Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Neointimal hyperproliferation and platelet activation/aggregation are two major cardiovascular abnormalities commonly observed in blood vessels after an insult such as cellular injury, mechanical or physiological stress, or overload due to peripheral resistance (Schwartz et al., 1986; Schwartz and Reidy, 1987). The exact molecular mechanism(s) of neointimal hyperproliferation in response to cell injury is poorly understood. It is generally held that cellular injury triggers an enhanced release of certain cytokines that induce hyperproliferation in aortic smooth muscle cells (Delafontaine 1998; Stouffer and Runge, 1998). Among the cytokines that have been discovered thus far, serotonin (5-HT), angiotensin II, endothelin, and platelet-derived growth factors (PDGFs) are primarily involved in cardiovascular remodeling (Nemecek et al., 1986; Saward and Zahradka, 1996). These agents act via specific cell surface receptors to trigger various intracellular signal transduction cascades involved in cellular hyperplasia. During mechanical cellular injury, for example, mitogen-activated protein kinase is activated, and its translocation from the cytosol to the nucleus provides the intracellular signal leading to stress-dependent induction of protooncogenes (mainly c-fos and c-jun) and hence cell proliferation (Seth et al., 1992; Davis, 1993; Janknecht et al., 1993; Gonzalez et al., 1993; Karin, 1995; Seewald et al., 1998).

Several attempts have been made in the past to develop selective inhibitors of vascular neointimal hyperplasia, with limited success (Weissberg et al., 1993). Recent studies have demonstrated that, after restenosis postangioplasty, mitogen-activated protein kinase activity is significantly increased in the pig coronary artery, and this could be used as an index of cell proliferation (Yau and Zahradka, 1997; Pyles et al., 1997). In the present study, we have explored the influence of 5-HT in rat aortic smooth muscle cell (RASMC) proliferation and the involvement of 5-HT receptor activation in this process using a novel receptor antagonist, sarpogrelate. The basic aim of this study was to establish the ability of sarpogrelate (also known as Anplag or MCI-9042) to function as a specific antiproliferative agent via 5-HT receptors. Our data demonstrate that sarpogrelate effectively inhibits RASMC proliferation at a concentration of 0.1 µM.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. Cell culture materials, including powdered Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and gentamicin (10 mg/ml), flasks and microtiter plates were purchased from GIBCO/BRL, Life Technologies (Mississauga, Canada). 5-HT, endothelin, and PDGF were purchased from Sigma-Aldrich Chemical Co. (Oakville, Canada). All other chemicals were of reagent grade quality and were purchased from Sigma-Aldrich Chemical Co. Radioisotopes for protein studies ([2,6-³H]phenylalanine, 55 Ci/mmol), RNA ([5,6-³H]uridine, 36 Ci/mmol), and DNA ([methyl-³H]thymidine, 2 Ci/mmol) were purchased from Amersham Canada (Oakville, Canada).

Rat Aortic Smooth Muscle Cell Culture. All experimental protocols described were approved by the local Animal Care Committee in accordance with the standards of the Canadian Council of Animal Care. Cultured RASMCs were prepared as described previously (Chamley et al., 1979). Briefly, aortae from male Sprague-Dawley rats were cleared of adventitial and intimal tissues before a skin biopsy punch was used to remove 1-mm segments for explant. The explants were incubated at 37°C in 5% CO₂ in DMEM (high glucose) containing 10% fetal bovine serum plus 1 µg/ml gentamicin with changes of medium every 72 h. To facilitate the adherence of explants, the flasks were turned upside down every day. Initial growth from the explants occurred within 1 to 2 weeks, and the cells were identified as smooth muscle cells (SMCs) by immunohistochemical detection of smooth muscle myosin and smooth muscle -actin (Saward and Zahradka, 1997). Furthermore, the ubiquitous myosin staining observed in these populations (Fig. 1) demonstrated the presence of >95% smooth muscle cells. Once the cells reached 70% confluence, they were placed into serum-free media supplemented with 5 µg/ml transferrin, 1 nM selenium, 0.2 mM ascorbic acid, and 10 nM insulin (Saward and Zahradka, 1997) for 30 h. This treatment was used to synchronize the cells before mitogen stimulation.

View larger version (53K): [in this window] [in a new window]   Fig. 1.   Immunohistochemical staining of explant-derived cell populations for SMCs. Verification of smooth muscle origin was demonstrated by staining for smooth muscle -actin (A) and smooth muscle myosin (C). Individual cells were identified within the same fields (B, actin; D, myosin) with Hoescht 33342, a nucleus-specific stain, and an estimate was made of the number of non-SMCs (<5% myosin-negative) in the population.

Macromolecular Synthesis. The rate of DNA, RNA, and protein synthesis in response to various agents was studied using 96-well microtiter plates (Fisher Scientific, Fairlawn, NJ) in DMEM + 10% FBS in 200 µl in the presence of (1 µCi/ml) [³H]thymidine, [³H]uridine, and [³H]phenylalanine, respectively. Logarithmic concentrations of 5-HT and sarpogrelate were prepared in PBS, and each experimental concentration was run in triplicate. The cells were collected at different time intervals (0, 4, 8, 12, and 24 h) using 0.25% trypsin-EDTA solution. The reaction was stopped using 10% trichloroacetic acid in PBS, and the samples were filtered through GF/B glass filters under vacuum. Radioactivity was counted using a Beckman LS10 scintillation counter.

Cell Proliferation Assay. Cell proliferation was measured using 3-(4,5-dimethylthiozol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) according to Saward and Zahradka (1996). MTT (5 mg/ml) was dissolved in RPMI 1640 without phenol red, filter sterilized, and stored at 4°C. The dye (200 µl) was added to the culture medium and incubated for 4 h. At the end of the incubation, the medium was removed, and the converted dye was solubilized with acidified isopropanol (0.04-0.1 N HCl in absolute propanol). The absorbance of the converted dye was measured at 570 nm with a background subtraction at 630 to 690 nm.

Flow Cytometry. RASMCs were incubated for 24 h in Falcon T2 flasks with 5-HT and/or sarpogrelate. The cellular monolayer was washed three times with PBS, and the cells were detached using trypsin-EDTA as described above. The number of cells was measured using a Coulter counter, and all viability was verified by trypan blue exclusion and phase-contrast microscopy. RASMCs (10⁴) were stained in propidium iodide prepared in Krishan buffer (containing Tris, EDTA, and Nonidet P-40 and RNase, 10 µg/ml). The cells were stained for 30 min at 4°C in ice and passed through a 0.2-µm Nitex filter. The cell suspension was fixed in 1% buffered formaldehyde, passed through a 27-gauge hypodermic needle (to avoid nozzle clogging), and collected in Falcon plastic tubes specially designed for the Becton-Dickinson flow cytometer. The samples were analyzed using a Becton-Dickinson fluorescence-activated cell sorter (FACS) Calibur machine with an argon laser at 488 nm spectral wavelength. Acquisition parameters used to acquire the dot plots, scatter plots, line plots, and three-dimensional plots are described in the legends to Figs. 4 and 5. Various phases of the cell cycle (such as G₀-S, G₁-S, and G₂-M) were recorded to analyze the rate of DNA synthesis and volume doubling time. Flow cytometric data was correlated and confirmed with the cell proliferation study made using phase-contrast microscopy and fluorescence microscopy.

Statistics. Data were typically averaged for six to nine determinations at each experimental dose using Origin (version 3.5) for statistical analysis and Excel for equation solving. SigmaPlot (version 4.02) computer software was used for plotting the concentration-response curves and determining the EC₅₀ and IC₅₀ values of compounds. The data were analyzed statistically using multiple measures ANOVA and Student's t test, and was considered significant when p < .05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Serotonin is a potent cytokine for RASMCs as demonstrated by a 5-HT-induced increase in cell proliferation. Serotonin stimulated a concentration-dependent increase in cell growth as measured using MTT (Fig. 2A). The contribution of the 5-HT2A receptor was indicated by the action of the 5-HT2A-specific antagonist sarpogrelate, which inhibited serotonin-induced cell growth in RASMCs (Fig. 2B). To distinguish between growth and proliferation, which can also be designated as hypertrophy and hyperplasia, respectively, cell numbers were measured. 5-HT clearly increased RASMC cell numbers after 24 h (Fig. 3). Although the experimental period was not long, there was a statistically significant difference in the values between control and 5-HT treatment conditions. Sarpogrelate blocked the 5-HT-dependent increase in cell number. Interestingly, treatment with sarpogrelate alone reduced cell number compared with the control. A contribution by 5-HT to serum-dependent proliferation may be indicated by these data. Alternatively, sarpogrelate treatment may affect cell viability; however, at concentrations <100 µM, trypan blue exclusion did not reveal any evidence of cell death, flow cytometry was unable to detect the presence of apoptotic cells, and proliferation resumed after removal of the agent (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effect of 5-HT on the proliferation of RASMCs. A, concentration dependence of 5-HT on RASMCs as measured with MTT without and with coincubation with sarpogrelate (1 µM). B, concentration dependence of sarpogrelate on 5-HT (1 µM)-induced cell proliferation in RASMCs. MTT cell proliferation was measured using a 96-well microtiter plate as described in Materials and Methods. Spectral wavelength was measured at 550 nm. Optical densities were measured using a SpectroTherm (Molecular Dynamics, Sunnyvale, CA) microplate reader. Data are presented as means ± S.E. for triplicate samples.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Effect of 5-HT and sarpogrelate on cell number. RASMCs were left untreated (control) or were treated with 5-HT (1 µM), sarpogrelate (1 µM), or a combination of 5-HT (1 µM) plus sarpogrelate (1 µM). After the treatment period (24 h), cells were released by trypsinization, and cell numbers were determined with a Coulter counter. Data are presented as means ± S.E.

Flow Cytometry and DNA Content. Flow cytometry serves as a link between morphological and radiotracer studies. We conducted these studies to further correlate and confirm serotonin-induced cell proliferation and/or hypertrophy and its inhibition by sarpogrelate. Stimulation of cell cycle progression by 5-HT was confirmed using propidium iodide as a fluorochrome, which binds to DNA and provides an estimate of cells in the early G₁-S (DNA replication) and late G₂-M (mitotic) phases of the cell cycle according to DNA content. A concentration-dependent increase in DNA content was observed in response to serotonin (10¹²-10⁶ M) as compared with control cells (Fig. 4). Preincubation of RASMCs with sarpogrelate significantly inhibited the serotonin-induced increase in DNA content (Fig. 4). These data were confirmed by evidence that sarpogrelate inhibited the serotonin-induced cell cycle progression as demonstrated by the number of cells present in the G₂-M phase (Fig. 5).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Measurement of serotonin-dependent effects on DNA synthesis in RASMCs as measured by flow cytometry. RASMCs were incubated for 24 h in the absence or presence of 5-HT with or without sarpogrelate. Cells were stained with propidium iodide and subjected to FACS analysis. The data are presented as fluorescence intensity (FL2-H), which indicates relative DNA content, versus number of cells (counts). The concentration provided on the left indicates the amount of 5-HT added in the center column and the amount of sarpogrelate (5-HT is constant at 1 µM) in the right-hand column. The area under the peak indicates DNA content for each cell.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Effect of sarpogrelate on 5-HT-induced cell cycle progression. Cells were stimulated with varying concentrations of 5-HT with or without sarpogrelate (1 µM) for 24 h and subjected to FACS analysis after labeling with propidium iodide. Fluorescence intensity is plotted relative to cell number. Peaks on the left-hand side represent the G1 phase (I), while right-shifted peaks indicate the G2 phase (II).

Radiotracer Studies. The increase in DNA content and cell number observed with 5-HT treatment indicates that both cell growth and division are stimulated by this agent. A 5-HT-induced, sarpogrelate-sensitive, concentration-dependent increase in [³H]thymidine incorporation, which peaked at approximately 20 h (Fig. 6), confirmed the previous observations. Similar increases in RNA and protein synthesis over time were indicated by incorporation of [³H]uridine and [³H]phenylalanine, respectively (Fig. 6). Both processes were inhibited by sarpogrelate, with sarpogrelate at 10 µM capable of inhibiting RNA and protein synthesis at any given concentration of 5-HT. To define the concentration-dependent responses of the RASMCs to 5-HT and sarpogrelate, cells were stimulated with 5-HT, and incorporation of thymidine, uridine, and phenylalanine was measured at 24 h posttreatment (Figs. 7-9). These data demonstrate that the maximal response to 5-HT, including DNA, RNA, and protein synthesis, occurs with a concentration of approximately 100 nM. Furthermore, 1 µM sarpogrelate inhibited the response by ~50% (Figs. 7-9A). The concentration response to sarpogrelate is illustrated in Figs. 7 to 9B, and these data indicate that 50% inhibition is obtained with 1 nM sarpogrelate. This is consistent for all parameters being measured.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effect of sarpogrelate on 5-HT-induced RASMC growth. Radiolabeled precursors were used to monitor DNA synthesis (thymidine incorporation), RNA synthesis (uridine incorporation), and protein synthesis (phenylalanine incorporation) over 24 h after stimulation of RASMCs with 1 µM 5-HT. The effectiveness of 10 µM sarpogrelate as an inhibitor was also tested. Data are presented as means ± S.E.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Concentration dependence of DNA synthesis to 5-HT and sarpogrelate. A, RASMCs were treated with varying concentrations of 5-HT with or without 1 µM sarpogrelate for 24 h in the presence of labeled precursor ([3H]thymidine). B, [3H]thymidine incorporation was measured over a 24-h incubation period after treatment with 1 µM 5-HT in the presence of varying concentrations of sarpogrelate. Data are presented as means ± S.E.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Concentration dependence of RNA synthesis to 5-HT and sarpogrelate. A, RASMCs were treated with varying concentrations of 5-HT with or without 1 µM sarpogrelate for 24 h in the presence of labeled precursor ([3H]uridine). B, [3H]uridine incorporation was measured over a 24-h incubation period after treatment with 1 µM 5-HT in the presence of varying concentrations of sarpogrelate. Data are presented as means ± S.E.

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Concentration dependence of protein synthesis to 5-HT and sarpogrelate. A, RASMCs were treated with varying concentrations of 5-HT with or without 1 µM sarpogrelate for 24 h in the presence of labeled precursor ([3H]phenylalanine). B, [3H]phenylalanine incorporation was measured over a 24-h incubation period after treatment with 1 µM 5-HT in the presence of varying concentrations of sarpogrelate. Data are presented as means ± S.E.

Specificity of Sarpogrelate. The results presented in this report indicate that 5-HT stimulates SMC proliferation. In addition, the inhibition observed with sarpogrelate suggests that 5-HT operates through the 5-HT2A receptor. Receptor-mediated stimulation of SMC growth and division also occurs in response to endothelin and PDGF. To evaluate 1) the specificity of sarpogrelate and 2) the relationship between 5-HT and other mitogenic cytokines, the SMC response to endothelin and PDGF was monitored in the presence and absence of sarpogrelate. The mitogenic effect of both agents was pronounced as shown by assays for DNA, RNA, and protein synthesis (Figs. 10 and 11). Although we have shown that 5-HT-induced DNA, RNA, and protein synthesis was partially suppressed when SMCs were preincubated with 1 µM sarpogrelate (Fig. 6), the endothelin- and PDGF-induced increases in DNA, RNA, and protein synthesis were not affected by concentrations of sarpogrelate up to 10 µM (Figs. 10 and 11).

View larger version (20K): [in this window] [in a new window]   Fig. 10.   Effect of sarpogrelate on the stimulation of RASMC growth by endothelin. DNA synthesis (A), RNA synthesis (B), and protein synthesis (C) were monitored over 24 h after stimulation with 10 nM endothelin-1 in the presence or absence of 10 µM sarpogrelate. Data are presented as means ± S.E.

View larger version (20K): [in this window] [in a new window]   Fig. 11.   Effect of sarpogrelate on the stimulation of RASMC growth by PDGF. DNA synthesis (A), RNA synthesis (B), and protein synthesis (C) were monitored over 24 h after stimulation with 10 µM PDGF in the presence or absence of 10 µM sarpogrelate. Data are presented as means ± S.E.

Comparison to 5-HT Receptor Antagonists. Sarpogrelate is considered a selective 5-HT2A receptor antagonist. Nevertheless, there are indications that sarpogrelate may also bind to other 5-HT receptor subtypes. This information is highly relevant to the current study, because it has not been clearly established whether 5-HT-dependent cell proliferation is mediated by 5-HT2A receptors alone or involves multiple subtypes. To address the issue of specificity, we compared the effect of sarpogrelate on 5-HT-mediated DNA synthesis with other well characterized 5-HT receptor antagonists. These data showed that the inhibition observed with sarpogrelate is greater than that observed with ketanserin (Fig. 12). The antagonists cinanserin, mianserin, and methysergide, in contrast, were much less effective. Interestingly, none of the antagonists are capable of completely inhibiting the stimulation by 5-HT. The implications of the latter observation, however, are unclear.

View larger version (28K): [in this window] [in a new window]   Fig. 12.   Comparative assessment of 5-HT receptor antagonists as inhibitors of 5-HT-stimulated DNA synthesis. RASMCs were exposed to varying concentrations of the 5-HT antagonists methysergide, mianserin, cinanserin, ketanserin, and sarpogrelate over the 24-h incubation period after addition of 5-HT. DNA synthesis was measured by incorporation of [3H]thymidine, and the data are presented as means ± S.E.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The stimulation of vascular SMC proliferation by 5-HT, first recognized by Nemecek et al. (1986), may be an important element in vascular pathophysiology (Fanburg and Lee, 1997). Although it is recognized that 5-HT is a potent vasoactive agent (Hollenberg, 1985), the possibility that 5-HT promotes SMC proliferation and migration leading to vascular obstruction has received considerable interest. The investigations by Crowley et al. (1994) and Pakala et al. (1994) are particularly relevant, because they demonstrate that SMC and endothelial cell proliferation in response to vascular injury is mediated by 5-HT released from adhering platelets. Based on these reports, it has been proposed that antagonists for 5-HT receptors could be used to restrict restenosis after angioplasty (Pakala et al., 1997).

Multiple receptors for 5-HT have been identified, and pharmacological characterization indicates that there are at least seven distinct subtypes (Zifa and Fillion, 1992; Foy et al., 1992; Fanburg and Lee, 1997). With respect to normal vascular physiology, both 5-HT1A and 5-HT2 receptors mediate SMC contraction (Foy et al., 1992). Additionally, 5-HT1A and 5-HT2, as well as the 5-HT1B receptors have been shown to stimulate SMC proliferation (Fanburg and Lee, 1997). Because numerous, apparently subtype-specific, 5-HT receptor antagonists have been developed, clarification of receptor involvement in SMC proliferation would assist in identifying those agents with the potential for therapeutic intervention.

The primary question addressed by this investigation was whether 5-HT-stimulated SMC proliferation is inhibited by sarpogrelate. Using SMC from rat aorta, we showed that cell growth, DNA replication, and mitosis occurred in response to 5-HT treatment. Although sarpogrelate has been reported as specific for the 5-HT2A receptor (Maruyama et al., 1991; Hara et al., 1991; Nishio et al., 1996; Takada et al., 1997), thus advocating a role for this receptor in the RASMC proliferative response, our data cannot dismiss the involvement of other receptor subtypes. Selectivity of receptor antagonists is often difficult to determine using standard pharmacological methodology. Difficulties arise from variation in the affinity of 5-HT and 5-HT receptor antagonists for the receptors, as well as the potency of the receptor in evoking a cellular response. In addition, there have been reports of heteromeric complexes of 5-HT receptors (Fletcher and Barnes, 1998; van Hooft et al., 1998), as well as altered antagonist-receptor interactions caused by modulation by G proteins (Pauwels et al., 1998). The comparison of various 5-HT receptor antagonists on 5-HT-stimulated DNA synthesis was intended to address the issue of multiple receptor subtype involvement (Fig. 12). The most potent antagonist, ketanserin, is a mixed 5-HT2A/B/C antagonist that may also bind to 5-HT1B/1D receptors. In contrast, cinanserin and mianserin apparently show greater selectivity for the 5-HT2 receptor family. The anomalous response to methysergide, a broad-spectrum antagonist that would be expected to be the most potent antagonist, may be explained by its action as a 5-HT1A receptor agonist (Newman-Tancredi et al., 1997). This information, therefore, strongly supports the view that multiple 5-HT receptors are involved in 5-HT-mediated RASMC proliferation. Multiple receptor involvement has already been demonstrated for other ligands (Yamazaki et al., 1997; Yau et al., 1996). Therefore, based on our observations, it is likely that the potency of sarpogrelate as an antiproliferative agent results from binding to multiple receptor subtypes, presumably similar to those identified for ketanserin. Based on the effective inhibition of RASMC proliferation we have observed, sarpogrelate is being evaluated as inhibitor of intimal proliferation postangioplasty.

The rationale for testing sarpogrelate under conditions of endothelin and PDGF stimulation was to determine whether 5-HT is a component of mitogenic pathways associated with other growth factors, as has been proposed for PDGF and epidermal growth factor (Crowley et al., 1994). Our observations indicate that sarpogrelate had no effect on the mitogenic actions of either PDGF or endothelin (Figs. 10 and 11). It is therefore evident that 5-HT does not operate in RASMCs as a paracrine/autocrine factor for either PDGF or endothelin. Although 5-HT may not be directly involved as a mediator of PDGF and endothelin mitogenesis, it does operate synergistically with other growth factors (Pakala et al., 1997; Araki et al., 1990). Given the high concentration of 5-HT in the dense granules of platelets, in addition to thromboxane A₂, PDGF, and transforming growth factor , it is apparent that 5-HT is a major contributor to SMC migration and proliferation through the 5-HT2 receptor family (Tamura et al., 1997; Fanburg and Lee, 1997). Inhibition of growth stimulation by 5-HT may thus have clinical application for the prevention of restenosis. Our data therefore suggest that sarpogrelate, alone or in combination with other agents (Pakala et al., 1997; Origuchi et al., 1997), is a good candidate for clinical evaluation as an inhibitor of neointimal proliferation. This perception is supported by evidence that sarpogrelate operates as a vasodilator (Pawlak et al., 1996) and thus would provide additional benefits to patients with reduced coronary flow (Tanaka et al., 1998).