Demonstration of stimulatory effects of platelet-derived growth factor on cultivated rat arterial smooth muscle cells

Experimental

Cell Research

Copyright @I 1983 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/83/060231-07$02.00/0

145 (1983) 231-237

Demonstration of Stimulatory Effects of Platelet-derived on Cultivated Rat Arterial Smooth Muscle Cells Differences

between

JAN NILSSON,’ THYBERG’

Growth Factor

Cells from Young and Adult Animals TOMASZ

KSIAZEK,’

CARL-HENRIK

HELDIN*

and

JOHAN,

‘Department of Histology, Karolinska Institutet, S-10401 Stockholm and ‘Institute of Medical and Physiological Chemistry, The Biomedical Center (BMC), University of Uppsala, S-751 23 Uppsala, Sweden

SUMMARY The effects of platelet-derived growth factor (PDGF) on DNA synthesis and proliferation in cultures of arterial smooth muscle cells obtained from young and adult ts, respectively, were measured. Addition of 10-20 rig/ml of PlXiF to medium MCDB 104 “k I duced DNA synthesis in quiescent cultures of cells from young animals to a similar extent as lO-20% whole blood serum (WBS). PDGF further stimulated proliferation of the cells in medium MCDB 104, although less markedly than 10% WBS. Antibodies against PDGF partially inhibited the growth response after stimulation with serum. This shows that PDCF is a major growth factor in serum for these cells and that PDGF can promote entrance into and passage through S phase and mitosis independent of plasma factors. Cells from adult animals were also found to respond to PDGF, although a higher concentration (25 @ml) was required to obtain a maximum effect. These cells, however, responded better than cells from young animals to stimulation with serum. Further, antibodies against PDCF did not inhibit the growth-stimulatory effect of serum to any appreciable extent. Thus, serum contains growth factors other than PDGF that stimulate preferentially the proliferation of smooth muscle cells from adult animals.

Platelet-derived growth factor (PDGF) has been identified as the major serum component responsible for proliferation of connective tissue cells in vitro [l-3]. It has been suggested that this factor plays a role in the pathogenesis of atherosclerosis [4]. PDGF is stored in the a-granules of platelets and released during blood clotting [5,6]. It is a highly cationic polypeptide that consists of two subunits with a total molecular weight of 30000-35 000 [7-l 11. Specific receptors for PDGF have been demonstrated on the surface of fibroblasts, smooth muscle cells, and glial cells [Q-16]. Early cellular effects of PDGF include tyrosine-specific phosphorylation of membrane proteins [17], increased amino acid uptake [18] synthesis of RNA [ 191 and certain cytoplasmic proteins [20], and stimulated endocytosis [21, 221. The exact relation of these early events to the initiation of DNA synthesis and mitosis is not known. Heldin et al. [23] recently found that PDGF when added to serum-free medium was able to promote proliferation of human glial cells. In contrast, it has been suggested that PDGF acts synergistically with plasma components, mainly somatomedins, to induce entry of quiescent 3T3 cells into S phase 12, 241. Previous investigations on arterial smooth muscle cells have shown that PDGF stimulates DNA synthesis, proliferation, and endocytosis in the presence of plasma-derived serum [21, 22, 251. In the present study we have investigated the effects of PDGF on smooth muscle cells obtained from young and adult rats, respectively. We show that

232 Nilsson

Exp Cell Res 145 (1983)

et al.

PDGF stimulates DNA synthesis, proliferation, and endocytosis in smooth muscle cells from young animals when cultured under serum-free conditions in medium MCDB 104. Smooth muscle cells from adult animals also respond to PDGF, but to a lesser extent. However, these cells showed a very good response to serum, in fact they responded better than cells from young animals. Thus, cells from adult animals have retained their growth potential, although growth factors other than PDGF seem to be responsible for the major part of the stimulatory effect in serum. MATERIALS

AND

METHODS

Cell culture Aortae from 5-day-old and &month-old Sprague-Dawley rats were cleaned from intima and adventitia under a dissection microscope. The remaining media was cut into small pieces and digested for 12-15 h at 37°C in 0.2 % collagenase (Sigma type I) in culture medium with 10 % newborn calf serum (NCS) on a gyratory shaker. The freed cells were passed through a nylon fdter, rinsed twice in culture medium, and counted. They were then plated out in 75 cm* Falcon plastic Basks (24x ld celIs/flask) in 10 ml of medium F-12 supplemented with 10 mM HEPES, 10 mM TBS, 50 pg/ml of gentamycin sulfate, 50 @ml of L-ascorbic acid, and 10 % NCS and kept at 37°C in an atmosphere of 5 % COJ95 % air. Medium was changed three times a week. After contluence had been reached, the cells were growth-arrested by transfer to medium containing 10% human plasmaderived serum (PDS [26]) for 48 h, trypsinized, and replated into 35 mm plastic Petri dishes. The cells were then used for the experiments either directly or after further growth in medium containing 10% human whole blood serum (WBS [26]). Experiments done under serum-free conditions were preceded by rinsing and 2x24 h incubation in serum-free media to arrest growth and to remove serum and plasma factors.

Platelet-derived

growth factor (PDGF)

PDGF was purified from human platelets as described [lo]. Experiments were performed either with pure PDGF or with partially purified PDGF (about 30% pure; this material was taken through the purification procedure except for the last step).

Antibodies

to platelet-derived

growth factor

Antibodies against PDGF were produced by injecting a rabbit in the popliteal lymph nodes biweekly with 15 ug of pure PDGF [lo], the first time together with Freund’s complete adjuvance and at subsequent occasions together with Freund’s incomplete adjuvance. The anti-PDGF immune serum used in this study was obtained after eight injections. The immunoglobulin ha&ion was purified by applying 4 ml of the immune serum to a 4 ml column of protein A-Sepharose (Pharmacia Fine Chemicals). After washing with phosphate-buffered saline (PBS), the column was desorbed by elution with 0.05 M citrate buffer, pH 3.0. Fractions (2.0 ml) were collected in test tubes containing 0.2 ml of 1.0 M Tris base. Those containing immunoglobulins were pooled and dialysed against PBS. This immunoglobulin fraction added at 40 pg/ml inhibited the stimulatory effect of 10 t&ml of PDGF on DNA synthesis in human tibroblasts by more than 90%.

Measurement

of DNA synthesis and cell growth

For assay of DNA synthesis the cells were exposed to [3H]tbyrnidine for 24 h and the incorporation of radioactivity into DNA determined by autoradiographic analysis of the percentage of labelled nuclei [27]. Proliferation was followed by trypsinization of the cultures and counting of the cells in an electronic cell counter [26].

Endocytosis

assay

Horseradish peroxidase (HBP; Sigma type II) was used as a fluid-phase marker to determine the rate of endocytosis. The cells were exposed to 1.0 mg/ml of the enzyme for 2 h, rinsed four times with PBS, reincubated in 10% NCS-medium, and rinsed another three times with PBS to remove enzyme adsorbed to the bottom of the Petri dishes [28]. They were then lysed in 0.05% Triton X-100 and assayed for peroxidase activity [28] and protein content [29].

Stimulation of smooth muscle cells by PDGF

Exp Cell Res 145 (1983)

so ‘a t t

10

20

30 40 PDOF l”g/mll

50

t----e-

10

30 WBS I%1

Fig. 1. Effects of various concentrations of (a) PDGF or (b) WBS on DNA synthesis of smooth muscle cells. Subconfluent, growth-arrested cultures were given experimental media and exposed to 2.5 uCi/ml of [‘Hlthymidine for 24 h. They were then fixed and processed for autoradiography. (a) Cells from 0, A, young; 0, adult animals given: 0, 0, medium MCDB 104 or A, DMEM after exposure to various concentrations of partly purified PDGF. (b) Cells from 0, young or 0, adult animals cultured in different concentrations of WBS. Each value is the mean of percentage labelled nuclei in triplicate cultures (SD < 10 %).

RESULTS Efict on DNA synthesis Subconfluent, growth-arrested cultures of arterial smooth muscle cells from young animals maintained in serum-free Dulbecco’s modified Eagle medium (DMEM) or medium MCDB 104 (Gibco 1301)were incubated with various concentrations of PDGF. As shown in fig. 1a, PDGF stimulated DNA synthesis in both media, although the effect was more prominent in medium MCDB 104. In this medium, 10 rig/ml of PDGF produced a maximum response of about 50% labelled nuclei, an effect comparable to that of lO-20% WBS (fig. 1b). Further, addition of an excess of PDGF antibodies (40 ug/ml) to medium containing 10% WBS reduced DNA synthesis to a level comparable to that obtained with 10% PDS (table 1). Smooth muscle cells from adult animals cultured under serum-free conditions Table 1. E#ect of antibodies to PDGF on DNA synthesis in cultured smooth muscle cells stimulated with serum Medium

% labelled nuclei of cells from Adult animals Young animals

10% PDS 10% WBS 10% WBS+PDGF

26.6 48.8 29.6

antibodies

31.2 54.1 39.5

Confluent, growth-arrested cultures were given 10% PDS medium or 10% WBS medium with or without 40 pg/ml of PDGF antibodies, exposed to 2.5 @.X/ml of [3H]thymidine for 24 h, and processed for autoradiographic analysis. Each value is the mean of triplicate cultures (SD
233

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Nilsson

et al.

Exp Cell Res

145 (1983)

3b 120

t

I

I 5 D.YS

10

5

IO

D-e

Fig. 2. Effect of PDGF on proliferation of smooth muscle cells. Sparse cultures of cells from 0, A, young; 0, ‘A, adult animals were given medium MCDB 104 0, 0, with or A, A, without 10 rig/ml of pure PDGF (medium changed three times a week) trypsinized at indicated times, and cell numbers determined using an electronic cell counter. Each value is the mean of triplicate cultures (SD
also responded to PDGF. However, a higher concentration (25 rig/ml) was required for maximum effect (55% labelled nuclei). These cells responded to WBS as well, in fact WBS produced a higher maximum effect on cells from adult auimals (80% labelled nuclei) compared with cells from young animals (50% labelled nuclei; fig. 1b). Antibodies against PDGF neutralized some, but not all, of the WBS-induced stimulation of DNA synthesis in cells from adult animals. E#ect on cell growth

Under serum-free conditions 10 &ml of pure PDGF in medium MCDB 104 produced a clear proliferative response during the first 5 days in sparse cultures of smooth muscle cells from young animals. Growth thereafter continued at a slower rate, with a doubling in the cell number at’ter about 15 days (fig. 2). Stimulation of the cells with 10% WBS gave a more prominent growth response than PDGF in serum-free medium, whereas 10% PDS essentially lacked effect. The proliferation induced by 10% WBS was partially inhibited by addition of antibodies against PDGF (fig. 3 a). The proliferative effect of PDGF on cells from adult animals under defined conditions was less pronounced (fig. 2). However, these cells proliferated even better than cells from young animals in the presence of 10% WBS. Furthermore, this proliferation was not to any appreciable extent affected by antibodies against PDGF (fig. 3 b). Effect on endocytosis

The effect of PDGF on endocytosis was studied using subconfluent and confluent cultures of cells maintained in PDS or serum-free medium. In cultures of smooth muscle cells from young animals, 10 rig/ml of partly purified PDGF enhanced

Exp Cell

Stimulation of smooth muscle cells by PDGF

Res 145 (1983)

Table 2. Uptake of HRP by cultured smooth muscle cells isolated from young animals HRP uptake Subcontluent cultures

Confluent cultures

Medium

ng/mg protein

@dish

ng/mg protein

&dish

F-12 F-12+10 rig/ml of PDGF

147 173

16 20

204 240

37 48

10% PDS 10% PDS+ 10 rig/ml of PDGF

188 224

29 36

160 214

44 74

10% WBS

225

36

225

74

Growth-arrested cultures were given experimental media for 24 h, exposed to 1.0 mg/ml of HRP in serum-free medium for 2 h, rinsed, and harvested for assay of peroxidase activity and protein content. Each value is the mean of triplicate cultures (SD
uptake of HRP by 2O-30% at both densities and in serum-free as well as in 10% PDS medium. In the latter medium, PDGF raised endocytosis to a similar level as in medium containing 10% WBS (table 2). In subconfluent cultures of smooth muscle cells from adult animals, PDGF stimulated HRP uptake by about 20% in serum-free medium and by about 10% in 10% PDS medium. Moreover, HRP uptake was 2530% higher in 10% WBS medium than in 10% PDS medium. In confluent cultures PDGF lacked effect on HRP uptake (table 3). Culture in serum-free medium for 24 h, i.e. the time used in the endocytosis experiments, led to a 3&50% decreased protein content per dish. This was largely due to a decreased protein content per cell, since no appreciable loss of cells occurred under these conditions (fig. 2). Therefore, HRP uptake, calculated Table 3. Uptake of HRP by cultured smooth muscle cells isolated from adult animals HRP uptake S&cot&tent

cultures

Confluent cultures

Medium

ng/mg protein

@dish

ng/mg protein

&dish

F-12 F-12+10 rig/ml of PDGF

201 242

22 28

218 219

20 24

10% PDS 10% PDS+lO rig/ml of PDGF 10% WBS

169 184 213

29 31 46

136 133 140

24 19 32

See footnote to table 2.

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et al.

Exp

Cell

as ng of enzyme per mg ceil protein, is deceptively high in serum-free cultures. Thus, HRP uptake per dish was 20-50% higher in PDS medium and 60-150% higher in WBS medium than in serum-free medium (cf tables 2 and 3). Summing up, PDGF had a moderate but consistent stimulatory effect on fluidphase endocytosis. Moreover, as in the studies on DNA synthesis and proliferation, cells from young animals responded better to PDGF than cells from adult animals. The effect was of similar magnitude in serum-free and PDS medium. It was, however, less marked than that reported by Davies & Ross [21, 221, using the latter type of medium and higher concentrations of PDGF. Thus, it seems likely that PDGF is not the only serum component that stimulates endocytosis. The enhanced endocytic rate may be part of a general activation of the cells. Its relation to passage through GO/G1 and entrance into S phase remains unknown. DISCUSSION The present study shows that low concentrations of PDGF (about 0.3 nM) stimulates both DNA synthesis and mitosis of rat arterial smooth muscle cells from young animals cultured in a chemically defined, serum-free medium. These results support previous observations on human glial cells [23], indicating that PDGF is in itself sufficient to promote quiescent cells to enter into and pass through S phase and mitosis. Contrarily, it has been proposed that PDGF alone does not stimulate cells to a complete cell cycle traverse, but only makes them competent and that plasma factors, e.g. somatomedins, are required for progress into S phase and division [2, 241. This concept of competence and progression was worked out in a system utilizing mouse 3T3 cells cultured in DMEM, a nutritionally poor medium. When using this medium, we obtained a distinctly lower stimulatory effect of PDGF on DNA synthesis than in MCDB 104, a medium in which the concentrations of different components have been optimized for growth of diploid fibroblasts at low serum concentration [30]. It is therefore possible that the need for plasma in cultures of 3T3 cells reflects a nutritional role rather than a supply of specific progression factors. It is also possible that somatomedins are required together with PDGF for the cells to grow and that smooth muscle and glial cells produce this hormone themselves in sufficient quantities. In support of such a possibility, synthesis of somatomedin C has been demonstrated in cultures of human fibroblasts. Moreover, this synthesis was stimulated by PDGF [31]. Also smooth muscle cells from adult animals responded to PDGF when cultivated under defined conditions. However, these cells required higher concentrations of PDGF than cells from young animals. Furthermore, antibodies against PDGF were less effective in neutralizing the effect of WBS on these cells compared with cells from young animals. This suggests that, whereas PDGF is a major mitogen in WBS for cells from young animals, other growth factors in WBS seems to be more important for the proliferation of cells from adult animals. We found, somewhat surprisingly, that cells from adult animals proliferated better in WBS than cells from young animals. This shows that arterial smooth muscle of adult animals has not lost its growth potential. This is in agreement

Res 145 (1983)

Exp

Cell

Res 145 (1983)

Stimulation

of smooth muscle cells by PDGF

with results recently reported by Sternerman et al. [32], who found a higher incorporation of [3H]thymidine into smooth muscle cells of adult than of young rat aortas after de-endothelialization in vivo. The observed higher susceptibihty of adult smooth muscle cells to growth stimulation may in part account for the higher frequency with advanced age of proliferative lesions involving smooth muscle cells, i.e. atherosclerosis [4, 321. The authors thank Karin Blomgren for expert technical assistance. Financial support was obtained from the Swedish Medical Research Council (proj. no. 06537), the Swedish Cancer Society, the King Gustaf V 80th Birthday Fund, the Swedish Society of Medical Sciences, the Bergvall Foundation, and from the Funds of Karolinska Institutet. T. K. is on leave from the Department of Histology and Embryology, Institute of Biostructure, Medical School, Warsaw, Poland.

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