(Circulation. 1997;96:2280-2286.)
© 1997 American Heart Association, Inc.
Articles |
Effect of Serotonin, Thromboxane A2, and Specific Receptor Antagonists on Vascular Smooth Muscle Cell Proliferation
From the Department of Internal Medicine, Division of Cardiology, University of Texas Houston Medical School.
Correspondence to Claude R. Benedict, MD, DPhil, Professor, Department of Internal Medicine, Division of Cardiology, The University of Texas Houston Medical School, 6431 Fannin, MSB 6.039, Houston, TX 77030.
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Abstract |
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Background Restenosis is a major complication that limits the long-term efficacy of coronary angioplasty. Migration and proliferation of activated medial smooth muscle cells (SMCs) is considered an important mechanism in this process. Because at sites of vascular injury, aggregating platelets release both serotonin (5-HT) and thromboxane A2 (TXA2), we examined whether 5-HT and TXA2 can induce SMC proliferation and whether there is synergistic interaction between these two mediators.
Methods and Results The mitogenic effects of 5-HT and TXA2 either alone or in combination was examined in serum-free medium on canine aortic SMCs by [3H]thymidine incorporation into DNA and by cell counting. 5-HT induced SMC proliferation at a concentration of 100 nmol/L, whereas the effect of TXA2 (U46619, a stable TXA2 mimetic) on inducing proliferation of SMCs was observed at a concentration of 100 nmol/L. When these two mediators were added together, there was a synergistic interaction on inducing SMC proliferation even at subthreshold concentrations. The mitogenic effect of 5-HT and its synergistic interaction with TXA2 on SMC proliferation was abolished by a 5-HT2 receptor antagonist, LY281067, without affecting the contribution of TXA2. Similarly, the TXA2 synthase inhibitor/receptor antagonist ridogrel abolished the mitogenic effect of TXA2 and the interaction between 5-HT and TXA2 without affecting the response to 5-HT. When LY281067 and ridogrel were used together, they abolished the mitogenic effects of 5-HT and TXA2.
Conclusions At sites of vascular injury, platelet-induced SMC proliferation may also be modulated by nonpeptide growth mediators. A combination of a 5-HT2 receptor antagonist and TXA2 synthase inhibitor/receptor may be useful for attenuation of restenosis after angioplasty.
Key Words: serotonin • thromboxane • restenosis • cells • muscle, smooth • platelets
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Introduction |
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Restenosis, defined as >50% decrease in luminal diameter at the site of previous balloon dilation, is a major clinical complication that limits the long-term efficacy of coronary angioplasty.1 2 3 Migration and proliferation of activated medial SMCs is considered to be an important pathophysiological mechanism in development of restenosis after angioplasty.4 5 6 PTCA leads to endothelial and medial injury with platelet aggregation at the site of vascular damage. Aggregating platelets release mediators that sustain continuing platelet aggregation, induce dynamic coronary artery vasoconstriction, and lead to SMC proliferation.7 Previous studies have suggested a specific role for peptide growth factors released by platelets in inducing SMC proliferation.1 2 8 9 10 However, recent studies indicate that vasoactive compounds, such as 5-HT released by platelets during platelet aggregation, may also be mitogenic for some cell types.11 12 13 14 Several studies have reported that 5-HT stimulates vascular SMC proliferation in the presence of serum or growth factors.15 16 17 18 19 20 21 Recently, we have shown that 5-HT is mitogenic to canine endothelial cells.22 However, there are conflicting reports regarding the mitogenic effect of TXA2 on SMC proliferation,23 24 25 26 27 28 and it is unknown whether there is an interaction between TXA2 and 5-HT in inducing SMC proliferation and whether this can be inhibited by specific receptor antagonists. In this study, we report that 5-HT and TXA2 stimulate SMC proliferation under serum-free conditions, and there is a significant synergistic interaction between the two mediators in inducing the same. The mitogenic effect of these mediators and their synergistic interaction was blocked by preincubation of SMCs with specific receptor antagonists.
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Methods |
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Materials
5-HT (as creatine sulfate), BSA, insulin, transferrin, and pargyline were purchased from Sigma Chemical Co; the TXA2 mimetic U46619 [(15S)-hydroxy-11
,9-(epoxymethano)-presta-5Z,13E-dienoic
acid] was obtained from Upjohn; LY281067 was a gift from Eli Lilly
Laboratories; MDL 73247EF was a gift from Marion Merrell Dow Research
Institute; methiothepin mesylate and NAN-190 hydrobromide were from
Research Biochemicals Inc; and ketanserin and ridogrel were a gift from
Jansen Pharmaceuticals. 5-HT was dissolved in 50 mmol/L
citrate buffer, pH 5.5. DMEM, FCS, and PBS were purchased from Gibco
BRL Life Technologies. Trypsin-EDTA and HBSS were purchased from Sigma,
and [3H]thymidine (specific activity, 20Ci/mol) was from
Dupont NEN Research Products. Other reagents were purchased from
local vendors.
Isolation, Culture, and Characterization of Primary SMCs
Canine aortic SMCs were isolated by the explant method. The
intima was first peeled off from the aorta, and then the media was
carefully stripped away from the adventitia and placed in a Petri dish
containing warmed DMEM (37°C). The medial layer was cut into 
1-mm
squares, which were transferred into a 25-cm2 tissue
culture flask and barely covered with DMEM supplemented with 10% FBS.
The blocks of tissue were cultured in a humidified atmosphere of 95%
air and 5% CO2 (vol/vol) at 37°C. After 1 to 2
weeks, the tissue blocks were removed, and the migrated SMCs were
cultured. After isolation, the identity of the SMCs was confirmed by
morphological examination and by staining for ß-actin.
Subcultures of SMCs were done once they became confluent; medium from the plates was aspirated, and the cells were washed with 10 mL PBS. Then, 2 to 3 mL of trypsin EDTA (0.05% trypsin, 0.53 mmol/L EDTA in Ca2+,Mg2+-free HBSS) was added to the cells and incubated at room temperature for 2 to 3 minutes. The action of trypsin was stopped by the addition of 7 to 8 mL DMEM containing 10% FBS. The cells were collected by centrifugation at 150g for 10 minutes. After the supernatant was removed, the pelleted cells were dispersed in 10 mL DMEM containing 10% FBS, and fresh cultures were initiated from these cells.
[3H]Thymidine Incorporation
The SMCs in the second or third passage were seeded in
35-mm-diameter tissue culture plates at a density of 65 000 to 75 000
cells per plate in DMEM containing 10% FBS and allowed to proliferate
for 72 hours. After 72 hours, the growth medium was replaced with 2
mL DMEM containing 0.1% FBS and incubated for 72 hours to arrest
the cell growth (synchronization). After synchronization, 2 mL DMEM
containing 500 µg/mL BSA, 10 µg/mL human transferrin,
10 µg/mL bovine insulin, 25 ng/mL selenium, 0.2
mmol/L ascorbate, 100 µmol/L pargyline, and the
given concentrations of 5-HT and/or TXA2 was added. The
cells were grown in the presence of compounds for 20 hours, and then
[3H]thymidine (1 µCi/plate; specific activity, 20
Ci/mmol) was added to the medium. Four hours after the addition of
[3H]thymidine, we terminated the experiments by
aspirating the medium and washing with ice-cold Dulbecco's PBS (pH
7.4) containing 1 mmol/L CaCl2, 1
mmol/L MgCl2, and 6% trichloroacetic acid.
Acid-insoluble [3H]thymidine was collected on glass fiber
filters. The filters were washed with 100% ethanol and air-dried, and
[3H]thymidine was quantified in a liquid scintillation
counter. All the experiments were performed in quadruplicate, and each
experiment was repeated a minimum of three times.
Determination of SMC Number
Canine aortic SMCs were synchronized and grown in the presence
of given concentrations of 5-HT and/or TXA2 as described in
the "[3H]Thymidine Incorporation" section. After 24
hours, 0.4 mL of 2% (wt/vol) crude pancreatic trypsin in Dulbecco's
PBS containing 152 mmol/L EDTA was added to each dish. The
dishes were incubated at room temperature for 2 minutes before addition
of 0.8 mL of horse serum. The contents of each dish were diluted to 20
mL with isotone II (Coulter Electronics), and the cell number was
determined with a Coulter counter. Triplicate counts were taken for
each plate, and quadruplicates were used for each determination.
Statistical Analyses
Data were analyzed by one-way ANOVA. In each figure,
mean values±SD are shown.
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Results |
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Effects of 5-HT on [3H]Thymidine Incorporation by Vascular SMCs
The effect of increasing concentrations of 5-HT on [3H]thymidine incorporation into the DNA of SMCs is shown in Fig 1A
. 5-HT at an added concentration
of 100 nmol/L induced proliferation of SMCs, and the effect
peaked at a concentration of 100 µmol/L. At
concentrations of 5-HT >150 µmol/L, there was a
reduction in the [3H]thymidine incorporation (Fig 1A),
indicating that higher concentrations may be cytotoxic to the SMCs. The
effect of 5-HT on SMC DNA synthesis response was also time dependent.
The cells required incubation with 5-HT for at least 4 hours before a
significant induction in [3H]thymidine incorporation was
observed (data not shown).
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Effects of TXA2 on [3H]Thymidine
Incorporation by Vascular SMCs
The effect of increasing concentration of TXA2
(U46619, a stable TXA2 mimetic) on
[3H]thymidine incorporation into SMCs is shown in Fig 1B
.
The stimulatory effect of U46619 was observed at concentrations as low
as 100 nmol/L of U46619 and was maximal at concentrations
between 10 and 30 µmol/L of U46619 added to the medium.
As with 5-HT, with higher concentrations of U46619, there was a
decrease in the [3H]thymidine incorporation into the
SMCs.
Combined Effects of 5-HT and TXA2 on
[3H]Thymidine Incorporation by Vascular SMCs
Because platelet aggregation at sites of vascular injury
releases both 5-HT and TXA2, we examined whether
there was an interaction between these two mediators in inducing
SMC proliferation. 5-HT alone at an added concentration of 50
µmol/L induced an 2.5-fold increase in
[3H]thymidine incorporation (Fig 1A). Similarly, when
0.75 µmol/L of U46619 was added alone to the medium,
there was an 3-fold increase in [3H]thymidine
incorporation (Fig 1B). When 5-HT and U46619 were added together at
these concentrations (50 µmol/L 5-HT and 0.75
µmol/L TXA2), there was a 10- to 15-fold increase
in the [3H]thymidine incorporation (instead of an
additive effect of 6-fold increase), which indicates a synergistic
effect on SMC DNA synthesis response (Fig 2). The synergism between 5-HT and U46619
was also observed at subthreshold concentrations, at which neither 5-HT
nor TXA2 alone induces cell proliferation. By itself, 5-HT
at an added concentration of 0.05 µmol/L or U46619 at an
added concentration of 0.0075 µmol/L did not induce
proliferation of SMCs. However, when they were added together at these
concentrations (0.05 µmol/L 5-HT and 0.0075
µmol/L TXA2), there was a 5- to 6-fold increase in
[3H]thymidine incorporation into SMCs.
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Combined Effects of 5-HT and TXA2 on Vascular SMC
Number
To determine whether the induction of DNA synthesis by 5-HT
or TXA2 resulted in an increase in cell number, the SMCs
were grown in serum-free medium alone (control) or medium containing
different concentrations of 5-HT or TXA2 alone or in
combination, and the number of cells was counted. The results show that
the induction of DNA synthesis by 5-HT and TXA2 leads to an
increase in cell number (Fig 3). The
cellular proliferation response appears to be a function of the 5-HT or
TXA2 concentration present in the medium. The
synergistic interaction between 5-HT and TXA2 on inducing
[3H]thymidine incorporation also resulted in a
significant increase in cell number. 5-HT at an added concentration of
50 µmol/L increased the cell number by 102 706±6039
compared with control (P<.001). Similarly, TXA2
at an added concentration of 0.75 µmol/L increased the
cell number by 125 559±4285 compared with control
(P<.001). When both 5-HT and TXA2 were added
together at the above concentrations, cell number increased by
350 919±3584 (Fig 3).
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Effect of 5-HT Receptor Antagonists on 5-HT–Induced
[3H] Incorporation Into Vascular SMCs
To determine the type of 5-HT receptors involved in inducing the
[3H]thymidine incorporation, canine aortic SMCs were
preincubated with various concentrations of the 5-HT2
receptor antagonists ketanserin (Fig 4A) or LY28106729 (data not
shown) before they were stimulated with 5-HT. The results show that the
mitogenic effect of 5-HT on SMCs was blocked in a
dose-dependent manner by both the 5-HT2 receptor
antagonists. In contrast, when the SMCs were preincubated
with MDL 73147EF (5-HT3 receptor antagonist)
(Fig 4B), NAN-190 hydrobromide (5-HT1A/5-HT1D
receptor antagonist) (Fig 4C), or methiothepin mesylate
(5-HT1/5-HT2 receptor antagonist)
(Fig 4D), they failed to block the mitogenic effect of 5-HT
on [3H]thymidine incorporation. None of the 5-HT receptor
antagonists were cytotoxic to the cells at the
concentrations tested. These results indicate that the
mitogenic effect of 5-HT may be mediated by activation of
5-HT2 receptors.
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Effect of TXA2 Receptor Antagonist Ridogrel
on U46619-Induced [3H]Thymidine Incorporation Into Canine
Aortic SMCs
To determine whether a TXA2 receptor
antagonist would inhibit the U46619-induced
[3H]thymidine incorporation, the SMCs were preincubated
for 2 hours with various concentrations of ridogrel (a combined
TXA2 synthase inhibitor and receptor
antagonist)30 before being stimulated with
U46619. The results indicate that the mitogenic effect of
U46619 on aortic SMCs was blocked by ridogrel in a dose-dependent
manner (Fig 5). The concentrations of
ridogrel used were not cytotoxic to the SMCs. Thus, it is likely that
the mitogenic effect of TXA2 is mediated by the
activation of TXA2 receptors.
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Inhibition of 5-HT–and TXA2-Induced SMC
Proliferation
Because there is a synergistic interaction between 5-HT and
TXA2 on SMC proliferation, we also examined whether the
receptor antagonists would inhibit the synergistic
interaction between 5-HT and TXA2 on SMC proliferation.
Aortic SMCs were preincubated for 2 hours with either LY281067 or
ridogrel or both and then stimulated with given concentrations of 5-HT
and TXA2. LY281067 at a concentration of 10 µg/mL
blocked the proliferative effect of 5-HT and the synergistic
interaction with TXA2 without inhibiting the cellular
proliferative response to TXA2 (Fig 6A). Similarly, ridogrel at a
concentration of 30 µg/mL blocked the proliferative effect of
TXA2 and the synergistic interaction with 5-HT without
inhibiting the mitogenic response to 5-HT (Fig 6B). When
SMCs were pretreated with both LY281067 and ridogrel, the proliferative
effects of both 5-HT and TXA2 as well as their synergistic
interaction with each other were inhibited (Fig 6C).
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Discussion |
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The data obtained in this study indicate that 5-HT and TXA2 are mitogenic to vascular SMCs at nanomolar concentrations. More importantly, the data also suggest a synergistic interaction between the two mediators in promoting SMC proliferation. Further, subthreshold concentrations of 5-HT and TXA2 added together induced a fivefold to sixfold increase in [3H]thymidine incorporation and an increase in cell number. It is also noteworthy that the mitogenic effects and the synergistic interactions between these mediators were blocked by specific receptor antagonists.
It is believed that the initial events after angioplasty include
platelet adhesion and aggregation and thrombus formation at the
site of vascular injury. Aggregating platelets release several
vasoactive mediators, including 5-HT and
TXA2.31 We have previously demonstrated that
after angioplasty in humans, high concentrations of 5-HT are
present in the coronary sinus blood samples.7
We also found that in a rabbit model of angioplasty involving the
femoral artery, there was ongoing platelet deposition at the site
of vascular injury with progressive accumulation of 5-HT at the same
site. The concentrations of 5-HT measured at the local site of injury
in our animal model (750 pmol/g tissue)22 or in
coronary sinus plasma of patients (0.12 to 0.20
µmol/L)7 exceeded the concentrations required in
vitro to stimulate SMC proliferation.
5-HT has growth-stimulatory effects on Chinese hamster lung fibroblasts,11 rat renal mesangial cells,12 rat jejunal crypt cells,13 neurons or neuroblasts,32 pancreatic carcinoid cells,14 aortic endothelial cells,22 and SMCs from different species.15 16 17 18 19 20 21 Serotonin mediates its effect by different receptor subtypes. Serotonergic receptors have been classified on the basis of the receptor coupling mechanisms.33 The major subtypes include those negatively coupled to adenylate cyclase (5-HT1 class of receptors), those coupled to phospholipase C (5-HT2 class of receptors), those that activate a ligand-gated ion channel (coupled to Ca2+ by unknown mechanisms) (5-HT3 class of receptors), and a new type that are G protein–coupled (5-HT4 class of receptors). Unlike 5-HT1 receptors, the 5-HT4 receptors are positively linked to adenylate cyclase. The growth-promoting effects of 5-HT on nontransformed Chinese hamster lung fibroblasts11 and human pancreatic carcinoid cells14 are mediated by the 5-HT1B receptor. In contrast, in vascular SMCs, the contractile,34 chemotactic,35 and mitogenic responses are mediated by 5-HT2 receptors.15 16 17 18 19 20 21 In bovine aortic SMCs, 5-HT stimulates [3H]thymidine incorporation,16 inhibits the hydrolysis of inositol phosphate, and increases the intracellular Ca2+ concentration, all via the 5-HT2 receptor.20 Corson et al36 showed that 5-HT induced the expression of mRNA for c-fos and stimulated intracellular Ca2+ mobilization in rat aortic SMCs but failed to increase the cell number. Further, these effects of 5-HT were inhibited by selective 5-HT2 receptor antagonists. In agreement with these earlier studies, we found that the mitogenic effect of 5-HT on SMCs was mediated by the 5-HT2 receptors, because both LY281067 and ketanserin, known 5-HT2 receptor antagonists, blocked the effect of 5-HT on these cells, whereas MDL 73147 EF (5-HT3 receptor antagonist), methiothepin mesylate (5-HT1/5-HT2 receptor antagonist), and NAN-190 hydrobromide (5-HT1A/5-HT1D receptor antagonist) were without any effect.
Conflicting reports exist about the effects of TXA2 mimetics on vascular SMC growth. Dorn et al26 reported that U46619 stimulates c-fos expression and protein synthesis as measured by [3H]leucine incorporation but did not stimulate DNA synthesis. Similarly, Crowley et al15 reported that TXA2 analogue failed to stimulate SMC growth and that antagonists to TXA2 had only minimal inhibitory effects on platelet-induced SMC proliferation. In contrast, Hanasaki et al27 reported that U46619 increased [3H]thymidine incorporation and cell proliferation; Ishimitsu et al24 demonstrated that 9,11-epithio-11,12-metharno-TXA2, another stable TXA2 analogue, stimulated DNA synthesis in rat vascular SMCs; and Sachinidis et al23 showed that U46619 and carbocyclic TXA2 stimulated [3H]thymidine incorporation and an increase in cell number, both of which were blocked by a selective TXA2/PGH2 receptor antagonist. Our findings support a proliferative role for TXA2 in canine aortic SMCs (an increase in both DNA synthesis and cell number), and this effect was blocked by a TXA2 synthase inhibitor/receptor antagonist. It is known that the proliferation of vascular SMCs in response to growth factors differs considerably and depends on several factors, such as isolation procedure, strain and age of the animal, type of vasculature, cultivating conditions, number of the passage in which cells are examined, cell seeding density, and culture time.37 It has been suggested that the capacity of vascular SMCs to proliferate depends on the phenotype of the cell. For SMCs, two phenotypes have been described: (1) a contractile phenotype in which the SMCs are unable to proliferate and (2) a synthetic phenotype in which SMCs respond to growth factors.37 Because the phenotype of SMCs is known to change with different passages, the difference between our results and those of others15 26 36 may reflect differences in phenotype of the SMCs examined.
Previous studies indicate that 5-HT, TXA2, or ADP released
from platelets can act synergistically with the known platelet-
or vessel wall–derived peptide growth factors, such as
platelet-derived growth factor, transforming growth factor-ß, and
epidermal growth factor.15 16 23 In this study, we found
that 5-HT and TXA2 act synergistically with each other and
induce SMC proliferation. Knezewic et al38 showed that the
G protein -subunit copurified with TXA2 receptor belongs
to the Gq subfamily, which suggest that the
TXA2 receptor may couple with the Gq subfamily
of G protein -subunit to exert its effects. The Gq
subfamily of G protein -subunit is known to be pertussis toxin
insensitive,39 40 which may explain the lack of effect of
pertussis toxin on TXA2-mediated
responses.23 41 42 However, it has been shown that the
Gq subfamily can also couple to several other receptors and
thereby modulate specific phospholipase Cß1,
which catalyzes the hydrolysis of phosphoinositide
bisphosphate, with subsequent formation of IP3 and
diacylglycerol and increase in Ca2+.39 42 In
addition, the activated TXA2 receptors in human
platelets have been shown to be coupled to the G12
subfamily of the G protein and one or more members of the
Gq subfamily.43 Serotonin-mediated
responses are known to be pertussis toxin sensitive, which indicates
that serotonergic receptors are coupled with the Gi
subfamily of the G protein -subunit, which also catalyzes the
hydrolysis of phosphoinositide
bisphosphate.11 22 Thus, the coupling of both
TXA2 and 5-HT receptors to the same class of -subunits
of G protein may explain the synergistic interaction between 5-HT and
TXA2 in our studies. Thus, platelet-derived 5-HT and
TXA2 not only have a direct mitogenic effect
but also at subthreshold concentrations induce proliferation due to
synergistic interaction between the two mediators. Thus, the effect of
platelet-derived mediators on vascular SMCs is not solely due to
peptide growth factors but is also most likely due to other nonpeptide
factors that are capable of amplifying the proliferative response.
It has been reported that profound thrombocytopenia markedly reduced intimal proliferation in mechanically injured rat and rabbit arteries.10 However, antiplatelet agents, such as aspirin,44 or anticoagulants, such as warfarin45 or heparin,46 that inhibit platelet aggregation and/or thrombus formation have not prevented the development of neointimal proliferation after coronary angioplasty. Serruys et al, in randomized, double-blind, placebo-controlled trials, showed that the TXA2 blocker GR 32191B47 and the 5-HT2 receptor antagonist ketanserin48 failed to prevent the development of restenosis. Data from the present study and others15 16 23 suggest a possible mechanistic explanation for the lack of success of these strategies. Specifically, even small local concentrations of peptide and nonpeptide (eg, 5-HT, TXA2, ADP) growth mediators, acting in concert with each other, may induce significant SMC proliferation. The antithrombotic or antiplatelet therapies may not be able to completely suppress the release and/or the mitogenic effects of these mediators, and specific serotonergic and TXA2 receptor antagonists should be used in combination with antiplatelet therapies to prevent the neointimal proliferative response. In fact, we have shown that a strategy of combined use of a 5-HT and a TXA2 synthase inhibitor/receptor antagonist may attenuate neointimal proliferation after vascular injury in vivo in experimental animal models.49
In summary, platelet-induced SMC proliferation may not depend solely on peptide growth factors but may also be modulated by nonpeptide growth factor mediators, such as 5-HT and TXA2. These nonpeptide growth factors may also function as amplification factors among themselves and/or potentiate cellular growth response in conjunction with other known peptide growth factors. The data obtained in this study suggest that inhibition of neointimal proliferation after vascular injury may require the combined use of multiple growth factor inhibitors to simultaneously block several critical cellular activation pathways.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health/National Heart, Lung, and Blood Institute grants RO1-HL-50179 and RO1-HL-54839 (Dr Willerson) and RO1-HL-39916, RO1-HL50653, and an American Heart Association Grant-in-Aid (Dr Benedict). We wish to thank Shirley McWhorter for typing the manuscript.
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Footnotes |
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Guest editor for this article was Valentin Fuster, MD, PhD, Mount Sinai Medical Center, New York, NY.
Received February 3, 1997; revision received May 1, 1997; accepted May 5, 1997.
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