(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
-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.
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) ()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.
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
-thrombin but also in response to stimulation with 10% fetal
bovine serum.
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 TEAAc (pH 6.5). Approximate yield following HPLC
purification was 50%.
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 10
cells/well; p <
0.05).
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 P-labeled cDNA probe. Similar results
were obtained in three independent
experiments.
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, -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.
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 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.
The ability of an
antisense sequence to the thrombin receptor to inhibit serum-induced
SMC proliferation probably reflects significant levels of
-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.
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.