European Journal of Clinical Investigation (1992) 22,46 1-468
Human macrophages modulate the phenotype of cultured rabbit aortic smooth muscle cells through secretion of platelet-derived growth factor N. MORISAKI, N. KOYAMA, M. KAWANO, S. MORI, K. UMEMIYA, T. KOSHIKAWA, Y. SAITO & S. YOSHIDA The Second Department of Internal Medicine, School of Medicine, Chiba University, 1-8-1 Inohana, Chiba 280, Japan Received 5 June 1991 and in revised form 3 February 1992; accepted 10 February 1992
Abstract. Phenotypic change of aortic smooth muscle cells (SMC) is a key step for their abnormal proliferation in atheromatous lesions. In this study modulation of the growth properties of SMC by macrophages was investigated to clarify the mechanism regulating the SMC phenotype. Cultured rabbit SMC preincubated with either macrophages derived from human peripheral monocytes, or conditioned medium from macrophages grew faster than control SMC in the absence of either macrophages or conditioned medium. SMC preincubated with purified plateletderived growth factor (PDGF) also grew faster than control SMC in the absence of PDGF, and their rapid growth was maintained for at least two passages. SMC preincubated with conditioned medium of macrophages plus anti-PDGF antibody did not grow faster than control SMC. Furthermore SMC preincubated with PDGF acquired the ability to secrete some mitogen, which differed from PDGF. These results suggest that macrophages modulate the phenotype of SMC by a mechanism mediated by PDGF. As a result the SMC grow faster and at the same time secrete some mitogen probably distinct from PDGF in an autocrine manner. Keywords. Autocrine, growth, phenotype, plateletderived growth factor, smooth muscle cells. Introduction Arterial smooth muscle cells show different phenotypes such as contractile and synthetic forms [I]. The latter form is suitable for proliferation in the arterial wall as in the atheromatous intima. Furthermore, we have found different phenotypes even among cells of the synthetic phenotype. For example, smooth muscle cells (SMC) cultured from atheromatous intimal lesions (intimal SMC) grow more rapidly than SMC cultured from normal media (medial SMC) [2]. This is Correspondence: Dr Nobuhiro Morisaki, The Second Department of Internal Medicine, School of Medicine, Chiba University, 1-8-1 Inohana, Chiba 280, Japan.
in part due to an acquired autocrine system of growth factors [3,4,5] and in part due to lack of negative feedback control of growth by PGI2 [6,7l. Moreover, the intimal SMC metabolize modified low density lipoprotein (LDL), probably by acquiring a so-called ‘scavenger receptor’ that the medial SMC d o not have [2]. These findings suggest that such a synthetic form of SMC has pathological phenotypes that contribute more to the formation of intimal thickening than the simple synthetic form of SMC such as medial SMC. It is unclear what factors are responsible for the conversion of the simple synthetic form of SMC to the pathological synthetic form in vivo. We have shown that coculture of SMC with endothelial cells leads to the appearance of SMC that grow more rapidly than before. Macrophages also often co-exist with SMC in early lesions, so in this paper we studied the following problems. Can macrophages modulate the growth phenotype of SMC? If so, what factor is responsible for this modulation? And what is the mechanism involved? Materials and methods Materials: Purified human PDGF was purchased from R & D System Inc. (Minneapolis, MN, USA). Recombinant PDGF A-A and B-B chains were kindly supplied by Dr Heldin (Ludwig Institute, Uppsala, Sweden). Interleukin- 1 was kindly supplied by 00tsuka Pharmaceutical Co. (Tokyo, Japan). A goat antihuman PDGF polyclonal antibody was purchased from Collaborative Research Inc. (Lot No. 88-1 527, Lexington, MA, USA). The sources of other materials for tissue culture were as reported previously [3,6,7]. Millicells for coculture were purchased from Japan Millipore Ltd. (Tokyo, Japan). These millicells have a membrane with pores of 45 pm at the bottom that allows macrophage-derived substances to pass through. Tissue cultures: SMC from rabbit thoracic aorta were obtained by an explant method as reported [8], and were used for experiments at the second and third passages. Monocyte-derived macrophages were pre46 I
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pared from 10 ml of blood from a healthy volunteer as reported previously [9]. Briefly, mixed mononuclear cells were plated in a 60 mm diameter petridish and incubated for 3 h to allow the monocytes to adhere to the dish. Non-adherent cells were discarded and the adherent cells were incubated with 4 ml of Dulbecco's modified Eagle's medium (DME) containing 20% fresh human serum for 7 days to obtain monocytederived macrophages. Conditioned medium (CM) was collected by incubating these macrophages with 2 ml of DME containing 10% fetal bovine serum (FBS) for 2 days, and was stored at 4°C. CM was collected with 2 ml of DME only when direct mitogenic effect of CM was measured. Experimental procedure: Three series of experiments were carried out to examine the effect of macrophage-derived factors on the growth phenotype of SMC (not the direct effect on growth): (1) Coculture-SMC were seeded into 24-well plates (Corning, NY, USA) at 3 x lo4 cells well-' and allowed to become attached to the wells. Then a millicell was hung into the well without its direct attachment to the bottom of the well. Macrophages were harvested in 4 ml of DME containing 10% FBS (culture medium) from the dishes described above, and volumes of 1 ml were seeded into the millicells. Each coculture contained a total of 2 ml of culture medium. After medium change, the cells were cocultured for 3 days. Control cultures contained millicells without macrophages. After coculture, the SMC were harvested from experimental and control wells, seeded into 24-well plates at a density of lo4 cells well-' and grown for 4-6 days. Cell numbers were counted on the indicated days. (2) Macrophage-CM-SMC were seeded into 24-well plates at 3 x lo4 cells well-' as described above. After they became attached they were cultured with 0-8 ml of the culture medium and 0.2 ml of CM for 3 days. Control cells were cultured in 1 ml of culture medium. In some experiments, anti-PDGF antibody was added during preincubation of SMC with CM. After culture, cell growth was measured as described above. (3) PDGF and interleukin-1-SMC seeded in 24-well plates as described above were cultured in 1 ml of culture medium containing either purified PDGF at 10 ng ml-' or interleukin-1 at 10 ng ml-' for 3 days. Control cells were cultured in 1 ml of culture medium. After culture, cell growth was measured as described above. At the end of this experiment, SMC at confluency in some wells were used for another cell growth experiment (2nd passage) or were incubated in 1 ml of DME well-' for 2 days for preparation of CM [3]. DNA synthesis was measured as previously reported [6] except that 0.1% FBS was added to the assay system besides growth factors. DNA synthesis was not affected by 0.1% FBS alone as judged by comparison with the effect of DME alone. SMC at the second to third passages were used as target cells, because at this passage they did not secrete any mitogen [3].
Statistics: Statistical analysis was performed using Student's t-test (two-tailed). Results Eflect of coculture on growth phenotype of SMC
Figure 1 shows the growth curve of SMC that had been preincubated with macrophages. In this experiment, macrophages were removed from the culture before assay of cell growth to avoid their direct effect on the SMC. After preincubation with macrophages, SMC attached to the wells at the same rate as control SMC (day 0) but grew significantly faster than control SMC (day 2-6; P
Human macrophages modulate the phenotype of cultured rabbit aortic smooth muscle cells through secretion of platelet-derived growth factor N. MORISAKI, N. KOYAMA, M. KAWANO, S. MORI, K. UMEMIYA, T. KOSHIKAWA, Y. SAITO & S. YOSHIDA The Second Department of Internal Medicine, School of Medicine, Chiba University, 1-8-1 Inohana, Chiba 280, Japan Received 5 June 1991 and in revised form 3 February 1992; accepted 10 February 1992
Abstract. Phenotypic change of aortic smooth muscle cells (SMC) is a key step for their abnormal proliferation in atheromatous lesions. In this study modulation of the growth properties of SMC by macrophages was investigated to clarify the mechanism regulating the SMC phenotype. Cultured rabbit SMC preincubated with either macrophages derived from human peripheral monocytes, or conditioned medium from macrophages grew faster than control SMC in the absence of either macrophages or conditioned medium. SMC preincubated with purified plateletderived growth factor (PDGF) also grew faster than control SMC in the absence of PDGF, and their rapid growth was maintained for at least two passages. SMC preincubated with conditioned medium of macrophages plus anti-PDGF antibody did not grow faster than control SMC. Furthermore SMC preincubated with PDGF acquired the ability to secrete some mitogen, which differed from PDGF. These results suggest that macrophages modulate the phenotype of SMC by a mechanism mediated by PDGF. As a result the SMC grow faster and at the same time secrete some mitogen probably distinct from PDGF in an autocrine manner. Keywords. Autocrine, growth, phenotype, plateletderived growth factor, smooth muscle cells. Introduction Arterial smooth muscle cells show different phenotypes such as contractile and synthetic forms [I]. The latter form is suitable for proliferation in the arterial wall as in the atheromatous intima. Furthermore, we have found different phenotypes even among cells of the synthetic phenotype. For example, smooth muscle cells (SMC) cultured from atheromatous intimal lesions (intimal SMC) grow more rapidly than SMC cultured from normal media (medial SMC) [2]. This is Correspondence: Dr Nobuhiro Morisaki, The Second Department of Internal Medicine, School of Medicine, Chiba University, 1-8-1 Inohana, Chiba 280, Japan.
in part due to an acquired autocrine system of growth factors [3,4,5] and in part due to lack of negative feedback control of growth by PGI2 [6,7l. Moreover, the intimal SMC metabolize modified low density lipoprotein (LDL), probably by acquiring a so-called ‘scavenger receptor’ that the medial SMC d o not have [2]. These findings suggest that such a synthetic form of SMC has pathological phenotypes that contribute more to the formation of intimal thickening than the simple synthetic form of SMC such as medial SMC. It is unclear what factors are responsible for the conversion of the simple synthetic form of SMC to the pathological synthetic form in vivo. We have shown that coculture of SMC with endothelial cells leads to the appearance of SMC that grow more rapidly than before. Macrophages also often co-exist with SMC in early lesions, so in this paper we studied the following problems. Can macrophages modulate the growth phenotype of SMC? If so, what factor is responsible for this modulation? And what is the mechanism involved? Materials and methods Materials: Purified human PDGF was purchased from R & D System Inc. (Minneapolis, MN, USA). Recombinant PDGF A-A and B-B chains were kindly supplied by Dr Heldin (Ludwig Institute, Uppsala, Sweden). Interleukin- 1 was kindly supplied by 00tsuka Pharmaceutical Co. (Tokyo, Japan). A goat antihuman PDGF polyclonal antibody was purchased from Collaborative Research Inc. (Lot No. 88-1 527, Lexington, MA, USA). The sources of other materials for tissue culture were as reported previously [3,6,7]. Millicells for coculture were purchased from Japan Millipore Ltd. (Tokyo, Japan). These millicells have a membrane with pores of 45 pm at the bottom that allows macrophage-derived substances to pass through. Tissue cultures: SMC from rabbit thoracic aorta were obtained by an explant method as reported [8], and were used for experiments at the second and third passages. Monocyte-derived macrophages were pre46 I
462
N. MORISAKI et al.
pared from 10 ml of blood from a healthy volunteer as reported previously [9]. Briefly, mixed mononuclear cells were plated in a 60 mm diameter petridish and incubated for 3 h to allow the monocytes to adhere to the dish. Non-adherent cells were discarded and the adherent cells were incubated with 4 ml of Dulbecco's modified Eagle's medium (DME) containing 20% fresh human serum for 7 days to obtain monocytederived macrophages. Conditioned medium (CM) was collected by incubating these macrophages with 2 ml of DME containing 10% fetal bovine serum (FBS) for 2 days, and was stored at 4°C. CM was collected with 2 ml of DME only when direct mitogenic effect of CM was measured. Experimental procedure: Three series of experiments were carried out to examine the effect of macrophage-derived factors on the growth phenotype of SMC (not the direct effect on growth): (1) Coculture-SMC were seeded into 24-well plates (Corning, NY, USA) at 3 x lo4 cells well-' and allowed to become attached to the wells. Then a millicell was hung into the well without its direct attachment to the bottom of the well. Macrophages were harvested in 4 ml of DME containing 10% FBS (culture medium) from the dishes described above, and volumes of 1 ml were seeded into the millicells. Each coculture contained a total of 2 ml of culture medium. After medium change, the cells were cocultured for 3 days. Control cultures contained millicells without macrophages. After coculture, the SMC were harvested from experimental and control wells, seeded into 24-well plates at a density of lo4 cells well-' and grown for 4-6 days. Cell numbers were counted on the indicated days. (2) Macrophage-CM-SMC were seeded into 24-well plates at 3 x lo4 cells well-' as described above. After they became attached they were cultured with 0-8 ml of the culture medium and 0.2 ml of CM for 3 days. Control cells were cultured in 1 ml of culture medium. In some experiments, anti-PDGF antibody was added during preincubation of SMC with CM. After culture, cell growth was measured as described above. (3) PDGF and interleukin-1-SMC seeded in 24-well plates as described above were cultured in 1 ml of culture medium containing either purified PDGF at 10 ng ml-' or interleukin-1 at 10 ng ml-' for 3 days. Control cells were cultured in 1 ml of culture medium. After culture, cell growth was measured as described above. At the end of this experiment, SMC at confluency in some wells were used for another cell growth experiment (2nd passage) or were incubated in 1 ml of DME well-' for 2 days for preparation of CM [3]. DNA synthesis was measured as previously reported [6] except that 0.1% FBS was added to the assay system besides growth factors. DNA synthesis was not affected by 0.1% FBS alone as judged by comparison with the effect of DME alone. SMC at the second to third passages were used as target cells, because at this passage they did not secrete any mitogen [3].
Statistics: Statistical analysis was performed using Student's t-test (two-tailed). Results Eflect of coculture on growth phenotype of SMC
Figure 1 shows the growth curve of SMC that had been preincubated with macrophages. In this experiment, macrophages were removed from the culture before assay of cell growth to avoid their direct effect on the SMC. After preincubation with macrophages, SMC attached to the wells at the same rate as control SMC (day 0) but grew significantly faster than control SMC (day 2-6; P
