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© 1995 American Heart Association, Inc.
Articles |
c-myc in Vasculoproliferative Disease
From Harvard University–Massachusetts Institute of Technology, Division of Health Sciences and Technology (E.R.E.), Cambridge; the Department of Medicine (E.R.E., M.S., R.D.R.), Harvard Medical School, Boston, Mass; and the Department of Biology (M.S., M.G.S., R.D.R.), Massachusetts Institute of Technology, Cambridge.
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Abstract |
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Abstract Antisense oligonucleotides to genes central to cellular proliferation have suppressed smooth muscle cell growth in vitro and in vivo. We now report that although the response of cultured smooth muscle cells to antisense oligonucleotides to c-myc and c-myb is identical, the response of the injured arterial wall to these oligomers depends on the kinetics of gene expression and oligonucleotide delivery. Two different antisense oligonucleotides to each oncogene were administered to the perivascular aspect of injured rat carotid arteries via polymer-based delivery systems. The acute release of antisense oligonucleotides from the Pluronic gels reduced in vitro cell growth 54.8% with c-myc and 56.9% with c-myb. The more sustained release from ethylene vinyl acetate copolymer (EVAc) matrices was slightly less efficient, inhibiting proliferation 47.3% and 43.3%, respectively. However, although both EVAc and Pluronic release of c-myb antisense oligonucleotide sequences inhibited intimal hyperplasia 2 weeks after injury, only the more prolonged EVAc matrix release of antisense oligonucleotide to c-myc was effective. The failure of the short course of c-myc oligomer release from Pluronic gels stemmed from early successful suppression with late loss of regulation and not from inactivation of the antisense oligonucleotide within the polymeric gel. Within 24 hours of injury, Pluronic-based release of c-myc antisense oligomers reduced mRNA levels in the tunica media 2.5-fold and immunocytochemical identification of c-myc expression by 98.8%. As a result, the number of proliferating cells was decreased 6.5-fold 3 days after injury. One week after injury, however, the effect of Pluronic gel–released c-myc antisense was lost on both the number of cells expressing the oncogene (only 29.6% suppression) and the extent of intimal hyperplasia (intimal-to-medial area ratio, 0.35±0.02 versus 0.5±0.1 in control arteries). In contrast, EVAc release of the same sequences obliterated protein expression, maintaining a 99.6±0.7% inhibition for the duration of the experiment, and reduced intimal hyperplasia 2 weeks after injury 11.1-fold. We conclude that the effectiveness of the antisense approach to suppression of neointimal proliferation depends on the time course of expression of the target gene as well as the kinetics of oligonucleotide delivery.
Key Words: antisense oligonucleotides • c-myb • c-myc • restenosis • polymer-based drug delivery
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The common theme of the accelerated arteriopathies that follow angioplasty, vascular bypass, or even organ transplantation is the accumulation of arterial wall smooth muscle cells within the intimal monolayer.1 2 3 4 A multitude of factors and mediators contribute to migration and proliferation of smooth muscle cells,5 including various growth factors, cytokines, the angiotensin axis, calcium, processes of oxidation and glycation, lipid infiltration, and cell-specific effects of leukocytes, monocytes, and platelets. Increasingly, the molecular controls of these and other events are being identified.
Two parallel lines of investigation have identified the extent and nature of this genetic control. In the first set of studies, a number of investigators have correlated the expression of various genes with specific cell-proliferative events. In this manner, gene expression has been temporally related to cell cycling and divided into three groups.6 The first group consists of immediate-early genes and includes the most quickly induced genes, such as c-myc, KC, JE, A21, L51, and the ornithine decarboxylase gene. These genes are thought to be related to the generation of second messengers resulting from growth factor–receptor interactions, and their expression is independent of protein synthesis. By virtue of their central importance to the initiation of cell events, they have been also classified as competence genes.7 Delayed-early genes represent a second group that does require new protein synthesis and, perhaps, the transcriptional regulation by one of the immediate-early genes. 2F1 and M11 are examples from this group. Late-G1 genes are induced at the terminal phase of G1, or the beginning of the S phase. Genes that encode for c-myb, thymidine kinase, or proliferating cell nuclear antigen fall into this category. In a second set of experiments designed to elucidate genetic control of vascular smooth muscle cell proliferation, selective inhibition of gene expression or inactivation of protein function has been used. Antisense oligonucleotides, generated to inhibit expression of specific genes, have successfully reduced and virtually eliminated smooth muscle cell proliferation in culture8 9 10 and smooth muscle cell accumulation in vivo.11
Both c-myc and c-myb have been identified as crucial to the control of smooth muscle cell growth in vitro.7 8 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 However, there is little information regarding the function of these genes in vivo. We report in the present study that although the responses of cultured vascular smooth muscle cells to synthetic antisense oligonucleotides to c-myc and c-myb are identical, the responses of arterial wall smooth muscle cells to these oligomers depend on the mode with which the oligonucleotides are delivered to the blood vessel wall. This may reflect the difference in the extent of gene expression or the time course over which these genes act. Our results indicate that an in-depth understanding of the kinetics of gene expression may be crucial to fully delineating the pathobiology of vascular injury and repair.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The two antisense phosphorothioate c-myc oligonucleotides (antisense 1 [GAA GCT CAC GTT GAG GGG], nucleotides 4 to 21 of rat c-myc; antisense 2 [TCA TAG TTC CTG TTA], nucleotides 22 to 36 of rat c-myc), the two c-myb oligonucleotides (antisense 1 [GTG TCG GGG TCT CCG GGC], nucleotides 4 to 21 of rat c-myb; antisense 2 [GCT GTG TCG GGG TCT CCG], nucleotides 22 to 39 of rat c-myb), and the scrambled control phosphorothioate c-myc and c-myb oligonucleotides (CTC GTA TTC ATA ATT GCC and CTG CGT TGG CGT GCG GCG) were synthesized in the Massachusetts Institute of Technology Biopolymers Laboratory. The oligonucleotides were deprotected on the column, dried down, resuspended in Tris-EDTA (10 mmol Tris [pH 7.4] and 1 mmol EDTA [pH 8.0]), and quantified by spectrophotometry.
Polymer Matrices
Oligonucleotides were embedded within EVAc-controlled release
matrices or Pluronic gels for the controlled release of these
compounds.
EVAc
As previously described,27 28 oligonucleotides were
combined with bovine serum albumin (BSA) at a ratio of 1:100 and then
mixed with a solution of ethylene vinyl acetate copolymer (EVAc, Dupont
Co) dissolved in dichloromethane (10% [wt/vol]) to achieve a final
ratio of 33% (wt/wt). The drug-polymer suspension was poured into
precooled glass molds, removed after hardening, and placed at -20°C
and then under vacuum (600 mtorr) for 2 days each. The resultant matrix
was a homogeneous dispersion of drug within a porous network of
EVAc.27 Smaller pellets were cut from the larger slabs and
coated with four layers of EVAc. Drug release was restrained to emanate
from a hole in the coating and near zero–order kinetics obtained in
this fashion.28 29 We have used these matrices extensively
in a range of in vitro and in vivo systems.28 29 30 31 32 33
Pluronic Gels
In other experiments, more rapid first-order release was
obtained when the oligonucleotides were mixed with the poloxamer
Pluronic F-127 gel (BASF Wyandotte Corp) and then laid over the exposed
artery.11 Oligonucleotide was added to a 25% (wt/vol)
solution of the gel at a concentration of 5 mg/mL and stored at 4°C.
After injury, 200 µL of the mixture was applied over the artery where
it hardened in situ.
In Vitro Controlled Release Kinetics
The kinetics of release from the Pluronic gels and EVAc matrices
was followed spectrophotometrically. Five identical gels or matrices
were placed in glass scintillation vials filled with 5 mL sterile
saline, which was removed in its entirety at set periods of time. The
vials were mounted on a shaking platform residing at 37°C. The
optical density of the saline solution was determined at 220 and 280
nm, and oligonucleotide release was extrapolated from the ratio of
oligomer to BSA as previously described.27 28 29
Effects on Smooth Muscle Cells in Culture
The effects of the oligonucleotides in culture were measured on
vascular SV40LT smooth muscle cells.34 Cells were cultured
in DMEM supplemented with 10% heat-inactivated (65°C, 45 minutes)
fetal bovine serum (10% FBS-DMEM). Cells were plated at
7.5x104 cells per well in a Belco cluster 12-well plate
and allowed to attach in 10% FBS-DMEM. After attachment, cells were
washed twice with phosphate-buffered saline (PBS) and growth-arrested
by replacing the medium with 0.5% FBS-DMEM. Cells were kept
growth-arrested for 96 hours and then released from G0 by
addition of 10% FBS-DMEM. Cells were simultaneously exposed to
antisense or control oligonucleotides released from EVAc matrices or
Pluronic gel. After an additional 72 hours, the cells were washed with
PBS and released from the dish by trypsinization, and cell number was
determined with a Coulter counter. Each data point is presented as
mean±SEM for three separate experiments performed with triplicate
wells at each dose.
Arterial Injury and Application of Oligomers
Endothelial denudation of the left common carotid artery in male
Sprague-Dawley rats (300 to 500 g, Charles River Breeding
Laboratories, Kingston, Mass) was performed with a 2F Fogarty balloon
catheter (American Edwards Laboratories).35 Rats were
anesthetized with intraperitoneal ketamine (0.04 mg/g body wt) and
xylazine (0.02 mg/g body wt). A midline incision exposed the distal
left common and external carotid arteries. The balloon catheter was
introduced into the external carotid artery and passed three times with
the balloon distended sufficiently with air to generate slight
resistance. On removal of the catheter, the external carotid artery was
ligated.
The wounds were bathed clean with sterile saline and wiped dry with a sterile cotton swab, and the controlled release devices containing the oligomers were placed around the artery as previously described.11 28
Tissue Processing and Analysis
Immediately after injury and on postoperative days 1, 3, 7, and
14, animals were euthanatized and perfused via the left ventricle with
Ringer's lactate solution. The carotid arteries were isolated,
removed, cut into three equal segments, and immersion-fixed in
Carnoy's fixative (60% methanol, 30% chloroform, and 10% glacial
acetic acid) or 10% formalin. The location of the implanted devices
was noted, and the devices were recovered with the arteries. The
arterial segments were paraffin-embedded and microtome-sectioned. Eight
to 12 sections along the length of each segment were obtained and
stained with hematoxylin/eosin or Verhoeff's elastin stain. The
intimal, medial, and adventitial areas, the ratio of the intimal to
medial area, and the percentage of luminal occlusion were calculated
for each arterial segment by using computerized digital planimetry with
a dedicated videomicroscope and individualized software. The averages
of all sections and segments were used for comparison. Edge-detection
software was further used to detect cell number within 8 to 32 sections
per media or intima and, when combined with area data, used to
determine cell density. All analyses were confirmed by visual
inspection, and the accuracy of the system was verified with a series
of matched manual cell-density determinations. Analysis was carried out
by an investigator blinded to the nature of the specimens.
Immunocytochemistry
Cell proliferation was followed by injecting the thymidine
analogue 5-bromo-2'-deoxyuridine (BrdU, New England Nuclear, Dupont
Corp) intraperitoneally, at 50 mg/kg, 1 hour before death.
Intracellular BrdU was identified immunocytochemically as previously
described by using a mouse IgG anti-BrdU antibody (New England Nuclear,
Dupont Corp) to BrdU diluted 1:50 and exposed for 60 minutes. The
expression of c-myc was similarly detected by using a mouse
monoclonal antibody to c-myc (Cambridge Research
Biochemicals). The sensitivity of this assay was examined by using a
range of dilutions from 1:50 to 1:2000, exposed for 30, 60, and 90
minutes. Antibody visualization was achieved with avidin-peroxidase
complex (Vector Laboratories)–3,3'-diaminobenzidine (Sigma Chemical
Co) reaction. The number of antibody-positive cells per unit area and
as a fraction of the total number of cells was determined by an
investigator blinded to the nature of the specimens.
mRNA Analysis
mRNA levels were determined from whole native and injured
arteries harvested 24 hours after balloon denudation. After
exsanguination and perfusion with normal saline as described above, the
arteries were snap-frozen in liquid nitrogen and pulverized in a
stainless steel chamber cooled in liquid nitrogen. The powder was taken
up in guanidine thiocyanate and sonicated. Total RNA was extracted by
using a Stratagene extraction kit (Stratagene Cloning Systems), and 20
µg per well of total RNA was loaded on a Hybond-N nylon membrane
(Amersham) by using a Slot-Blot apparatus (Schleicher & Schuell). The
RNA was cross-linked with ultraviolet radiation (Stratalinker,
Stratagene Cloning Systems) and prehybridized at 65°C with QuikHyb
solution (Stratagene Cloning Systems) supplemented with 100 µg/mL
sonicated salmon sperm DNA for 2 hours. Murine c-myc coding
sequence (courtesy of Dr E. Ruley, Vanderbilt University) and human
ß-actin cDNA were radiolabeled to a specific activity of
109 cpm/µg by using a Boehringer-Mannheim random priming
kit, purified on G-25 spin columns (Boehringer-Mannheim), and added to
the QuikHyb solution. The hybridization was carried out at 65°C for 2
hours. The blots were then washed in 2x standard saline citrate
(SSC)/0.1% sodium dodecyl sulfate (SDS) solution at room temperature
and twice in 0.2x SSC/1.0% SDS solution at 65°C, visualized by
autoradiography, and quantified by a Betascope 603 blot analyzer
(Betagen).
Statistics
Data are presented as mean±SEM. Statistical comparisons
were performed with nonpaired t tests for groups of unequal
sample sizes, and data were rejected as not significantly different if
values of P>.05 were observed.
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Results |
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Controlled Release of Oligonucleotides
Both polymeric systems used in the present study sustained the release of the oligonucleotides. However, the kinetics of release was markedly different between these two systems. Pluronic poloxamer gels are hydrophilic and rapidly degrade in an aqueous environment. Thus, release from Pluronic gels was complete within hours (Fig 1
). In contrast, EVAc is a stable hydrophobic material,
and when drug is incorporated within matrices of this material, release
was sustained for weeks (Fig 1).
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) or ethylene vinyl
acetate copolymer (EVAc) matrices (
) was determined
spectrophotometrically as described in the text. The cumulative
percentage of the total amount of oligonucleotide incorporated within
the devices released is plotted against time for both formulations.
Although release from the hydrophilic and biodegradable poloxamer
proceeds to completion within hours, a more prolonged pattern is
observed from the matrices of the nondegradable hydrophobic EVAc
matrices.
Tissue Culture Effects of Antisense to c-myb and
c-myc
Previous experiments have documented that the addition of
antisense oligonucleotides to both c-myb8 and
c-myc10 26 36 directly to culture media
suppressed vascular smooth muscle cell proliferation in vitro.
Consequently, we tested the growth-inhibitory ability of polymer-based
delivery of oligonucleotides. The controlled release of antisense
oligonucleotides to both c-myb and c-myc
inhibited SMC proliferation to a similar extent in culture (Fig 2). At 72 hours after exposure to both antisense
sequences (antisense 1 and 2) for c-myc or c-myb,
the cell number was significantly reduced. The data reproduced for
antisense 1 in Fig 2 reveal a 54.8% decrease in cell number after the
acute release of antisense from the Pluronic gels for c-myc
and a 56.9% decrease for c-myb. EVAc matrix release of
these same oligonucleotides was slightly less efficient, inhibiting
proliferation 47.3% and 43.3%, respectively. All of these values were
statistically significantly reduced from control (P<.0001),
whereas scrambled oligonucleotides had no effect on proliferation.
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In Vivo Effects of Antisense c-myb and
c-myc Oligonucleotides
In a previous investigation, we demonstrated that Pluronic
gel–based delivery of antisense c-myb oligonucleotides
suppressed smooth muscle cell accumulation in the neointima
2 weeks after arterial injury.11 Although both EVAc and
Pluronic release of antisense oligonucleotide to c-myb
inhibited intimal hyperplasia 2 weeks after injury, only the more
prolonged EVAc matrix release of antisense oligonucleotide to
c-myc was effective in this regard (Fig 3,
left). Although the differences are most apparent at 2
weeks, Pluronic gel–based delivery of antisense c-myc was
already ineffective 7 days after application. Pluronic gel and EVAc
matrix delivery of c-myb, on the other hand, maintained
their effectiveness (Fig 3, right). At 1 week, neointimal
thickening in the animals treated with c-myc antisense
released from Pluronic gels was 7.3 times that seen in similar animals
exposed to the antisense when released from EVAc matrices, and at 2
weeks, this difference still remained at 6.5-fold. The same response
was noted for both antisense oligonucleotides to each of the genes,
supporting the sequence specificity of the effect. Release of two
different scrambled sequences did not have an inhibitory effect.
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Verification of Viability of Pluronic-Based Release of
c-myc Antisense Sequence
The absence of effect with Pluronic-based release might have
occurred because the oligomer was inactivated in the course of
dissolution within the Pluronic gel. To determine whether this was the
case, we examined the biological effect of the release of sense and
antisense oligonucleotides on c-myc mRNA expression, protein
synthesis, and cell proliferation in four animals 24 hours after
denuding arterial injury. Quantitative mRNA analysis showed that
the Pluronic-based release of antisense c-myc oligomers
resulted in a 2.5-fold reduction in c-myc mRNA expression
normalized for ß-actin control levels 24 hours after injury
(3464±101 cpm [scrambled control–treated animals] versus 1386±768
cpm [antisense-treated animals], P<.04, data not shown).
Hybridization with a ß-actin probe demonstrated relatively equal RNA
loading in both groups. Alteration in mRNA expression was accompanied
by a comparable 3.7-fold reduction in immunocytochemical identification
of c-myc expression within the media at the same time point
and a 6.5-fold decrease in cellular proliferation as determined by BrdU
uptake 3 days later.
Time Course of c-myc Expression and Antisense
Regulation In Vivo
Given the retention of biological activity of the released
antisense strand over the course of its release from the Pluronic gel
and its specific effects in tissue culture (Fig 2
), the short course of
c-myc antisense oligonucleotide administration might not
suppress intimal hyperplasia if the continued expression of this gene
is continued long after the initial injury and termination of therapy.
There are certainly tissue culture data to support this
view6 12 14 15 17 18 22 and even some data involving
animal models of vascular injury similar to our own.37
Therefore, we examined c-myc expression over the course of
the entire experiment. Six arterial sections were taken from the length
of the denuded carotid arteries in four animals subjected to denuding
arterial injury and exposed to the short course of Pluronic gel release
of antisense 1 c-myc oligonucleotide or the more prolonged
continuous administration of the oligomer from EVAc matrices. Sense
sequences were released from the EVAc matrices in a set of control
animals. As depicted in Fig 4, c-myc
expression was virtually undetectable at baseline in noninjured
arteries. After vascular injury, protein levels rose and peaked at 7
days. The continuous EVAc matrix administration of antisense
oligonucleotide to c-myc suppressed gene expression. The
minor rise at day 7 may indicate that the maximum signal for protein
synthesis might break through even this form of oligomer release,
although it remained at least 14 times lower than values in the control
animals (Fig 4). Pluronic gel release suppressed gene expression in the
first day but was progressively less effective with time. By day 14,
protein expression in arterial sections from animals treated with this
form of therapy actually exceeded that observed in the control
animals.
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Discussion |
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Both c-myb and c-myc are nuclear proto-oncogenes deemed to play a role in the proliferation of vascular smooth muscle cells in culture.7 8 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Although we found that antisense suppression of expression of these proto-oncogenes yielded similar effects on vascular smooth muscle cell proliferation in culture (Fig 2
), in vivo potency was very much dependent on the way in
which the compounds were delivered to the blood vessel wall (Figs 3 and 4). Antisense c-myb oligonucleotides inhibited smooth muscle
cell accumulation within the blood vessel wall whether they were
administered for only the first few hours after injury from Pluronic
gels or for the full duration of the animal's life after injury when
released from EVAc matrices. c-myc oligonucleotides
inhibited cultured smooth muscle proliferation to an extent equal to
that of their c-myb counterparts. Yet although the transient
release of these same oligonucleotides reduced vascular wall
c-myc expression 24 hours after administration and medial
smooth muscle cell proliferation at 72 hours, by 2 weeks no overall
effect on intimal hyperplasia was noted (Fig 3). It was not until the
oligomers were released for a much longer period of time (Fig 1) that
prolonged suppression of c-myc expression was noted (Fig 4)
and reduction of intimal hyperplasia was achieved (Fig 3). The effect
of both c-myb and c-myc oligonucleotides is
sequence specific, as demonstrated by similar results with two
different antisense sequences and the absence of effect with scrambled
control sequences.
Single-Dose Versus Continuous Administration
Antisense oligonucleotides to c-myc have been used to
discern important mechanisms of cell cycling,12 14 23
proliferation,10 23 24 26 36 38 and
migration10 39 in a diverse set of cells and disease
states.10 12 14 23 24 26 36 38 39 40 41 Literature is already
accumulating involving the role of c-myc in vascular disease
as well, and these experiences mirror our own. Bennett et
al26 examined the effects of antisense c-myc
oligonucleotide complementary to the first five codons of the human
c-myc mRNA on the growth of vascular smooth muscles in vitro
and in the denuded rat artery.26 This oligomer halted
smooth muscle cell proliferation in culture 48-fold but only reduced
intimal hyperplasia by 
44±13% when released from Pluronic gel in
the perivascular space of injured blood vessels.26
Although the antisense sequence used was different from our own, the in
vivo effects were similar to those we observed. Pluronic gel release of
our antisense oligomers to c-myc only inhibited 30.0±1.7%
of the proliferation 1 week after denuding injury (Fig 3). However, we
also noted that although the continuous EVAc matrix release of the
antisense oligomer was only modestly effective in culture (Fig 2), this
method of administration provided a dramatic reduction in intimal
hyperplasia (Fig 3). The overall effect 2 weeks after injury was almost
twice as great as the early effects on cell proliferation, implying a
continued effect. Pluronic gel release inhibited the first phase of
smooth muscle cell proliferation 6.3-fold, and EVAc release reduced
overall intimal hyperplasia 11.1-fold. The added effect from continuous
release emphasizes the potential continuous role of c-myc in
smooth muscle proliferation during the latter phases of vascular injury
(Fig 4) and may have had an even greater effect if used with Bennett's
sequence as well.26
The expression of c-myc beyond the immediate postinjury
period (Fig 4) is an important, but not unexpected, finding. Indeed, it
has been shown that although c-myb is expressed early and
transiently in the course of the proliferation of vascular smooth
muscle cells, c-myc has a more prolonged and cyclic
expression.6 12 14 22 Gadeau et al6
demonstrated that the mRNA level of c-myc began to increase
30 minutes after serum stimulation, reached its maximum 30 to 60
minutes after that, declined until the 6th hour, and then fluctuated
during further progression through the cell cycle. Other studies have
shown that as cells proceed through the cell cycle, c-myc
continues to be expressed at a constant level15 17 18 and
may even be reinduced at the end of the first cycle.6
These same effects were noted in the wall of balloon-denuded artery:
Miano et al37 showed a biphasic pattern of expression with
peaks at both 6 hours and 7 days after arterial injury. The
reactivation of c-myc at 1 week coincides with the rapid
phase of intimal hyperplasia. c-myb, on the other hand, is
initially expressed much later after cell proliferation. Its peak
expression is not noted until 18 hours after injury and returns to
baseline within an additional 6 hours. Thus, it is possible that only
early inhibition of c-myb expression is required to block
its full effect, whereas continued therapy is required for
c-myc.
There are further data that would argue against the efficiency of a single dose or brief exposures of cells and tissues to the oligonucleotide. Two separate groups have shown that cultured smooth muscle cells begin to proliferate normally after exchange of the media containing antisense oligomers for fresh media devoid of the antisense sequences.9 42 A single dose of c-myc antisense oligonucleotide had no effect on the growth of smooth muscle cells virally transfected with human c-myc cDNA and constitutively expressing the protein.26 This suggested to the authors that elevated RNA levels can titrate out the growth-inhibitory effect of the antisense oligonucleotide and lends further support to the sequence-specific interaction of the oligomer with the complementary mRNA strand.26 It is possible that continuous release of any of the antisense oligomers used would inhibit growth of even these cells. Dose-response experiments could examine a part of this question. If increased amounts of one oligonucleotide could achieve the same effects as lesser amounts of another, then one could attribute the variability in response to dose and potency alone. The use of perivascular release precludes this experiment. Less than 0.1% of the drug that is administered from this region of the blood vessel comes in contact with the blood vessel wall; the remainder is diluted within the perivascular space and rest of the body.43 Alterations in dose do little to change the amount deposited. It appears that increased amounts of delivered compound simply lead to increased amount lost systemically.
The final important note to be made in comparing our data with data of Bennett et al26 is that there is a difference in overall effect both in culture and in vivo. A variety of possibilities arise to explain this discrepancy, and all of them further highlight the complexity of working with these compounds and the lack of full understanding of the basic biological mechanisms behind vascular injury and repair. The use by Bennett et al of an oligonucleotide different from ours is critical. Epstein and colleagues9 10 44 have demonstrated that only a two- or three-base shift in the antisense target can be critical to whether the gene is effectively inhibited by the oligonucleotide. They noted that target dependence may be related to secondary structures of the mRNA strand, which could interfere with hybridization of the oligomer with the corresponding mRNA target, changes in cellular uptake efficiency, variability of the ability of the ribosome to read through the mRNA:oligonucleotide duplex, or differences in resistance to degradation.9 It is quite possible that sequence differences could lead to differences in uptake and/or retention in the blood vessel wall. Along these lines it is interesting to note that at the dose equivalent to what was used in our present study, Bennett et al demonstrated a far more significant inhibition of proliferation of rat aortic smooth muscle cells in culture than the 1.8-fold inhibition that we observed but only a slightly greater effect in vivo.
Clinical Significance
Antisense oligonucleotides might be used as a therapeutic modality
of significance for the complex lesions of the human accelerated
arteriopathies, which include restenosis after angioplasty, bypass
surgery, and organ transplantation, if these oligomers can be
administered in an efficient and expeditious manner. Thus, the
requirement for continued therapy with c-myc beyond the
initial dosing is clinically disturbing as well as scientifically
intriguing. Although it is interesting from a fundamental point of view
that the single-dose suppression of some proto-oncogenes can forever
prevent cell cycling23 26 45 and other genes may require
more prolonged inhibition to achieve the same effect, on a practical
level any need for prolonged and/or continuous therapy greatly
complicates potential clinical uses. There is increasing evidence that
single-dose therapy fails for other classes of drugs proposed to combat
proliferative vascular disease. We recently demonstrated that although
continuous intravenous or perivascular administration of heparin
virtually obliterated intimal hyperplasia after vascular injury,
periodic administration of the same heparin compound at the same total
dose made injury worse.33 The latter mode of
administration is precisely how heparin was provided in the clinical
trials that failed to demonstrate clinical benefit after vascular
injury,46 47 and when animals were treated as humans were
in the clinical trials, vascular injury was exacerbated, not
ameliorated.33
It would be logistically prohibitive to require continuous or periodic dosing in humans. The technology of implantable devices has yet to reach the stage where prolonged continuous delivery from these types of devices is possible, and repeated injections are doomed to noncompliance and failure. Moreover, implantable devices are problematic in the setting of percutaneous procedures. The kind of innovative approaches proposed by Morishita et al45 may be needed in this setting. The cellular uptake of oligonucleotides encapsulated within liposomes coated with inactivated hemagglutinating virus was markedly enhanced and significantly stabilized and sustained.45 Luminal delivery could be obtained, and a host of oligonucleotides were used within this liposomal vehicle.
Summary
The increasing use of antisense technology has contributed greatly
to our understanding of basic mechanisms of vascular
disease.6 8 9 10 11 23 24 26 36 38 39 40 41 42 44 45 48 49 50 Yet we are
clearly still not entirely cognizant of the full force of the power of
gene expression regulation after vascular injury and how best to
suggest molecular modification of the response to injury and attempts
at repair.44 51 The present study, together with the
wealth of data to date, suggests that we must try to couple our
increased understanding of the molecular signals for growth and repair
with increasingly innovative means of delivering molecular modifiers to
proliferating cells.
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Acknowledgments |
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This study was supported in part by National Institutes of Health grants HL-33014 (Dr Rosenberg), HL-41484 (Dr Rosenberg), GM/HL-49039 (Dr Edelman), and AG-00294 (Dr Edelman); a Burroughs-Welcome Fund Award in Experimental Therapeutics (Dr Edelman); a Whitaker Foundation grant in Biomedical Engineering (Dr Edelman); American Heart Association Grant CSA 9100420 (Dr Simons); and a Canadian Heart and Stroke Foundation Fellowship (Dr Sirois). Dr Chris Reilly supplied the SV40LT smooth muscle cells used in the tissue culture assays. Oligonucleotide synthesis was performed in the Massachusetts Institute of Technology Biopolymers Laboratory.
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Footnotes |
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Reprint requests to Dr Elazer R. Edelman, Harvard University–Massachusetts Institute of Technology, Division of Health Sciences and Technology, Cambridge, MA 02139.
This manuscript was sent to Leslie A. Leinwand, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received July 25, 1994; accepted October 10, 1994.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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