting pellet was resuspended in 1.5 ml TE buffer, homogenized using a dounce glass
homogenizer and assayed for protein content with the Lowry method. 100 jg of membrane
proteins were incubated with 200 pM of the radiolabeled AngII analog 12I-Sar-lle-AngI in the
presence of absence of 1 pM cold Angi, Losartan or PD 123319 to determine the nonspecific,
total, AT-1 and AT-2 binding, respectively. The membrane proteins were incubated in
triplicates with the receptor ligands in 500 gl of incubation medium containing TE buffer, ImM
bacitracin, 10 pM 0-phenantrolene and 0.1% bovine serum albumin for 90 minutes on room
temperature. At the end of the incubation procedure, membrane-bound ligand was collected
on Whatman filter paper using a Brandel cell harvester, and radioactivity was determined with
a Beckman gamma-counter.
Receptor Binding Assay on Cultured NG108-15 Cells
NG108-15 cells were grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin and 1
gig/ml Fungizone in 100 mm plates in a humidified atmosphere of 5% CO2 and 95% air at
37C. Oligonucleotides were administered to 1-day-old cell cultures (60-70% confluent) in
serum-free DMEM at final concentrations of 2.5 or 5 gM. After 24 hours ofoligonucleotide
treatment, differentiation was induced by replacing the DMEM with medium containing 1.5%
dymethylsulfoxyde (DMSO) and 0.5% FBS. 48 hours after the initiation of oligonucleotide
treatment the differentiating medium was removed, the cells were washed with cold phosphate
buffered saline (PBS) and collected in TE buffer (4 ml/plate) by mechanical dissociation. Cells
were homogenized with a dounce glass homogenizer and centrifuged on 50,000 g for 30
minutes on 4 C. The resulting pellet was resuspended in 1.5 ml TE buffer, rehomogenized and
assayed for protein content. Receptor binding assay was performed as described above.
NG108-15 cells were treated with oligonucleotides and differentiated as described
above. 48 hours after the initiation of oligonucleotide treatment, cells were washed with PBS,
and total RNA was extracted and purified with TRI-Reagent. 12 gig RNA/lane was run on a
1.2% agarose-formaldehyde gel on 30 V for 16 hours. RNA was transferred to a nylon
membrane and stabilized by UV-crosslinking. A 3'P-labeled riboprobe was generated by in
vitro transcription of the linearized AT-la cDNA using the Promega Gemini System II. The
AT-la clone pKSCalSb was a gift from T.G. Murphy (Emory University, Atlanta, GA).
Membranes were prehybridized in 50% formamide, 5X SSPE, 5X Demhardt's solution, 0,8%
SDS and 10 mg salmon sperm DNA for 4 hours at 56 C, then hybridized in the same solution
with approximately 106 cpm/ml AT-la riboprobe for 15-18 hours. The following day the
membranes were washed with 1 x SSPE, 0.5% SDS for 3X15 minutes at 65 C and in 0.1 x
SSPE, 0.5% SDS for 30 minutes at 65 C. The membranes then were wrapped in plastic foil
and exposed to X-ray film, which was developed 3-7 days later.
Detection of Fluorescent Labeled Oligonucleotides
AS and S phosphorothioated oligonucleotides were labeled on both 5' and 3' ends with
fluorescein isothiocyanate (FITC) at the DNA Synthesis Laboratory of the University of
Florida. Rats equipped with intracerebroventricular guide cannulas were injected with 50 p g
FITC labeled oligonucleotides dissolved in 4 p.1 saline. 1 hour later the rats were sacrificed, the
brains were removed and cryostat-sectioned into 20 pm-thick slices. The slide-mounted brain
slices were viewed with a BioRad 600 MRC laser-scanning confocal microscope. For cellular
uptake experiments, NG108-15 cells were cultured on 1.2 cm diameter glass cover slips, and
1-day-old cell cultures were treated with 5 or 25 pM FITC-labeled oligonucleotides for various
time periods. At the end of the incubation, the cells were washed with PBS, the cover slip
carrying the cells was removed from the culture dish and mounted upside down onto a glass
microscope slide using paraphenyline diamine mounting medium. The slides were stored in the
dark until viewed with laser-scanning confocal microscope.
Expresion Vector Experiments
Expression Vector Construction
pJDT95, a pBR322-derived plasmid containing the AAV-2 genome with truncated cap
gene was provided by E.M. Meyer (University of Florida, Gainesville). pTR-UF, a plasmid
carrying the terminal repeats but lacking the rep and cap genes of AAV was provided by N.
Muzyczka (University of Florida, Gainesville). pKSCal8b was carried in Esherichia Coli
DH5a, whereas pJDT95 was carried inKColi HB101. Both strains of bacteria were grown in
ampiciUin (50 g.g/ml) containing Terrific Broth. The bacterial transformation, growth and
alkaline lysis of bacteria was performed according to Sambrook et al.(189). Plasmids were
purified using Magic Maxipreps (Promega). The strategy for constructing the AT-1 antisense
expression vector paATI is summarized in Fig. 15. pJDT95 was linearized by restriction
enzyme digestion with HIindUl and KpnI. A 855 bp fragment of the AT-1 cDNA including the
first 761 bp of the coding region and 94 bp upstream of the transcription initiation codon
(ATG) was copied with polymerase chain reaction (PCR) using a 3' primer which incorporated
a HindiH recognition sequence on its 5' flanking region. The resulting PCR product was
digested with KpnI and HIindlH to generate compatible ends with pJDT95. This AT-1 cDNA
fragment was ligated into the linearized pJDT95 using T-4 ligase. The different recognition
sequences of Hindin and KpnI assured the proper antisense orientation of the AT-1 fragment.
The resulting recombinant products were analyzed by restriction enzyme digestion with NdeI
and subsequent 1% agarose gel electrophoresis. DNA fragments were visualized by ethidium
bromide staining and were photographed under ultraviolet illumination with Polaroid camera.
Restriction and modifying enzymes were purchased from Promega (Madison, WI).
Transfection ofNG108-15 Cells with Expression Vector
The antisense vector paATI was delivered to the cells using the cationic lipid
Transfectam (Promega). The positively charged Transfectam binds the vector DNA and it also
shows affinity to the cell membrane lipids. The exact mechanism of cellular uptake of plasmid
DNA is, however, unclear. Transfectam has been shown to deliver plasmid DNA effectively
into a variety of cell types. We mixed 15 pg of antisense vector DNA with 30 pl of
Transfectam in serum-free medium, and overlayed it onto 65-75% confluent NG108-15 cells
grown on 100 mm poly-L-lysine coated plates. The cells were incubated on 37C overnight,
then the medium was replaced with differentiating medium containing 1.5% DMSO and 0.5%
FBS. Differentiation ofNG108-15 cells continued for 48 hours, after which cells were washed
and subjected to receptor binding assay or immunocytochemistry. Control cells received
treatment with either expression vector without antisense sequence completed with
Transfectam, or Transfectam alone, or no treatment.
Helper Virus Superinfection of AAV-transfected Cells
A heat-sensitive mutant adenovirus ts149 was used as helper-virus to enhance antisense
RNA production, ts149 was grown in 293 cells, a human transformed kidney cell line.
Approximately 90 % confluent 293 cells were infected with 10 m.o.i. (multiplicity of infection)
ts149. Infected 293 cells were grown in DMEM supplemented with 10% calf serum (CS) and
100 U/ml penicillin/streptomycin on 32 C, which is the permissive temperature for ts149.
Four days later 293 cells were harvested, the cells were disrupted by brief sonication and the
cell debris was separated by centrifugation. The ts149 viruses were isolated by density gradient
centrifugation in CsCI2 gradient. Virus stocks were stored in 50% glycerol on -20 C.
Upon transfection of NG108-15 cells with the antisense vector paATI, 5-10 m.o.i. (5-10
helper virus/cell) ts149 was added to the transfection medium along with the expression vector.
Transfection of Brain Cells in Vivo
SHRs were equipped with guide cannulas aiming at the anteroventral third ventricle
(AV3V) area of the rat brain. The coordinates were: 1 mm lateral, 0.3 mm rostral from the
bregma, 8.1 mm deep in 7 degrees angle in a 250-275 g rat. The nose bar was set at -3.3 mm.
Following recovery, a mixture of 0.5 gg paATI, 1 Wl Transfectam and 0.5 pIl (5x105) helper
virus (ts149) was slowly (0.5 pl/minute) infused into the AV3V region of the brain of SHRs.
Systolic blood pressure was recorded before and 3 days after the infusion using the tail cuff
method. After the last blood pressure recordings, the rats were sacrificed and the brains were
removed for immunohistochemical detection of expressed viral rep proteins.
Rats were anesthetized and the heart was exposed. The brain was perfused after
clamping the descending aorta and the inferior vena cava, opening the right auricle and
inserting a needle into the left ventricle. 50 ml saline containing 100 U/ml heparin was infused
through the heart followed by 100 ml Zamboni's fixative. The brain was removed and stored in
Zamboni's fixative overnight. The anterior hypothalamic region was sectioned into 40 pm
thick slices and washed in PBS. The slices were then pre-treated with 3% hydrogen peroxide,
10% ovalbumin, 3% goat serum and 0.2% Triton 100X in PBS. The primary rep antibody
IF811 was diluted 1:1000 in PBS containing 0.2% Triton 100X and 1% goat serum, and
incubated with brain slices on a shaking table on 4 C overnight. The next day the brain slices
were washed in PBS, and incubated with rabbit anti-mouse IgG for 45 minutes on 4 C. The
slices were washed again, and incubated with peroxidase-antiperoxidase for 45 minutes on 4
C. The complex was stained with diaminobenzidine (DAB) in hydrogen peroxide, the brain
slices were mounted on microscope slides, coverslipped and viewed under light microscope.
For detecting rep expression in cultured cells, NG108-15 cells were grown on poly-L-
lysine coated 35 numm culture dishes to 50-60% confluence, and treated with the expression
vector paATI and helper virus ts149 as described above. Cells were incubated on 37 C for 48
hours, then were washed three times with PBS. Cells were fixed with 3.5% formaldehyde in
PBS on room temperature for 30 minutes, then rinsed with PBS three times. Cells were
permeabilized by incubating them with 0.2% Triton 100X and rinsed with PBS. The primary
antibody IF811 was diluted 1:25,000 in PBS, and incubated with the cells overnight on 4 C.
Following three rinses with PBS the cells were incubated with the secondary antibody, and
stained with anti-peroxidase and DAB as described above. The primary antibody IF811 (a
generous gift of Dr. Nick Muzyczka, University of Florida, Gene Therapy Center) was raised
in mouse and it recognizes all four rep proteins.
All numerical values are expressed as mean + SEM. Statistical analysis was performed
using Sigmastat (Jandel Scientific) computer software. One-way ANOVA was used to
determine treatment effect in the receptor binding studies, whereas two-way ANOVA was
used to analyze blood pressure data. Duncan multiple range test was used for individual
comparisons. A probability (p) value of<0.05 was considered statistically significant.
The Plan of the Study
Antisense Oligonucleotide Experiments In Vivo
1. First we designed antisense olgonucleotides targeting the AT-1 receptor mRNA.
Two oligonucleotides were designed: the first was a 18-mer phosphodiester targeting bases 1-
18, while the second was a 15-mer phosphorothioate targeting bases 63-77. Corresponding
sense and scrambled oligonucleotides had also been synthesized.
2. These oligonucleotides were then tested for their ability to lower blood pressure
upon their injection into the lateral brain ventricle of SHRs.
3. Autoradiography or membrane receptor binding was performed on oligonucleotide-
treated rats to detect any changes in AngI receptor number.
4. Functional activity of brain AT-1 receptors following antisense oligonucleotide
treatment was tested by measuring drinking response to i.c.v. AngI injection.
5. Oligonucleotide uptake was studied using fluorescein-labeled oligonucleotides.
Antisense Oligonucleotide Experiments In Vitro
6. A glioma-neuroblastoma hybrid cell line NG108-15 was used to study the cellular
mechanism of antisense inhibition. Membrane receptor binding experiments established the
effectiveness of antisense oligonucleotides in inhibiting AT-1 receptor synthesis.
7. Northern blotting was performed to detect changes in the AT-1 mRNA.
8. The cellular uptake of fluorescein labeled oligonucleotides was studied with laser-
scanning confocal microscope.
Antisense Expression Vector Experiments
1. Antisense expression vectors were constructed, and their ability to downregulate
AT-1 receptor or angiotensinogen synthesis was tested by receptor binding or RIA,
2. Expression of transcripts from the AT-1 antisense vector was detected by
3. The AT-1 antisense expression vector was injected into SHRs, followed by blood
pressure recording and immunohistochemical detection of a viral protein.
Antisense Oligonucleotide Experiments In Vivo
Blood pressure of antisense oligonucleotide treated SHRs
First an 18-base long ("18-mer") antisense oligonucleotide was tested for its ability to
lower blood pressure in the SHR. The 18-mer oligonucleotide targeted bases -6 to +12 of the
AT-la mRNA, thus covering the AUG translation initiation codon (Fig. 1). This
oligonucleotide contained natural phosphodiester bonds with the exception of the 17th, which
was thioated to resist 3' exonucleases. Spontaneously hypertensive rats were equipped with
guide cannulas to the right lateral brain ventricle. The 18-mer oligonucleotides were
administered every 12 hours for 3 days in a dose of 50 tg/ 4 gl i.c.v. Systolic blood pressure
decreased on the 2nd day after the beginning of the injections and remained low on the third
day as determined by tail cuff blood pressure recordings (Fig. 2).
Antisense: 5'-GTT AAG GGC CAT TTT GTT-3'
Sense: 5'-AAC AAA ATG GCC CTT AAC-3'
Antisense: 5'-TAA CTG TGC CTG CCA-3'
5'-TGG CAG GCA CAG TTA-3'
Scrambled: 5'-CTT ACT AGC CTA GGC-3'
Figure 1. Base sequence of oligonucleotides. (A) The 18 bases long (18-mer) antisense
oligonucleotide was complementary in sequence to bases -6 to +12 of the AT-la mRNA
(target sequence). A sense oligonucleotide served as control, the base sequence of which was
similar to the target sequence. The 18-mers were phoshodiester oligonucleotides, except the
17th base, which was modified by phosphorothioation. (B) The second set of oligonucleotides
were 15-mers and were targeted to bases +63 to +77. Besides the antisense oligonucleotide, a
sense oligonucleotide, which has similar base composition as the target sequence on the AT-1
mRNA, and scrambled oligo having the same base composition as the antisense but in a
random order, served as control.
I-- I Control
baseline 1 2 3
Figure 2. Blood pressure lowering effect of a 18-mer phosphodiester antisense oligonucleotide
targeted to the translation initiation site of the AT-1 receptor mRNA. 50 gig oligonucleotide
was injected every 12 hours for 3 days into the lateral brain ventricle of spontaneously
hypertensive rats. Systolic blood pressure was monitored daily with a tail cuff blood pressure
recorder (*: p<0.05, two-way ANOVA followed by Duncan multiple range test; n=3-4).
Then fully phosphorothioated antisense, sense and scrambled oligonucleotides were
synthesized corresponding to nucleotides +63 to +77 of the AT-la receptor mRNA. SHRs
were fitted with carotid catheters for direct blood pressure recording, and were injected with
50 p.g of sense or antisense oligonucleotide dissolved in 4 ,Ll saline through an i.c.v. cannula.
Baseline blood pressure data was recorded 1 hour before oligonucleotide injection. Three
measurements were taken 10 minutes apart and averaged. The mean arterial pressure (MAP)
was calculated using the equation MAP=0.43(S-D)+D, where S and D represent systolic and
diastolic pressure, respectively. Antisense oligonucleotide injection into the lateral brain
ventricle resulted in a significant fall of 27 + 8 mmHg in the mean arterial pressure of SHR
(Fig. 3). For the antisense oligonucleotide treated rats, the pre-treatment mean blood pressure
was 178 + 5 mmHg, and the 24 h post-treatment mean blood pressure was 150 + 6 mmHg
(n=7). Saline, scrambled or sense oligonucleotide injections did not alter blood pressure in
Drinking response to AngHi stimulation
SHRs treated with antisense or scrambled oligonucleotides or saline i.c.v. 24 hours
earlier were stimulated to drink by i.c.v. injections of 50 ng/ 2jgl AngI. The amount of water
consumed over a 30 minutes period immediately after the AngII injection decreased from the
control levels (11.7+0.5 ml/30 minutes) to 5.0 + 0.8 ml/30 minutes in the antisense
oligonucleotide treated rats (p<0.01) (Fig. 4). The water intake in the scrambled
oligonucleotide treated rats showed no significant difference from that of the animals without
100 MIT= -II
Control Scrambled Sense Antisense
Figure 3. Decrease in mean arterial pressure in SHR 24 hours after a single i.c.v. injection of
50 gg of AS-ODN specific for AT, receptor mRNA. Baseline blood pressure data was
recorded prior to the injection. All blood pressure data were obtained by direct measurement
through a carotid catheter. The mean arterial pressure was calculated using the equation:
[0.43(S-D)]+D where S and D represent systolic and diastolic pressure respectively (*: p<0.05,
two-way ANOVA followed by Duncan multiple range test; n=6-7).
8_ -- -
Control Scrambled Antisense
Figure 4. Effect of AT-1 antisense oligonucleotides on drinking in response to i.c.v. AngII.
Spontaneously hypertensive rats were administered 50 ng AngIl 24 hour after saline (0.90/%),
scrambled or antisense oligonucleotide treatment. The water intake of each rat was measured
for 30 minutes after AngII injection. (**:p<0.01, ANOVA followed by Duncan multiple range
Autoradiography of AT-1 receptors after antisense oligonucleotide treatment
After the 24 h blood pressure measurements, rat were sacrificed, the brains were
removed and cryostat-sectioned for autoradiography. 12I-Sar-Ile-AngII binding was decreased
by 16% in the paraventricular nucleus (PVN), 9 % in the suprachiasmatic nucleus (SCN) and
17 % in the median preoptic nucleus (MnPO) in the antisense oligonucleotide (50 Rg/4 jgl)
injected rat brains compared to saline-injected controls. Binding in the sense and scrambled
controls in the MnPO, however, was also decreased to some extent (about 10%). Similar
tendency was observed in the SON, which showed a decrease of 14 % after antisense
treatment and 8-9 % decrease after sense or scrambled oligonucleotide treatment. There were
no changes in the PVN in the sense or scrambled oligo treated rats (Fig. 5). Receptor numbers
in the mediodorsal thalamic nucleus and in the lateral septum, areas that are known to have
type-2 Angl (AT-2) receptors, did not decrease after antisense injection.
In an effort to improve the delivery of antisense oligonucleotides to their proposed site
of action, the hypothalamus, we injected 50 pgg/ 2 gl oligonucleotides into the third brain
ventricle once a day for three days (coordinates: 1mm lateral, 1mm caudal to the bregma, 8.5
mm ventral in 8 degree angle). We observed 14% decrease in the PVN, 14% in the OVLT and
10% in the MnPO 3 hours after the third antisense oligonucleotide injection. Similar treatment
with sense oligonucleotides resulted in no change in the PVN, 14% decrease in the OVLT and
29 % increase in the MnPO. Direct injections of 25 gg/ 2 pl antisense oligonucleotides in
the right PVN (coordinates: 1 mm lateral, 1.8 mm caudal to the bregma, 6.5 mm ventral)
resulted in an apparent decrease in AT-1 receptor binding when compared to the contralateral
PVN (Fig. 6)., but the controls showed almost the same change.
Figure 5. Computer-enhanced images of representative autoradiograms showing reduced
AngIl receptor density in the antisense oligonucleotide treated rat brain. 24 hours after the
i.c.v. oligonucleotide injection the brains were removed, and 20 glm sections were incubated
with 500 pM '2I-Sar-]le-Angl for 2 hours in the presence or absence of excess AngI. The
images show coronal sections of scrambled (SCR), sense (S) and antisense (AS) treated rat
brains in the level of the paraventricular nucleus of the hypothalamus.
Figure 6. 12I-Sar-Ile-Angll binding in the brain injected with oligonucleotides directly into the
tissue. 25 gig antisense oligonucleotide in 2 gl saline was slowly infused into the right
paraventricular nucleus. Bar shows the color code for signal intensity.
Long-term effect ofantisense treatment in SHR
It was noticed upon testing some of the surviving rats that the depressor effect of the
antisense oligonucleotide injection lasted for more than 24 hours. A long-term experiment was
designed where the blood pressure of antisense treated SHRs was monitored daily for 9 days.
A baseline blood pressure recording was taken, and 50 g.g/4 Il antisense or scrambled
oligonucleotide was injected into the lateral brain ventricle. Daily blood pressure recordings
were taken between 10:00-11:00 am. every day for 9 days. There was significant decrease
in the mean arterial pressure (MAP) between days 1 and 7 after antisense oligonucleotide
injection when compared to scrambled oligonucleotide treated rats. MAP decreased to
normotensive levels at day 1 (137.8 + 3.5 mmHg) in the antisense oligonucleotide treated rats,
reached its lowest value on day 2 (133.1 + 7.8 mmHg) and remained below control levels
through day 7 (Fig 7). Blood pressure gradually returned back to hypertensive levels, but it
reached the MAP of scrambled oligonucleotide treated rats only at day 8 after treatment.
Scrambled oligonucleotide injection (50 p.g / 4 p1) did not cause significant changes in MAP of
SHRs throughout the 9-day monitoring period. Heart rate did not show significant changes
from their pre-treatment values (antisense: 435 + 51 /minutes; scrambled: 408 + 12 /minutes).
In the antisense oligonucleotide treated rats, however, there was a tendency of elevated heart
rate when compared to the heart rate of scrambled treated rats (Fig. 8), which might be a
interpreted as a consequence of baroreflex compensation for the lowered blood pressure.
Angiotensin receptor binding on membrane fractions ofhypothalamic cells
The time point when there was the greatest difference in MAP between antisense and
scrambled oligonucleotide treated rats was chosen for receptor binding experiments. At day 3
110- H Antisense
100 I I I i 1 I I i
0 1 2 3 4 5 6 7 8 9
Figure 7. Time course of the blood pressure decrease in SHR following a single injection of
AT-I antisense oligonucleotide. Rats received 50 jig of antisense or scrambled
oligonucleotides in 4 ll saline into the right lateral brain ventricle on day 0. Blood pressure
recordings were made prior and every day after the injection for 9 days between 10 11 am
(*:p<0.05, **:p<0.01, two-way ANOVA, followed by Duncan multiple range test; n=4-1 1).
I I I I I I I I I I
0 1 2 3 4 5 6 7 8 9
Figure 8. Heart rate of SHR after i.c.v. injection of 50 g.g of AS-ODN or 50 jtg of SC-ODN.
Heart rate was measured simultaneously with the daily blood pressure recordings using a
Statham transducer and Gould 2400 recorder (n=4-1 1).
post-treatment the rats were sacrificed, the brains were removed, and the hypothalamic
blockcontaining the septum, thalamus and hypothalamus was dissected, homogenized and
centrifuged for radioligand binding. MI-Sar-Ile-AngII binding was determined in the presence
or absence of unlabeled Angl, Losartan or PD123319 to determine nonspecific, total, AT-1
and AT-2 receptor binding, respectively.
Two doses of oligonucleotides were tested. Both with 25 pg and 50 pg
oligonucleotides there was a significant decrease in AT-1 binding in the antisense
oligonucleotide treated rat hypothalami (894 46 cpm/lOO1g tissue homogenate and 850.8 +
46.1 cpm/100gg tissue homogenate for the lower and the higher dose, respectively) when
compared to scrambled oligonucleotide treated rat hypothalami (1044 + 32 cpm/100lg tissue
homogenate and 1039.2 + 50.9 cpm/100gg tissue homogenate). There was no change in AT-
2 binding (Fig. 9).
Oligonucleotide uptake by brain cells
Antisense oligonucleotides were labeled on 5' and 3' ends with fluorescein-
isothiocyanate (FITC). 50 gg/ 4 gl labeled oligonucleotides were injected in the lateral brain
ventricle of SHRs. 1 hour later rats were sacrificed, the brains were removed and cryostat-
sectioned into 20 mm thick slices. The brain slices were mounted onto glass slides, and viewed
with confocal microscope. Fluorescent signal was detected in the lateral brain ventricles on
both sides, in the third ventricle as well as in the fourth ventricle. Fluorescence was also
detected in the cortex. The oligonucleotides diffused well in the cerebrospinal fluid; there was
no apparent difference between the strength of the fluorescent signal in the lateral ventricles or
Antisense (25p g)
Figure 9. AngIl receptor binding assay on cell membranes prepared from the hypothalamic
region including the hypothalamus, thalamus and septum of SHRs. Binding of 'OI-Sar-Ile-
AngIl was determined in the presence of the AT-1 and AT-2 subtype-specific AngI receptor
antgonists losartan and PD 123319, respectively. The assay was performed 3 days after
injection of two different doses (25 jig or 50 gig) oligonucleotide (*:p<0.05, t-test, n=5).
the in third ventricle. Oligonucleotides diffused into the brain tissue in 1-2 mm deep from the
ventricles and from under the dura mater. The highest concentrations of oligonucleotides were
found in the tissue bordering the ventricles, and the signal decreased as the distance from the
ventricles increased (Fig. 10).
When the same amount of FITC-labeled antisense oligonucleotides were injected into
the third ventricle, a similar pattern of oligonucleotide distribution was observed: equal spread
of fluorescent signal in the lateral and in the third ventricle, whereas less signal was detected in
the fourth ventricle and on the surface of the cortex.
Antisense Oligonucleotide Experiments In Vitro
Receptor binding in antisense oligonucleotide treated NG108-15 cells
NG108-15 cells, a rat glioma mouse neuroblastoma cell line was grown to 65-75%
confluence on poly-L-lysine treated 100 mm tissue culture dishes. 3 x 106 cells / dish were
treated with 2.5 or 5 pM sense or antisense oligonucleotides or with saline in serum-free tissue
culture media (DMEM). On the following day, the culture media was replaced with
differentiating media, and the cells were differentiated for 24 hours. At the end of the
differentiation period, cells were harvested, and receptor binding was performed on cell
5 pM antisense oligonucleotide treatment resulted in a significant decrease in 1'25I-Sar-
Ile-AngII binding (35% decrease when compared to the saline treated control cells) when
compared to the sense oligonucleotide treated cells (10% decrease compared to control). 2.5
pM antisense oligonucleotide treatment did not cause significant change in receptor binding in
NG108-15 cells (Fig. 11).
Figure 10. Distribution of FITC-labeled antisense oligonucleotides in the brain of a Sprague-
Dawley rat one hour after injection of 50 jig oligonudcleotide into the right lateral brain
ventricle. The sagittal gap represents the third ventricle in coronal section.
S2.5uM A2.5uM S 5uM A5uM
Figure 11. Receptor binding on membrane preparates of NG108-15 cells shows 35% decrease
in '2I-Sar-lIe-AngI binding in cells treated with 5 pM antisense ODNs for 24 hours when
compared to sense ODN treated cells. The cells were differentiated for another 24 hours after
oligonucleotide treatment, then processed for receptor binding. 100 jgg of the membrane
homogenates was incubated with 200 pM '2I-Sar-IIe-AnglI in the presence or absence of 1
pM unlabeled AngI for 2 hours. Membrane-bound ligand was separated using a Brandel cell
harvester. Radioactivity was measured with a Beckman gamma counter. Values are shown as
percent of control (1885359 cpm/100 pig cell homogenate), where control cells received
0.9% saline treatment (*:p<0.05, t-test, n=3-4).
Detection of AT-1 mRNA with Northern blot after oligonucleotide treatment
In order to detect any possible changes in the amount or length of the mRNA of the
AT-1 receptor after antisense treatment, we treated NG108-15 cells with 5 AM sense or
antisense oligonucleotides or saline vehicle, and after 24 hours of cell differentiation total
cellular RNA was extracted, run on 1.2 % agarose gel, blotted onto nylon membrane and
hybridized overnight with a 32P-labeled riboprobe. The membranes were washed and exposed
with X-ray films for 3-7 days. The resulting autoradiograms were analyzed with a digital
The pooled results of three such experiments showed that there was no significant
difference in the amount of AT-1 mRNA detected in the control, sense or antisense treated
cells (Fig 12). The probe hybridized with a single band of RNA in all treatment groups. The
estimated size of the RNA in this band is in good agreement with the expected size of the AT-1
mRNA (2.3 kb).
Cellular uptake of oligonucleotides
To determine the transfection efficiency and the intracellular localization of
oligonucleotides, we administered 5 or 25 pM FITC-labeled sense or antisense
oligonucleotides to NG108-15 cells and incubated them for 15 minutes, 30 minutes, 1 hour, 6
hours and 24 hours on 37 C. At the end of the incubation period the cells were fixed,
mounted onto glass slides, and examined with laser-scanning confocal microscope.
Examination of the cells with laser-scanning confocal microscope showed ring-shaped
intracellular localization of the fluorescent labeled oligonucleotides after 15 minutes and 1 hour
r 0 0
e 2.0 C
Control Sense Antisense
Figure 12. Computer-aided densitometric readings of the pooled results of three Northern
blots. NG108-15 cells were treated with 5 gM sense or antisense ODNs for 24 hours, then
differentiated for an additional 24 hours. Cells were harvested and RNA was extracted using
TRI-REAGENT (Molecular Research Center, Inc.). Twelve microgram of the extracted RNA
was ran on agarose-formaldehyde gel and transferred to nylon membrane. A radiolabeled
runoff transcript of the plasmid pKSCal8b carrying the full length AT, receptor cDNA was
used for Northern hybridization. Control cells received 0.9% saline treatment (n=3). A
representative Northern blot is also shown.
incubation. After 6 hour incubation, however, the fluorescent signal was detected almost
exclusively in the center of the cells (Fig. 13). As it was confirmed with light microscopy, this
spherical image corresponded well to the shape and localization of the nucleus of NG108-15
cells, and the ring-shaped signal detected in the first 1 hour represented cytoplasmic
localization. The nuclear signal was maintained through 24 and 48 hours. Based on the
number of cells showing fluorescent staining, we estimate that the transfection efficiency is 75-
95% inNG108-15 cells.
To examine whether base sequence influences cellular trafficking of oligonucleotides,
both sense and antisense oligonucleotides were incubated with NG108-15 cells for the time
periods described above. Both sense and antisense oligonucleotides were taken up by the cells
with similar efficiency, and both oligonucleotides were transported through the cytoplasm to
the nucleus with similar rate. There was no apparent difference in the strength or the duration
of the fluorescence between sense and antisense oligonucleotide treated cells.
Antisense Expression Vector Experiments
Inhibition of AT-1 receptor with antisense expression vector
AT-1 antisense expression vector was constructed by inserting 855 bp of the AT-1
receptor cDNA (29) into the AAV-derived expression vector pJDT95 downstream to the
AAV promoter p40 in antisense direction (Fig. 14). The resulting plasmid paATI was
analyzed by digesting it with NdeI restriction enzyme to confirm the insertion and the antisense
orientation of the AT-1 fragment.
NG108-15 cells were grown on poly-L-lysine coated plates to 65-75% confluence. 1
day after plating the cells, the growth medium was removed, the cells were washed 3 times
Figure 13. Laser scanning confocal microscopic images ofNG108-15 cells after 15 minutes, 1
hour, 6 hours, and 24 hours of incubation with FITC-labeled antisense ODNs. Cells were
grown to 40-50% confluence on glass coverslips coated with poly-L-Lysine. Following the
incubation with 25 pM F1TC-labeled ODNs the tissue culture medium was removed, and the
cells on the coverslips were mounted on microscope slides using paraphynyline polyvinyl
alcohol-glycerol mounting medium. Images were acquired using a BioRad MRC-600 laser
scanning confocal microscope.
HindMll, KpnI digest
KpnI, EcoRI digest
generated by PCR
K ATH antisense
K AT, antisense
Figure 14. Strategy for antisense expression vector cloning targeting angiotensin type-1
receptor. The plasmid pJDT95 carrying recombinant adeno-associated virus (AAV) DNA was
linearized by digestion with HindUI and KpnI. The plasmid carrying the full length AT-1
cDNA was digested with KpnI and EcoRI, which released a 855 bp long fragment of the 5' end
of the AT-1 cDNA including the translation initiation site. The EcoRI end was modified to
HindlIl by performing polymerase chain reaction with one primer containing the HindII site.
The resulting fragment was ligated in the antisense direction into the linearized pJDT95.
with DMEM and incubated with 15 gg ofpaATI completed with 30 W1 Transfectam in 4.5 ml
DMEM. The next day the transfection medium was removed and differentiation medium was
added for 48 hours. At the end of this incubation period, NG108-15 cells were harvested, and
membrane receptor binding was performed. Control cells received either no treatment, or 30
Jl Transfectam alone, or 15 gg control vector containing no antisense sequence (pJDT95dlk)
completed with 30 gl Transfectam.
NG108-15 cells transfected with paATI showed 20% decrease in I-Sar-]le-Angll
binding when compared to mock-treated cells (p<0.05). Transfectamn treatment alone, or
pJDT95dlk control vector treatment resulted in no difference in receptor binding when
compared to control cells (Fig. 15).
Coinfection of AAV-transduced cells with adenovirus activated AAV, resulting in an
enhanced antisense effect. Immediately after transfecting NG108-15 cells as described above,
5X106 adenovirus (tsl149) was added to the transfection medium of paATI treated cells.
Differentiation and receptor binding was performed as described above. '22-Sar-Ile-AngII
binding decreased by 63 % compared to control vector treated levels in the paATI plus
adenovirus treated cells. Adenoviral enhancement of antisense activity was observed only in
the AT-1 subtype of AngII receptors, whereas the AT-2 subtype remained unaffected (Fig.
Immunocytochemical detection of AAV transcripts
Besides making AT-i antisense RNA from its p40 promoter, paATI also produces
viral rep proteins, albeit in smaller amounts. The rep proteins are not essential for the antisense
Control Transfectam Mock Antisense
Figure 15. The AT-1 antisense expression vector paAT produces 20%/ decrease in receptor
binding in NG108-15 cells when compared to mock vector treated cells. A glioma-
neuroblastoma hybrid cell line, NG108-15 was grown to 65-75 % confluence and was
transfected with the angiotensin type-1 receptor antisense vector paATI using the synthetic
transfection agent Transfectam. 20 hours later the cells were placed on differentiating medium,
which induced receptor synthesis. 48 hours later the cells were harvested, homogenized and
centrifuged to separate the membrane fraction. Radioligand binding assay was performed
using '2I-Sar-Ile-Angll. The membrane bound ligand was separated by filtering the reaction
through a Whatman filter paper (*:p<0.05, one-way ANOVA, Duncan multiple range test;
Figure 16. A representative figure showing that infection of paATl-transfected cells with
adenovirus further decreased AnglI receptor binding. Adenovirus (2-3/cell) was added to the
differentiating medium of NG108-15 cells for 48 hours following transfection with paAT1.
Adenovirus (Ad), a helper virus for adeno-associated virus (AAV) enhances the activity of
AAVs p40 promoter which drives the expression of the antisense mRNA. Receptor binding
was performed as described above, using Losartan and PD123319, the AT-1 and AT-2 specific
receptor inhibitors to differentiate between AnglI receptor subtypes.
effect, but we used them as markers for monitoring AAV transcriptional activity. Following
transfection with paATI and adenovirus as described above, NG108-15 cells were incubated
with the rep antibody IF811 and stained with diaminobenzidine (DAB). For controls,
uninfected cells, Transfectamn-only treated cells, control plasmid plus Transfectam treated cells
and paATI transfected cells without adenovirus were also stained. As a control vector, pTR-
UF, an AAV-derived expression vector lacking the rep gene was used.
Cells infected with paATI and adenovirus showed strong immunoreactivity in the
nucleus. Approximately 10-20% of the cells were stained with the rep antibody (Fig. 17).
Uninfected controls were devoid of any staining, whereas Transfectam-treated or control
vector treated cells showed uniform, light nonspecific staining. Cells transfected with paATI
but without adenovirus did not show specific staining.
Inhibition of angiotensinogen synthesis by antisense vector
It was shown in our laboratory that antisense oligonucleotides targeted to
angiotensinogen mRNA decrease angiotensinogen synthesis in the hypothalamus and decrease
the mean arterial pressure (190). An antisense expression vector was constructed by inserting
the complete cDNA of the angiotensinogen gene into the AAV-derived expression vector
pJDT95 in the antisense direction (Fig 18). The resulting construct (paAo) was tested on the
hepatoma cell line H-4 for inhibition of angiotensinogen production. 1 gg or 2 pgg of the
vector DNA was mixed with 10 1t or 20 jgl Lipofectin, a cationic transfection agent and
incubated with the H-4 cells for 20 hours. After the transfection medium was removed, cells
^ ~'tril ,*
Figure 17. Immunocytochemical staining of the viral rep protein in NG108-15 cells following
liposomal gene delivery. NG108-15 cells were transfected with 15 gg paATI antisense vector
DNA mixed with 30 gl Transfectamn and tsl49 adenovirus (2-3 adenovirus/cell). Two days
after the transfection the cells were fixed and stained with the rep antibody IF811.
Hind III, Acc65 (Kpn I)
Hind III, Acc65 I (Kpn 1)
Hind III, Acc65 I (Kpn I)
Figure 18. Construction of the angiotensinogen antisense expression vector paAo
(pJDT95aAo). The adeno-associated virus derived expression vector pJDT95 and the plasmid
pGEM4 carrying the complete angiotensinogen cDNA was digested with Hindll and Acc65I.
The digestion products were separated on low melting gel and the larger fragment of pJDT95
and the smaller fragment ofpGEM4Ao were in-gel ligated with T4 ligase.
were incubated for an additional 72 hours in EMEM supplemented with insulin, transferrin and
selenium. At the end of the incubation period the medium was collected for radioimmunassay.
The amount of secreted angiotensinogen decreased in the cells transfected with 2 jig
paAo plus 20 jig Lipofectin to 15+2.2 pg/mg protein whereas the cells transfected with similar
amounts of Lipofectin alone secreted 22.9+5 pg/mg protein. 1 jig paAo completed with 10 g1
Lipofectin showed less inhibition in angiotensinogen production (Fig 19).
Transfection of SHR brains with antisense vector
SHRs were equipped with indwelling guide cannulas to the median preoptic area of the
anterior hypothalamus. After baseline blood pressure recordings, one rat was injected with 1
gg paATI mixed with 2 jil Transfectam and 105 adenovirus (ts149). The control rats received
the same dose of Transfectam and ts149 with or without control plasmid DNA (pJDT95dlk).
Systolic blood pressure was measured again three days after treatment with tail cuff blood
pressure recorder. After sacrificing the rats, the brains were removed and processed for
immunohistochemistry using the rep antibody. A limited number of cells along the cannula
tract in the cortex and the septum of the vector-treated (paATI and pJDT95dlk) rat showed
positive immunostaining (Fig. 20). There was no change in systolic blood pressure in the
antisense vector treated rat (181.5 mmHg pre-treatment vs. 185 mmHg post-treatment).
control Lipo (10) Lipo (20) aAo (1) aAo (2)
Figure 19. Transfection of H-4 hepatoma cells with the angiotensinogen antisense expression
vector paAo resulted in 30-34% decrease in angiotensinogen production. Hepatoma cells were
grown to 50-60%o confluence in EMEM+10%FBS+10%/oCS. 1 jg or 2 gg vector DNA was
mixed with 10 pIl or 20 gl Lipofectin and overlaid onto cells in serum-free EMEM for 20
hours. After the incubation period the cells were maintained in EMEM+ITS (insulin-
transferrin-sodium-selenite serum supplement) for 72 hours, after which the medium was
collected for angiotensinogen radioimmunoassay.
Figure 20. Immunohistochemical localization of the rep protein in SHR brains transfected with
paATl. 1 gg of the AT-1 antisense expression vector paATI was completed with 2 pl
Transfectam and mixed with 105 adenovirus. SHRs were injected in the median preoptic area.
Three days later the animals were sacrificed, and 40 pm thick brain section were processed for
Summary of the Results
1. Both phosphodiester and phosphorothioate antisense oligonucleotides effectively
decreased high blood pressure in SHRs when injected i.c.v. The effect of a single dose of
phosphorothioate oligonucleotide lasted for 7 days.
2. Membrane receptor binding revealed significant decrease in AT-1 receptor binding in
the hypothalamic region, whereas AT-2 receptor binding remained unaffected.
Autoradiography showed that the most affected areas were in the anterior hypothalamus: the
paraventricular nucleus, suprachiasmatic nucleus and the median preoptic nucleus.
3. Antisense oligonucleotide treatment markedly inhibited drinking response of AT-I
receptors to i.c.v. AnglI injection.
4. Oligonucleotides diffused throughout the ventricular system and into the brain tissue
from the ventricles within 1 hour.
5. Antisense oligonucleotides decreased receptor binding in cultured NG108-15 cells.
6. There was no change in the amount or in the length of the AT-1 mRNA in NG108-15
cells following antisense oligonucleotide treatment at doses which were effective in decreasing
7. Oligonucleotides are taken up by NG108-15 cells rapidly, and the majority of them are
transported to the nucleus within 6 hours.
8. Antisense expression vectors can decrease AT-1 receptor and angiotensinogen
synthesis in cultured cells.
9. Liposomal delivery of the antisense vector to the rat brain was insufficient in eliciting
any change in systolic blood pressure.
Antisense Oligonucleotide Design
At the time of the writing of this manuscript the theory of antisense oligonucleotide
design has not been completely worked out (191). This is probably a direct consequence of the
fact that the question of the mechanism of action of antisense oligonucleotides is also a matter
of debate: several potential action sites exist for antisense, and it is not unlikely that different
mechanisms will be active under different circumstances.
When our project started, however, even less was known about how to use antisense
molecules, and to our knowledge, there was but one published article on applying antisense
oligonucleotides to living animals (177). Therefore we started out testing different
oligonucleotides, including the one which followed the most common example, the antisense
oligonucleotide targeting the initiation codon AUG and neighboring nucleotides on the AT-1
mRNA, as well as the one which is used in most of the experiments and targeted bases +63 to
+77 downstream to the initiation codon.
Even though our initial screening of antisense oligonucleotides was not entirely
systematic, the picture emerged that multiple injections of the phosphodiester antisense
oligonucleotide was needed to elicit a response similar to that of a single injection of the
phosphorothioated oligonucleotide. This effect was most likely to be due to the shorter half-
life of the phosphodiester oligonucleotide (141). On the other hand, the two antisense oligos
targeting different areas on the AT-1 mRNA appeared to be similar in their ability to decrease
high blood pressure.
Lowering Blood Pressure with Antisense Oligonucleotides
Blood pressure measurements have been a favorite subject for physiologists, since it
provides numerical data on an essential life function, and it is the outcome of the interplay
between several biological systems. Our system of interest was the brain and its influence on
Our result of lowering the blood pressure of SHRs by i.c.v. injection lends itself to the
explanation that the AT-1 antisense oligonucleotides entered brain cells, bound to AT-1
mRNA which resulted in decreased AT-1 receptor synthesis, the consequence of which was a
decreased effectiveness of brain AngII and, eventually, lower blood pressure.
Let's examine this proposed chain of events step-by-step:
Our results show that 1 hour after the injection of 50 gg/4 gl FITC-labeled
oligonucleotide into the right lateral ventricle there was fluorescent signal in the wall of both
lateral ventricles as well as around the third ventricle, and the signal could be followed to about
2 mm into the tissue. We detected signal around the fourth ventricle as well as in the cerebral
and cerebellar cortex, although with decreased density.
This spread of the oligonucleotides seems to be sufficient, since most of the AT-1-
containing brain nuclei are located around the ventricular system. We have also identified
individual cells showing fluorescent signal by confocal microscopy (190). That the FITC label
remains attached to the oligonucleotide after entering the cell has been shown by others in our
laboratory (B. Kimura, personal communication). Other investigators using biotin-labeled
oligonucleotides reported similar distribution in the brain after i.c.v. injection (192).
Inhibition of AT-i mRNA
Our investigations of the effects of antisense on its target mRNA focused on
measurements of the amount and the size of AT-1 mRNA. Our northern blots did not show
decrease in the amount or in the size of the AT-1 mRNA. This suggested to us that the target
mRNA was unlikely to be degraded or cleaved upon antisense treatment, but probably other
mechanisms were involved in inhibiting normal translation (see below). Direct measurements
of the binding of antisense oligonudcleotide to AT-1 mRNA (e.g. nuclease protection assay)
were not conducted.
Inhibition of AT-1 Protein Synthesis
Receptor binding on freshly prepared hypothalamic-thalamic cell membranes supported
the notion that the number of AT-1 binding sites was decreased after i.c.v. antisense injection.
The effect appeared to be specific to the AT-1 receptor subtype since the AT-2 receptors
remained unaffected. The single-point binding assays performed do not typically allow one to
draw numerical conclusions about the number of binding sites, but since it was not anticipated
that antisense treatment would in any way change receptor affinity, we concluded that the
decreases seen in our assay were proportional to the decrease of the number of AT-1
We employed autoradiography to localize the brain areas affected by the antisense
injections. Besides lateral ventricular injections we tested the effect of antisense
oligonucleotides injected into the third ventricle. Although not statistically significant, the
results suggested that the decreases were localized in the anterior hypothalamus and
anteroventral third ventricle (AV3V), the most prominent decrease being detected in the PVN.
Whereas both sets of experiments, the one using third ventricle and the other using
lateral ventricle injections, suggested a decrease in the AV3V region, they differ in that after
third ventricular injections the receptor number in the OVLT tended to be decreased, whereas
after lateral ventricular injections a decrease was seen in the MnPO. A possible explanation for
this difference might be that the oligonucleotides had better access to the OVLT after injection
in the third ventricle than after lateral ventricular injections. An even more striking difference
occurred in another circumventricular organ, the SFO, where all injected oligonucleotides
tended to increase the AT-1 receptor number after being injected into the third ventricle. Since
this tendency for increase was seen with all oligonucleotides including antisense, sense and
scrambled, it appeared to be non-specific. Finally, the results of the autoradiography suggested
that some decrease, although to a variable extent, may have also occurred in the SCN and
AT-1 receptors in sites that were farther away from the ventricles, such as piriform
cortex or NTS in the brainstem showed no tendency to be decreased by antisense, thus
suggesting a rather localized action of antisense oligonucleotides. This is in agreement with the
experiments showing relatively restricted distribution and tissue penetration of fluorescent
labeled oligonucleotides. In nuclei containing exclusively AT-2 receptors (mediodorsal
thalamic nucleus, lateral septum or inferior olive), there was also no sign of decrease by
antisense treatment. This might be attributed to the specificity of the antisense inhibition to the