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Ann Thorac Surg 2001;71:1856-1864
© 2001 The Society of Thoracic Surgeons

Original article: cardiovascular

Sarpogrelate inhibits serotonin-induced proliferation of porcine coronary artery smooth muscle cells: implications for long-term graft patency

Sushil K. Sharma, PhDa, Dario F. Del Rizzo, MD, PhDa, Peter Zahradka, PhDa, Sukhinder K. Bhangu, BSca, Jeffrey P. Werner, BSca, Hideo Kumamoto, DVMb, Nobuakira Takeda, MD, PhDb, Naranjan S. Dhalla, PhD, MD, (Hon)a

a Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, University of Manitoba, Faculty of Medicine, Winnipeg, Manitoba, Canada
b Department of Internal Medicine, Aoto Hospital, Jikei University School of Medicine, Tokyo, Japan

Accepted for publication January 5, 2001.

Address reprint requests to Dr Del Rizzo, Laboratory for Experimental Cardiovascular Surgery, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Taché Ave, Winnipeg, MB, Canada R2H 2A6
e-mail: delrizzo{at}cc.umanitoba.ca
e-mail: ddelrizzo{at}exchange.hsc.mb.ca


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. Serotonin can induce proliferation of vascular smooth muscle cells. We assessed the ability of a specific serotonin receptor antagonist, sarpogrelate, to inhibit proliferation of cultured porcine coronary artery smooth muscle cells.

Methods. Cell proliferation and mitotic activity were measured using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide. To determine the effect of sarpogrelate on DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and protein synthesis, radioactive incorporation of 3H-thymidine, 3H-uridine, and 3H-phenylalanine, respectively, was used. Synthesis of DNA was also assessed by flow cytometry with propidium iodide as a fluorochrome.

Results. Serotonin, platelet-derived growth factor, endothelin, and angiotensin II all induced proliferation of porcine coronary artery smooth muscle cells. Sarpogrelate specifically inhibited the serotonin-induced cytokine trigger but did not influence platelet-derived growth factor–, endothelin–, or angiotensin II–induced cell proliferation. Sarpogrelate inhibited the serotonin-induced increase in intracellular free ionized calcium concentration, prevented mitogen-activated protein kinase activation, and down-regulated expression of the protooncogenes c-fos and c-jun. Sarpogrelate acted at the G1 phase of the cell cycle.

Conclusions. These data suggest that sarpogrelate could be used as a therapeutic agent to inhibit serotonin-induced neointimal hyperplasia and improve patency of coronary artery bypass grafts.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Although coronary artery bypass grafting is a widespread and established means of treating advanced coronary artery disease, the late benefits of the procedure are influenced by the fate of the bypass conduits. Proliferation of the smooth muscle cells (SMCs) within the conduit is thought to be a major cause of both early and late graft failure. Serotonin (5-HT), angiotensin II (Ang II), endothelin, and platelet-derived growth factor (PDGF) have all been implicated in this process [1, 2]. These growth factors induce intracellular signaling involved in cell proliferation by way of specific cell surface receptors.

As a result of metabolic- or mechanical-induced cellular injury, mitogen-activated protein (MAP) kinase is activated and undergoes translocation from the cytosol to the nucleus. This molecular event is a key element in signal transduction. Activation of MAP kinase provides the intracellular signal that leads to the stress-dependent induction of protooncogenes (mainly c-fos and c-jun) with the resultant induction of cellular proliferation [3–7]. Therefore, MAP kinase activity can be used as an intracellular marker of cell proliferation [8, 9].

Several attempts have been made in the past to develop selective inhibitors of vascular neointimal hyperplasia, but success has been limited [10]. In the present study, we explored the effect of 5-HT on porcine coronary artery smooth muscle cell (PCASMC) proliferation in culture. Furthermore, we investigated the contribution of 5-HT receptor activation to the induction of PCASMC proliferation with the use of a selective 5-HT receptor antagonist, sarpogrelate. The basic aim of this study was to establish the ability of sarpogrelate to function as a specific antiproliferative agent by the blockade of 5-HT receptors. We also wished to establish whether or not the effects of sarpogrelate-induced inhibition of PCASMC proliferation were independent of Ang II, PDGF, or endothelin. Finally, we examined the effect of sarpogrelate on the 5-HT–induced increase in intracellular free ionized calcium concentration [Ca+2i] in PCASMCs, the induction of MAP kinase, and the stimulation of protooncogene expression.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Cell culture materials, including powdered Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum, Roswell Park Memorial Institute 1640, gentamicin sulfate (1 µg/mL), flasks, and microtiter plates, were purchased from GIBCO-BRL (Mississauga, ON, Canada). Serotonin, endothelin, PDGF, dimethyl sulfoxide, propidium iodide, and DNase (deoxyribonuclease)-free RNase (ribonuclease) were purchased from Sigma-Aldrich Chemical Co (Oakville, ON, Canada). All other chemicals were of reagent-grade quality and were purchased from Sigma-Aldrich Chemical Co. Radioisotopes for protein ([2,6 3H]-phenylalanine, 55 Ci/mmol), RNA (ribonucleic acid) ([5,6 3H]-uridine, 36 Ci/mmol), and DNA (deoxyribonucleic acid) ([methyl 3H]-thymidine, 2 Ci/mmol) studies were purchased from Amersham Canada (Oakville, ON, Canada). Fura-2 (acetoxymethyl ester) was purchased from Molecular Probes (Eugene, OR). Sarpogrelate (MCI-9042, Anplag, (±)-1-[2-2(3-methoxyphenyl)ethyl]phenoxy]-3-(dimethylamino)-2-propyl hydrogen succinate hydrochloride) was generously provided by the Mitsubishi-Tokyo Pharmaceutical Co Ltd (Tokyo, Japan).

Tissue culture techniques
Porcine hearts were obtained from the local abattoir and transported to the laboratory on ice. Segments of the left anterior descending coronary artery were isolated and placed into culture within 2 hours of sacrifice. Cultured PCASMCs were prepared from free-floating explants as previously described [11]. The explants were incubated in a humidified chamber maintained at 37°C in 5% carbon dioxide in DMEM (high glucose) containing 20% fetal bovine serum with 1 µg/mL of gentamicin. The culture medium was changed every 72 hours. Cellular origin was determined by immunocytochemical detection of smooth muscle myosin, smooth muscle {alpha}-actin, and h-caldesmon [11]. All cell preparations consisted of greater than 95% smooth muscle cells [11].

MTT cell proliferation assay
Porcine coronary artery smooth muscle cells were grown in Falcon T2 flasks. After 5 days of growth, the cells reached the confluent stage. The adherent cells were trypsinized and centrifuged, and the cell pellet was washed three times in phosphate-buffered saline solution (PBS) (pH 7.4). Thereafter the cells were reconstituted in DMEM containing 10% fetal bovine serum and 1 µg/mL of gentamicin. The cells were then seeded into 96-well microtiter plates (200 µL containing 104 cells per well), and cell growth over time was examined with phase-contrast microscopy. Cell proliferation was measured with the use of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as previously described [2]. The MTT was dissolved in Roswell Park Memorial Institute 1640 without phenol red, filter-sterilized, and stored at 4°C. The medium was removed by aspiration, and cells were washed three times in PBS and incubated with MTT (100 µL per well) for 4 hours at 37°C. The reaction was stopped with acidified isopropanol (100 µL). Purple blue color was developed by placing the microtiter plates in a dark chamber for 30 minutes. Quantitative measurements were obtained by absorption spectroscopy with a microtiter plate reader at 570 nm with background subtraction at 630 to 690 nm.

DNA, RNA, and protein synthesis
Porcine coronary artery smooth muscle cells were grown as described. The cells were harvested by trypsinization and, after centrifugation at 1,200 rpm for 6 minutes at 4°C, the pellet was washed three times in PBS (pH 7.4). The cell suspension was prepared in 10 mL of DMEM supplemented with 10% fetal bovine serum and 1 µg/mL of gentamicin. Cell number was determined with the aid of a Coulter counter and a hemocytometer. The cells (104/200 µL) were added to microtiter plates and synchronized for 24 hours in serum-free defined medium (DMEM supplemented with high glucose, selenium, transferrin, insulin, and ascorbate). The cells were examined by phase-contrast microscopy, and cell viability was checked with the trypan blue exclusion method. Subsequently, the cells were grown in a humidified incubator (37°C, 5% carbon dioxide) for 96 hours in the presence of 1 µCi/mL of radiotracer (3H-thymidine, 3H-uridine, or 3H-phenylalanine). The incubation was terminated by the addition of 10% trichloroacetic acid in PBS. The trichloroacetic acid precipitable fraction was captured on Millipore (GF/B) filters and counted in a liquid scintillation counter using a Scintiverse-II scintillation cocktail.

Flow cytometry
Porcine coronary artery smooth muscle cells were synchronized with a 72-hour incubation in serum-free defined medium (DMEM supplemented with 5 µg/mL transferrin, 10-9 mol/L selenium, 2 x 10-4 mol/L ascorbate, and 10-8 mol/L insulin) before adding cytokines, sarpogrelate, or both. The cells were washed three times in PBS and detached with trypsin-EDTA (ethylenediamine tetraacetic acid). The cells were stained for 30 minutes at 4°C on ice with propidium iodide prepared in Krishan buffer (containing Tris, EDTA, NP-40, and 10 µg/mL of DNase-free RNase) and then passed through a 0.2 µm Nitex filter. The cell suspension was fixed in 1% buffered formaldehyde, passed through a 27-gauge hypodermic needle, and analyzed with a BD FACS Calibur machine with an argon laser at 488 nm. Acquisition parameters used to acquire the dot plots, scatter plots, line plots, and three-dimensional plots are described in the figure legend. Phases of the DNA cell cycle (G0 + G1, S, and G2 + M) were identified and used to assess the rate of DNA synthesis and cell-doubling time. Flow cytometric data were correlated and confirmed with cell proliferation studies obtained by both phase-contrast and fluorescence microscopy.

[Ca2+]i measurements
Porcine coronary artery smooth muscle cells were grown to confluence as already described in large Falcon flasks. The cellular monolayer was washed three times with HEPES buffer. The adherent monolayer was then detached with a trypsin-EDTA solution (3 minutes at 37°C). The cell suspension was spun at 1,200 rpm for 6 minutes at 4°C in a Beckman GS-15 centrifuge. The cell pellet was washed three times in HEPES buffer. The washed cells were resuspended in HEPES buffer and then incubated in the presence of 1 µmol/L Fura-2 for 45 minutes at 37°C. Cells were collected by centrifugation as just described and washed three times to remove any unincorporated Fura-2. The washed Fura-2-loaded cells were resuspended in HEPES buffer and maintained at room temperature with continuous bubbling of 95% oxygen and 5% carbon dioxide. Intracellular free ionized calcium was measured with an Aminco spectrofluorometer.

Reverse transcriptase–polymerase chain reaction
Porcine coronary artery smooth muscle cells were grown in Falcon T2 flasks in the absence or presence of 5-HT (1 µmol/L), sarpogrelate (1 µmol/L), or both for 96 hours, washed three times in PBS (pH 7.4), and harvested by trypsinization as already described. The cell pellet was collected by centrifugation and washed again three times in PBS. Ribonucleic acid was extracted with 4 N-guanidine isothiocyanate, and complementary (c) DNA was prepared by reverse transcription. Reverse transcribed cDNA was amplified by employing specific forward and reverse primers along with 5 U/µL Taq DNA polymerase and dNTPs (deoxynucleotide triphosphate) (including 1 Ci/mL of 32P-dCTP [deoxycytidine triphosphate]) in a multihead RoboCycler thermal cycler (Stratagene, La Jolla CA). The specific forward and reverse primers were as follows:
c-fos: Forward 5'-CACGACCATGATGTTCTCGG-3'
Reverse 5'-AGTAGATTGGCAATCTCGGT-3'
c-jun: Forward 5'-TGAAGCAGAGCATGACCTTG-3'
Reverse 5'-GACACTGGGCAGCGTATTCT-3'
GAPDH: Forward 5'-TTCAACGGCACAGTCAAGG-3'
Reverse 5'-CATGGACTGTGGTCATGAG-3'

The thermal cycler was programmed for 2 minutes of denaturation at 94°C, annealing at 54°C for 1 minute, and elongation at 72°C for 1 minute. After 30 thermal cycles, preparations were subjected to 10 minutes of extended elongation at 72°C and then cooled to 4°C for 30 minutes. The amplified products were visualized under ultraviolet illumination, and quantification was determined with a Bio-Rad GS-670 imaging densitometer (Bio-Rad Canada, Mississauga, ON, Canada) equipped with Molecular analyst software. Subsequently, the bands were excised, and {alpha}-dCTP incorporation was determined by a liquid scintillation counter.

Immunoblotting
Cells were prepared and treated as described in earlier sections. Cells were lysed with sodium dodecyl sulfate gel sample buffer at specific time points after treatment, briefly sonicated, and heated for 5 minutes at 95°C. Samples were applied to 10% polyacrylamide gels and subjected to electrophoresis at 20 mA. The proteins were then transferred to poly(vinylidene difluoride) (PVDF) membranes by electrophoresis. Membranes were blocked for 60 minutes at ambient temperature with 3% bovine serum albumin dissolved in TBS-T (10 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, and 0.1% Tween-20) and subsequently incubated for 60 minutes with primary antibody (diluted in 1% bovine serum albumin/TBS-T). Each membrane was washed extensively with TBS-T, incubated for 60 minutes with horseradish peroxidase–coupled secondary antibody, and washed extensively again. Bound antibody was detected by chemiluminescence and recorded with Kodak X-Omat X-ray film.

Statistical analyses
Data analyses were carried out using Origin version 3.5 (Microcal Software Inc, Northampton, MA) or Microsoft Excel (Office 2000; Microsoft Corp, Seattle, WA) statistical software packages. Plotting the concentration–response curves and determining the effective concentration that will produce 50% of maximal response (EC50) and the inhibitory concentration that will produce 50% inhibition (IC50) values of compounds was performed with SigmaPlot version 4.02 (Jandel Scientific, Corte Madera, CA) computer software. Concentration–response curves were generated by regression analysis. The [Ca2+]i was estimated by exporting the data ASC-II files and subsequently importing these data into the Origin computer software program for further analysis. In addition, the data were also analyzed with SigmaPlot software. Continuous variables are presented as the mean ± one standard deviation. Values were derived from six to nine determinations at each experimental dose, unless otherwise indicated. The data were analyzed by either analysis of variance or Student’s t test. In all instances, a p value of less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Specificity of sarpogrelate
Treatment of PCASMCs with mitogenic factors increases the rate of cell growth, thereby leading to cell division. Serotonin has been identified as an important growth factor capable of modulating SMC proliferation. Receptor-mediated stimulation of PCASMC growth and cell division also occurs in response to other mitogens including Ang II, endothelin, and PDGF.

To determine the specificity of sarpogrelate and the relationship between 5-HT and other mitogenic cytokines, the SMC response to 5-HT, endothelin, Ang II, and PDGF was monitored in the presence and absence of sarpogrelate. This analysis, which consisted of measuring cell number 96 hours after addition of the growth factors, demonstrated that the mitogenic effect of all four agents (at 10 µmol/L concentration) was pronounced (Fig 1). Stimulation with 5-HT, Ang II, PDGF, and endothelin all caused a significant increase in cell number relative to control (p < 0.05). Preincubation (10 minutes) with sarpogrelate (1 µmol/L) inhibited the effect of 5-HT but had no effect on the cellular response to endothelin, Ang II, and PDGF. These results suggest that the negative regulatory effects of sarpogrelate on the proliferation of PCASMCs are exerted specifically through antagonism of 5-HT receptors.



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  		Fig 1. Effect of sarpogrelate on smooth muscle cell response to mitogenic stimulation. Cell number was measured using a Coulter counter 96 hours after treatment in the absence (open bars) and in the presence (solid bars) of sarpogrelate (1 µmol/L): no mitogen (A), serotonin (5-HT) (1 µmol/L) (B), angiotensin II (1 µmol/L) (C), platelet-derived growth factor (1 µmol/L) (D), and endothelin (1 µmol/L) (E). Data represented the mean ± the standard error of the mean of five determinations. In response to 5-HT, sarpogrelate significantly reduced cell number (p < 0.05). No significant differences were seen in the other groups.

 
Antimitogenic actions of sarpogrelate
The results presented in Figure 1 confirm that PCASMC number is increased by treatment with 5-HT and that this effect of 5-HT can be blocked by sarpogrelate. Although cell number is useful for indicating increased cell proliferation, it alone is insufficient to exclude the possibility that sarpogrelate inhibits cell proliferation by another mechanism. Additional indicators of growth and proliferation were therefore measured.

Radiotracer studies that measured RNA, DNA, and protein synthesis were used to measure the stimulation of PCASMC growth by 5-HT. A concentration-dependent increase in 3H-thymidine incorporation was observed with 5-HT, with maximal incorporation at 1 µmol/L concentration. Similar increases in 3H-uridine and 3H-phenylalanine incorporation were also maximal with 1 µmol/L 5-HT (data not shown). Pretreatment with sarpogrelate inhibited these 5-HT–dependent increases in 3H-thymidine, 3H-uridine, and 3H-phenylalanine incorporation (Fig 2). Examination of the concentration-dependent inhibition curves demonstrated that sarpogrelate inhibited the response to 5-HT with an IC50 of 2 nmol/L. These data demonstrated that the mitogenic effects of 5-HT on PCASMCs as assessed by DNA, RNA, and protein synthesis are inhibited by sarpogrelate.



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  		Fig 2. Rate of (A) DNA (deoxyribonucleic acid), (B) RNA (ribonucleic acid), and (C) protein synthesis in response to serotonin (5-HT) treatment in the presence of increasing concentrations of sarpogrelate. Synthesis of DNA, RNA, and protein was estimated over a 96-hour period in the presence of increasing log concentrations of sarpogrelate and fixed concentrations of 5-HT (1 µmol/L) using 3H-thymidine, 3H-uridine, and 3H-phenylalanine incorporation (1 µCi/mL) in growth-arrested, synchronized quiescent cells as described in Material and Methods section. (CPM = count per minute.)

 
Elevated rates of RNA, DNA, and protein synthesis are indicative of increased cell growth, but these variables cannot be used to distinguish between hypertrophic and hyperplastic growth. Smooth muscle can undergo either form of growth, and the response depends on a number of factors. Whereas the increase in cell number previously reported (see Fig 1) is indicative of a hyperplastic process, the MTT cell proliferation assay verified that there was a concentration-dependent increase in cell number after exposure to 5-HT for 72 hours (Fig 3). The presence of 1 µmol/L sarpogrelate reduced the effect of 5-HT over this concentration range (Fig 3B), and sarpogrelate showed a concentration-dependent inhibition at a single dose of 5-HT (Fig 3A). These data indicate that sarpogrelate inhibits 5-HT–induced proliferation of PCASMCs. The inability of 5-HT to readily displace sarpogrelate from the 5-HT2A receptor implies this agent (sarpogrelate) is a high-affinity (insurmountable) antagonist of this receptor (see Fig 3B).



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  		Fig 3. MTT cell proliferation assay of porcine coronary artery smooth muscle cells (PCASMCs) in presence of log dose of serotonin (5-HT) and sarpogrelate. (A) Concentration curve of sarpogrelate with 1 µmol/L 5-HT. (B) Response of PCASMCs to varying concentrations of 5-HT without (•) or with sarpogrelate (1 µmol/L) ({circ}). Data points are presented as the mean ± the standard error of the mean of samples conducted in triplicate.

 
The data thus far presented show that sarpogrelate exerts its negative regulatory effects on cellular proliferation by the inhibition of the 5-HT receptor, but the possibility still exists that sarpogrelate also affects cell viability. The subsequent experiments were carried out to assess this hypothesis. Cell death results in breakdown of the cell membrane. The loss of membrane integrity allows the entry of trypan blue dye into the cells. We found no trypan blue in PCASMCs treated with sarpogrelate to a concentration of 200 µmol/L. Furthermore, flow cytometry was unable to detect the presence of apoptotic cells (data not shown). On the basis of these studies, we concluded that inhibition of cell proliferation by sarpogrelate does not occur as a result of toxicity. This was further confirmed by establishing that proliferation resumed after withdrawal of sarpogrelate (data not shown).

Flow cytometry
To further correlate and confirm 5-HT–induced cell proliferation and its inhibition by sarpogrelate, flow cytometric analysis was conducted on synchronized cells. In this method, propidium iodide, a fluorochrome that binds to DNA, provides an estimate of the proportion of cells in the prereplicative (G0 + G1), replicative (S), and postreplicative (G2 + M) phases of the cell cycle, as determined by their respective DNA content. This technique confirmed that 5-HT induces cellular proliferation in SMCs. We found that 5-HT increased the proportion of cells that were in the G2 + M phases (Fig 4B) relative to control (Fig 4A). Preincubation of PCASMCs with sarpogrelate was associated with a reduction in the number of cells in the postreplicative phase concomitant with an increase in cell numbers in the prereplicative G0 + G1 phases (Fig 4C). These data confirm our previous findings that sarpogrelate can counteract the stimulatory effects of 5-HT. Furthermore, the results of these experiments suggest that sarpogrelate may exert its negative regulatory effects on SMC proliferation by preventing the passage of these cells through the G1 phase of the cell cycle.



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  		Fig 4. Flow cytometric analysis of serotonin (5-HT)–stimulated porcine coronary artery smooth muscle cells. Quiescent cells were treated with (B) 5-HT (1 µmol/L) alone or (C) with sarpogrelate (1 µmol/L) for 96 hours and then stained with propidium iodide for FACS analysis as described in Material and Methods section. Peaks a and b represent cells in the G1 and G2 phases, respectively, of the cell cycle. (DNA = deoxyribonucleic acid.)

 
[Ca2+]i and sarpogrelate
The experimental data to this point confirm that 5-HT is a growth factor capable of inducing cellular proliferation of PCASMCs in culture and that sarpogrelate inhibits the stimulatory effects of 5-HT on these cells. However, these data do not provide information concerning the subcellular processes affected by sarpogrelate. To investigate possible mechanisms by which sarpogrelate exerts its negative regulatory effects, we carried out experiments that examined three critical events that lead to cell proliferation. Exposure of PCASMCs to 5-HT resulted in a rapid increase in Ca2+ and [Ca2+]i remained elevated for more than 400 seconds after treatment (Fig 5A). Thus, calcium entry exhibited a biphasic signal consisting of a fast transient influx followed by a sustained plateau. The 5-HT–induced increase in [Ca2+]i was concentration dependent for both peak height and plateau with an EC50 of 20 nmol/L. Preincubation of PCASMCs with increasing concentrations of sarpogrelate inhibited the 5-HT–induced increase in [Ca2+]i (Fig 5B). Inhibition by sarpogrelate (with an IC50 of 2 nmol/L) was concentration dependent. Inhibition of the 5-HT–induced increase in [Ca2+]i suggests that 5-HT–dependent increases in intracellular calcium are primarily routed through 5-HT2 receptor activation.



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  		Fig 5. Effect of serotonin (5-HT) and sarpogrelate on intracellular free ionized calcium [Ca2+]i of porcine coronary artery smooth muscle cells. Cells were loaded with Fura-2 for measurements of [Ca2+]i as described in Material and Methods section. (A) Cells were treated with 5-HT (1 µmol/L) ± sarpogrelate, and the change in [Ca2+]i was plotted relative to time. Shown is a typical profile in the presence or absence of sarpogrelate (100 nmol/L). (B) Peak height was measured from the time profiles under the conditions shown and compared with untreated control (set to 100%). The data points correspond to the following: control (no sarpogrelate) (•); 0.1 µmol/L sarpogrelate ({circ}), 1.0 µmol/L sarpogrelate ({blacktriangledown}); and 10 µmol/L sarpogrelate ({triangledown}). Each data point represents the mean ± the standard error of the mean for six replicates.

 
Activation of MAP kinase by 5-HT
Mitogen-activated protein kinase is an essential element in signal transduction pathways that transmit information from the cytosol to the nucleus, thus resulting in the induction of gene expression. Activated MAP kinase can be recognized by an immunoblotting technique that employs an antibody that specifically recognizes the activated (tyrosine-threonine–specific dually phosphorylated) form of this enzyme. Immunoblotting demonstrated that MAP kinase activation occurred in PCASMCs that were exposed to 5-HT (Fig 6). Although this technique identifies phosphorylated MAP kinase within the cell, the assay cannot quantitatively assess enzyme activity. However, previous work in our laboratory [8, 12] has confirmed a functional relationship between phosphorylation and MAP kinase activity. Our present experiments demonstrated that phosphorylation of MAP kinase occurred within 10 minutes after exposure to 5-HT (see Fig 6, lane 2). Furthermore, when the cells were pretreated with sarpogrelate before exposure to 5-HT, a significant reduction in MAP kinase phosphorylation resulted (see Fig 6, lane 4). These experiments demonstrated that sarpogrelate is capable of interfering with the transmission of signals from the cytosol into the nucleus in response to 5-HT.



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  		Fig 6. Effect of serotonin (5-HT) and sarpogrelate on mitogen-activated protein (MAP) kinase activation. Porcine coronary artery smooth muscle cells were harvested for Western blot analysis 10 minutes after treatment. Blots were probed with antibodies specific for dual tyrosine-threonine phosphorylated forms of MAP kinase (p42 and p44). Lane designations are as follows: 1 = untreated; 2 = 5-HT (1 µmol/L); 3 = sarpogrelate (1 µmol/L) and; 4 = 5-HT (1 µmol/L) + sarpogrelate (0.1 µmol/L). The positions of the MAP kinase bands were verified with molecular mass markers run in a parallel lane.

 
Induction of protooncogene expression by 5-HT
Experiments were conducted to determine the effect of 5-HT on the expression of the protooncogenes c-fos and c-jun. The techniques of reverse transcriptase-polymerase chain reaction amplification of messenger (m) RNA (Fig 7) and immunoblotting of proteins (Fig 7C) were used for this purpose. Serotonin (1 µmol/L) increased c-fos and c-jun mRNA expression in PCASMCs (see Figs 7A, 7B, column B) compared with control (see Fig 7B, column A). In contrast, sarpogrelate had no effect on either c-fos and or c-jun mRNA expression (see Fig 7B, column C). However, coincubation of sarpogrelate with 5-HT inhibited the 5-HT–induced increase in c-fos and c-jun mRNA expression (see Fig 7B, column D). These data corresponded to the results obtained with immunoblotting of the Fos and Jun proteins (see Fig 7C), although it could be seen that sarpogrelate alone might increase Jun protein levels (see Fig 7C, lane 3). This latter observation could indicate that sarpogrelate has a direct effect on PCASMCs independent of its role as a 5-HT receptor antagonist. Alternatively, 5-HT may have an autocrine/paracrine function for these cells. Additional experiments will be necessary to distinguish between these possibilities.



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  		Fig 7. Analysis of c-fos and c-jun levels in response to serotonin (5-HT) and sarpogrelate. (A) Total RNA (ribonucleic acid) was extracted from cells left untreated (A) or treated with 1 µmol/L 5-HT (B), 0.1 µmol/L sarpogrelate (C), or 1 µmol/L 5-HT + 0.1 µmol/L sarpogrelate (D). The RNA was amplified in the presence of 32P-dCTP as described in Material and Methods section and subsequently analyzed by agarose gel electrophoresis after staining with ethidium bromide. Lane M shows the molecular mass markers used to verify specific c-fos and c-jun amplification (upper gel). The amplification of GAPDH (lower gel) controls for variation in RNA loading. (B) Bands were excised, and incorporation of 32P-dCTP was measured by scintillation counting. Empty and shaded bars indicate c-fos and c-jun levels of 32P-dCTP incorporation, respectively. Data points are shown as the mean ± the standard error of the mean of five sample replicates. Samples A through D are the same as in 7A. (CPM = count per minute.) Immunoblotting of c-fos and c-jun gene products was used to monitor protein levels. Cells were treated with serotonin (5-HT) ± sarpogrelate (both at 1 µmol/L) and harvested in gel sample buffer. Samples were subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the proteins were transferred to poly (vinylidene difluoride) (PVDF) membrane. The membrane was subsequently probed with antibody specific to either the Fos or Jun proteins. Treatment conditions were as follows: untreated control (A), 1 µmol/L 5-HT (B), 0.1 µmol/L sarpogrelate (C), and 1 µmol/L 5-HT + 0.1 µmol/L sarpogrelate (D).

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
The problem of vein graft attrition after coronary artery bypass grafting is complex and multifactorial. However, SMC proliferation is considered a key component in this process [13–16]. Early proliferation of SMCs results in neointimal thickening (also called neointimal proliferation or intimal hyperplasia) and is responsible for early graft failure. Late graft failure is the result of atherosclerosis. However, it is still beneficial to consider atherosclerosis as a pathological condition whose development is secondary to the uncontrolled proliferation of the SMCs within the vessel wall [17]. Under normal circumstances, SMCs in the walls of arteries and veins exhibit a nonproliferative contractile phenotype. As a result of injury, mitogenic stimulation, or altered metabolic states within the host, these cells begin to grow. They acquire a synthetic phenotype through a process that is referred to as phenotypic modulation. The result is a decrease in levels of actin and myosin; the SMCs become motile and assume an appearance similar to that of fibroblasts. At this point, these cells begin to secrete collagen and other proteins that eventually result in the formation of an extracellular matrix. The matrix becomes the scaffold for calcification. This is known as vascular remodeling [15]. The evidence to date suggests that SMCs from arterial or venous origins can proliferate, can undergo phenotypic modulation, and are essential in the development of vascular occlusive disease. Thus, the study of mammalian SMCs from various sources may give insight into the regulatory pathways that control the proliferation of these cells.

In the current work, we elected to study the effect of 5-HT on the proliferation of SMCs derived from porcine coronary arteries. There were many reasons why we chose to study this particular mitogen in the porcine model. We [18] had undertaken a previous study in which we found that 5-HT induced proliferation of SMCs from rat aorta and that the mitogenic effects of 5-HT could be inhibited by sarpogrelate. However, that study was primarily observational and gave no insight into the signal transduction pathways that potentially regulate this process. The current study was undertaken to build on this foundation. We chose the pig (versus the rat) as our model this time because of its closer relationship to humans along the phylogenetic tree. In addition, we explored in much greater detail the mechanisms by which 5-HT may induce SMC proliferation. Finally, we hope this work may eventually become clinically relevant to the problem of vessel occlusion of both the native circulation and bypass conduits.

Recently Sperti and colleagues [19] investigated the response of the radial artery and the internal mammary artery to 5-HT in patients 1 year after coronary artery bypass grafting. They found that 5-HT caused major vasoconstriction of radial artery grafts but not internal mammary artery conduits. Further evidence of why 5-HT may be an important factor in the genesis of vascular occlusive disease comes from studies of hypercholesterolemia-induced neointimal hyperplasia in experimental vein grafts [20]. In New Zealand White rabbits fed a 1% cholesterol diet, accelerated atherosclerosis developed in a reversed vein common carotid artery bypass model. Of particular relevance to our current work, these grafts demonstrated a significant increase in vasomotor tone when exposed to 5-HT. There is a correlation between altered vasomotor reactivity and the development of vascular atherosclerotic disease. Beyond its effect on vasomotor reactivity, there is evidence that 5-HT induces cellular proliferation of a variety of cells in culture, including vascular SMCs [1, 21]. As noted by Fanburg and Lee [21]: "A hypertrophic, as well as a proliferative response, has been shown to occur in some smooth muscle cells. It has been proposed that, through these signaling pathways, serotonin plays an important role in remodeling of both the pulmonary and systemic circulations." Furthermore, the proliferative effect of 5-HT is synergistic with that of more conventional growth-producing polypeptides.

The proliferation of smooth muscle and endothelial cells in response to vascular injury is mediated by the release of 5-HT from platelets [22, 23]. Furthermore, the vasoconstrictor response to 5-HT can be reversed by the 5-HT2 blocker ketanserin [24]. These studies suggest that antagonists to 5-HT receptors may be useful agents to prevent restenosis after both coronary artery bypass grafting and percutaneous transluminal coronary angioplasty. Multiple receptors for 5-HT have been identified, and pharmacological characterization suggests that there are at least seven distinct subtypes [21, 25, 26]. One of the objectives of this study was to explore whether stimulation of SMC proliferation by 5-HT is inhibited by a 5-HT2A receptor antagonist, sarpogrelate. In PCASMCs, we found that cell growth, DNA synthesis, and mitosis occurred in response to 5-HT treatment (see Figs 1 through 4). Multiple 5-HT receptor subtypes could potentially be involved in the induction of SMC proliferation. Other growth factors have already been demonstrated to exert their mitogenic effect through multiple receptors [27, 28].

The rationale for testing sarpogrelate under conditions of Ang II, endothelin, or PDGF stimulation was to determine whether 5-HT is a component of the mitogenic pathways associated with other growth factors, as has been proposed for PDGF and epidermal growth factor [22]. In our experiments, sarpogrelate had no effect on the mitogenic action of Ang II, PDGF, and endothelin (see Fig 1). Thus our data suggest that 5-HT does not operate in PCASMCs as a paracrine/autocrine factor for Ang II, PDGF, or endothelin. Nevertheless, it remains possible that PDGF and Ang II may be involved in the later stages of vascular remodeling triggered by 5-HT, since 5-HT operates synergistically with other growth factors (see Fig 1) [29, 30].

Serotonin may exert its proliferative effects through its regulation of thromboxane A2, PDGF, and transforming growth factor ß, but it is also apparent that 5-HT exerts a direct effect on both proliferation and migration mediated through a ligand-receptor interaction by way of the 5-HT2 receptor family [21, 31]. Undoubtedly then, an interaction between 5-HT and its receptor must activate a series of signal transduction pathways that ultimately induce proliferation. Our findings that sarpogrelate inhibited the 5-HT–induced increase in [Ca2+]i, which could act as a primary signaling event involved in DNA synthesis and hence cell proliferation, are totally consistent with this hypothesis (see Fig 5). Sarpogrelate also inhibited the 5-HT–induced increase in the expressions of c-fos and c-jun mRNA expression in PCASMCs (see Fig 7). A strong correlation between [Ca2+]i and protooncogene expression has been demonstrated, and both are likely early events mediating the mitogenic actions of 5-HT (see Figs 5, 7).

Serotonin is an important mediator of cellular proliferation of coronary artery SMCs. The data presented here support the concept that intervention to block 5-HT receptors may aid in the prevention of restenosis. A live animal study is warranted, and if the in vitro results are corroborated, a clinical investigation of sarpogrelate may be justified.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
Financial support was provided by a grant from the Medical Research Council of Canada (MRC) Group in Experimental Cardiology. Dr Dhalla holds the MRC Pharmaceutical Research and Development Chair in Cardiovascular Research supported by Merck Frost, Canada.

We thank Mr Jin Shiomura of the Mitsubishi-Tokyo Pharmaceutical Co Ltd, Tokyo, Japan, for his continued interest in our studies and for providing the sarpogrelate used.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

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