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The Modulation of Collagen Synthesis in Cultured Arterial Smooth Muscle Cells by Platelet-Derived Growth Factor Yoshikatsu Okada*, Shogo Katsuda, Yutaka Matsui and Isao Nakanishi Department of Pathology, School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 920, Japan. *Corresponding author

Abstract Changes on collagen synthetic activity of cultured arterial smooth muscle cells of rabbits induced with purified platelet-derived growth factor (PDGF) were examined. PDGF treatment (final concentration was 5 units/ml) decreased the total collagen synthesis per cell, while the rate of collagen synthesis against total protein synthesis was raised by PDGF. Type analysis of collagen revealed substantial reduction of type IV collagen and relative increase of type V collagen in the PDGF-treated cells. By immunofluorescence study using anti-type IV collagen antibody, the lacework fluorescence was decreased with PDGF supplement. These findings indicate that PDGF induces the decrease of type IV collagen synthesis with the simultaneous diminution of basement membrane formation probably in association with phenotypic modulation of smooth muscle cells. Introduction PDGF is a cationic glycoprotein of approximately 30,000 Mr. consisting of PDGF-A chain and -B chain, which is secreted from various cells including platelets, macrophages, endothelial cells and smooth muscle cells (Ross et al., 1986). PDGF is a potent mitog6n for mesenchymal cells as well as a candidate for factor(s) enhancing total collagen synthesis in smooth muscle cells (Stavenow et al., 1981) and type V collagen synthesis in fibroblasts (Narayanan and Page, 1983). We present here the changes of collagen synthesis in cultured arterial smooth muscle cells in response to PDGF, especially on type IV collagen which is one of a major component of a basement membrane (Glanville, 1987) and type V collagen, and discuss briefly the relationship between phenotypic modulation and collagen synthesis.

Materials and Methods Cell culture Smooth muscle cells were obtained from the aortic media of male rabbits weighing about 2.5 kg. Culture technique was similar to that described by Ross (1971). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), Kanamycin (100 Ixg/ml) and Amphotericin B (5 lxg/ml) until they reached confluence. Then, they were trypsinized and subcultured. The cells in the first or second passage were used for the experiments.

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The cells were seeded in 35 mm Petri dishes at a concentration of 1 x 105 cells per each dish containing 2 ml of the medium. Cell counts were made in duplicate for each dish using a hemacytometer. Five half-maximal units of PDGF (Collaborative Research, USA) were added to the medium on day 7 when the cells become confluent. The amount of PDGF had been determined effective on the growth stimulation in our previous work (Katsuda et al., 1988). The cells were cultured with PDGF and 1% of FBS for 24 h. Cells cultured with no PDGF were also made. Metabolic labeling was performed using 50 p.Ci/ml of L-[2,3-3H]-proline (Amersham, USA), 0.1 mM sodium ascorbate and 0.5 mM 13-aminopropionitrile fumarate, with or without PDGF, for 24 h. Then, the cells were harvested together with the culture medium and disrupted by a Branson Sonifier Cell Disruptor 185 (Branson, USA). A part of the sonicated solution which was the sample for quantitative analysis was boiled to inactivate protease activity. The rest of the solution which was the sample for collagen type analysis was mixed with protease inhibitors (0.25 M ethylenediamine tetraacetic acid disodium, 10 mM phenylmethane sulfonyl fluoride, 0.1 M N-ethylmaleimide). Quantitative and type analysis of collagen Quantitative and type analyses of collagen synthesized by cultured cells were performed as we have already described (Okada et al., 1990). Collagen as a percent of total protein was determined by the following formula (Peterkofsky et al., 1982): cpm in collagen x 100 cpm in collagen + 5.4 x cpm in non-collagenous protein For type analysis of collagen, samples were digested with pepsin (Behringer Manheim GmbH, West Germany) (Hata et al., 1980). Electrophoresis was performed as described by Weber and Osbom (1975), using 3% stacking gel (pH 6.8) and 5% separating gel (pH 8.8), and followed by fluorography (Chamberlain, 1979). The densities of the bands corresponding collagen chains in non-reduced condition were measured with a Sakura PDS 15 densitometer. Immunoblotting Immunoblotting using a monospecific antibody against type IV collagen was performed based on the method described by Towbin et al. (1979). Briefly, the proteins in polyacrylamide gel were transferred to a nitrocellulose sheet (0.45 I.tm pore size) with a Horizeblot AE 6670 (Atto, Japan) at 250 mA for 2 h. Subsequently, the nitrocellulose sheet was washed in a tray containing TBS buffer (10 mM Tris-HC1 buffer pH 7.6, 0.9% NaCI, 0.05% Tween 20). Following the blocking of any nonspecific reaction with TBS buffer containing 1% bovine serum albumin in a moist chamber at room temperature for 30 min, the nitrocellulose sheet was immunostained with the avidin-biotin-peroxidase complex method using monospecific anti-human type IV collagen rat IgG as a primary antibody which was prepared in our laboratory (Minamoto et al., 1988), and anti-rat IgG rabbit IgG (DAKO) as a secondary antibody.

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Indirect immunofluorescence study The cells seeded in Lab-Tek chamber slides (No. 4820; Miles Scientific, USA) were fixed in situ with acetone for 10 min at 4°C, washed with phosphate-bufferd saline (PBS), and subsequently immersed in 10% normal goat serum (DAKO, Denmark). Then the samples were incubated with the primary antibody against human type IV collagen diluted 1 : 10 for 90 min at room temperature, followed by FITCconjugated anti-rat IgG goat IgG (DAKO) diluted 1 : 10 for 60 min at room temperature. Immunofluorescense was observed using a Vanox AH microscope (Olympus, Japan). Results

The number of the cells cultured with PDGF was slightly increased (34.2 + 1.3 × 10 4) compared with that of the cells cultured with no PDGF (31.7 + 1.4 x 104) (Table 1). Smooth muscle cells cultured with PDGF showed decreased collagen synthesis per cell (1.85 + 0.24 cpm x 10-l) compared with those cultured with no PDGF (2.19 + 0.17 cpm x 10 -l) (Table 1). Synthesis of non-collagenous proteins per cell was also decreased in the cells cultured with PDGF. The rate of collagen synthesis to total protein synthesis, however, was increased in the ceils cultured with PDGF (11.6 + 0.9%) compared with those in the cells cultured with no PDGF (9.1 + 0.5%) (Table 1). Electrophoresis followed by fluorography revealed that smooth muscle cells synthesized types I, III and V collagen and the electrophoretic pattern of collagens showed no significant difference whether they were cultured with PDGF or not (Fig.l). Besides those collagen types, an unknown band was seen at the site just below the top (about 500,000 dalton in molecular weight) in non-reduced condition (Fig. 1, arrow), and this protein migrated to the sites corresponding to about 185,000 dalton in molecular weight (Fig. 1, upper arrowhead) and to about 85,000 dalton (Fig 1, lower arrowhead) due to the reduction. Immunoblotting using type IV specific antibody reveal,ed that all these bands are type IV collagen or their fragments (Fig. 2). Table 1. Synthesis of total collagenous and non-collagenous proteins in arterial smooth muscle cells cultured with or without PDGF cpa No PDGF PDGF

2.19 + 0.17 1.85+0.24

NCpb

%c

4.03 + 0.09 9.1 + 0.5 2.61+0.14 11.6+0.9

Cell numberd 31.7 + 1.4 34.2+1.3

aCollagenous protein (cpm) / cell (x 10). bNon-collagenous protein (cpm)/cell (x 10). CRate of collagenous protein synthesis to total protein synthesis, see Materials and Methods. dCell number (x 10 -4) Each value shows mean + S. E. M. of duplicate determinations.

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2 3 4

DTT PDGF

--

-t--

-F

+

--

+

Fig. 1. Fluorograms of [3H]proline-labeled collagen chains synthesized by smooth muscle cells cultured with platelet-derived growth factor (PDGF) (lanes 2, 4) or with no PDGF (lanes 1,3) for 24 h. Samples were electrophoresed under a non-reduced condition (lanes 1,2) or a reduced condition (lanes 3,4). The band indicated by the arrow has migrated to a site (upper arrow head) above the al(V) band and to a site (lower arrow head) below the o.2(1) band after reduction. DTT, dithiothreitol.

1 2

FrontDTT

--

-F

Fig. 2. Immunoblotting for type IV collagen. Nitrocellulose membranes to which the collagens (lane 1, non-reduced; lane 2, reduced) synthesized by cultured smooth muscle cells were transferred. The bands indicated by the arrow and the arrow heads correspond to those indicated by the arrow and the arrow heads in Fig. 1. D'Iq', dithiothreitol.

In an immunofluorescence study using anti-type IV collagen antibody, fluorescence was observed around the cells showing a lacework or network-like pattem as well as in the cytoplasm showing a granular pattern (Fig. 3A). Although fluorescent images of the cells cultured with PDGF were similar to those with no PDGF, the intensity of the fluorescence of the cells, particularly that around the cells, cultured with PDGF was evidently decreased (Fig. 3B). Densitometric analysis of the bands corresponding to the collagen types showed that the smooth muscle cells cultured with PDGF decreased type IV collagen synthesis approximately to 3.9% of the total collagen, while the cells cultured with no PDGF synthesized type IV collagen approximately at 5.9% of the total collagen (Table 2). Thus, in estimation of type IV collagen synthesis, there was significant difference between 7.22 + 0.94 in PDGF and 12.92 + 1.00 in non-PDGF. On the other hand, the rate of type V collagen synthesis in the cells cultured with PDGF was relatively increased compared with that in the cells cultured with no PDGF (Table 2).

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3[3 Fig. 3. Indirect immunofluorescent study using anti-type IV collagen antibody on cultured smooth muscle cells cultured with no PDGF (A) or 5 units/ml of PDGF (B). A. Distinct fluorescence showing a lacework pattern is observed in the cell cultured with no PDGF implicating well developed basement membrane around the cell surface. Focal dense deposits of type IV collagen also can be seen (x 215). B. Intensity of the fluorescence is weakened and lacework pattern is obscure (x 215). Table 2. Type analysis of the collagen synthesized in arterial smooth muscle cells cultured with or without PDGF Collagen type (%) I

No PDGF PDGF

79.0 + 0.5 76.0 + 0.3

III

IV

V

12.9 + 0.9 14.1 + 1.2

5.9 + 0.3 3.9 + 0.1

2.2 + 0.2 6.0 + 1.6

Each value shows mean + S.E.M. of duplicate determinations. Discussion

The present study showed that cultured smooth muscle cells synthesize types I, III, IV and V collagen with or without PDGF supplement. In biochemical and immunofluorescent analyses, however, type IV collagen was significantly reduced by the addition of 5 units of PDGF. Since type IV collagen is one of the major component of the basement membrane of various cells (Glanville, 1987) including arterial smooth muscle cells (Heickendorff, 1988), decreased synthesis of type IV collagen would presumably lead to diminished formation of the basement membrane. According to Campbell et al (Chamley-Cmpbell et al., 1979; Campbell et al., 1987), smooth muscle cells take two different phenotypes, one of which is a contractile phenotype and another is a synthetic phenotype. The cytoplasm of the former is

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predominated with myofilaments, and that of the latter is filled with rough endoplasmic reticulum and free ribosomes and contains sparse myofilaments. We have already shown in vitro that contractile smooth muscle cells are enveloped with basement membrane ultrastructurally while synthetic cells have fragmented basement membrane and synthesize less type IV collagen (Katsuda et al., 1987). Thus, the activity of type IV collagen synthesis seems to give a strong influence on smooth muscle cell phenotype. Thyberg et al (1983) reported that PDGF enhanced phenotypic modulation of smooth muscle cells from a contractile to a synthetic state morphologically. Although the mechanism of this phenotypic modulation remains obscure, the present results seem to suggest that the decreased synthesis of type IV collagen is well correlated with the phenotypic changes of smooth muscle cells to the synthetic state modulated by PDGF. PDGF is generally known to be an enhancing factor for collagen synthesis in smooth muscle cells (Stavenow et al., 1984). This appears to be inconsistent with our result that showed slight decrease in collagen synthesis of smooth muscle cells supplement of PDGF. In the previous work by Stavenow et a1.(1984), however, supernatant of cultured platelets was used as a substitute for purified PDGF, which was employed in the present study. Thus, we must be careful to compare the previous result with ours, since it is possible that the crude platelet supernatant could be contaminated with transforming growth factor (TGF) 13(Assoian and Sporn, 1986; Ignotz and Massague, 1986) and/or unknown factor(s) affecting the collagen metabolism. At least, relative synthesis of collagenous proteins per total proteins rather increased with addition of PDGF. It was noted that synthesis of type V collagen was enhanced slightly in the smooth muscle cells stimulated with PDGF compared to that in the cells cultured with no PDGF. Such an increased synthesis of type V collagen was also reported in human gingival fibroblasts treated with PDGF by Narayanan and Page (1983). Since type V collagen is known to increase not only in atherosclerotic lesions (Murata et al., 1986; Ooshima, 1981), but also in such fibrosing lesions as scar tissues (Ehrlich and White, 1981) and scirrhous carcinoma (Minamoto et al., 1988), type V collagen secreted by smooth muscle cells or fibroblasts may play a role in the progression of tissue remodeling. Then, PDGF would be implicated in deposition of extracellular matrix components including type V collagen. Another major function of PDGF is cell proliferation activity and it is known as one of the major growth factors to play an important role on the development of atherosclerosis (Ross, 1986) as well as a promoter of collagenolytic enzymes (Yanagi et al., 1992). Therefore, PDGF acts not only on cell growth,, cell migration of medial smooth muscle cells but also modulates the collagen metabolism in association with phenotypic changes of smooth muscle cells.

References Assoian, R. K. and Sporn, M. B. (1986). Type [3 transforming growth factor in human platelets: Release during platelet degranulation and action on vascular smooth muscle cells. J. Cell Biol. 102: 1217-1223.

Campbell, J. H., Campbell, G. R., Kocher, O. and Gabbiani, G. (1987). Cell biology of smooth muscle in culture: Implications for atherogenesis. Int. Angiol. 6: 73-79. Chamberlain, J. P. (1979). Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal. Biochem. 98: 132-135. Chamley-Campbell, J., Campbell, G. R. and Ross, R. (1979). The smooth muscle cell in culture. Physiol. Rev. 59: 1-61.

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Ehrlich, H.P. and White, B.S. (1981). The identification of otA and orB collagen chains in hypertrophic scar. Exp. Mol. Pathol. 34: 1-8. Glanville, R. W. (1987). Type IV collagen, in: Mayne, R. and Burgeson, R.E. (eds.). Structure and function of collagen types. Orlando: Academic Press. Hata, R., Ninomiya, Y., Nagai, Y. and Tsukada, Y. (1980). Biosynthesis of interstitial types of collagen by albumin-producing rat liver parenchymal cell (hepatocyte) clones in culture. Biochemistry 16: 169-176. Heickendorff, L. (1988). Laminin, fihronectin and type IV collagen in BM-like ma[erial from cultured arterial smooth muscle cells. Int. J. Biochem. 20: 381386. Ignotz, R. A. and Massague, J. (1986). Transforming growth factor-~ stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J. Biol. Chem. 261: 4337-4345. K a t s u d a , S., Okada, Y., Minamoto, T. and Nakanishi, I. (1987). Enhanced synthesis of type IV collagen in cultured arterial smooth muscle cells associated with phenotypic modulation by dimethyl sulfoxide. Cell Biol. Int. Rep. 11: 861-870. Katsuda, S., Okada, Y., Nakanishi, I., and Tanaka, J. (1988). Inhibitory effect of dimethyl sulfoxide on the proliferation of cultured smooth muscle cells: Relationship to the cytoplasmic microtubules. Exp. Mol. Pathol. 48: 48-59.

Minamoto, T., Ooi, A., Okada, Y., Mai, M., Nagai, Y. and Nakanishi, I. (1988). Desmoplastic reaction of gastric carcinoma. A light- and electronmicroscopic immunohistochemical analysis by using collagen type-specific antibodies. Hum. Pathol. 19" 815-821. M u r a t a , K., Motayama, T. and Kotake, C. (1986). Collagen types in various layers of the human aorta and their changes with the atherosclerotic process. Atherosclerosis 60:251-262. Narayanan, A. S. and Page, R. C. (1983). Biosynthesis and regulation of type V collagen in diploid human fibroblasts. J. Biol. Chem. 258:1169411699.

Okada, Y., Katsuda, S., Matsui, Y., Watanabe, H. and Nakanishi, I. (1990).- Collagen synthesis by cultured arterial smooth muscle cells during spontaneous phenotypic modulation. Acta Pathol. Jpn. 40: 157-164. Peterkofsky, B., Chojkier, M. and Bateman, J. (1982). Determination of collagen synthesis in tissue and cell culture systems, In: Furthmayr, H. (ed.). Immunocytochemistry of the extracellular matrix. Vol. 2. Boca Raton: CRC Press. Ross, R., Raines, E. W. and Bowen-Pope, D. F. (1986). The biology of platelet-derived growth factor. Cell 46: 155-169. Ross, R. (1986). Pathogenesis of atherosclerosis: An update. N. Engl. J. Med. 314: 488-500. Ross, R. (1971). The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J. Cell Biol. 50: 172-186. Stavenow, L., Kjelistr~Jm, T. and Malmquist, J. (1981). Stimulation of collagen production in growth-arrested myocytes and fibroblasts in culture by growth factor(s) from platelets. Exp. Cell Res. 136:321-325.

Thyherg, J., Palmberg, L., Nilsson, J., Ksiazek, T. and SjiJlund, M. (1983). Phenotype modulation in primary cultures of arterial smooth muscle cells. On the role of platelet-derived growth factor. Differentiation 25: 156-167. Weber, K. and Osborn, M. (1975). Proteins and sodium dodecyl sulfate: Molecular weight determination on polyacrylamide gels and related procedures,

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in: Neurath, H., Hill, R. L., and Boeder, C. L. (eds.). The proteins. New York: Academic Press.

Yanagi, H., Sasaguri, Y., Sugama, K., Morimatsu, M. and Nagase, H. (1992). Production of tissue collagenase (matrix metalloproteinase 1) by human aortic smooth muscle cells in response to platelet-derived growth factor. Atherosclerosis, 91:207-216. Paper received 22.02.92.

Revised paper accepted 14.08.92.