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Effects of smooth muscle cell derived growth factor (SDGF) in combination with other growth factors on smooth muscle cells
Atherosclerosis, 78 (1989) 61-67 Elsevier Scientific Publishers Ireland,
61 Ltd.
ATH 04333
Effects of smooth muscle cell derived growth factor (SDGF) in combination with other growth factors on smooth muscle cells Nobuhiro Morisaki, Noriyuki Koyama, Seijiro Mori, Tetsuto Kanzaki, Tomoko Koshikawa, Yasushi Saito and Sho Yoshida Second Department
of Internal Medicine, School of Medicine,
Chiba University, Chiba 280 (Japan)
(Received 1 August, 1988) (Revised, received 18 January, 1989) (Accepted 9 February, 1989)
Recently intimal thickening was shown to be due to stimulation of proliferation of arterial smooth muscle cells (SMC) by autocrine secretion of growth factor(s). We have reported that cultured rabbit aortic SMC secrete a growth factor (SDGF) distinct from other known growth factors. This paper reports on studies on the biological characteristics of SDGF. Putative “competence” and “progression” growth factors synergistically stimulated DNA synthesis of cultured rabbit aortic SMC. Conditioned medium (CM) from SMC containing SDGF stimulated DNA synthesis in SMC synergistically with either the competence factors or the progression factors. This synergistic effect was also observed in the presence of optimal concentrations of both competence and progression factors. The continuous presence of CM was essential for its stimulation of DNA synthesis whereas the presence of PDGF for only the first 4 h of culture was sufficient to induce maximum stimulation of DNA synthesis. These results suggest that SDGF is a new growth factor distinct from either competence or progression factors and that it stimulates a different pathway in SMC from those stimulated by other known growth factors.
Key uvords: Cell proliferation; Smooth muscle cell; Progression factor; Atherosclerosis
Autocrine;
Introduction
matous lesions [l-3]. We recently reported an autocrine mechanism for SMC proliferation [4]. Rabbit aortic medial SMC were found to secrete a factor named SDGF (smooth muscle cell derived growth factor) after numerous passages. This factor was physicochemically, immunologically, and biologically different from known growth factors such as platelet derived growth factor (PDGF), fibroblast growth factor (FGF), epidermaf growth
Proliferation of aortic smooth muscle cells (SMC) is important in the formation of athero-
Correspondence to: Dr. Nobuhiro Morisaki, The Second Department of Internal Medicine, School of Medicine, Chiba University, 1-8-1 Inohana, Chiba 280, Japan. 0021-9150/89/$03.50
0 1989 Elsevier Scientific
Publishers
Ireland,
Ltd.
Growth
factor;
Competence
factor;
62 factor (EGF), and somatomedin C, and could stimulate DNA synthesis in the absence of other growth factors. We were interested to know the effect of SDGF in combination with the other growth factors mentioned above in the pathogenesis of atherosclerosis. Two kinds of growth factor are essential for proliferation of quiescent cells, at least in relation to the 3T3 cell line [5]. The first includes the competence factors, which stimulate G, stage cells to enter the G, stage of the cell cycle, and the other is represented by the progression factors, which stimulate G, stage cells to progress to the S stage. The former includes PDGF and FGF, and the latter EGF and somatomedin C. SMC, which are mesenchymal cells like 3T3 cells, should require both kinds of growth factor for proliferation, although this has not yet been established. EGF and somatomedin C are present in vivo in circulating blood. Therefore, the limiting factor for the proliferation of SMC should be a competence factor(s) in the arterial wall [6]. Paracrine and autocrine secretions have both been reported to be sources of competence factors. PDGF is derived from clotted platelets, and PDGF-like factor(s) are also secreted by endothelial cells [7], macrophages [8] and rat arterial SMC [9-111. FGF is secreted by macrophages. In this study, we attempted to obtain biological characterization of SDGF by investigating its effects in combination with the other growth factors mentioned above on the growth of cultured rabbit aortic SMC.
Materials and Methods Materials PDGF and FGF were purchased from R & D System Inc. (Minneapolis, MN). EGF was from Collaborative Research Inc. (Lexington, MA). Interleukin-1 (IL-l) was from Genzyme Inc. (Boston, MA). Somatomedin C (99% purity) and tumor necrotizing factor (TNF, 99.6% purity) were kindly supplied by Fujisawa Pharmaceutical Co. (Tokyo, Japan). The sources of 6-[ 3H]thymidine, Dulbecco’s modified Eagle’s medium (DME), and fetal bovine serum (lot No. 27 N3541) were as reported previously [13].
Tissue culture Primary cultures of SMC were established from the medial layer of the thoracic aorta of male Japanese white rabbits (Oryctologus cuniculus var. domesticus) by an explant method [14]. Cells from primary cultures were subcultured to confluency in T-75 flasks, harvested, seeded at a density of 3 X lo4 cells/well in 1 ml of DME supplemented with 10% FBS in 24-well plates (16~mm wells), and incubated in a CO, (5%) incubator for about 4 days until they reached confluency. The medium was renewed every 2 or 3 days. For collection of conditioned medium (CM), SMC at the 2nd passage were further subcultured at a 1 : 2 split ratio in T-75 flasks [14]. Conditioned medium (CM) without plasma was collected at the 4th to 6th passage as reported [12]. Pooled CM was used for experiments. DNA synthesis The assay method was described previously [13]. Briefly, confluent SMC at the 3rd passage in 24-well plates (cell number, about 1.2 X 105/well) were synchronized to the G, stage by serum depletion for 24 h. Then the cells were treated with growth factor(s) and [ 3H]thymidine incorporation into DNA was measured over 24 h incubation. Cells were used at the 3rd passage because at this passage SMC secreted little material with rnitogenie activity into the medium [12]. Results Effects of combinations of putative competence and progression factors PDGF or FGF, and EGF or somatomedin C were used as competence and progression factors, respectively. Figs. 1 and 2 show the effects of combinations of PDGF and EGF or somatomedin C on DNA synthesis in SMC. EGF in the absence of PDGF increased DNA synthesis only slightly (maximum, about 2.5-fold over the control value at 5 or 10 ng/ml), but in the presence of sufficient PDGF (5 ng/ml) it increased DNA synthesis markedly and dose-dependently (maximum, about 16-fold the control value at 10 or 20 ng/ml) (Fig. 1). The effect of PDGF in combination with somatomedin C was similar to that of PDGF in combination with EGF (Fig. 2), although the ex-
63
1
2
4
8
Somatomedin 1
1
10 EGF
20
(ng/ml)
C (w/ml)
Fig. 2. Dose dependence of effect of somatomedin C on DNA synthesis of SMC in the presence or absence of PDGF. PDGF was added at 5 ng/ml. See legend to Fig. 1 for other condi-
Fig. 1. Dose dependence of effect of EGF on DNA synthesis of SMC in the presence or absence of PDGF. Confluent SMC in 24-well plates were synchronized to the Go stage by serum depletion for 24 h. Then cells were treated with growth factor(s) and 13H]thymidine incorporation into DNA during 24 h incubation was measured. PDGF was added at 5 ng/ml. Points are means for duplicate cultures. The 2 values at each point did not differ by more than 10% from each other.
tent of growth stimulation by these factors was different. This difference was mainly due to the use of different primary cultures [15]. Figs. 3 and 4 show the effects of combinations of PDGF and EGF, and FGF and somatomedin C, respectively, as functions of the concentrations of PDGF and FGF, respectively. In the absence of EGF, PDGF stimulated DNA synthesis only slightly (maximum, about 2.5-fold the control value at 2.5 or 5 ng/ml), but in the presence of sufficient EGF (10 ng/ml) PDGF stimulated DNA synthesis markedly and dose-dependently (maximum, 9.5-fold the control value at 5 ng/ml) (Fig. 3). Similarly, FGF alone increased DNA synthesis to only about 3-fold the control level, whereas in the presence of sufficient somatomedin
I
0.5
1
I
I
5
2.5
PDG F (ng/ml
)
Fig. 3. Dose dependence of effect of PDGF on DNA synthesis of SMC in the presence or absence of EGF. EGF was added at 10 ng/ml. See legend to Fig. 1 for other conditions.
somatomedin
i-
C (+I
somatomedin
(
L L 0
2.5
10
5 FGF
(ng/ml)
Fig. 4. Dose dependence of effect of FGF on DNA synthesis of SMC in the presence or absence of somatomedin C. Somatomedin C was added at 4 ng/ml. See legend to Fig. 1 for other conditions.
tors alone stimulated DNA synthesis slightly or moderately and in their presence 100% CM caused synergistic stimulation (maximum, 15fold the control level with EGF and 1Zfold with somatomedin C). Fig. 8 shows the interaction of CM with the competence factor FGF. FGF alone stimulated DNA synthesis only slightly (maximum, 3-fold of control values at 5 to 10 ng/ml) and 100% CM had a synergistic effect (maximum, ll-fold the control level with 5 to 10 ng/ml of FGF). Fig. 9 shows the effect of CM with PDGF in combination with an effective concentration of 10 ng/ml of the progression factor EGF. PDGF caused synergistic and dose-dependent stimulation of DNA synthesis over the control level with EGF alone (maximum, 5-fold at 5-10 ng/ml of PDGF). CM alone at 100% increased DNA synthesis to g-fold the control level with EGF alone. CM at 100% had a synergistic effect with PDGF at O-2.5 ng/ml in stimulating DNA synthesis (maximum, 21.5-fold the control level with EGF). With PDGF concentrations of over 2.5 ng,/ml, DNA synthesis in the presence of CM was less than that with 2.5 ng/ml of PDGF.
(4 ng/ml), it markedly stimulated DNA synthesis in a dose-dependent manner (maximum, about 13-fold the control value at 10 ng/ml). These findings show that competence and progression factors have synergistic effects on SMC.
C
I
conditioned
medium (_t)
Effect of CM in combination with FBS FBS, which is believed to contain both competence and progression factors, stimulated DNA synthesis dose-dependently at concentrations of up to 10% (Fig. 5). CM alone increased DNA synthesis up to about 6.5fold of the control level, as previously reported 1121. Stimulation of DNA synthesis by FBS was greater in the presence of CM than in its absence, although the combined effect of CM and FBS was less than the sum of their individual effects.
Effect of CM in combination with progression or competence factors Figs. 6 and 7 show the effects of CM in combination with the progression factors EGF and somatomedin C, respectively. The progression fac-
conditioned
s P
medium H
1 I 0
2.5 Fetal
1 5 bovine
I 10 serum
(%I
Fig. 5. Dose dependence of effect of fetal bovine serum on DNA synthesis of SMC in the presence or absence of conditioned medium. 100% conditioned medium was added. See legend to Fig. 1 for other conditions.
65
‘“I
conditioned
,f ._ t
conditioned
medium (4
medium H
5
conditioned
medium (1-1
conditioned
medium (-4
5 i
2
. I
a
1
z
0
5
EG F (ng/ml
10
1
Fig. 6. Dose dependence of effect of EGF on DNA synthesis of SMC in the absence or presence of conditioned medium. 100% of conditioned medium was added. See legend to Fig. 1 for other conditions.
Effects of CM or PDGF on DNA synthesis as functions of the period of stimulation 3T3 cells in the G, stage soon entered the competent G, stage of the cell cycle when stimulated with a competence factor [4], and once they became competent, they did not need the competence factor for progression to the S stage. We examined whether SDGF in CM acts like known competence factors, such as PDGF (Table 1). Exposure of SMC to a sufficient concentration of the progression factor EGF (10 ng/ml) for the entire incubation period (24 h) increased their DNA synthesis only 3.6-fold. Exposure of G, stage SMC to the competence factor PDGF for 1 or 2 h resulted in little increase in DNA synthesis over that in the control with EGF. However, 4-h exposure of SMC to PDGF (5 ng/ml) markedly increased DNA synthesis (from 3.6- to 31.9-fold over the control with DME). Exposure of SMC to PDGF for 24 h resulted in a much greater increase
P
1
2
4
8
Somatomedin
C (ng/ml
)
Fig. 7. Dosedependence of effect of somatomedin C on DNA synthesis of SMC in the absence or presence of conditioned medium. 100% of conditioned medium was added. See legend to Fig. 1 for other conditions
conditioned
medium &)
conditioned
medium (--I
10
5
F GF
(ng/ml)
Fig. 8. Dose dependence of effect of FGF on DNA of SMC in the absence or presence of conditioned 100% of conditioned medium was added.
synthesis medium.
66 than exposure for O-2 h, but less than that induced by 4-h exposure. Exposure of SMC to 100% pooled CM for l-4 h caused less than 2-fold increase in DNA synthesis over that of the control with EGF, and exposure to CM for 24 h increased DNA synthesis to 6-fold that of the control with EGF.
TABLE
Discussion
1) DME 2) DME + EGF 3) DME+EGF+PDGF (1 h) 4)DME+EGF+PDGF(2 h) 5) DME + EGF + PDGF (4 h) 6) DME + EGF + PDGF (24 h) 7) DME + EGF + CM (1 h) 8) DME + EGF + CM (2 h) 9) DME + EGF + CM (4 h) 10) DME+EGF+CM (24 h)
As shown in Figs. 1-4, competence factors such as PDGF or FGF have synergistic effects with progression factors, such as EGF or somatome&n C, in increasing DNA synthesis in SMC as in 3T3 cells. Table 1 shows that exposure of SMC in the G, stage to PDGF for 4 h was sufficient to induce their entry into the S stage in the presence of EGF. These results strongly indicate that growth factors can be classified into two
(EGF
6))
conditioned medium , (EGF (id)
0
5 PDG
H
10
F (w/ml)
Fig. 9. Dose dependence of effect of PDGF on DNA synthesis of SMC in the presence of EGF and the presence or absence of conditioned medium. 10 ng/mI of EGF and 100% conditioned medium were added. See legend to Fig. 1 for other conditions.
1
EFFECTS OF CONDITIONED MEDIUM (CM) AND PDGF ON DNA SYNTHESIS BY SMOOTH MUSCLE CELLS AS FUNCTIONS OF THEIR PERIODS OF STIMULATION Condition
DNA synthesis (dpm/well)
Increased stimulation over control
2470 8 920 13300 11800 78 800 38 900 13700 14200 11900 53200
1.0 3.6 5.4 4.8 31.9 15.7 5.5 5.7 4.8 21.5
value
EGF (10 ng/ml) was added to the cultures at 0 h of incubation (No. 2-lo), and again with medium change at the times of incubation indicated in parentheses (Nos. 3-5, 7-9). PDGF (5 ng/mI) or 100% conditioned medium was added to the cultures at 0 h of incubation and removed on medium change at the times of incubation indicated in parentheses (Nos. 3-5, 7-9). In this experiment 1 PCi [3H]thymidine was added to cultures after incubation for 4 h. The medium was not changed throughout the incubation for Nos. 6 and 10. Values are means for duplicate cultures. The 2 values at each condition did not differ by more than 10% from each other.
categories, competence and progression factors, in rabbit aortic SMC as in 3T3 cells. CM had synergistic effects with both competence and progression factors in increasing DNA synthesis in SMC (Figs. 6-9). We have reported that the growth factor(s) in CM differ(s) from PDGF, FGF, EGF, and somatomedin C [12]. They also differ from IL-l and TNF, because these factors do not stimulate DNA synthesis. IL-l at concentrations of l-10 U/ml had no effect on DNA synthesis of SMC in the presence or absence of 10e5 M indomethacin. TNF at 10K1’ to lo-’ M also did not stimulate DNA synthesis, and instead inhibited it to 50% of the control level with DME (Morisaki et al. unpublished data). These findings suggest that CM may contain both competence and progression factors and that these competence and progression factors in CM interact. This is unlikely, however, because CM had synergistic effects with optimal doses of competence and progression factors in increasing
67 DNA synthesis (Fig. 9), and because the presence of the factor(s) in CM in the culture medium for more than 4 h, probably for the whole period, was necessary for its stimulation of DNA synthesis. From these findings we propose that the factor (SDGF) in CM is a multifunctional growth factor that cannot be classified as either a competence or progression factor and the pathway by which GO stage cells are stimulated to enter the S stage by SDGF is different from the pathways stimulated by known competence and progression factors. Purification of SDGF is now in progress in order to examine this possibility. Our current idea on the role of SDGF in the pathogenesis of atherosclerosis is that it stimulates development of lesions rather than initiating them. This idea is based on the following observations: (1) SDGF is secreted from medial SMC at high passage level (i.e., for earlier stage of proliferation, a paracrine growth factor(s) is essential) [12]. (2) Culture of intimal SMC from atheromatous lesions secreted growth factors from an early stage of passage [16]. (3) SDGF alone [12] or in combination with other growth factors stimulated cell proliferation. Significant findings in the present study were that SDGF could stimulate SMC proliferation in the absence of growth factors derived from platelets, macrophages, or endothelial cells, but that the effect of SDGF was amplified by EGF or somatomedin C, both of which are present in the blood. Acknowledgements Part of this work was supported by grants from the Ministry of Education, Japan (Nos. 61570300, 62570276). References 1 Ross, R., The pathogenesis of atherosclerosis - an update, N. Engl. J. Med., 314 (1986) 488. 2 Nilsson, J., Growth factors and the pathogenesis of atherosclerosis, Atherosclerosis, 62 (1986) 185. 3 Schwartz, SM., Campbell, G.R. and Campbell, J.H., Replication of smooth muscle cells in vascular disease, Circ. Res., 58 (1986) 427.
4 Morisaki, N., Kanzaki, T., Koshikawa, T., Saito, Y. and Yoshida, S., Secretion of a new growth factor, smooth muscle cell derived growth factor, distinct from platelet derived growth factor by cultured rabbit aortic smooth muscle cells, FEBS Lett., 230 (1988) 186. 5 Scher, C.D., Antoniades, H.N. and Stiles, C.D., Platelet-derived growth factor and the regulation of the mammalian fibroblast cell cycle, B&him. Biophys. Acta, 560 (1979) 217. 6 Antoniades, H.N., Human platelet-derived growth factor and the sis-PDGF-2 gene: In: Guroff, G. (Ed.), ‘Oncogenes, Genes, and Growth Factors’, John Wiley & Sons, Inc., New York, 1987, pp. l-40. 7 DiCorleto, P.E. and Bowen-Pope, D.F., Cultured endothelial cells produce a platelet-derived growth factor-like protein, Proc. Natl. Acad. Sci. USA, 80 (1983) 1919. 8 Shimokado, K., Raines, E.W., Madtes, D.K., Barrett, T.B., Benditt, E.P. and Ross, R., A significant part of macrophage-derived growth factors consists of at least two forms of PDGF, Cell, 43 (1985) 277. 9 Seifert, R.A., Schwartz, SM. and Bowen-Pope, D.F., Developmentally regulated production of platelet-derived growth factor-like molecules, Nature, 311 (1984) 669. 10 Nilsson, J., Sjolund, M., Palmberg, L., Thyberg, J. and Heldin, C.-H., Arterial smooth muscle cells in primary culture produce a platelet-derived growth factor-like protein, Proc. Natl. Acad. Sci. USA, 82 (1985) 4418. 11 Walker, L.N., Bowen-Pope, D.F., Ross, R. and Reidy, M.A., Production, of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury, Proc. Natl. Acad. Sci. USA, 83 (1986) 7311. 12 Sejersen, T., Betcholtz, C., Sjolund, M., Heldin, C.-H., Westermark, B. and Thyberg, J., Rat skeletal myoblasts and arterial smooth muscle cells express the gene for the a chain but not the gene for the B chain (c-sis) of platelet-derived growth factor (PDGF) and produce a PDGF-like protein, Proc. Natl. Acad. Sci. USA, 83 (1986) 6844. 13 Morisaki, N., Kanzaki, T., Motoyama. N., Saito, Y. and Yoshida, S. Cell cycle-dependent inhibition of DNA synthesis by prostaglandin I, in cultured rabbit aortic smooth muscle cells, Atherosclerosis, 71 (1988) 165. 14 Morisaki, N., Kanzaki, T., Fujiyama, Y., Oosawa, I., Shirai, K., Matsuoka, N., Saito, Y. and Yoshida, S., Metabolism of n -3 polyunsaturated fatty acids and modification of phospholipids in cultured rabbit aortic smooth muscle cells, J. Lipid Res., 24 (1985) 930. 15 Morisaki, N., Kanzaki, T., Saito, Y. and Yoshida, S., Lack of inhibition of DNA synthesis by prostaglandin I, in cultured intimal smooth muscle cells from rabbits, Atherosclerosis, 73 (1988) 67. 16 Morisaki, N., Saito, Y. and Yoshida, S., Autocrine mechanism of cell proliferation of the arterial smooth muscle cells, J. Jap. Atheroscler. Sot., 16 (1989) 1185.
61 Ltd.
ATH 04333
Effects of smooth muscle cell derived growth factor (SDGF) in combination with other growth factors on smooth muscle cells Nobuhiro Morisaki, Noriyuki Koyama, Seijiro Mori, Tetsuto Kanzaki, Tomoko Koshikawa, Yasushi Saito and Sho Yoshida Second Department
of Internal Medicine, School of Medicine,
Chiba University, Chiba 280 (Japan)
(Received 1 August, 1988) (Revised, received 18 January, 1989) (Accepted 9 February, 1989)
Recently intimal thickening was shown to be due to stimulation of proliferation of arterial smooth muscle cells (SMC) by autocrine secretion of growth factor(s). We have reported that cultured rabbit aortic SMC secrete a growth factor (SDGF) distinct from other known growth factors. This paper reports on studies on the biological characteristics of SDGF. Putative “competence” and “progression” growth factors synergistically stimulated DNA synthesis of cultured rabbit aortic SMC. Conditioned medium (CM) from SMC containing SDGF stimulated DNA synthesis in SMC synergistically with either the competence factors or the progression factors. This synergistic effect was also observed in the presence of optimal concentrations of both competence and progression factors. The continuous presence of CM was essential for its stimulation of DNA synthesis whereas the presence of PDGF for only the first 4 h of culture was sufficient to induce maximum stimulation of DNA synthesis. These results suggest that SDGF is a new growth factor distinct from either competence or progression factors and that it stimulates a different pathway in SMC from those stimulated by other known growth factors.
Key uvords: Cell proliferation; Smooth muscle cell; Progression factor; Atherosclerosis
Autocrine;
Introduction
matous lesions [l-3]. We recently reported an autocrine mechanism for SMC proliferation [4]. Rabbit aortic medial SMC were found to secrete a factor named SDGF (smooth muscle cell derived growth factor) after numerous passages. This factor was physicochemically, immunologically, and biologically different from known growth factors such as platelet derived growth factor (PDGF), fibroblast growth factor (FGF), epidermaf growth
Proliferation of aortic smooth muscle cells (SMC) is important in the formation of athero-
Correspondence to: Dr. Nobuhiro Morisaki, The Second Department of Internal Medicine, School of Medicine, Chiba University, 1-8-1 Inohana, Chiba 280, Japan. 0021-9150/89/$03.50
0 1989 Elsevier Scientific
Publishers
Ireland,
Ltd.
Growth
factor;
Competence
factor;
62 factor (EGF), and somatomedin C, and could stimulate DNA synthesis in the absence of other growth factors. We were interested to know the effect of SDGF in combination with the other growth factors mentioned above in the pathogenesis of atherosclerosis. Two kinds of growth factor are essential for proliferation of quiescent cells, at least in relation to the 3T3 cell line [5]. The first includes the competence factors, which stimulate G, stage cells to enter the G, stage of the cell cycle, and the other is represented by the progression factors, which stimulate G, stage cells to progress to the S stage. The former includes PDGF and FGF, and the latter EGF and somatomedin C. SMC, which are mesenchymal cells like 3T3 cells, should require both kinds of growth factor for proliferation, although this has not yet been established. EGF and somatomedin C are present in vivo in circulating blood. Therefore, the limiting factor for the proliferation of SMC should be a competence factor(s) in the arterial wall [6]. Paracrine and autocrine secretions have both been reported to be sources of competence factors. PDGF is derived from clotted platelets, and PDGF-like factor(s) are also secreted by endothelial cells [7], macrophages [8] and rat arterial SMC [9-111. FGF is secreted by macrophages. In this study, we attempted to obtain biological characterization of SDGF by investigating its effects in combination with the other growth factors mentioned above on the growth of cultured rabbit aortic SMC.
Materials and Methods Materials PDGF and FGF were purchased from R & D System Inc. (Minneapolis, MN). EGF was from Collaborative Research Inc. (Lexington, MA). Interleukin-1 (IL-l) was from Genzyme Inc. (Boston, MA). Somatomedin C (99% purity) and tumor necrotizing factor (TNF, 99.6% purity) were kindly supplied by Fujisawa Pharmaceutical Co. (Tokyo, Japan). The sources of 6-[ 3H]thymidine, Dulbecco’s modified Eagle’s medium (DME), and fetal bovine serum (lot No. 27 N3541) were as reported previously [13].
Tissue culture Primary cultures of SMC were established from the medial layer of the thoracic aorta of male Japanese white rabbits (Oryctologus cuniculus var. domesticus) by an explant method [14]. Cells from primary cultures were subcultured to confluency in T-75 flasks, harvested, seeded at a density of 3 X lo4 cells/well in 1 ml of DME supplemented with 10% FBS in 24-well plates (16~mm wells), and incubated in a CO, (5%) incubator for about 4 days until they reached confluency. The medium was renewed every 2 or 3 days. For collection of conditioned medium (CM), SMC at the 2nd passage were further subcultured at a 1 : 2 split ratio in T-75 flasks [14]. Conditioned medium (CM) without plasma was collected at the 4th to 6th passage as reported [12]. Pooled CM was used for experiments. DNA synthesis The assay method was described previously [13]. Briefly, confluent SMC at the 3rd passage in 24-well plates (cell number, about 1.2 X 105/well) were synchronized to the G, stage by serum depletion for 24 h. Then the cells were treated with growth factor(s) and [ 3H]thymidine incorporation into DNA was measured over 24 h incubation. Cells were used at the 3rd passage because at this passage SMC secreted little material with rnitogenie activity into the medium [12]. Results Effects of combinations of putative competence and progression factors PDGF or FGF, and EGF or somatomedin C were used as competence and progression factors, respectively. Figs. 1 and 2 show the effects of combinations of PDGF and EGF or somatomedin C on DNA synthesis in SMC. EGF in the absence of PDGF increased DNA synthesis only slightly (maximum, about 2.5-fold over the control value at 5 or 10 ng/ml), but in the presence of sufficient PDGF (5 ng/ml) it increased DNA synthesis markedly and dose-dependently (maximum, about 16-fold the control value at 10 or 20 ng/ml) (Fig. 1). The effect of PDGF in combination with somatomedin C was similar to that of PDGF in combination with EGF (Fig. 2), although the ex-
63
1
2
4
8
Somatomedin 1
1
10 EGF
20
(ng/ml)
C (w/ml)
Fig. 2. Dose dependence of effect of somatomedin C on DNA synthesis of SMC in the presence or absence of PDGF. PDGF was added at 5 ng/ml. See legend to Fig. 1 for other condi-
Fig. 1. Dose dependence of effect of EGF on DNA synthesis of SMC in the presence or absence of PDGF. Confluent SMC in 24-well plates were synchronized to the Go stage by serum depletion for 24 h. Then cells were treated with growth factor(s) and 13H]thymidine incorporation into DNA during 24 h incubation was measured. PDGF was added at 5 ng/ml. Points are means for duplicate cultures. The 2 values at each point did not differ by more than 10% from each other.
tent of growth stimulation by these factors was different. This difference was mainly due to the use of different primary cultures [15]. Figs. 3 and 4 show the effects of combinations of PDGF and EGF, and FGF and somatomedin C, respectively, as functions of the concentrations of PDGF and FGF, respectively. In the absence of EGF, PDGF stimulated DNA synthesis only slightly (maximum, about 2.5-fold the control value at 2.5 or 5 ng/ml), but in the presence of sufficient EGF (10 ng/ml) PDGF stimulated DNA synthesis markedly and dose-dependently (maximum, 9.5-fold the control value at 5 ng/ml) (Fig. 3). Similarly, FGF alone increased DNA synthesis to only about 3-fold the control level, whereas in the presence of sufficient somatomedin
I
0.5
1
I
I
5
2.5
PDG F (ng/ml
)
Fig. 3. Dose dependence of effect of PDGF on DNA synthesis of SMC in the presence or absence of EGF. EGF was added at 10 ng/ml. See legend to Fig. 1 for other conditions.
somatomedin
i-
C (+I
somatomedin
(
L L 0
2.5
10
5 FGF
(ng/ml)
Fig. 4. Dose dependence of effect of FGF on DNA synthesis of SMC in the presence or absence of somatomedin C. Somatomedin C was added at 4 ng/ml. See legend to Fig. 1 for other conditions.
tors alone stimulated DNA synthesis slightly or moderately and in their presence 100% CM caused synergistic stimulation (maximum, 15fold the control level with EGF and 1Zfold with somatomedin C). Fig. 8 shows the interaction of CM with the competence factor FGF. FGF alone stimulated DNA synthesis only slightly (maximum, 3-fold of control values at 5 to 10 ng/ml) and 100% CM had a synergistic effect (maximum, ll-fold the control level with 5 to 10 ng/ml of FGF). Fig. 9 shows the effect of CM with PDGF in combination with an effective concentration of 10 ng/ml of the progression factor EGF. PDGF caused synergistic and dose-dependent stimulation of DNA synthesis over the control level with EGF alone (maximum, 5-fold at 5-10 ng/ml of PDGF). CM alone at 100% increased DNA synthesis to g-fold the control level with EGF alone. CM at 100% had a synergistic effect with PDGF at O-2.5 ng/ml in stimulating DNA synthesis (maximum, 21.5-fold the control level with EGF). With PDGF concentrations of over 2.5 ng,/ml, DNA synthesis in the presence of CM was less than that with 2.5 ng/ml of PDGF.
(4 ng/ml), it markedly stimulated DNA synthesis in a dose-dependent manner (maximum, about 13-fold the control value at 10 ng/ml). These findings show that competence and progression factors have synergistic effects on SMC.
C
I
conditioned
medium (_t)
Effect of CM in combination with FBS FBS, which is believed to contain both competence and progression factors, stimulated DNA synthesis dose-dependently at concentrations of up to 10% (Fig. 5). CM alone increased DNA synthesis up to about 6.5fold of the control level, as previously reported 1121. Stimulation of DNA synthesis by FBS was greater in the presence of CM than in its absence, although the combined effect of CM and FBS was less than the sum of their individual effects.
Effect of CM in combination with progression or competence factors Figs. 6 and 7 show the effects of CM in combination with the progression factors EGF and somatomedin C, respectively. The progression fac-
conditioned
s P
medium H
1 I 0
2.5 Fetal
1 5 bovine
I 10 serum
(%I
Fig. 5. Dose dependence of effect of fetal bovine serum on DNA synthesis of SMC in the presence or absence of conditioned medium. 100% conditioned medium was added. See legend to Fig. 1 for other conditions.
65
‘“I
conditioned
,f ._ t
conditioned
medium (4
medium H
5
conditioned
medium (1-1
conditioned
medium (-4
5 i
2
. I
a
1
z
0
5
EG F (ng/ml
10
1
Fig. 6. Dose dependence of effect of EGF on DNA synthesis of SMC in the absence or presence of conditioned medium. 100% of conditioned medium was added. See legend to Fig. 1 for other conditions.
Effects of CM or PDGF on DNA synthesis as functions of the period of stimulation 3T3 cells in the G, stage soon entered the competent G, stage of the cell cycle when stimulated with a competence factor [4], and once they became competent, they did not need the competence factor for progression to the S stage. We examined whether SDGF in CM acts like known competence factors, such as PDGF (Table 1). Exposure of SMC to a sufficient concentration of the progression factor EGF (10 ng/ml) for the entire incubation period (24 h) increased their DNA synthesis only 3.6-fold. Exposure of G, stage SMC to the competence factor PDGF for 1 or 2 h resulted in little increase in DNA synthesis over that in the control with EGF. However, 4-h exposure of SMC to PDGF (5 ng/ml) markedly increased DNA synthesis (from 3.6- to 31.9-fold over the control with DME). Exposure of SMC to PDGF for 24 h resulted in a much greater increase
P
1
2
4
8
Somatomedin
C (ng/ml
)
Fig. 7. Dosedependence of effect of somatomedin C on DNA synthesis of SMC in the absence or presence of conditioned medium. 100% of conditioned medium was added. See legend to Fig. 1 for other conditions
conditioned
medium &)
conditioned
medium (--I
10
5
F GF
(ng/ml)
Fig. 8. Dose dependence of effect of FGF on DNA of SMC in the absence or presence of conditioned 100% of conditioned medium was added.
synthesis medium.
66 than exposure for O-2 h, but less than that induced by 4-h exposure. Exposure of SMC to 100% pooled CM for l-4 h caused less than 2-fold increase in DNA synthesis over that of the control with EGF, and exposure to CM for 24 h increased DNA synthesis to 6-fold that of the control with EGF.
TABLE
Discussion
1) DME 2) DME + EGF 3) DME+EGF+PDGF (1 h) 4)DME+EGF+PDGF(2 h) 5) DME + EGF + PDGF (4 h) 6) DME + EGF + PDGF (24 h) 7) DME + EGF + CM (1 h) 8) DME + EGF + CM (2 h) 9) DME + EGF + CM (4 h) 10) DME+EGF+CM (24 h)
As shown in Figs. 1-4, competence factors such as PDGF or FGF have synergistic effects with progression factors, such as EGF or somatome&n C, in increasing DNA synthesis in SMC as in 3T3 cells. Table 1 shows that exposure of SMC in the G, stage to PDGF for 4 h was sufficient to induce their entry into the S stage in the presence of EGF. These results strongly indicate that growth factors can be classified into two
(EGF
6))
conditioned medium , (EGF (id)
0
5 PDG
H
10
F (w/ml)
Fig. 9. Dose dependence of effect of PDGF on DNA synthesis of SMC in the presence of EGF and the presence or absence of conditioned medium. 10 ng/mI of EGF and 100% conditioned medium were added. See legend to Fig. 1 for other conditions.
1
EFFECTS OF CONDITIONED MEDIUM (CM) AND PDGF ON DNA SYNTHESIS BY SMOOTH MUSCLE CELLS AS FUNCTIONS OF THEIR PERIODS OF STIMULATION Condition
DNA synthesis (dpm/well)
Increased stimulation over control
2470 8 920 13300 11800 78 800 38 900 13700 14200 11900 53200
1.0 3.6 5.4 4.8 31.9 15.7 5.5 5.7 4.8 21.5
value
EGF (10 ng/ml) was added to the cultures at 0 h of incubation (No. 2-lo), and again with medium change at the times of incubation indicated in parentheses (Nos. 3-5, 7-9). PDGF (5 ng/mI) or 100% conditioned medium was added to the cultures at 0 h of incubation and removed on medium change at the times of incubation indicated in parentheses (Nos. 3-5, 7-9). In this experiment 1 PCi [3H]thymidine was added to cultures after incubation for 4 h. The medium was not changed throughout the incubation for Nos. 6 and 10. Values are means for duplicate cultures. The 2 values at each condition did not differ by more than 10% from each other.
categories, competence and progression factors, in rabbit aortic SMC as in 3T3 cells. CM had synergistic effects with both competence and progression factors in increasing DNA synthesis in SMC (Figs. 6-9). We have reported that the growth factor(s) in CM differ(s) from PDGF, FGF, EGF, and somatomedin C [12]. They also differ from IL-l and TNF, because these factors do not stimulate DNA synthesis. IL-l at concentrations of l-10 U/ml had no effect on DNA synthesis of SMC in the presence or absence of 10e5 M indomethacin. TNF at 10K1’ to lo-’ M also did not stimulate DNA synthesis, and instead inhibited it to 50% of the control level with DME (Morisaki et al. unpublished data). These findings suggest that CM may contain both competence and progression factors and that these competence and progression factors in CM interact. This is unlikely, however, because CM had synergistic effects with optimal doses of competence and progression factors in increasing
67 DNA synthesis (Fig. 9), and because the presence of the factor(s) in CM in the culture medium for more than 4 h, probably for the whole period, was necessary for its stimulation of DNA synthesis. From these findings we propose that the factor (SDGF) in CM is a multifunctional growth factor that cannot be classified as either a competence or progression factor and the pathway by which GO stage cells are stimulated to enter the S stage by SDGF is different from the pathways stimulated by known competence and progression factors. Purification of SDGF is now in progress in order to examine this possibility. Our current idea on the role of SDGF in the pathogenesis of atherosclerosis is that it stimulates development of lesions rather than initiating them. This idea is based on the following observations: (1) SDGF is secreted from medial SMC at high passage level (i.e., for earlier stage of proliferation, a paracrine growth factor(s) is essential) [12]. (2) Culture of intimal SMC from atheromatous lesions secreted growth factors from an early stage of passage [16]. (3) SDGF alone [12] or in combination with other growth factors stimulated cell proliferation. Significant findings in the present study were that SDGF could stimulate SMC proliferation in the absence of growth factors derived from platelets, macrophages, or endothelial cells, but that the effect of SDGF was amplified by EGF or somatomedin C, both of which are present in the blood. Acknowledgements Part of this work was supported by grants from the Ministry of Education, Japan (Nos. 61570300, 62570276). References 1 Ross, R., The pathogenesis of atherosclerosis - an update, N. Engl. J. Med., 314 (1986) 488. 2 Nilsson, J., Growth factors and the pathogenesis of atherosclerosis, Atherosclerosis, 62 (1986) 185. 3 Schwartz, SM., Campbell, G.R. and Campbell, J.H., Replication of smooth muscle cells in vascular disease, Circ. Res., 58 (1986) 427.
4 Morisaki, N., Kanzaki, T., Koshikawa, T., Saito, Y. and Yoshida, S., Secretion of a new growth factor, smooth muscle cell derived growth factor, distinct from platelet derived growth factor by cultured rabbit aortic smooth muscle cells, FEBS Lett., 230 (1988) 186. 5 Scher, C.D., Antoniades, H.N. and Stiles, C.D., Platelet-derived growth factor and the regulation of the mammalian fibroblast cell cycle, B&him. Biophys. Acta, 560 (1979) 217. 6 Antoniades, H.N., Human platelet-derived growth factor and the sis-PDGF-2 gene: In: Guroff, G. (Ed.), ‘Oncogenes, Genes, and Growth Factors’, John Wiley & Sons, Inc., New York, 1987, pp. l-40. 7 DiCorleto, P.E. and Bowen-Pope, D.F., Cultured endothelial cells produce a platelet-derived growth factor-like protein, Proc. Natl. Acad. Sci. USA, 80 (1983) 1919. 8 Shimokado, K., Raines, E.W., Madtes, D.K., Barrett, T.B., Benditt, E.P. and Ross, R., A significant part of macrophage-derived growth factors consists of at least two forms of PDGF, Cell, 43 (1985) 277. 9 Seifert, R.A., Schwartz, SM. and Bowen-Pope, D.F., Developmentally regulated production of platelet-derived growth factor-like molecules, Nature, 311 (1984) 669. 10 Nilsson, J., Sjolund, M., Palmberg, L., Thyberg, J. and Heldin, C.-H., Arterial smooth muscle cells in primary culture produce a platelet-derived growth factor-like protein, Proc. Natl. Acad. Sci. USA, 82 (1985) 4418. 11 Walker, L.N., Bowen-Pope, D.F., Ross, R. and Reidy, M.A., Production, of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury, Proc. Natl. Acad. Sci. USA, 83 (1986) 7311. 12 Sejersen, T., Betcholtz, C., Sjolund, M., Heldin, C.-H., Westermark, B. and Thyberg, J., Rat skeletal myoblasts and arterial smooth muscle cells express the gene for the a chain but not the gene for the B chain (c-sis) of platelet-derived growth factor (PDGF) and produce a PDGF-like protein, Proc. Natl. Acad. Sci. USA, 83 (1986) 6844. 13 Morisaki, N., Kanzaki, T., Motoyama. N., Saito, Y. and Yoshida, S. Cell cycle-dependent inhibition of DNA synthesis by prostaglandin I, in cultured rabbit aortic smooth muscle cells, Atherosclerosis, 71 (1988) 165. 14 Morisaki, N., Kanzaki, T., Fujiyama, Y., Oosawa, I., Shirai, K., Matsuoka, N., Saito, Y. and Yoshida, S., Metabolism of n -3 polyunsaturated fatty acids and modification of phospholipids in cultured rabbit aortic smooth muscle cells, J. Lipid Res., 24 (1985) 930. 15 Morisaki, N., Kanzaki, T., Saito, Y. and Yoshida, S., Lack of inhibition of DNA synthesis by prostaglandin I, in cultured intimal smooth muscle cells from rabbits, Atherosclerosis, 73 (1988) 67. 16 Morisaki, N., Saito, Y. and Yoshida, S., Autocrine mechanism of cell proliferation of the arterial smooth muscle cells, J. Jap. Atheroscler. Sot., 16 (1989) 1185.