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Serotonin and thromboxane A2 stimulate platelet-derived microparticle-induced smooth muscle cell proliferation
Cardiovascular Radiation Medicine 5 (2004) 20 – 26
Serotonin and thromboxane A2 stimulate platelet-derived $ microparticle-induced smooth muscle cell proliferation Rajbabu Pakala * Department of Internal Medicine, Division of Cardiology, University of Texas Health Science Center Medical School, Houston, TX 77030, USA Received 19 December 2003; accepted 22 December 2003
Abstract
Introduction: At the sites of vascular injury, activated and aggregating platelets release small vesiculated structures called platelet microparticles (PMPs). Apart from PMPs they also release several vasoactive mediators including serotonin and thromboxane A2 (TXA2). PMPs, serotonin, and TXA2 have been shown to stimulate vascular smooth muscle cell (VSMC) proliferation. Thus, this study is designed to examine the interaction between PMPs and serotonin or TXA2 in inducing rabbit VSMC proliferation. Methods: Growth-arrested rabbit SMCs were incubated in serum-free medium with different concentrations of PMPs with or without serotonin or TXA2. VSMC proliferation was examined by increase in incorporation of [3H]thymidine into DNA and by increase in cell number. Results: PMPs stimulated DNA synthesis in a dose-dependent manner; up to an added concentration of 30 Ag/ml (1489F90%) they stimulated SMC proliferation in a logarithmic fashion. Serotonin at 50 AM (345F21%) and TXA2 at 7.5 AM (900F36%) had their maximal effect. When added together, PMPs (10 Ag/ml) and serotonin (5 AM), synergistically induced DNA synthesis (581F36% and 211F11% when added alone and 1201F95% when added together), whereas PMPs (10 Ag/ml) and TXA2 (5 AM) additively induced DNA synthesis (581F36% and 781F56% when added alone and 1262F115% when added together). These increases in DNA synthesis were paralleled by increase in cell number. Conclusion: PMPs, serotonin, and TXA2 are mitogenic to SMC, and function as amplification factors to each other, suggesting 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. D 2004 Elsevier Inc. All rights reserved.
Keywords:
Platelet microparticles; Serotonin; Thromboxane A2; Smooth muscle cells; Proliferation; Restenosis
1. Introduction When platelets are stimulated with agonists such as thrombin, collagen, or calcium ionophore, A23187, or exposed to high-shear stress they release small (< 1 Am) vesiculated structures called platelet microparticles (PMPs) [1– 6]. PMPs are detectable in serum of normal donors [7]
$ There is no financial relationship between the author and the subject matter. * Cardiovascular Research Institute, Washington Hospital Center, GHRB # 313, 108 Irving Street, NW, Washington, DC, USA. Tel.: +1202-877-3867; fax: +1-202-877-3997. E-mail address: [email protected] (R. Pakala).
1522-1865/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.carrad.2003.12.002
and their levels increase in pathological states such as sepsis [8], heparin-induced thrombocytopenia [9], cerebrovascular events [10], unstable angina [11], acute myocardial infarction [12], and cardiopulmonary bypass [13]. PMPs express several platelet –endothelium attachment receptors on their surface, for example, glycoprotein IIb/IIIa (CD41), Ib and IaIIa, and P-selectin (CD62P) [14,15]. They also contain bioactive lipids including sphingosine 1-phosphate (SPP-1) and arachidonic acid (AA) [14,16]. It has been reported that after interaction with target cells PMPs trigger some biological responses; for example, they may activate endothelial cells [15], polymorphonuclear leukocytes [17], and monocytic U-937 cells [14,17], modulate monocyte – endothelial cell interactions [18] and leukocyte – leukocyte interactions
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[19], inhibit apoptosis of polymorphonuclear leukocytes [19, 20], and induce chemotaxis of U-937 cells [14,16]. The functional role of PMPs is still largely unknown. PMPs contain both pro- and anticoagulant proteins [5,21], and it has been suggested that the effects of PMPs on blood coagulation are related to the negative charge derived from exposed membrane phospholipids. During thrombosis, an association of PMPs with fibrin has been described [22], indicating a possible role of PMPs in the hemostatic process. Since PMPs have been shown to activate endothelial cells, leukocytes, and platelets and up-regulate interaction between these cells, they may play an important role in restenosis. PMPs have been shown to stimulate proliferation of endothelial cells, smooth muscle cells (SMCs), and hematopoietic cells [23 – 25]. Because flow dynamics would result in the accumulation of PMPs and other plateletreleased factors such as serotonin and thromboxane A2 (TXA2) at the sites of endothelial injury we wanted to test if PMPs, serotonin, and TXA2 will interact with each other in stimulating SMC proliferation.
2. Materials and methods 2.1. Materials Bovine serum albumin (BSA), insulin, transferrin, N-(2hydroxyethyl) piperazine-NV-(2-ethanesulfonic acid), 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), pargyline, and serotonin as creatinine sulfate were purchased form Sigma, St. Louis, MO. TXA2 mimetic U 46619 [(15S)-hydroxy-11a,9a-(epoxymethano)-presta5Z,13E-dioic acid] was obtained from Upjohn, Kalamazoo, MI. Platelet-derived growth factor (PDGF) was obtained from Amersham Life Science, Arlington Heights, IL. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin –EDTA, Hank’s balanced salt solution (HBSS), and phosphate buffered saline (PBS) were purchased from Gibco BRL Life Technologies, Gaithersburg, MD. [3H]Thymidine (specific activity 20 Ci/ mol) was from Dupont NEN Research Products, Boston, MA; Tissue culture plastics were from Falcon Labware, Lincoln Park, NJ. Other reagents were purchased from local vendors. 2.2. Isolation of platelets Platelet pellets were prepared according to the method previously reported [15]. Briefly, using 18-gauge needles nine volumes of blood were collected into one volume of citrate buffer (3.8% sodium citrate). The blood samples were centrifuged at 100g for 15 min at room temperature and the supernatant platelet-rich plasma (PRP) was carefully collected with a plastic pipette. All the preparations were done at room temperature. Prostaglandin I2 was added to PRP at the final concentration of 5 ng/ml for
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the purpose of cytoprotection and PRP was centrifuged at 650g for 13 min. The platelet pellet was resuspended in a HEPES buffer containing 142 mM NaCl, 6.2 mM KCl, 2.4 mM MgSO4, and 6.5 mM HEPES, pH 7.4. Then, after the second addition of prostaglandin I2 at the final concentration of 300 ng/ml to the suspension, platelets were centrifuged at 650g for 13 min. The same washing procedure was repeated and platelets resuspended in the HEPES buffer without prostaglandin I2. The final platelet count was adjusted to be 3108/ml. The suspension thus obtained was found to be free of any contaminants by a microscopic examination. 2.3. Preparation of PMPs Washed platelets were suspended in HEPES-buffered saline containing 1 mM CaCl2. Preparation of PMPs was done according to the modified method previously reported [15,16]. Briefly, washed platelets were stimulated with thrombin (0.5 U/ml) plus collagen (10 mg/ml) at 37 jC for 10 min without stirring. The reaction was terminated by addition of EGTA (final concentration of 5 mM) and cooling on an ice bath. PMPs were separated from the activated platelets by centrifugation at 1000g for 15 min at 4 jC. From the resulting supernatant, PMPs were pelleted by centrifugation at 10,000g at 4 jC for 15 min. PMPs were washed with HEPES buffer, pelleted again, and resuspended in HEPES buffer. The protein content of PMPs was determined by the protein assay of Bradford according to the manufacturer’s instructions (Bio-Rad). 2.4. Arterial SMC isolation Rabbit aortic SMCs were isolated using the explant method [26,27]. The intima was first peeled off from the aorta, and then the media carefully stripped away from the adventitia and placed in a Petri dish containing warmed HEPES-buffered DMEM (37 jC). The medial layer was cut into approximately 1-mm2 squares, which were transferred to a 25-cm2 tissue culture flask and barely covered with DMEM supplemented with 20% FBS. The blocks of tissue were cultured at 37 jC in a humid atmosphere of 5% CO2 and 95% air (vol/vol). After 3 – 4 weeks, the tissue blocks were removed and the migrated VSMCs were cultured, followed by subculture using trypsinization. The identity of the SMC was confirmed by morphological examination and by staining for a-actin. 2.5. DNA synthesis DNA synthesis was performed by measurement of [3H]thymidine incorporation into the cellular DNA as described previously [26,27]. Primary SMC in the first or second passage were seeded in 35-mm-diameter tissue culture plates and grown to semiconfluence in DMEM containing 10% FBS. Then, the growth medium was
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replaced with 2 ml DMEM containing 0.1% FBS and incubated for approximately 72 h for growth arrest and synchronization of the cells. After that, the medium was replaced with DMEM containing 500 Ag/ml BSA, 10 Ag/ml bovine insulin, 20 Ag/ml human transferrin, and 25 ng/ml selenium. For experiments containing serotonin, 100 Ag/ml pargyline (a monoamine oxidase inhibitor) was added to inhibit serotonin breakdown. Following this, experiments were divided into six groups based on the absence or presence of treatment. Group 1: To find out the effect of PMPs, serotonin, or TXA2, SMCs were incubated with indicated concentrations of PMPs, serotonin, or TXA2 mimetic U 46619 for 24 h and DNA synthesis measured as described below. Group 2: To evaluate the interaction between PMPs and serotonin, SMCs were simultaneously incubated with indicated concentrations of PMPs and serotonin for 24 h and the DNA synthesis measured as described below. Group 3: To evaluate the interaction between PMPs and TXA2, SMCs were simultaneously incubated with indicated concentrations of PMPs and U 46619 for 24 h and the DNA synthesis measured as described below. For all the experiments, SMCs were exposed to [3H]thymidine at a concentration of 1 ACi/plate for the last 4 h in the 24-h incubation period. At the end of this period, medium was removed, and the plates were washed with ice-cold PBS. Subsequently, 6% trichloroacetic acid was added to the cells, and the acid-insoluble [3H]thymidine was collected on glass fiber filters (Fisher Scientific, Fair Lawn, NJ). The filters were washed with 100% ethanol and air-dried, and [3H]thymidine was quantified in a liquid scintillation counter (Packard 2200CA, Packard Instrument, Downers Grove, IL). All the experiments were performed in triplicate and each experiment was repeated at least three times.
in triplicate in each of the five plates and each experiment was repeated at least three times. 2.7. Statistical analysis Data are presented as meanFstandard error (S.E.), and expressed as a percentage change from the basal value for the unstimulated cells ( = 100%). Data were analyzed by one-way analysis of variance (ANOVA) and Bonferroni/ Dunn test was employed to identify differences among the groups when the overall F statistics were significant. P < .05 was considered to be statistically significant.
3. Results 3.1. Effect of PMPs, TXA2, and serotonin on SMC DNA synthesis The concentration-dependent effect of PMPs on [3H]thymidine incorporation into SMC DNA is shown in Fig. 1. PMPs significantly increased [3H]thymidine incorporation in a concentration-dependent manner, reaching a plateau at around 30 Ag/ml (1489 F 90%, P < .0001 vs. the control) (Fig. 1). The effects obtained by PMPs significantly exceeded the effect of PDGF (10 ng/ml) (Fig. 1). Thus, the potency of PMPs to stimulate DNA synthesis in SMCs is higher than that of PDGF. PMPs also stimulated SMC proliferation, as shown by an increase in cell number (Fig. 5).
2.6. Cell number Primary SMCs in passage 1 or 2 were seeded in 35-mmdiameter tissue culture plates at a density of 90,000 to 110,000 cells per plate in DMEM containing 10% FBS. After 96 h, the growth medium was replaced with 2 ml DMEM containing 0.1% FBS and incubated for approximately 72 h for growth arresting and synchronization. Then, the medium was replaced with DMEM containing 500 Ag/ml BSA, 10 Ag/ml bovine insulin, 20 Ag/ml human transferrin, 25 ng/ml selenium, and 100 Amol/l pargyline, and the indicated concentrations of PMPs, U46619, serotonin, alone or together were added; cells were counted 48 h after addition of the compounds. The experiments was terminated by aspiration of the medium; then plates were washed with PBS and 1 ml of 2% (wt/vol) crude pancreatic trypsin in Dulbecco’s PBS containing 152 Amol/l EDTA was added to each dish. The dishes were incubated at room temperature for 5 min. The contents of each dish were diluted to 10 ml with isotone II (Coulter Electronics, Hialeah, FL) and the cell number was determined with an electronic cell counter (Coulter Counter ZM, Coulter, Hialeah). Counting was done
Fig. 1. Effect of PMPs on SMC DNA synthesis. Growth-arrested SMCs were stimulated with given concentrations of PMPs or 20 ng/ml PDGF in serum-free medium or 10 serum for 24 h and the amount of [3H]thymidine incorporated into the DNA was measured as described in Materials and Methods. A value of 100% is the baseline value (unstimulated) for [3H]thymidine incorporation into DNA: 100% = 3426 F 397 CPM/106 cells. Results are presented as percentage increase from the baseline value. Experiments were performed with three different batches of cells, and each batch was tested in quadruplicate. Each value is expressed as mean F S.E. *P < .01 vs. unstimulated cells.
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lated [3H]thymidine incorporation into the DNA of VSMCs in a dose-dependent manner. The mitogenic effect of U 46619 peaked at 7.5 AM (900 F 36%, P < .005). 3.2. Interaction between PMPs and U 46619 or serotonin on SMC DNA synthesis
Fig. 2. Effect of serotonin or TXA2 on SMC DNA synthesis. Growth-arrested SMCs were stimulated with given concentrations of serotonin or stable TXA2 mimetic U 46619 in serum-free medium for 24 h and the amount of [3H]thymidine incorporated into the DNA was measured as described in Materials and Methods. A value of 100% is the baseline value (unstimulated) for [3H]thymidine incorporation into DNA: 100% =4017F456 CPM/106 cells. Results are presented as percentage increase from the baseline value. Experiments were performed with three different batches of cells, and each batch was tested in quadruplicate. Each value is expressed as mean F S.E. *P < .01 vs. unstimulated cells.
Serotonin and TXA2 stimulated [3H]thymidine incorporation into the DNA of SMCs in a dose-dependent manner. The mitogenic effect of serotonin peaked at 50 AM (345F21%, P<.005). At concentrations greater then 50 AM, there is a decrease in the amount of [3H]thymidine incorporated, suggesting that at higher concentrations serotonin may be cytotoxic (Fig. 2). TXA2 mimetic U 46619 also stimu-
Fig. 3. Interaction between PMPs and serotonin in inducing SMC DNA synthesis. Growth-arrested SMCs were stimulated with given concentrations of PMPs and serotonin in serum-free medium for 24 h and the amount of [3H]thymidine incorporated into the DNA was measured as described in Materials and Methods. A value of 100% is the baseline value (unstimulated) for [3H]thymidine incorporation into DNA: 100% =3212 F 218 CPM/106 cells. Results are presented as percentage increase from the baseline value. Experiments were performed with three different batches of cells, and each batch was tested in quadruplicate. Each value is expressed as mean F S.E. #P < .01 vs. respective controls, *P < .01 vs. respective controls.
Because PMPs, TXA2, and serotonin are released by activated platelets and possibly present at the sites of vascular injury, we examined whether there was an interaction between these mediators in inducing SMC proliferation. Coincubation of growth-arrested SMCs with different concentrations of PMPs (1, 5, and 10 Ag/ml) with mitogenic and nonmitogenic concentrations of serotonin (0.1, 0.5, 1 and 5 AM) resulted in a concentration-dependent synergistic increase in the [3H]thymidine incorporation (Fig. 3). For example, when 1 Ag/ml PMPs or nonmitogenic concentrations of serotonin (0.1 AM) was added to the growth-arrested SMCs, PMPs increased the [3H]thymidine incorporation by c250% and serotonin had no effect. However, when they were added together they increased the amount of [3H]thymidine incorporated into the DNA to 320 F 9% ( P < .05), suggesting a synergistic interaction between PMPs and serotonin in inducing [3H]thymidine incorporation (Fig. 3). Similar synergistic interaction could be also observed when higher concentrations of PMPs and serotonin were added together. When 10 Ag/ml PMPs or 5 AM serotonin was added to the growth-arrested SMCs there was a 581F36% and 211F11% increase in the [3H]thymidine incorporation, respectively. However, when the same concentrations of
Fig. 4. Interaction between PMPs and TXA2 in inducing SMC DNA synthesis. Growth-arrested SMCs were stimulated with given concentrations of PMPs and TXA2 mimetic U 46619 in serum-free medium for 24 h and the amount of [3H]thymidine incorporated into the DNA was measured as described in Materials and Methods. A value of 100% is the baseline value (unstimulated) for [3H]thymidine incorporation into DNA: 100% = 3658F352 CPM/106 cells. Results are presented as percentage increase from the baseline value. Experiments were performed with three different batches of cells, and each batch was tested in quadruplicate. Each value is expressed as mean F S.E. #P < .01 vs. respective controls, *P < .01 vs. respective controls.
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PMPs and serotonin were added together there was a 1201F95% increase in the [3H]thymidine incorporation (instead of 790% if it was an additive effect) (Fig. 3). Coincubation of growth-arrested SMCs with different concentrations of PMPs (1, 5, and 10 Ag/ml) with mitogenic and nonmitogenic concentrations of U 46619 (0.05, 0.5, 1, and 5 AM) resulted in a concentration-dependent additive increase in the [3H]thymidine incorporation (Fig. 4). For example, when 1 Ag/ml PMPs or 0.05 AM U 46619 was added to the growth-arrested SMCs, PMPs increased the [3H]thymidine incorporation by c250% and U 46619 increased the [3H]thymidine incorporation by c150%; when they were added together they increased the amount of [ 3 H]thymidine incorporated into the DNA to c320 ( P < .05), suggesting an additive effect (Fig. 3). This additive effect was sustained even when higher concentrations of PMPs and U 46619 were added together. When 10 Ag/ml PMPs or 5 AM U 46619 was added to the growth-arrested SMCs there was a c550% and c750% increase in the [3H]thymidine incorporation, respectively. When the same concentrations of PMPs and U 46619 were added together there was a c1250%, increase in the [3H]thymidine incorporation (Fig. 4). The effect of PMPs and/or serotonin, PMPs and/or U 46619, was further examined in a cell proliferation assay. Growth-arrested SMCs were stimulated with PMPs and/or serotonin, PMPs and/or U 46619, and the number of cells counted. The results show that the induction of DNA synthesis by PMPs, and their interaction with serotonin or U 46619 leads to an increase in cell number (Fig. 5). At the time of the termination of the experiment in control culture the cell number increased to 290,600 F11,400. PMPs, serotonin, and
Fig. 5. Effect of PMPs, serotonin and TXA2 on SMC number. Growtharrested SMCs were stimulated with given concentrations of PMPs and/or serotonin or TXA2 mimetic U 46619, or PDGF in serum-free medium or 10% FBS (serum). After 48 h, cells were washed and the cell numbers determined as described in Materials and Methods. Experiments were performed with three different batches of cells, and each batch was tested in quadruplicate. Each value is expressed as meanFS.E. *P<.01 vs. unstimulated cells.
U46619 increased the cell number to 510,600 F25,400, 360,700 F12,400, and 540,700 F29,600, respectively (Fig. 5). When added together, PMPS and serotonin and PMPs and U 46619 increased the cell number to 1,010,700 F 65,600 and 1,052,7100F89,700, respectively (Fig. 5).
4. Discussion PMPs, serotonin, and TXA2 are released by activated platelets at the sites of vascular injury and have been shown to be mitogenic to SMC [23,26,27]. The present study demonstrates that PMPs and serotonin interact synergistically in inducing SMC proliferation, and PMPs and TXA2 interact additively in inducing SMC proliferation. Plateletderived mitogens are considered to be important for SMC proliferation in atherosclerosis and restenosis [28]. However, PDGF, which is considered to be the most potent plateletderived mitogen in vitro [29], has only weak mitogenic properties in vivo [30]. Thus, the mechanisms of plateletmediated mitogenesis are not clear. Studies in animal models have suggested that the pathophysiologic mechanisms of restenosis are regulated in large part by the vasoactive compounds derived from different sources within the site of injury [31]. After balloon catheterization of arteries, or due to endothelial dysfunction, subendothelial surface is exposed to the elements of blood, resulting in rapid activation and aggregation of platelets that results in thrombus formation [32]. The present study and earlier studies provide evidence that apart from PDGF, other factors released by platelets such as serotonin, TXA2, adenosine diphosphate, and PMPs are mitogenic to SMC [23,26,27,30] and they act in concert with each other and other known SMC growth factors to further induce SMC proliferation [26,27,33,34]. The concentrations of microparticles that stimulated SMC mitogenesis are comparable with microparticle concentrations that are necessary to evoke transcellular platelet activation [15] and monocyte migration [18]. The mechanisms leading to the stimulation of SMC mitogenesis by microparticles are unknown. Most likely, PMPs contain components that, alone or in combination, are responsible for the effects observed in our studies; however, the particular components that are responsible for these various effects remain to be elucidated. Sphingosine 1-phosphate and AA, which are present in the membranes of PMPs, have been shown to regulate SMC proliferation [35,36]. A transcellular AA transfer leads to an enhanced prostaglandin formation by SMC, as demonstrated for platelets and endothelial cells [15]. It has been reported that PMPs may activate certain signaling pathways in human cells; for example, PMPs were found to stimulate PKC, PI-3K, MAPK p42/44, p38, and JNK1 pathways in U-937 cells [16], MAPKp42/44 and PI3K-AKT pathways in malignant and normal human hematopoietic cells [25], and MAPKp42/44 pathway in SMC [23], providing molecular evidence that PMPs interact with
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the target cells and activate several signaling cascades that increase both cell survival and proliferation. A specific interaction of PMPs and SMCs is another attractive hypothesis to explain the mitogenic effects of PMPs. Further studies are needed to clarify this issue. The mitogenic and chemotactic effect of serotonin on cultured VSMCs is mediated predominantly via the 5-HT2 receptors [26,33,34,36,]. Serotonergic receptors have been divided into ligand-gated and Gi-protein-coupled receptors [26]. The 5-HT2 belongs to the latter class. In an earlier study, we have demonstrated that serotonin induces SMC proliferation via Gi-protein-coupled receptors, and TXA2 via Gqprotein-coupled receptors [26]. The possible explanation for the interaction between PMPs and serotonin or TXA2 is that the serotonin and TXA2may exert their mitogenic effects via the Gi-protein- and Gq-protein-coupled receptor pathways and PMPs via MAPKp42/44 pathway. Obviously, because the concentrations of serotonin TXA2 and factor PMPs used in our experiments exceed by far the concentrations physiologically occurring in blood vessels, one has to question the physiological relevance of the findings of this study derived from cultured cells. The need for the high concentrations of the agonists in cell culture studies can be explained by the assumption that cultured cells may express less intact receptors than the cells in native vessels. Nevertheless, cultured cells provide a useful model for studying the effects and interactions of growth factors and their intracellular signaling transduction pathways.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
5. Conclusion [14]
PMPs and other platelet-derived factors such as serotonin and thromboxine, which can be accumulated at the sites of vascular injury following platelet activation and aggregation, can stimulate VSMC proliferation. Moreover, these factors can interact with each other and function as amplification factors among themselves and/or potentiate cellular growth responses in conjunction with other known 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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Serotonin and thromboxane A2 stimulate platelet-derived $ microparticle-induced smooth muscle cell proliferation Rajbabu Pakala * Department of Internal Medicine, Division of Cardiology, University of Texas Health Science Center Medical School, Houston, TX 77030, USA Received 19 December 2003; accepted 22 December 2003
Abstract
Introduction: At the sites of vascular injury, activated and aggregating platelets release small vesiculated structures called platelet microparticles (PMPs). Apart from PMPs they also release several vasoactive mediators including serotonin and thromboxane A2 (TXA2). PMPs, serotonin, and TXA2 have been shown to stimulate vascular smooth muscle cell (VSMC) proliferation. Thus, this study is designed to examine the interaction between PMPs and serotonin or TXA2 in inducing rabbit VSMC proliferation. Methods: Growth-arrested rabbit SMCs were incubated in serum-free medium with different concentrations of PMPs with or without serotonin or TXA2. VSMC proliferation was examined by increase in incorporation of [3H]thymidine into DNA and by increase in cell number. Results: PMPs stimulated DNA synthesis in a dose-dependent manner; up to an added concentration of 30 Ag/ml (1489F90%) they stimulated SMC proliferation in a logarithmic fashion. Serotonin at 50 AM (345F21%) and TXA2 at 7.5 AM (900F36%) had their maximal effect. When added together, PMPs (10 Ag/ml) and serotonin (5 AM), synergistically induced DNA synthesis (581F36% and 211F11% when added alone and 1201F95% when added together), whereas PMPs (10 Ag/ml) and TXA2 (5 AM) additively induced DNA synthesis (581F36% and 781F56% when added alone and 1262F115% when added together). These increases in DNA synthesis were paralleled by increase in cell number. Conclusion: PMPs, serotonin, and TXA2 are mitogenic to SMC, and function as amplification factors to each other, suggesting 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. D 2004 Elsevier Inc. All rights reserved.
Keywords:
Platelet microparticles; Serotonin; Thromboxane A2; Smooth muscle cells; Proliferation; Restenosis
1. Introduction When platelets are stimulated with agonists such as thrombin, collagen, or calcium ionophore, A23187, or exposed to high-shear stress they release small (< 1 Am) vesiculated structures called platelet microparticles (PMPs) [1– 6]. PMPs are detectable in serum of normal donors [7]
$ There is no financial relationship between the author and the subject matter. * Cardiovascular Research Institute, Washington Hospital Center, GHRB # 313, 108 Irving Street, NW, Washington, DC, USA. Tel.: +1202-877-3867; fax: +1-202-877-3997. E-mail address: [email protected] (R. Pakala).
1522-1865/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.carrad.2003.12.002
and their levels increase in pathological states such as sepsis [8], heparin-induced thrombocytopenia [9], cerebrovascular events [10], unstable angina [11], acute myocardial infarction [12], and cardiopulmonary bypass [13]. PMPs express several platelet –endothelium attachment receptors on their surface, for example, glycoprotein IIb/IIIa (CD41), Ib and IaIIa, and P-selectin (CD62P) [14,15]. They also contain bioactive lipids including sphingosine 1-phosphate (SPP-1) and arachidonic acid (AA) [14,16]. It has been reported that after interaction with target cells PMPs trigger some biological responses; for example, they may activate endothelial cells [15], polymorphonuclear leukocytes [17], and monocytic U-937 cells [14,17], modulate monocyte – endothelial cell interactions [18] and leukocyte – leukocyte interactions
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[19], inhibit apoptosis of polymorphonuclear leukocytes [19, 20], and induce chemotaxis of U-937 cells [14,16]. The functional role of PMPs is still largely unknown. PMPs contain both pro- and anticoagulant proteins [5,21], and it has been suggested that the effects of PMPs on blood coagulation are related to the negative charge derived from exposed membrane phospholipids. During thrombosis, an association of PMPs with fibrin has been described [22], indicating a possible role of PMPs in the hemostatic process. Since PMPs have been shown to activate endothelial cells, leukocytes, and platelets and up-regulate interaction between these cells, they may play an important role in restenosis. PMPs have been shown to stimulate proliferation of endothelial cells, smooth muscle cells (SMCs), and hematopoietic cells [23 – 25]. Because flow dynamics would result in the accumulation of PMPs and other plateletreleased factors such as serotonin and thromboxane A2 (TXA2) at the sites of endothelial injury we wanted to test if PMPs, serotonin, and TXA2 will interact with each other in stimulating SMC proliferation.
2. Materials and methods 2.1. Materials Bovine serum albumin (BSA), insulin, transferrin, N-(2hydroxyethyl) piperazine-NV-(2-ethanesulfonic acid), 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), pargyline, and serotonin as creatinine sulfate were purchased form Sigma, St. Louis, MO. TXA2 mimetic U 46619 [(15S)-hydroxy-11a,9a-(epoxymethano)-presta5Z,13E-dioic acid] was obtained from Upjohn, Kalamazoo, MI. Platelet-derived growth factor (PDGF) was obtained from Amersham Life Science, Arlington Heights, IL. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin –EDTA, Hank’s balanced salt solution (HBSS), and phosphate buffered saline (PBS) were purchased from Gibco BRL Life Technologies, Gaithersburg, MD. [3H]Thymidine (specific activity 20 Ci/ mol) was from Dupont NEN Research Products, Boston, MA; Tissue culture plastics were from Falcon Labware, Lincoln Park, NJ. Other reagents were purchased from local vendors. 2.2. Isolation of platelets Platelet pellets were prepared according to the method previously reported [15]. Briefly, using 18-gauge needles nine volumes of blood were collected into one volume of citrate buffer (3.8% sodium citrate). The blood samples were centrifuged at 100g for 15 min at room temperature and the supernatant platelet-rich plasma (PRP) was carefully collected with a plastic pipette. All the preparations were done at room temperature. Prostaglandin I2 was added to PRP at the final concentration of 5 ng/ml for
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the purpose of cytoprotection and PRP was centrifuged at 650g for 13 min. The platelet pellet was resuspended in a HEPES buffer containing 142 mM NaCl, 6.2 mM KCl, 2.4 mM MgSO4, and 6.5 mM HEPES, pH 7.4. Then, after the second addition of prostaglandin I2 at the final concentration of 300 ng/ml to the suspension, platelets were centrifuged at 650g for 13 min. The same washing procedure was repeated and platelets resuspended in the HEPES buffer without prostaglandin I2. The final platelet count was adjusted to be 3108/ml. The suspension thus obtained was found to be free of any contaminants by a microscopic examination. 2.3. Preparation of PMPs Washed platelets were suspended in HEPES-buffered saline containing 1 mM CaCl2. Preparation of PMPs was done according to the modified method previously reported [15,16]. Briefly, washed platelets were stimulated with thrombin (0.5 U/ml) plus collagen (10 mg/ml) at 37 jC for 10 min without stirring. The reaction was terminated by addition of EGTA (final concentration of 5 mM) and cooling on an ice bath. PMPs were separated from the activated platelets by centrifugation at 1000g for 15 min at 4 jC. From the resulting supernatant, PMPs were pelleted by centrifugation at 10,000g at 4 jC for 15 min. PMPs were washed with HEPES buffer, pelleted again, and resuspended in HEPES buffer. The protein content of PMPs was determined by the protein assay of Bradford according to the manufacturer’s instructions (Bio-Rad). 2.4. Arterial SMC isolation Rabbit aortic SMCs were isolated using the explant method [26,27]. The intima was first peeled off from the aorta, and then the media carefully stripped away from the adventitia and placed in a Petri dish containing warmed HEPES-buffered DMEM (37 jC). The medial layer was cut into approximately 1-mm2 squares, which were transferred to a 25-cm2 tissue culture flask and barely covered with DMEM supplemented with 20% FBS. The blocks of tissue were cultured at 37 jC in a humid atmosphere of 5% CO2 and 95% air (vol/vol). After 3 – 4 weeks, the tissue blocks were removed and the migrated VSMCs were cultured, followed by subculture using trypsinization. The identity of the SMC was confirmed by morphological examination and by staining for a-actin. 2.5. DNA synthesis DNA synthesis was performed by measurement of [3H]thymidine incorporation into the cellular DNA as described previously [26,27]. Primary SMC in the first or second passage were seeded in 35-mm-diameter tissue culture plates and grown to semiconfluence in DMEM containing 10% FBS. Then, the growth medium was
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replaced with 2 ml DMEM containing 0.1% FBS and incubated for approximately 72 h for growth arrest and synchronization of the cells. After that, the medium was replaced with DMEM containing 500 Ag/ml BSA, 10 Ag/ml bovine insulin, 20 Ag/ml human transferrin, and 25 ng/ml selenium. For experiments containing serotonin, 100 Ag/ml pargyline (a monoamine oxidase inhibitor) was added to inhibit serotonin breakdown. Following this, experiments were divided into six groups based on the absence or presence of treatment. Group 1: To find out the effect of PMPs, serotonin, or TXA2, SMCs were incubated with indicated concentrations of PMPs, serotonin, or TXA2 mimetic U 46619 for 24 h and DNA synthesis measured as described below. Group 2: To evaluate the interaction between PMPs and serotonin, SMCs were simultaneously incubated with indicated concentrations of PMPs and serotonin for 24 h and the DNA synthesis measured as described below. Group 3: To evaluate the interaction between PMPs and TXA2, SMCs were simultaneously incubated with indicated concentrations of PMPs and U 46619 for 24 h and the DNA synthesis measured as described below. For all the experiments, SMCs were exposed to [3H]thymidine at a concentration of 1 ACi/plate for the last 4 h in the 24-h incubation period. At the end of this period, medium was removed, and the plates were washed with ice-cold PBS. Subsequently, 6% trichloroacetic acid was added to the cells, and the acid-insoluble [3H]thymidine was collected on glass fiber filters (Fisher Scientific, Fair Lawn, NJ). The filters were washed with 100% ethanol and air-dried, and [3H]thymidine was quantified in a liquid scintillation counter (Packard 2200CA, Packard Instrument, Downers Grove, IL). All the experiments were performed in triplicate and each experiment was repeated at least three times.
in triplicate in each of the five plates and each experiment was repeated at least three times. 2.7. Statistical analysis Data are presented as meanFstandard error (S.E.), and expressed as a percentage change from the basal value for the unstimulated cells ( = 100%). Data were analyzed by one-way analysis of variance (ANOVA) and Bonferroni/ Dunn test was employed to identify differences among the groups when the overall F statistics were significant. P < .05 was considered to be statistically significant.
3. Results 3.1. Effect of PMPs, TXA2, and serotonin on SMC DNA synthesis The concentration-dependent effect of PMPs on [3H]thymidine incorporation into SMC DNA is shown in Fig. 1. PMPs significantly increased [3H]thymidine incorporation in a concentration-dependent manner, reaching a plateau at around 30 Ag/ml (1489 F 90%, P < .0001 vs. the control) (Fig. 1). The effects obtained by PMPs significantly exceeded the effect of PDGF (10 ng/ml) (Fig. 1). Thus, the potency of PMPs to stimulate DNA synthesis in SMCs is higher than that of PDGF. PMPs also stimulated SMC proliferation, as shown by an increase in cell number (Fig. 5).
2.6. Cell number Primary SMCs in passage 1 or 2 were seeded in 35-mmdiameter tissue culture plates at a density of 90,000 to 110,000 cells per plate in DMEM containing 10% FBS. After 96 h, the growth medium was replaced with 2 ml DMEM containing 0.1% FBS and incubated for approximately 72 h for growth arresting and synchronization. Then, the medium was replaced with DMEM containing 500 Ag/ml BSA, 10 Ag/ml bovine insulin, 20 Ag/ml human transferrin, 25 ng/ml selenium, and 100 Amol/l pargyline, and the indicated concentrations of PMPs, U46619, serotonin, alone or together were added; cells were counted 48 h after addition of the compounds. The experiments was terminated by aspiration of the medium; then plates were washed with PBS and 1 ml of 2% (wt/vol) crude pancreatic trypsin in Dulbecco’s PBS containing 152 Amol/l EDTA was added to each dish. The dishes were incubated at room temperature for 5 min. The contents of each dish were diluted to 10 ml with isotone II (Coulter Electronics, Hialeah, FL) and the cell number was determined with an electronic cell counter (Coulter Counter ZM, Coulter, Hialeah). Counting was done
Fig. 1. Effect of PMPs on SMC DNA synthesis. Growth-arrested SMCs were stimulated with given concentrations of PMPs or 20 ng/ml PDGF in serum-free medium or 10 serum for 24 h and the amount of [3H]thymidine incorporated into the DNA was measured as described in Materials and Methods. A value of 100% is the baseline value (unstimulated) for [3H]thymidine incorporation into DNA: 100% = 3426 F 397 CPM/106 cells. Results are presented as percentage increase from the baseline value. Experiments were performed with three different batches of cells, and each batch was tested in quadruplicate. Each value is expressed as mean F S.E. *P < .01 vs. unstimulated cells.
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lated [3H]thymidine incorporation into the DNA of VSMCs in a dose-dependent manner. The mitogenic effect of U 46619 peaked at 7.5 AM (900 F 36%, P < .005). 3.2. Interaction between PMPs and U 46619 or serotonin on SMC DNA synthesis
Fig. 2. Effect of serotonin or TXA2 on SMC DNA synthesis. Growth-arrested SMCs were stimulated with given concentrations of serotonin or stable TXA2 mimetic U 46619 in serum-free medium for 24 h and the amount of [3H]thymidine incorporated into the DNA was measured as described in Materials and Methods. A value of 100% is the baseline value (unstimulated) for [3H]thymidine incorporation into DNA: 100% =4017F456 CPM/106 cells. Results are presented as percentage increase from the baseline value. Experiments were performed with three different batches of cells, and each batch was tested in quadruplicate. Each value is expressed as mean F S.E. *P < .01 vs. unstimulated cells.
Serotonin and TXA2 stimulated [3H]thymidine incorporation into the DNA of SMCs in a dose-dependent manner. The mitogenic effect of serotonin peaked at 50 AM (345F21%, P<.005). At concentrations greater then 50 AM, there is a decrease in the amount of [3H]thymidine incorporated, suggesting that at higher concentrations serotonin may be cytotoxic (Fig. 2). TXA2 mimetic U 46619 also stimu-
Fig. 3. Interaction between PMPs and serotonin in inducing SMC DNA synthesis. Growth-arrested SMCs were stimulated with given concentrations of PMPs and serotonin in serum-free medium for 24 h and the amount of [3H]thymidine incorporated into the DNA was measured as described in Materials and Methods. A value of 100% is the baseline value (unstimulated) for [3H]thymidine incorporation into DNA: 100% =3212 F 218 CPM/106 cells. Results are presented as percentage increase from the baseline value. Experiments were performed with three different batches of cells, and each batch was tested in quadruplicate. Each value is expressed as mean F S.E. #P < .01 vs. respective controls, *P < .01 vs. respective controls.
Because PMPs, TXA2, and serotonin are released by activated platelets and possibly present at the sites of vascular injury, we examined whether there was an interaction between these mediators in inducing SMC proliferation. Coincubation of growth-arrested SMCs with different concentrations of PMPs (1, 5, and 10 Ag/ml) with mitogenic and nonmitogenic concentrations of serotonin (0.1, 0.5, 1 and 5 AM) resulted in a concentration-dependent synergistic increase in the [3H]thymidine incorporation (Fig. 3). For example, when 1 Ag/ml PMPs or nonmitogenic concentrations of serotonin (0.1 AM) was added to the growth-arrested SMCs, PMPs increased the [3H]thymidine incorporation by c250% and serotonin had no effect. However, when they were added together they increased the amount of [3H]thymidine incorporated into the DNA to 320 F 9% ( P < .05), suggesting a synergistic interaction between PMPs and serotonin in inducing [3H]thymidine incorporation (Fig. 3). Similar synergistic interaction could be also observed when higher concentrations of PMPs and serotonin were added together. When 10 Ag/ml PMPs or 5 AM serotonin was added to the growth-arrested SMCs there was a 581F36% and 211F11% increase in the [3H]thymidine incorporation, respectively. However, when the same concentrations of
Fig. 4. Interaction between PMPs and TXA2 in inducing SMC DNA synthesis. Growth-arrested SMCs were stimulated with given concentrations of PMPs and TXA2 mimetic U 46619 in serum-free medium for 24 h and the amount of [3H]thymidine incorporated into the DNA was measured as described in Materials and Methods. A value of 100% is the baseline value (unstimulated) for [3H]thymidine incorporation into DNA: 100% = 3658F352 CPM/106 cells. Results are presented as percentage increase from the baseline value. Experiments were performed with three different batches of cells, and each batch was tested in quadruplicate. Each value is expressed as mean F S.E. #P < .01 vs. respective controls, *P < .01 vs. respective controls.
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PMPs and serotonin were added together there was a 1201F95% increase in the [3H]thymidine incorporation (instead of 790% if it was an additive effect) (Fig. 3). Coincubation of growth-arrested SMCs with different concentrations of PMPs (1, 5, and 10 Ag/ml) with mitogenic and nonmitogenic concentrations of U 46619 (0.05, 0.5, 1, and 5 AM) resulted in a concentration-dependent additive increase in the [3H]thymidine incorporation (Fig. 4). For example, when 1 Ag/ml PMPs or 0.05 AM U 46619 was added to the growth-arrested SMCs, PMPs increased the [3H]thymidine incorporation by c250% and U 46619 increased the [3H]thymidine incorporation by c150%; when they were added together they increased the amount of [ 3 H]thymidine incorporated into the DNA to c320 ( P < .05), suggesting an additive effect (Fig. 3). This additive effect was sustained even when higher concentrations of PMPs and U 46619 were added together. When 10 Ag/ml PMPs or 5 AM U 46619 was added to the growth-arrested SMCs there was a c550% and c750% increase in the [3H]thymidine incorporation, respectively. When the same concentrations of PMPs and U 46619 were added together there was a c1250%, increase in the [3H]thymidine incorporation (Fig. 4). The effect of PMPs and/or serotonin, PMPs and/or U 46619, was further examined in a cell proliferation assay. Growth-arrested SMCs were stimulated with PMPs and/or serotonin, PMPs and/or U 46619, and the number of cells counted. The results show that the induction of DNA synthesis by PMPs, and their interaction with serotonin or U 46619 leads to an increase in cell number (Fig. 5). At the time of the termination of the experiment in control culture the cell number increased to 290,600 F11,400. PMPs, serotonin, and
Fig. 5. Effect of PMPs, serotonin and TXA2 on SMC number. Growtharrested SMCs were stimulated with given concentrations of PMPs and/or serotonin or TXA2 mimetic U 46619, or PDGF in serum-free medium or 10% FBS (serum). After 48 h, cells were washed and the cell numbers determined as described in Materials and Methods. Experiments were performed with three different batches of cells, and each batch was tested in quadruplicate. Each value is expressed as meanFS.E. *P<.01 vs. unstimulated cells.
U46619 increased the cell number to 510,600 F25,400, 360,700 F12,400, and 540,700 F29,600, respectively (Fig. 5). When added together, PMPS and serotonin and PMPs and U 46619 increased the cell number to 1,010,700 F 65,600 and 1,052,7100F89,700, respectively (Fig. 5).
4. Discussion PMPs, serotonin, and TXA2 are released by activated platelets at the sites of vascular injury and have been shown to be mitogenic to SMC [23,26,27]. The present study demonstrates that PMPs and serotonin interact synergistically in inducing SMC proliferation, and PMPs and TXA2 interact additively in inducing SMC proliferation. Plateletderived mitogens are considered to be important for SMC proliferation in atherosclerosis and restenosis [28]. However, PDGF, which is considered to be the most potent plateletderived mitogen in vitro [29], has only weak mitogenic properties in vivo [30]. Thus, the mechanisms of plateletmediated mitogenesis are not clear. Studies in animal models have suggested that the pathophysiologic mechanisms of restenosis are regulated in large part by the vasoactive compounds derived from different sources within the site of injury [31]. After balloon catheterization of arteries, or due to endothelial dysfunction, subendothelial surface is exposed to the elements of blood, resulting in rapid activation and aggregation of platelets that results in thrombus formation [32]. The present study and earlier studies provide evidence that apart from PDGF, other factors released by platelets such as serotonin, TXA2, adenosine diphosphate, and PMPs are mitogenic to SMC [23,26,27,30] and they act in concert with each other and other known SMC growth factors to further induce SMC proliferation [26,27,33,34]. The concentrations of microparticles that stimulated SMC mitogenesis are comparable with microparticle concentrations that are necessary to evoke transcellular platelet activation [15] and monocyte migration [18]. The mechanisms leading to the stimulation of SMC mitogenesis by microparticles are unknown. Most likely, PMPs contain components that, alone or in combination, are responsible for the effects observed in our studies; however, the particular components that are responsible for these various effects remain to be elucidated. Sphingosine 1-phosphate and AA, which are present in the membranes of PMPs, have been shown to regulate SMC proliferation [35,36]. A transcellular AA transfer leads to an enhanced prostaglandin formation by SMC, as demonstrated for platelets and endothelial cells [15]. It has been reported that PMPs may activate certain signaling pathways in human cells; for example, PMPs were found to stimulate PKC, PI-3K, MAPK p42/44, p38, and JNK1 pathways in U-937 cells [16], MAPKp42/44 and PI3K-AKT pathways in malignant and normal human hematopoietic cells [25], and MAPKp42/44 pathway in SMC [23], providing molecular evidence that PMPs interact with
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the target cells and activate several signaling cascades that increase both cell survival and proliferation. A specific interaction of PMPs and SMCs is another attractive hypothesis to explain the mitogenic effects of PMPs. Further studies are needed to clarify this issue. The mitogenic and chemotactic effect of serotonin on cultured VSMCs is mediated predominantly via the 5-HT2 receptors [26,33,34,36,]. Serotonergic receptors have been divided into ligand-gated and Gi-protein-coupled receptors [26]. The 5-HT2 belongs to the latter class. In an earlier study, we have demonstrated that serotonin induces SMC proliferation via Gi-protein-coupled receptors, and TXA2 via Gqprotein-coupled receptors [26]. The possible explanation for the interaction between PMPs and serotonin or TXA2 is that the serotonin and TXA2may exert their mitogenic effects via the Gi-protein- and Gq-protein-coupled receptor pathways and PMPs via MAPKp42/44 pathway. Obviously, because the concentrations of serotonin TXA2 and factor PMPs used in our experiments exceed by far the concentrations physiologically occurring in blood vessels, one has to question the physiological relevance of the findings of this study derived from cultured cells. The need for the high concentrations of the agonists in cell culture studies can be explained by the assumption that cultured cells may express less intact receptors than the cells in native vessels. Nevertheless, cultured cells provide a useful model for studying the effects and interactions of growth factors and their intracellular signaling transduction pathways.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
5. Conclusion [14]
PMPs and other platelet-derived factors such as serotonin and thromboxine, which can be accumulated at the sites of vascular injury following platelet activation and aggregation, can stimulate VSMC proliferation. Moreover, these factors can interact with each other and function as amplification factors among themselves and/or potentiate cellular growth responses in conjunction with other known 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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