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Platelet-Induced Migration of Smooth Muscle Cells under Shear Stress
Microvascular Research 58, 177–182 (1999) Article ID mvre.1999.2172, available online at http://www.idealibrary.com on
BRIEF COMMUNICATION Platelet-Induced Migration of Smooth Muscle Cells under Shear Stress Tatsu Nakazawa, 1 Hiroshi Yasuhara, Kunihiro Shigematsu, and Hiroshi Shigematsu Division of Vascular Surgery, Department of Surgery, The University of Tokyo, Tokyo, Japan Received February 19, 1999
Key Words: endothelial cell; smooth muscle cell; platelet; platelet-derived growth factor; interleukin-1; interleukin-1 receptor antagonist.
INTRODUCTION Excessive migration and proliferation of smooth muscle cells (SMC) are the mechanisms of formation of atherosclerotic lesions and anastomotic intimal hyperplasia. Atherosclerotic plaques contain macrophages which produce inflammatory cytokines, such as interleukin-1 (IL-1) and leukotriene B 4 (Hsueh et al., 1985; Rola-Pleszczynski et al., 1985; Shimokado et al., 1981). A recent study demonstrated that local application of IL-1 in porcine coronary arteries enhanced intimal thickening, and it was blocked by a neutralizing antibody to either IL-1 or PDGF (Shimokawa et al., 1996). Thus, SMC motility in myointimal hyperplasia is affected by various inflammatory mediators produced by macrophages, endothelial cells (EC), and 1
To whom correspondence should be addressed at Division of Vascular Surgery, Department of Surgery, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Fax: 81(3)-38116822. 0026-2862/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
blood-borne platelets (Greenberg et al., 1978; Morisaki et al., 1989). The migration of SMC is influenced not only by the above-mentioned chemotactic mediators but also by mechanical force. We have demonstrated that shear stress on EC induces their production of PDGF in a previous research (Shigematsu et al., 1997). However, it has not yet been determined how blood-borne cells, such as platelets, affect SMC migration under shear stress. In this study, we investigated the effect of platelets on SMC migration through a pathway involving IL-1 and PDGF under shear stress.
MATERIALS AND METHODS
Cell Culture EC and SMC harvested from human abdominal aortas (Clonetics, San Diego, CA) were used. The culture of passages was carried out in modified MCDB151 medium (Clonetics) in a 37°C, 5% CO 2 environment. We used EC at passage 9 and SMC at passage 7 in this study.
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Materials An interleukin-1 receptor antagonist (IL-1ra) from Escherichia coli (Pepro Tech EC, London, England) was used. This is a naturally occurring protein which inhibits the activity of IL-1 a and IL-1b by competitively blocking binding to their type I and type II receptors. Murine anti-IL-1b antibody and rabbit anti-PDGF-BB antibody (Genzyme, Cambridge, MA) were also used in this study.
Preparation of Platelet Suspension Blood samples were collected by venipuncture from healthy donors who had not taken any medication for at least 1 month and were diluted with 12.5% ACD-A solution (Terumo, Tokyo, Japan) containing 2.2% sodium citrate, 0.8% citric acid, and 2.2% glucose. Platelet-rich plasma isolated from samples was washed three times with normal saline, and platelets derived from it were resuspended in medium.
Pulsatile Turbulent Flow and Shear Stress Loading Device The rotator (Multishaker MMS-300, Tokyo Rikakikai, Tokyo, Japan) was designed to rotate plastic wells horizontally over a 3.5-cm circumference. Then, plastic wells in which EC had been cultured were placed on the rotating apparatus and rotated. With the pulsatile changes in the height of media, the bottoms of the wells were exposed to pulsatile turbulent flow. The hemodynamic analysis in this model was described in detail previously. When EC are exposed to rotating movement at a speed of 160 rotations per minute (rpm), the exposed peak shear stress is in the range from 18 dyne/cm 2 at the edge of the dish to 90 dyne/ cm 2 at the center of the dish, with means of 12 and 60 dyne/cm 2, respectively. The shear stress created in this set up was comparable to that observed at peripheral arterial anastomoses (Shigematsu et al., 1997).
Measurement of Spreading Distance of SMC We marked a 3-mm square on the bottom of a plastic dish which was 3.5 cm in diameter (Costar,
Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Brief Communication
Cambridge, MA). Each dish was coated with 1 ml type I collagen gel (Cellmatrix Type-A kit, Nitta Gelatin, Osaka, Japan). A silicone plate with a 3-mm-square hole was placed on the collagen gel in accordance with the above-mentioned marked square. SMC (3.6 3 10 3) were put into this hole, and the silicone plate was removed after the SMC colonies had reached confluence. Using a microscope, the distance between the marked line and the tip of the cell colony was measured on all four sides. The spreading distance in the dish was the mean distance of the four sides.
Experimental Design Study 1: Influence of platelet mixture on SMC migration. Shear-loaded EC and platelets were used to enhance SMC migration. EC were cultured to confluence in a 3.5-cm-diameter plastic dish coated with 1 ml type I collagen gel and exposed to pulsatile turbulent flow for 24 h as described above using MCDB151 medium containing 15% fetal calf serum. The enhancing effect of platelets on SMC migration was determined in comparison with the effect of shear stress alone. We used three types of conditioned media: the supernatant of shear-loaded EC dishes, the supernatant of shear-loaded EC dishes and 5.0 3 10 4/ml platelets, and the supernatant of static EC dishes, which served as a control. In each medium, the supernatant of these dishes was filtered to prevent the contamination of EC and platelet. The SMC spreading distance was measured in each group every 24 h for 4 days. Study 2: Inhibitory effects of anti-PDGF, IL-1ra, and anti-IL-1 antibody on SMC mitogenic activity derived from shear-loaded EC. In the same way as in Study 1, we devised five types of conditioned media: the supernatant of shear-loaded EC dishes (Shear-EC), the supernatant of shear loaded EC dishes with 5.0 3 10 4/ml platelets (Shear-EC 1 PL), Shear-EC 1 PL medium containing 10 mg/ml anti-PDGF antibody (antiPDGF), Shear-EC 1 PL medium containing 100 ng/ml IL-1ra (IL-1ra), and Shear-EC 1 PL medium containing 1 ng/ml anti-IL-1 antibody in the (anti-IL-1). In the anti-IL-1 group, the medium was kept static for an hour to neutralize IL-1 in the supernatant after addition of anti-IL-1 antibody. The SMC spreading dis-
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FIG. 1. Additional effect of platelets on SMC mitogenic activity. SMC migration was induced significantly by conditioned medium from the supernatant of shear-loaded EC dishes (Shear-EC), compared to control conditioned medium from the supernatant of static EC dishes (Control) (*P , 0.05). Addition of platelets to the former supernatant induced greater SMC migration. SMC in the medium from the mixture of shear-loaded EC and platelets (Shear-EC 1 PL) spread significantly more than SMC in the medium from shear-loaded EC alone (**P , 0.0001). Error bars show standard deviation (n 5 6).
tance was determined as in Study 1. In each group, the concentration of IL-1 in the conditioned medium was also measured by ELISA. In this experiment, the spreading distance was regarded as an index of SMC migration. In another set of experiments, the degree to which proliferation affected the spreading distance of SMC in the present study was determined. We assessed the isolated proliferative activity of SMC, using Non-Radioactive Cell proliferation assay (CellTiter 96TM, Promega, Madison, WI). We added 50 ml of various culture media containing 5.0 3 10 3 SMC to each well of a 96-well plate (Falcon 3072, Becton Dickinson and Company, Lincoln Park, NJ). Then, after 4 days of incubation, we added 0.15 ml of Dye solution to each well in all groups and incubated these wells for 4 h in a 37°C, 5% CO 2 environment. Then, we added 1.0 ml Stop solution and incubated these wells overnight. Finally, the absorbance of each well at a wavelength of 570 nm was recorded using an ELISA reader (Model 450 Microplate Reader, Bio-Rad, Hercules, CA).
Statistical Analysis All data are presented as means 6 standard deviation. Statistical analysis was carried out by analysis of variance (ANOVA) with post hoc testing using Fisher’s protected least-significant difference test. Differences with P values less than 0.05 were considered significant.
RESULTS In Study 1, SMC migration was induced significantly by the conditioned medium derived from shear-loaded EC (2200 6 191 mm vs 1820 6 399 mm; P , 0.05). Furthermore, the mixture of platelets with shear-loaded EC further enhanced SMC spreading (3040 6 420 mm vs 2200 6 191 mm; P , 0.0001) (Fig. 1). In Study 2, the spreading distance was 2260 6 186 mm on Shear-EC dishes, 3130 6 311 mm on Shear-
Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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FIG. 2. Inhibitory effects of anti-PDGF antibody, IL-1ra, and anti-IL-1 antibody on SMC mitogenic activity derived from EC and platelets under pulsatile turbulent flow. There was a statistically significant difference in spreading distance on Day 4, between Shear and Shear-platelet groups (**P , 0.0001). Anti-PDGF suppressed SMC colony spread, which was enhanced by the conditioned medium of shear-loaded EC (*P , 0.05). Moreover, blocking the IL-1 pathway by anti-IL-1 or IL-1ra also suppressed enhanced SMC spreading activity (*P , 0.05). Error bars show standard deviation (n 5 6).
EC 6 PL dishes, 2400 6 175 mm on anti-PDGF antibody dishes, 2680 6 103 mm on IL-1ra dishes, and 2600 6 83 mm on anti-IL-1 dishes. SMC colonies spread further in platelet suspension under shear stress (P , 0.0001). This spreading activity was suppressed by anti-PDGF antibody (P , 0.05) or by blocking IL-1, using an IL-1 receptor antagonist or anti-IL-1 antibody (P , 0.05). However, these spreading activities were not suppressed to the control level by blocking IL-1. The spreading distances were still significantly longer in the IL-1ra and anti-IL-1 groups than in the Shear-EC group (P , 0.05) (Fig. 2). In the experiment to determine the isolated proliferative activity on SMC, the corrected absorbance on Day 0 was 0.473 6 0.048, which served as a control. The corrected absorbance at Day 4 was 0.466 6 0.088 in the Control group, 0.534 6 0.081 in the Shear-EC group, 0.522 6 0.055 in the Shear-EC 1 PL group, 0.520 6 0.067 in the anti-PDGF group, 0.542 6 0.028 in the IL-1ra group, and 0.492 6 0.045 in the anti-IL-1 group. There were no significant differences among these groups. The IL-1 level in each conditioned medium was the below detection limit (,3 pg/ml) by ELISA.
Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
DISCUSSION Several studies have shown that IL-1 and PDGF are involved in the development of myointimal hyperplasia (Funayama et al., 1998; Sterpetti et al., 1998). In vivo, various cells such as EC, macrophages, and platelets produce large quantities of various cytokines (Ross, 1993) and growth factors. It has not yet been determined how the blood-borne cells are precisely involved in myointimal hyperplasia. In the present study, SMC migration was enhanced by platelets added to shear-loaded EC. Our results demonstrate that cytokine-dependent mechanisms take place in the absence of macrophages in the development of myointimal hyperplasia. There appeared to be several sources of PDGF in the present study. Previous work demonstrated that shear-loaded EC produced PDGF (Hsieh et al., 1992; Malek et al., 1993; Shigematsu et al., 1997). The PGDF production of platelets may be increased by mechanical stimulation or through interaction with EC (DiCorleto et al., 1986; Ross, 1993), although the mechanisms remain to be elucidated. To examine the
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enhancing effect of platelets on SMC migration, we conducted Study 1. The additional enhancing effect of platelets on SMC migration suggests that PDGF is produced by platelets. The finding that either IL-1ra or a neutralizing IL-1 antibody suppressed SMC migration indicates the possibility of the existence of IL-1 in the supernatant of shear-loaded EC and platelets. EC produce various cytokines such as IL-1 and IL-6, even under a minimal shear stress of 6 dyne/cm 2 (Sterpetti et al., 1993). To determine the origin of IL-1 in this model, we measured IL-1 concentration in each supernatant of shearloaded EC and SMC. Although IL-1 concentration was below the detection limit of the ELISA (,3 pg/ml), the supernatant may contain undetectable endogenous IL-1 which is adequate for physiological activity. There is another possibility that SMC retains IL-1 in association with their surface and intracellularly, rather than releasing it in soluble form, and the surface-associated IL-1 has biological activity (Loppnow et al., 1992). Growth factors in the supernatant of shear-loaded EC and platelets may stimulate SMC to produce IL-1 in a paracrine or autocrine fashion. Regardless of the minimal level of IL-1 in the supernatant, SMC may have transcribed IL-1 genes and produced IL-1 at its surface. Further quantification of IL-1 mRNA of EC and SMC is needed to determine the origin of IL-1. A recent study demonstrated that platelet-derived IL-1 enhanced cytokine production of SMC, but not proliferation (Loppnow et al., 1998). Platelet-derived IL-1 may enhance SMC migration in this model. A complete block of the induced spreading activity of SMC could not be achieved using anti-PDGF antibody, anti-IL-1 antibody, or IL-1ra. These results suggest that several mechanisms take place in the control of SMC spreading in vivo. However, our results support the idea that blocking IL-1 activity is another potential treatment for preventing platelet-related myointimal hyperplasia. Especially, IL-1ra might be applied in clinical use because it is a protein that originates from the human body and has few side effects. Local application of high-dose IL-1ra may be suitable for clinical use to overcome inevitable feedback by the regulatory system.
REFERENCES
DiCorleto, P. E., and Chisolm, G. M. (1986). Participation of the endothelium in the development of the atherosclerotic plaque. Prog. Lipid Res. 25, 365–374. Funayama, H., Ikeda, U., Takahashi, M., Sakata, Y., Kitagawa, S., Takahashi, Y., Masuyama, J., Furukawa, Y., Miura, Y., Kano, S., Matsuda, M., and Shimada, K. (1998). Human monocyte-endothelial cell interaction induces platelet-derived growth factor expression. Cardiovasc. Res. 37, 216 –224. Greenburg, G. B., and Hunt, T. K. (1978). The proliferative response in vitro of vascular endothelial and smooth muscle cells exposed to wound fluids and macrophages. J. Cell. Physiol. 97, 353–360. Heldin, C. H., Westermark, B., and Wasteson, A. (1981). Specific receptors for platelet-derived growth factors on cells derived from connective tissue and glia. Proc. Natl. Acad. Sci. USA 78, 366 –368. Hsieh, H. J., Li, N. Q., and Frangos, J. A. (1992). Shear-induced platelet-derived growth factor gene expression in human endothelial cells is mediated by protein kinase C. J. Cell. Physiol. 150, 552–558. Hsueh, W., Sun, F. F., and Henderson, S. (1985). The biosynthesis of leukotriene B 4, the predominant lipoxygenase product in rabbit alveolar macrophage, is enhanced during immune activation. Biochem. Biophys. Acta 835, 92–97. Loppnow, H., and Libby, P. (1992). Functional significance of human vascular smooth muscle cell-derived interleukin 1 in paracrine and autocrine regulation pathways. Exp. Cell Res. 198, 283– 290. Loppnow, H., Bil, R., Hirt, S., Schonbeck, U., Herzberg, M., Werdan, K., Rietschel, E. T., Brandt, E., and Flad, H. D. (1998). Plateletderived interleukin-1 induces cytokine production, but not proliferation of human vascular smooth muscle cells. Blood 91, 134 – 141. Malek, A. M., Gibbons, G. H., Dzau, V. J., and Izumo, S. (1993). Fluid shear stress differentially modulates expression of gene encoding basic growth factor and platelet-derived growth factor B chain in vascular endothelium. J. Clin. Invest. 92, 2013–2021. Morisaki, N., Koyama, N., Mori, S., Kanzaki, T., Koshikawa, T., Saito, Y., and Yoshida, S. (1989). Effects of smooth muscle cell derived growth factor (SDGF) in combination with other growth factors on smooth muscle cells. Atherosclerosis 78, 61– 67. Rola-Pleszczynski, M., and Lemaire, I. (1985). Leukotrienes augment interleukin 1 production by human monocytes. J. Immunol. 135, 3958 –3961. Ross, R. (1993). The pathogenesis of atherosclerosis: A perspective for the 1990s. Nature 362, 801– 809. Shigematsu, K., Yasuhara, H., Shigematsu, H., and Muto, T. (1997). Anti-angiogenic drug AGM 1470 suppresses smooth muscle cell migration induced by endothelial PDGF. Eur. J. Vasc. Endovasc. Surg. 14, 290 –298.
Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
182 Shimokado, K., Raines, E. W., Madtes, D. K., Benditt, E. P., and Ross, R. (1981). A significant part of macrophage-derived growth factor consists of at least two forms of PDGF. Cell 43, 277–286. Shimokawa, H., Ito, A., Fukumoto, Y., Kadokami, T., Nakaike, R., Sakuta, M., Takayanagi, T., Egashira, K., and Takeshita, A. (1996). Chronic treatment with interleukin-1b induced coronary intimal lesions and vasospastic responses in pigs in vivo. J. Clin. Invest. 97, 769 –776.
Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Brief Communication
Sterpetti, A. V., Cucina, A., Moreana, A. R., Di Donna, S., D’Angelo, L. S., Cavalarro, A., and Stipa, S. (1993). Shear stress increases the release of interleukin-1 and interleukin-6 by aortic endothelial cells. Surgery 114, 911–914. Sterpetti, A. V., Cucina, A., Lepidi, S., Randone, B., Corvino, V., D’Angelo, L. S., and Carvallaro, A. (1998). Formation of myointimal hyperplasia and cytokine production in experimental vein grafts. Surgery 123, 461– 469.
BRIEF COMMUNICATION Platelet-Induced Migration of Smooth Muscle Cells under Shear Stress Tatsu Nakazawa, 1 Hiroshi Yasuhara, Kunihiro Shigematsu, and Hiroshi Shigematsu Division of Vascular Surgery, Department of Surgery, The University of Tokyo, Tokyo, Japan Received February 19, 1999
Key Words: endothelial cell; smooth muscle cell; platelet; platelet-derived growth factor; interleukin-1; interleukin-1 receptor antagonist.
INTRODUCTION Excessive migration and proliferation of smooth muscle cells (SMC) are the mechanisms of formation of atherosclerotic lesions and anastomotic intimal hyperplasia. Atherosclerotic plaques contain macrophages which produce inflammatory cytokines, such as interleukin-1 (IL-1) and leukotriene B 4 (Hsueh et al., 1985; Rola-Pleszczynski et al., 1985; Shimokado et al., 1981). A recent study demonstrated that local application of IL-1 in porcine coronary arteries enhanced intimal thickening, and it was blocked by a neutralizing antibody to either IL-1 or PDGF (Shimokawa et al., 1996). Thus, SMC motility in myointimal hyperplasia is affected by various inflammatory mediators produced by macrophages, endothelial cells (EC), and 1
To whom correspondence should be addressed at Division of Vascular Surgery, Department of Surgery, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Fax: 81(3)-38116822. 0026-2862/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
blood-borne platelets (Greenberg et al., 1978; Morisaki et al., 1989). The migration of SMC is influenced not only by the above-mentioned chemotactic mediators but also by mechanical force. We have demonstrated that shear stress on EC induces their production of PDGF in a previous research (Shigematsu et al., 1997). However, it has not yet been determined how blood-borne cells, such as platelets, affect SMC migration under shear stress. In this study, we investigated the effect of platelets on SMC migration through a pathway involving IL-1 and PDGF under shear stress.
MATERIALS AND METHODS
Cell Culture EC and SMC harvested from human abdominal aortas (Clonetics, San Diego, CA) were used. The culture of passages was carried out in modified MCDB151 medium (Clonetics) in a 37°C, 5% CO 2 environment. We used EC at passage 9 and SMC at passage 7 in this study.
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Materials An interleukin-1 receptor antagonist (IL-1ra) from Escherichia coli (Pepro Tech EC, London, England) was used. This is a naturally occurring protein which inhibits the activity of IL-1 a and IL-1b by competitively blocking binding to their type I and type II receptors. Murine anti-IL-1b antibody and rabbit anti-PDGF-BB antibody (Genzyme, Cambridge, MA) were also used in this study.
Preparation of Platelet Suspension Blood samples were collected by venipuncture from healthy donors who had not taken any medication for at least 1 month and were diluted with 12.5% ACD-A solution (Terumo, Tokyo, Japan) containing 2.2% sodium citrate, 0.8% citric acid, and 2.2% glucose. Platelet-rich plasma isolated from samples was washed three times with normal saline, and platelets derived from it were resuspended in medium.
Pulsatile Turbulent Flow and Shear Stress Loading Device The rotator (Multishaker MMS-300, Tokyo Rikakikai, Tokyo, Japan) was designed to rotate plastic wells horizontally over a 3.5-cm circumference. Then, plastic wells in which EC had been cultured were placed on the rotating apparatus and rotated. With the pulsatile changes in the height of media, the bottoms of the wells were exposed to pulsatile turbulent flow. The hemodynamic analysis in this model was described in detail previously. When EC are exposed to rotating movement at a speed of 160 rotations per minute (rpm), the exposed peak shear stress is in the range from 18 dyne/cm 2 at the edge of the dish to 90 dyne/ cm 2 at the center of the dish, with means of 12 and 60 dyne/cm 2, respectively. The shear stress created in this set up was comparable to that observed at peripheral arterial anastomoses (Shigematsu et al., 1997).
Measurement of Spreading Distance of SMC We marked a 3-mm square on the bottom of a plastic dish which was 3.5 cm in diameter (Costar,
Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Brief Communication
Cambridge, MA). Each dish was coated with 1 ml type I collagen gel (Cellmatrix Type-A kit, Nitta Gelatin, Osaka, Japan). A silicone plate with a 3-mm-square hole was placed on the collagen gel in accordance with the above-mentioned marked square. SMC (3.6 3 10 3) were put into this hole, and the silicone plate was removed after the SMC colonies had reached confluence. Using a microscope, the distance between the marked line and the tip of the cell colony was measured on all four sides. The spreading distance in the dish was the mean distance of the four sides.
Experimental Design Study 1: Influence of platelet mixture on SMC migration. Shear-loaded EC and platelets were used to enhance SMC migration. EC were cultured to confluence in a 3.5-cm-diameter plastic dish coated with 1 ml type I collagen gel and exposed to pulsatile turbulent flow for 24 h as described above using MCDB151 medium containing 15% fetal calf serum. The enhancing effect of platelets on SMC migration was determined in comparison with the effect of shear stress alone. We used three types of conditioned media: the supernatant of shear-loaded EC dishes, the supernatant of shear-loaded EC dishes and 5.0 3 10 4/ml platelets, and the supernatant of static EC dishes, which served as a control. In each medium, the supernatant of these dishes was filtered to prevent the contamination of EC and platelet. The SMC spreading distance was measured in each group every 24 h for 4 days. Study 2: Inhibitory effects of anti-PDGF, IL-1ra, and anti-IL-1 antibody on SMC mitogenic activity derived from shear-loaded EC. In the same way as in Study 1, we devised five types of conditioned media: the supernatant of shear-loaded EC dishes (Shear-EC), the supernatant of shear loaded EC dishes with 5.0 3 10 4/ml platelets (Shear-EC 1 PL), Shear-EC 1 PL medium containing 10 mg/ml anti-PDGF antibody (antiPDGF), Shear-EC 1 PL medium containing 100 ng/ml IL-1ra (IL-1ra), and Shear-EC 1 PL medium containing 1 ng/ml anti-IL-1 antibody in the (anti-IL-1). In the anti-IL-1 group, the medium was kept static for an hour to neutralize IL-1 in the supernatant after addition of anti-IL-1 antibody. The SMC spreading dis-
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FIG. 1. Additional effect of platelets on SMC mitogenic activity. SMC migration was induced significantly by conditioned medium from the supernatant of shear-loaded EC dishes (Shear-EC), compared to control conditioned medium from the supernatant of static EC dishes (Control) (*P , 0.05). Addition of platelets to the former supernatant induced greater SMC migration. SMC in the medium from the mixture of shear-loaded EC and platelets (Shear-EC 1 PL) spread significantly more than SMC in the medium from shear-loaded EC alone (**P , 0.0001). Error bars show standard deviation (n 5 6).
tance was determined as in Study 1. In each group, the concentration of IL-1 in the conditioned medium was also measured by ELISA. In this experiment, the spreading distance was regarded as an index of SMC migration. In another set of experiments, the degree to which proliferation affected the spreading distance of SMC in the present study was determined. We assessed the isolated proliferative activity of SMC, using Non-Radioactive Cell proliferation assay (CellTiter 96TM, Promega, Madison, WI). We added 50 ml of various culture media containing 5.0 3 10 3 SMC to each well of a 96-well plate (Falcon 3072, Becton Dickinson and Company, Lincoln Park, NJ). Then, after 4 days of incubation, we added 0.15 ml of Dye solution to each well in all groups and incubated these wells for 4 h in a 37°C, 5% CO 2 environment. Then, we added 1.0 ml Stop solution and incubated these wells overnight. Finally, the absorbance of each well at a wavelength of 570 nm was recorded using an ELISA reader (Model 450 Microplate Reader, Bio-Rad, Hercules, CA).
Statistical Analysis All data are presented as means 6 standard deviation. Statistical analysis was carried out by analysis of variance (ANOVA) with post hoc testing using Fisher’s protected least-significant difference test. Differences with P values less than 0.05 were considered significant.
RESULTS In Study 1, SMC migration was induced significantly by the conditioned medium derived from shear-loaded EC (2200 6 191 mm vs 1820 6 399 mm; P , 0.05). Furthermore, the mixture of platelets with shear-loaded EC further enhanced SMC spreading (3040 6 420 mm vs 2200 6 191 mm; P , 0.0001) (Fig. 1). In Study 2, the spreading distance was 2260 6 186 mm on Shear-EC dishes, 3130 6 311 mm on Shear-
Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
180
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FIG. 2. Inhibitory effects of anti-PDGF antibody, IL-1ra, and anti-IL-1 antibody on SMC mitogenic activity derived from EC and platelets under pulsatile turbulent flow. There was a statistically significant difference in spreading distance on Day 4, between Shear and Shear-platelet groups (**P , 0.0001). Anti-PDGF suppressed SMC colony spread, which was enhanced by the conditioned medium of shear-loaded EC (*P , 0.05). Moreover, blocking the IL-1 pathway by anti-IL-1 or IL-1ra also suppressed enhanced SMC spreading activity (*P , 0.05). Error bars show standard deviation (n 5 6).
EC 6 PL dishes, 2400 6 175 mm on anti-PDGF antibody dishes, 2680 6 103 mm on IL-1ra dishes, and 2600 6 83 mm on anti-IL-1 dishes. SMC colonies spread further in platelet suspension under shear stress (P , 0.0001). This spreading activity was suppressed by anti-PDGF antibody (P , 0.05) or by blocking IL-1, using an IL-1 receptor antagonist or anti-IL-1 antibody (P , 0.05). However, these spreading activities were not suppressed to the control level by blocking IL-1. The spreading distances were still significantly longer in the IL-1ra and anti-IL-1 groups than in the Shear-EC group (P , 0.05) (Fig. 2). In the experiment to determine the isolated proliferative activity on SMC, the corrected absorbance on Day 0 was 0.473 6 0.048, which served as a control. The corrected absorbance at Day 4 was 0.466 6 0.088 in the Control group, 0.534 6 0.081 in the Shear-EC group, 0.522 6 0.055 in the Shear-EC 1 PL group, 0.520 6 0.067 in the anti-PDGF group, 0.542 6 0.028 in the IL-1ra group, and 0.492 6 0.045 in the anti-IL-1 group. There were no significant differences among these groups. The IL-1 level in each conditioned medium was the below detection limit (,3 pg/ml) by ELISA.
Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
DISCUSSION Several studies have shown that IL-1 and PDGF are involved in the development of myointimal hyperplasia (Funayama et al., 1998; Sterpetti et al., 1998). In vivo, various cells such as EC, macrophages, and platelets produce large quantities of various cytokines (Ross, 1993) and growth factors. It has not yet been determined how the blood-borne cells are precisely involved in myointimal hyperplasia. In the present study, SMC migration was enhanced by platelets added to shear-loaded EC. Our results demonstrate that cytokine-dependent mechanisms take place in the absence of macrophages in the development of myointimal hyperplasia. There appeared to be several sources of PDGF in the present study. Previous work demonstrated that shear-loaded EC produced PDGF (Hsieh et al., 1992; Malek et al., 1993; Shigematsu et al., 1997). The PGDF production of platelets may be increased by mechanical stimulation or through interaction with EC (DiCorleto et al., 1986; Ross, 1993), although the mechanisms remain to be elucidated. To examine the
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enhancing effect of platelets on SMC migration, we conducted Study 1. The additional enhancing effect of platelets on SMC migration suggests that PDGF is produced by platelets. The finding that either IL-1ra or a neutralizing IL-1 antibody suppressed SMC migration indicates the possibility of the existence of IL-1 in the supernatant of shear-loaded EC and platelets. EC produce various cytokines such as IL-1 and IL-6, even under a minimal shear stress of 6 dyne/cm 2 (Sterpetti et al., 1993). To determine the origin of IL-1 in this model, we measured IL-1 concentration in each supernatant of shearloaded EC and SMC. Although IL-1 concentration was below the detection limit of the ELISA (,3 pg/ml), the supernatant may contain undetectable endogenous IL-1 which is adequate for physiological activity. There is another possibility that SMC retains IL-1 in association with their surface and intracellularly, rather than releasing it in soluble form, and the surface-associated IL-1 has biological activity (Loppnow et al., 1992). Growth factors in the supernatant of shear-loaded EC and platelets may stimulate SMC to produce IL-1 in a paracrine or autocrine fashion. Regardless of the minimal level of IL-1 in the supernatant, SMC may have transcribed IL-1 genes and produced IL-1 at its surface. Further quantification of IL-1 mRNA of EC and SMC is needed to determine the origin of IL-1. A recent study demonstrated that platelet-derived IL-1 enhanced cytokine production of SMC, but not proliferation (Loppnow et al., 1998). Platelet-derived IL-1 may enhance SMC migration in this model. A complete block of the induced spreading activity of SMC could not be achieved using anti-PDGF antibody, anti-IL-1 antibody, or IL-1ra. These results suggest that several mechanisms take place in the control of SMC spreading in vivo. However, our results support the idea that blocking IL-1 activity is another potential treatment for preventing platelet-related myointimal hyperplasia. Especially, IL-1ra might be applied in clinical use because it is a protein that originates from the human body and has few side effects. Local application of high-dose IL-1ra may be suitable for clinical use to overcome inevitable feedback by the regulatory system.
REFERENCES
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Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Brief Communication
Sterpetti, A. V., Cucina, A., Moreana, A. R., Di Donna, S., D’Angelo, L. S., Cavalarro, A., and Stipa, S. (1993). Shear stress increases the release of interleukin-1 and interleukin-6 by aortic endothelial cells. Surgery 114, 911–914. Sterpetti, A. V., Cucina, A., Lepidi, S., Randone, B., Corvino, V., D’Angelo, L. S., and Carvallaro, A. (1998). Formation of myointimal hyperplasia and cytokine production in experimental vein grafts. Surgery 123, 461– 469.