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(Received for publication, January 10, 1995) From the
The capacity of antisense sequences to the thrombin receptor to
selectively inhibit thrombin receptor expression and limit mitogenic
responses in vascular wall cells was investigated in vitro.
Eight phosphorothioate oligodeoxynucleotides based on the sequences of
the rat thrombin receptor (including sense, antisense, scrambled, and
missense controls) were synthesized, characterized, and purified by
high performance liquid chromatography. The antisense
oligodeoxynucleotide (ODN 4) inhibitory effect was sequence-specific
and both time- and concentration-dependent. A reduction in serum or
Recent investigations have led researchers to postulate that
thrombin may play a significant role in normal vessel wall healing
under physiological conditions(1, 2) . Hatton et
al.(3) have demonstrated that following catheter
denudation of the rabbit aorta, thrombin activity remains elevated at
the site of smooth muscle cell (SMC) ( The thrombin receptor has been cloned and classified
as a member of the G-protein-coupled family of receptors. This is
consistent with observations of phospholipase C-mediated
phosphoinositide turnover(11, 12) , phospholipase D
activation(13, 14) , protein
phosphorylation(15) , and inhibition of adenylate cyclase
activity (13, 14) after thrombin stimulation.
Furthermore, expression of the thrombin receptor gene has been
identified in proliferating arterial smooth muscle cells in
vitro(16) , and enhanced expression of this gene after
arterial wall injury in vivo has been documented(17) .
This suggests that both thrombin receptor gene regulation and receptor
activation contribute to vascular wall healing under normal conditions.
Likewise, thrombin receptor overexpression could well be a primary
event in facilitating maladaptive responses associated with neointimal
hyperplasia, both after bypass grafting and in restenosis following
balloon catheter angioplasty. Although several research groups have
synthesized oligopeptides and monoclonal antibodies as thrombin
receptor antagonists, these attempts have met with limited success.
Thus, the importance of thrombin receptor activation on vascular SMC
after mechanical vascular wall injury has yet to be determined.
Antisense strategies have had some success in the reduction of
eukaryotic cell surface receptors, including the reduction by nearly
90% of the epidermal growth factor receptor (18) and dramatic
inhibition of the activity of the muscarinic receptor(19) ,
luteinizing hormone receptor(20) , and acetylcholine receptor
subunits(21) . With regard to thrombin, the relative
contributions to vascular smooth muscle cell mitogenesis of thrombin
receptor activation, thrombin-mediated platelet activation, and fibrin
generation, as well as binding or cleavage of other cell surface
proteins by this serine protease, are unknown. Thus, the major
advantage of using an antisense approach to inhibit thrombin
receptor-mediated events in vascular cells is mechanistic, to determine
the effect of inhibiting thrombin receptor expression without affecting
the role of thrombin in coagulation or the effects of thrombin that
result from its binding to other proteins on or near the cell surface,
including thrombomodulin(22) , antithrombin III(23) ,
and protease nexin(24) . We report that specific reduction
of thrombin receptor-mediated events is possible using a
receptor-specific antisense DNA sequence. An important determinant of
specificity in the system we studied was the purity of the
nuclease-resistant phosphorothioate oligodeoxynucleotide sequences.
HPLC purification was required to minimize degenerate fragments and the
potential of nonspecific gene suppression. The down-regulation of
receptor expression in this model is produced by a pretranslational
mechanism. Further, we report that when thrombin receptor expression is
inhibited using an antisense sequence specific for nucleotides
4-20 of the rat thrombin receptor, there is a significant
reduction of smooth muscle cell mitogenesis, not only in response to
Figure 1:
Oligonucleotide
purification. A, pre-HPLC profile. B, post-HPLC
profile. Oligonucleotides were purified by reverse-phase HPLC utilizing
a linear gradient of acetonitrile buffered in 35-50 mM TEA
Figure 2:
SMC proliferation. Growth-arrested SMC
were incubated with DME- 10% FBS alone, DME-BSA alone, or DME-10% FBS
plus PBS or the indicated ODN sequences (30 µM). After 72
h of incubation, cells were counted using a hemocytometer. Antisense
oligonucleotide, 5`ASTR-2 (30 µM) reduced serum-induced
proliferation of SMC (82%; 6.17 ± 1.01 versus 34.08
± 3.89
Figure 3:
Percent inhibition of serum-induced SMC
proliferation: sense (sequence 3) versus antisense (sequence
4) oligonucleotides. At a concentration of 30 µM, sequence
4 reduced the SMC proliferative responses to 10% FBS at 3 days, an
effect notably enhanced after a 6-day exposure period. The data are
displayed as percent maximal proliferation (no oligonucleotides
= 100% proliferation) ± S.D.
Figure 4:
SMC proliferation: dose-response effect
of sequence 4. Inhibition of mitogenic responses was not observed over
the dose range of 0.1-5 µM. A reduction in SMC
proliferation was noted beginning at 15 µM after 4 days in
culture (19%; p < 0.05), and concentration dependence at
higher doses was noted.
Figure 5:
Anti-bromodeoxyuridine immunoperoxidase
staining. Detection of proliferating vascular smooth muscle cells using
bromodeoxyuridine immunohistochemistry after a 3-day exposure to: A, DME, 10% FBS; B, DME, 0.1% BSA; C, DME,
10% FBS plus 5`-STR-2; D, DME, 10% FBS plus 5`MSTR-2; E, DME, 10% FBS plus 5`-ASTR-2. Oligonucleotide sequences were
used at a final concentration of 30
µM.
Figure 6:
Down-regulation of thrombin receptor mRNA
by antisense oligonucleotides. SMC were treated for 72 h with 30
µM of indicated antisense or sense oligonucleotides and
total cellular RNA was isolated. Ten µg of RNA from each treatment
were analyzed for thrombin receptor transcripts by Northern blotting
using the respective
Figure 7:
Decreased thrombin receptor protein in
antisense oligonucleotide-treated SMC. Cells were treated for 72 h with
30 µM of antisense (5`ASTR-2) or sense (5`-STR-2)
oligonucleotides and cell surface associated thrombin receptor protein
content was measured by adherent cell ELISA assay. Similar results were
obtained in three separate experiments.
Increasing evidence suggests that thrombin generation may
contribute to normal vessel wall healing following arterial injury, as
well as to those maladaptive responses which lead to atherosclerosis,
neointimal hyperplasia, or restenosis. For example, Sarembock et
al.(27, 28) and, more recently, Gorog et al.(29) have reported that antithrombin treatment with
recombinant hirudin or
[sca]d-phenylalanine-proline-arginine-chloromethyl ketone, an
irreversible active site thrombin inhibitor) reduces restenosis after in vivo balloon catheter angioplasty in a rabbit model.
Nonetheless, no means exist to block thrombin receptor activation
selectively in order to delineate precisely the role of
receptor-mediated events in this complex pathway. Indeed, although it
is tempting to speculate that thrombin initiates neointimal hyperplasia
via a direct effect on smooth muscle mitogenesis, other thrombin
receptor-mediated phenomena may well hold as great or greater
significance, including: (a) the initiation of extracellular
matrix production by SMC(30, 31) ; (b) the
activation of platelet aggregation and
degranulation(32, 33) ; (c) the stimulation
of endothelial cells(34, 35, 36) ; or (d) the generation and release of cytokines by neutrophils,
monocytes, and T cells(37, 38) . Further, the
functional relevance of other known cell surface thrombin-binding
proteins is unknown. These questions cannot be adequately addressed
until selective blocking of the thrombin receptor is achieved. To date,
both small peptide sequences and antibodies have been investigated as
possible thrombin receptor antagonists without significant success in vivo. ( We have observed that an antisense
oligodeoxynucleotide which follows the initiation codon of the thrombin
receptor mRNA selectively inhibits the mitogenic responses of vascular
smooth muscle cells to fetal bovine serum, Based on our data, the
specific inhibition of mitogenic responses at lower concentrations and
at early time points is most likely a consequence of a direct decrease
in receptor expression. Indeed, as determined by adherent cell ELISA
assay, thrombin receptor protein was decreased significantly in
antisense oligonucleotide-treated cells. The effect of the antisense
ODN appears to be at the level of pretranslation as it affected the
thrombin receptor mRNA levels. A similar effect of antisense
oligonucleotides on the down-regulation of the respective mRNA was
reported for protein kinase C The ability of an
antisense sequence to the thrombin receptor to inhibit serum-induced
SMC proliferation probably reflects significant levels of
We have demonstrated selective inhibition of
thrombin receptor expression using an antisense approach. Admittedly,
the flexibility of this approach is limited by the requirement for ODN
supplementation to culture media and the inevitable nonspecific
reduction in cell proliferation after prolonged incubation.
Nonetheless, this work provides an important first step by which the
role of these receptor-mediated events in atherosclerosis and
injury-induced vascular restenosis can be precisely defined. This work was
presented in abstract form at the 1993 American Heart Association
Scientific Conference on the Molecular Biology of the Vascular Wall,
Boston.
Volume 270,
Number 13,
Issue of March 31, 1995 pp. 7431-7436
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-thrombin-induced smooth muscle cell (SMC) proliferation was noted
as early as 3 days at 30 µM (82%; 6.17 ± 1.01 versus 34.08 ± 3.89 
10
cells/well; p < 0.05) and at a dose as low as 15 µM after
4 days in culture (19%; p < 0.05). Nonspecific effects were
enhanced after prolonged exposure of SMC to the antisense
oligodeoxynucleotide (
6 days). A reduction of inositol phosphate
generation greater than 50% (p < 0.05) was detected after
exposure of SMC to antisense but not to sense or scrambled nucleotide
sequences. This was observed after stimulation with both thrombin and
SFFLRN (thrombin receptor peptide agonist). Northern blot analysis and
enzyme-linked immunosorbent assays revealed 50 and 22% decreases,
respectively, in thrombin receptor mRNA and protein (cell surface)
levels in antisense oligonucleotide-treated (72 h) SMC as compared to
untreated cells, suggesting that thrombin receptor down-regulation
occurred at the pretranslational level. Thus, thrombin
receptor-specific antisense sequences inhibit growthrelated effects
both of serum and thrombin on smooth muscle cells, potentially
providing a new strategy for selective inhibition of receptor-mediated
arterial injury responses.
)proliferation for up
to 10 days, and Bar-Shavit et al.(4) have confirmed
that thrombin bound to the subendothelial extracellular matrix remains
functionally active and protected from inactivation by antithrombin
III. In support of a role for thrombin in vascular lesion formation,
Okazaki et al.(5) have reported that thrombin, but
not other vasoactive agonists or growth factors, produces a pattern of
transiently increased platelet-derived growth factor-A and decreased
platelet-derived growth factor-
receptor mRNA in vascular smooth
muscle cells both in vitro and in vivo, a pattern
similar to that observed after vascular injury. Finally, a number of in vitro and in vivo studies have documented smooth
muscle proliferation following thrombin
exposure(6, 7, 8, 9) , and thrombin
inhibitors have been noted to reduce the mitogenic response of cultured
smooth muscle cells (10) . Despite these reports and our
general knowledge that thrombin mediates procoagulant, mitogenic,
vasoactive, and inflammatory effects (all of which may potentially
promote a proliferative vascular wall response), we know little of the
mechanism by which thrombin initiates the events leading to arterial
wall healing.-thrombin but also in response to stimulation with 10% fetal
bovine serum.
Cell Culture
Primary vascular SMC were isolated
from the thoracic aortas of 250-300-g male Sprague-Dawley rats
using a modification of the method of Travo et
al.(25) . Briefly, aortas were dissected, and the
adventitia and endothelium were mechanically removed and digested with
collagenase and elastase. The identity of cultured cells as SMC was
confirmed by staining with an anti--smooth muscle actin antibody
(antibody A2547 from Sigma). Cells were grown in Dulbecco's
modified Eagle's medium (DME) supplemented with 10% fetal bovine
serum, used in passages 2-5, and maintained in culture at 37
°C in a humidified 5% CO
atmosphere.Oligonucleotide Synthesis
Phosphorothioate
oligodeoxynucleotides were synthesized on an Applied Biosystems Inc.
(Foster City, CA) 380B DNA synthesizer. Oligodeoxynucleotides (ODNs)
were deprotected in saturated aqueous NHOH (12-15
ml/10 µmol of ODNs) for 16-24 h at 55 °C and then dried
in a vacuum concentrator. ODNs were then resuspended in 50 mM triethylamine-acetate buffer (TEA
Ac) (pH 6.5) and purified
by reverse-phase HPLC on a Water's Delta Prep 3000 system
(Milford, MA). A Vydac C8 column (22 250 mm, 10-µm particle
size, 300-Å pore size) was utilized and eluted with a linear
gradient of acetonitrile buffered in 35-50 mM TEAAc (pH 6.5). Fractions were collected and desiccated.
After partial evaporation, an aliquot was run on a microbore
reverse-phase HPLC using an ABI (Foster City, CA) VeloSep cartridge
(3.4 40 mm, C-8 silica, 3-µm particle size) for purity
verification.Proliferation Assay
SMC were seeded at a density
of 25,000 cells/well in six-well plates (Costar, Cambridge, MA). The
following day cells were washed twice with phosphate-buffered saline
(PBS) and the medium replaced with 0.1% BSA-DME (growth-arrest medium).
The cells were maintained in growth-arrest medium for 48 h, after which
the media was changed to DME with 10% fetal bovine serum (DME, 10% FBS)
and synthetic oligodeoxynucleotides added. The medium and
oligodeoxynucleotides were changed every 48 h. Cells were trypsinized
and counted manually using a hemocytometer after exposure to ODNs for
3, 4, and 6 days. Each experiment was carried out in triplicate and
repeated at least two additional times. Data are expressed as mean
± S.D.D-myo-Inositol Phosphate Assay
SMC
were plated onto 35-mm dishes, allowed to reach 90% confluence, then
growth arrested for 48 h. The medium was then changed to DME, 10% FBS,
and oligodeoxynucleotides were added for 96 h. During the last 24 h of
the 96-h incubation, myo-[
H]inositol (15
µCi/ml) was added. Unincorporated label was removed by washing
cultures in a warm balanced salt solution (termed Na
solution): 130 mM NaCl, 5 mM KCl, 1 mM MgCl, 1.5 mM CaCl, 20 mM HEPES (buffered to pH 7.4 with Tris base), 10 mM dextrose, 10 mM LiCl. SMC were incubated in 1 ml of
Na+ solution for 20 min at 37 °C and then stimulated with 140
nM -thrombin, 25 µM SFFLRN, or PBS for 30
min. The reaction was terminated by rapid aspiration of the buffer and
addition of 1 ml of chloroform/methanol/HCl (20:40:1, by volume). A
second wash with 500 µl of chloroform/methanol/HCl was performed.
Organic and aqueous phases were separated by the addition of 900 µl
of distilled water and 500 µl of chloroform. After centrifugation
(500 g for 10 min at 4 °C) and two chloroform
washes, the organic phases were pooled and evaporated to dryness under
N. Inositol phosphates in the aqueous phase were analyzed
by ion-exchange chromatography on Dowex AG-1X8 resin and quantified by
liquid-scintillation spectrometry.Immunohistochemistry
For bromodeoxyuridine (BrdU)
immunohistochemistry, cells were incubated at 37 °C for 2 h with
bromodeoxyuridine (30 µg/ml). Cells were then washed in PBS and
fixed in acid ethanol. After pretreatment with 10% sheep serum and
washes in PBS, cells were incubated with mouse anti-bromodeoxyuridine
monoclonal antibody (Amersham Corp.) for a 90-min incubation. This and
subsequent steps were performed according to the manufacturer's
specifications. The cells were again washed with PBS, incubated for an
additional 30 min with peroxidase-conjugated anti-mouse antibody, and
stained with diaminobenzidine tetrahydrochloride (Amersham).Northern Blot Analysis
Growth-arrested SMC were
treated for 72 h with DME containing 10% FBS in the presence or absence
of antisense (ODN 4) or sense (ODN 3) oligonucleotides, and total
cellular RNA was isolated using Tris reagent (Molecular Research
Center, Inc., Newark, NJ) using the manufacturer's protocol. Ten
µg of RNA were size fractionated by electrophoresis on 1.2%
agarose, 2% formaldehyde gel and then transferred to a Nytron filter
(Schleicher & Schuell). RNA was cross-linked to the filter using UV
irradiation (Stratalinker, Stratagene, La Jolla, CA). After a 4 h
prehybridization in 50% (v/v) formamide, 5 SSC (1 SSC
= 0.15 M NaCl, 0.015 M sodium citrate), 5
Denhardt's (1 Denhardt's = 0.02%
(w/v) each of Ficoll, polyvinyl pyrrolidone, and bovine serum albumin),
50 mM sodium phosphate (pH 6.5) and 250 µg/ml of sheared
salmon sperm DNA at 42 °C, the Nytran filter was hybridized in the
above buffer containing 10% (w/v) dextran sulfate and 1
10
counts/min/ml of 
P-labeled rat thrombin
receptor cDNA probe for 16 h at 42 °C. The filter was washed in 0.1
SSC containing 0.1% SDS for 60 min at 60 °C (with two
changes of solution), and the bands were visualized and quantitated by
the use of a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The
same filter was rehybridized with P-labeled human rDNA
probe to demonstrate equal amounts of RNA in each lane. Labeling of
probes with [-P]dCTP was done using a
multiprime labeling system kit (Amersham,).Adherent Cell ELISA Assay
Growth-arrested SMC were
treated as described above and washed with DME/Ham's F-12 (1:1)
medium containing 0.1% BSA. Cells were then incubated with rabbit
anti-rat thrombin receptor polyclonal antibodies. After incubation of
cells with horseradish peroxidase-conjugated goat anti-rabbit secondary
antibodies, the color was developed with ABST peroxidase substrate
according to the manufacturer's protocol (KPL, Gaithersburg, MD)
and the ODs were read at 405 nm.
Synthetic Oligonucleotides
Eight DNA sequences
were synthesized and are summarized in Table 1. Comparison of
sequence 7 with sequence 4 shows four mismatched nucleotides. Sequence
8 is a scrambled version of sequence 4, containing the same nucleotide
composition overall but in a random sequence not found in the Genbank
data base. The approximate yield following HPLC purification was 50% (Fig. 1).
Ac (pH 6.5). Approximate yield following HPLC
purification was 50%.
Inhibition of Mitogenic Responses by Antisense
DNA
Cell proliferation was specifically inhibited by the use of
antisense DNA to the first six codons that follow the initiation codon
of the thrombin receptor. Serum-induced mitogenesis was reduced after a
72-h exposure to media containing 30 µM of sequence 4, but
was minimally affected by incubation with the remaining seven
oligodeoxynucleotide sequences (Fig. 2). This inhibitory effect
was both dose- and time-dependent. Inhibition of mitogenic responses
was not observed over the dose range of 0.1-5 µM for
any sequence. However, a reduction in SMC proliferation was noted
beginning at 15 µM after 4 days in culture and at 10
µM after 6 days in culture. At a concentration of 30
µM, SMC proliferative responses were reduced at 3 days.
There was almost complete inhibition of SMC proliferation after a 6-day
exposure period (Fig. 3), although at this time there was also a
significant inhibitory effect by the sense oligodeoxynucleotide.
Concentration dependence was noted at both 4 days (Fig. 4) and 6
days (not shown) in culture. Uniformly, at higher oligodeoxynucleotide
doses and longer incubation periods, nonspecific inhibition of
serum-induced mitogenesis was observed. For confirmation, tritiated
thymidine uptake in response to SFFLRN and -thrombin was reduced,
and the increase in c-fos mRNA expression, typical of
thrombin-induced mitogenesis(26) , did not occur following
incubation with sequence 4 (data not shown).
10 cells/well; p <
0.05).
Inositol Phosphate Analysis
Following exposure of
SMC to either -thrombin or SFFLRN, an increase in inositol
phosphate production occurs. Depending on the timing of the assay, this
increase represents an approximate 30-150% increase over
base-line total inositol phosphate levels. As a measure of the effect
of inhibiting thrombin receptor expression using antisense
oligodeoxynucleotides, we measured total inositol phosphates in
cultured SMC following stimulation with either -thrombin or
SFFLRN. Total inositol phosphates did not increase following
stimulation with either -thrombin or SFFLRN of arterial smooth
muscle cells treated with antisense sequence 4 (Table 2). The
response of SMC treated with either the scrambled or missense sequence
was not significantly different from that of SMC treated with PBS.
Immunohistochemistry
Proliferating vascular smooth
muscle cells were detected in vitro using BrdU
immunohistochemistry. Representative micrographs of BrdU uptake under
various culture conditions are presented in Fig. 5. Treatment of
cells with antisense sequence 4, but not sense or missense sequences,
reduced BrdU uptake in a manner which paralleled the reduction of SMC
proliferation noted by either direct cell counting or tritiated
thymidine uptake.
Northern Blot Analysis
To determine whether the
growth-inhibitory effect of sequence 4 is due to a decreased synthesis
of thrombin receptor, we first analyzed its effects on thrombin
receptor mRNA levels. Growth-arrested SMC were treated with DME, 10%
FBS for 72 h in the presence or absence of 30 µM antisense
(ODN 4) or sense (ODN 3) oligonucleotides and total cellular RNA was
isolated. An equal amount of RNA (10 µg) from each condition was
then analyzed for thrombin receptor transcripts as described under
``Experimental Procedures.'' Incubation of SMC with antisense
oligonucleotide (ODN 4) caused a 50% decrease in thrombin receptor mRNA
levels as compared with the amounts in untreated cells (Fig. 6).
Neither antisense nor sense oligonucleotides affected rRNA levels,
suggesting that the effect of antisense oligonucleotide on thrombin
receptor mRNA is specific.
P-labeled cDNA probe. Similar results
were obtained in three independent
experiments.
Adherent Cell ELISA Assay
To further prove that
the decrease in thrombin receptor mRNA levels in SMC treated with
antisense sequence also resulted in a comparable reduction in the
protein, cell surface thrombin receptor protein levels were quantitated
in cells treated identically to those described above using adherent
cell ELISA assay. As evident from Fig. 7, SMC treated with
antisense oligonucleotide (ODN 4) had significantly lower levels of
thrombin receptor antigen compared to untreated cells.
)-thrombin, and the
thrombin receptor agonist peptide, SFFLRN. The observed nonspecific
inhibitory effects could be due to the presence of contaminating short
nucleic acid sequences following ODN synthesis because removal of these
sequences by HPLC minimized the nonspecific effects. A significant and
specific reduction in proliferation, whether measured by direct cell
counting or tritiated thymidine and BrdU incorporation, was noted
following a 72-96-h exposure of vascular SMC to ODN sequence 4,
but not to sense, missense, or scrambled nucleotide sequences. However,
nonspecific inhibition of cell proliferation by phosphorothioate
oligonucleotides was observed under conditions of either high
concentration or prolonged incubation. in human A549 lung carcinoma
cells(39) . Thrombin receptor-dependent signal transduction, as
measured by inositol phosphate generation and mitogenesis, is decreased
when SMC are exposed to antisense sequence 4. These findings thus show
a correlation between thrombin receptor down-regulation and decreased
growth in antisense oligonucleotide-treated SMC.-thrombin in the serum. This hypothesis may be supported further
by the fact that hirudin, an inhibitor of thrombin, reduced
serum-induced growth of SMC by only 30%, yet hirudin completely blocked
-thrombin-induced SMC proliferation (data not shown). In addition,
Melzig et al.(10) documented that heparin and
synthetic inhibitors of thrombin decreased the rate of division of
porcine vascular smooth muscle cells in culture supplemented with 10%
calf serum. Our own studies have shown low levels of natural thrombin
inhibitors, including antithrombin III, in commercial serum. ()The inability to completely abolish the serum-induced SMC
proliferation no doubt reflects the presence of other well-known smooth
muscle cell mitogens in the serum which are unaffected by the presence
of antisense sequences.
)))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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