EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
52, 179-191 (1990)
Effects of Collagen Matrix on Proliferation and Differentiation of Vascular Smooth Muscle Cells in Vitro NORIYUKI SAKATA, KOHJI KAWAMURA,AND Department
of
Pathology, School
of
SHIGEO TAKEBAYASHI
Medicine, Fukuoka University, Fukuoka, Japan
Received August 29, 1989, and in revised
form
December 18, 1989
In an attempt to better define the relationship between collagen matrices and vascular smooth muscle cells in vitro, proliferation of smooth muscle cells was observed in the early stages of culture. Cells spread on collagen gels had a longer doubling time and less incorporation of r3H]thymidine into DNA on the fust day of culture than did cells grown on a plastic substrate. Cells on collagen gels were more elongated than were those on the plastic substrate and showed a “hills and valleys” arrangement from the first day in culture on the collagen type III gel. All cells were identified as smooth muscle having definite microfilaments, dense bodies, and pinocytotic vesicles. They had a distinct actin filament running from end to end when labeled with nitrobenzoxadiazole-phallacidin. Cells on the collagen gels had a larger number of actin filaments traveling parallel to the direction of the major axis of their cytoplasm than did those on the glass substrate. Therefore, cultured smooth muscle cells in the more physiological environment for cells in vitro, i.e., on collagen gels, show a suppression of cellular proliferation and an enhancement of differentiation in the early stages of culture. The effects of collagens on the differentiation of cells vary with the collagen phenotype. 8 I’BO Academic Press. Inc.
INTRODUCTION Vascular smooth muscle cells seem to interact with extracellular matrices such as various phenotypes of collagens, glycosaminoglycans, and glycoproteins (Gospodarowicz et al., 1980; Schor et al., 1983; Herman, 1987). While collagen constituents are the major components of extracellular substances in arteries, the precise collagen-cell interaction remains obscure. Type I and III collagens are present in the arterial wall, the former being predominant in atherosclerotic lesions and the latter in the media (McCullagh et al., 1979). On the other hand, in the early stages of intimal thickening, type III collagen predominates and is the most important of the collagen species in atherogenesis (Gay and Balleisen, 1977). According to current studies, type I collagen is the predominant species in both atherosclerotic lesions and media and there is little change of the type III/type I ratio between them. Moreover, type V collagen is increased in the atherosclerotic plaques, compared to findings in the media and adventitia (Ooshima, 1981; Morton and Barnes, 1982). We examined the influence of type I and III collagens on the proliferation and differentiation of vascular smooth muscle cells, in vitro, and our findings are reported herein. MATERIALS
AND METHODS
1. Cell Culture Cultured smooth muscle cells were derived from explants isolated from the abdominal aortic media of a 6-month-old pig, under aseptic conditions. All cells were cultured at 37°C in a humidified, 5% CO, and 95% air atmosphere. Cultivation was done in Dulbecco’s modified Eagle’s medium (DME, GIBCO) containing 179 0014-4800/90 $3.00 Copyri& 0 1990 by Academic Press, Inc. AU ligbfs of reproduction in any form reserved.
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10% fetal calf serum and antibiotics, 100 k/ml penicillin (GIBCO) and 100 t&ml streptomycin (GIBCO), adjusted to pH 7.4. The cells were dispersed using 0.05% trypsina.53 m&f EDTA (GIBCO) and were subcultured into 75cm* tissue culture flasks (Falcon). Cultured cells between the third and seventh population doubling were used for the experiments. 2. Preparation
of Collagen
Gels
Type I and III collagens (Koken Co., Japan) were prepared from calf dermis and placenta, respectively. Briefly, tissues were digested by 0.5 M acetic acid containing pepsin. Collagens were extracted with incubation for 24 hr at 4°C. The isolation of type I and III collagens was made by the differential salt precipitation method described by Ooshima (1981). Purity of the type I and III collagens was checked by 5% SDS-PAGE according to Laemmli (1970). The samples for electrophoresis were redissolved in the sample buffer, which contained the final concentrations 62.5 mM Tris-HCL buffer (pH 6.8), 10% glycerol, 2% SDS, and 5% 2-mercaptoethanol. The proteins were dissociated by immersing the samples for 3 min in boiling water. Electrophoresis was performed with a constant current of 25 mA using a 3% stacking gel (pH 6.8) and a 5% separation gel (PH 8.9). After staining by Coomassie brilliant blue, the density of the band was determined by densitometry using a Dual-Wavelength TLC Scanner (CS-900, Shimadzu Co., Japan) and Chromatopack C-RIB (Shimadzu Co.). The SDS-PAGE pattern of collagen preparation is presented in Fig. 1. Several protein bands corresponded mainly to CX-,B-, and y-chains. The purity of collagen preparations exceeded 83% with densitometry. Type I and III collagens were prepared in 0.2% solutions by rapidly mixing 2 vol of 0.3% pepsin solubilized type I and III collagen solutions (Koken Co.) with 1 vol of 3x concentrated DME-12.5 mM Hepes (PH 7.4), on ice; 0.5 ml of 0.2% collagen solution was added to each dish (35 mm, Falcon); and the preparation was incubated at 37°C for 2 hr for gelation of the collagen. The polymerized collagen gels were then equilibrated overnight with complete media. 3. Growth
Curve
Cell monolayers were prepared by plating 2-ml aliquots of a cell suspension of smooth muscle cells (5 x IO4 cells/ml) onto plastic (plastic group) and type I (type I group) and type III (type III group) collagen gel substrata. The cultured cells were maintained in DME-12.5 mM Hepes (pH 7.4) containing 10% FCS and antibiotics for 9 days and the medium was changed every third day. After 1, 3, 5, 7, and 9 days in culture, the cells were observed under a phase contrast microscope (Nikon, TMD, Japan) and then washed twice with phosphate-buffered saline (PBS). Cells grown on plastic substrata were trypsinized with 0.05% trypsin0.53 mM EDTA at 37°C for 15 min. Cells grown on collagen gels were removed from dishes by incubation with 0.2% collagenase (Worthington Biochemical Corp. )-I mM CaCl, at 37°C for 20 min. The suspension was then pipetted to disperse the cells and a counting chamber was used for enumeration. After 24 hr of cultivation, cells on the plastic, glass, and collagen type I and type III substrates were detached and counted by the above-mentioned methods, to estimate plating efficiency. of DNA Synthesis Cultured smooth muscle cells were incubated in the media containing
4. Assay
2 t~Ci/ml
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OF COLLAGEN
ON
DIFFERENTIATION
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FIG. 1. SDS-PAGE (5%) patterns of type I and III collagen solutions. The gel was stained with Coomassie brilliant blue R-250. Molecular weight standards (lane 3) include a,-macroglobulin (170 kDa) and phosphorylase b (97 kDa). Both type I (lane 2) and III (lane 1) collagen solutions consisted mainly of protein bands that corresponded to a-chain, P-chain, and y-chain. F, front. T, top.
[3H]thymidine at 37°C for 2 hr on the first, third, and fifth days of culture. The media were removed and the cell sheet was rinsed three times with cold PBS containing 50 p,M thymidine (Wako Chemical Co., Japan). Cultured smooth muscle cells were harvested by incubation with 0.05% trypsin-0.53 mM EDTA-50 @4 thymidine in PBS in the case of uncoated plastic dishes or 0.2% collagenase-1 m&f CaClz50 l.~A4thymidine in PBS in the case of the collagen-coated dishes. A cell suspension was pipetted and a sample was taken for cell counting. Cell pellets, obtained by centrifugation at 1000 rpm for 10 min, were suspended in 1 ml of distilled water and then disrupted for 20 set at 0°C with a sonicator (Model W-10) tuned to 5, at the maximum power. One milliliter of 10% TCA was added to the sonicated cell suspension, and a TCA-precipitate was collected by centrifugation at 3000 t-pm for 15 min and then washed three times with 2 ml of cold 5% TCA. Precipitates were suspended in 1 ml of cold 10% TCA, incubated at 95°C for 15 min, and then placed on ice for 30 min. After centrifugation at 3000 rpm for 15 min, 0.1 ml of the hot TCA-soluble fraction was added to 6 ml of a scintillator (ACS II, Amersham, Canada). The amount of the radioactivity was determined with a liquid scintillation counter. A value was expressed as dpm per lo4 cells during 1 hr. 5. Electron Microscopic
Observation
After 1 and 3 days in culture,
the smooth muscle cells were fixed in 1.4%
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glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4) for 2 hr at 4”C, rinsed three times in the same buffer at 4°C and postfixed in 1% osmium tetroxide in the same buffer at 4°C for 2 hr. They were then dehydrated in a graded series of ethanols and embedded in Epon 812. Ultrathin sections were double-stained with uranyl acetate and lead citrate. All sections were observed under a JEM-1OOCX electron microscope. 6. Observation
of Actin Filaments
To visualize actin filaments, we used fluorophore nitrobenzoxadiazole (NBDb phallacidin (Molecular Probes), a specific probe for F-actin (Barak et al., 1980). A collagen-coated or uncoated coverslip (18 x 18 mm) was put in each dish (35 mm). Cells were inoculated onto the glass or collagen-coated coverslip in a dish at an initial plating density of 2 X 104/dish and cultivated with 2 ml of DME-12.5 mM Hepes containing 10% FCS and antibiotics. After 3,6, 12, and 24 hr in culture, the cultured cells were fixed in 4% paraformaldehyde in PBS at room temperature for 10 min. After rinsing twice with PBS at 4°C they were extracted with acetone at - 20°C for 5 min and then air-dried. Cells were incubated with NBD-phallacidin (1.6 x lop6 M) for 45 min at room temperature in a dark moist chamber, followed by washing twice in PBS for 3 min each time. Another coverslip was pretreated with a 50-fold excess of unlabeled phallacidin and then stained with NBDphallacidin, under identical conditions. The latter coverslip was considered to be a negative control. Cells were mounted in glycerol and viewed through a fluorescence microscope (Olympus, Japan). The frequency of cells with the distinct actin filaments in the cytoplasm was compared among the glass and collagen I and III groups. 7. Statistical
Analysis
Data were expressed as the means + SD. A statistical analysis was carried out by unpaired Student’s t test and a value of P < 0.05 was considered to be statistically significant. RESULTS 1. Phase Contrast Microscopy Figure 2 shows the phase contrast microscopic views of cultured smooth muscle cells on the first and ninth days of culture. Cells on the collagen gels were more elongated than those on plastic subtrata, and in particular the bipolar elongation was considerable on the type III collagen gel (Fig. 2F). Cells on the type III collagen gel aligned in the “hills and valleys” arrangement on the first day of culture, a characteristic of the cultured smooth muscle cell (Fig. 2C), and maintained this arrangement during the culture (Fig. 2F). Cells on type I collagen gel showed a similar structure on the first and third days of culture, but not as clearly as that on the type III collagen gel (Fig. 2B). In contrast, cells on plastic substrata did not show the hills and valleys alignment on the first and third days of culture (Fig. 2A). After 7 days, all the cells had the hills and valleys arrangement (Figs. 2D-2F). 2. Electron Microscopic Electron
microscopic
Observation observations
of the smooth muscle cells cultured
for 1
EFFECTS
OF
COLLAGEN
ON
DIFFERENTIATION
183
FIG. 2. Phase contrast micrographs of cultured smooth muscle cells. (A-C) On the fust day. (D-F) On the ninth day. (A,D) Plastic group. (B,E) Type I group. (C,F) Type III group. x24.
and 3 days are shown in Fig. 3. Smooth muscle cells on both collagen gel and plastic substrata had a rough endoplasmic reticulum (rER), a Golgi apparatus, and polysomes, in addition to discrete microtilament bundles, dense patches, and pinocytotic vesicles beneath the adjacent plasma membrane. The cells were connected through junctional formations. Smooth muscle cells on collagen gels were more hypertrophic and enriched with rER, polysomes, and glycogens than were those grown on the plastic substratum (Fig. 3). In addition, numerous vacuoles containing residual body-like substances were observed within the cytoplasm of cells cultured on the collagen gels (Fig. 3). There was no definitive difference in the development of microfilament bundles between cells on the collagen gels and plastic substrata on first and third days of culture. 3. Cell Growth To test for the effect of collagen matrices on smooth muscle cell proliferation, cell number and incorporation of [3H]thymidine of each group were determined
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FIG. 3. Smooth muscle cells grown on collagen type I gel for 1 day. Cells show a hypertrophic cytoplasm rich in endoplasmic reticulum, polysomes, and lysosome-like vacuoles (L). Microfilament bundles (MF) are also present beneath the plasma membrane. X 11,120.
during culture. Figure 4 shows the growth curve of each group. The cell number of the plastic group was much greater than those of type I and III groups during the culture period (*P < 0.001, **P < 0.02 vs type III on the first day). However, the difference between plastic and collagen groups diminished during the process of the culture. There was no difference in cell number between type I and III groups, except for the first day of culture. The plating efficiencies were affected by the substrata; that is, the mean values of relative plating efficiencies of the glass and collagens type I and type III to the plastic substrate were 85.2,69.7 and 83.8%, respectively. The population doubling time of smooth muscle cells of the plastic group was shorter (32.6 hr) from the first to the third day of culture than those of the type I (56.7 hr) and III (64.1 hr) groups, which was longer (80.2 hr)
0:
plastic
A: collagen
I
O:collagen
III
FIG. 4. Effect of collagen gels on growth of cultured smooth muscle cells. There was a statistically significant difference in counts of cells grown on plastic and type I or III groups during the experiment. The data are mean values + SD of two separate experiments done in triplicate. *P < 0.001, **P < 0.02, plastic group vs type III group.
EFFECTS OF COLLAGEN
185
ON DIFFERENTIATION
from the third to the fifth day of culture than those of types I (45.2 hr) and III (49.8 hr) (Fig. 5). In contrast, the doubling time of cells grown on collagen type I and III gels was shorter from the third to the fifth day rather than from the first to the third day of culture. The incorporation of [3H]thymidine into DNA was compared between the cells on the plastic and the collagen gel-coated dishes (Fig. 6). DNA synthetic activity of cells on both type I and III collagen gels was remarkably suppressed on the first day of culture, compared to that on the plastics (Fig. 6). Namely, the mean values of [3H]thymidine incorporation of cells on plastic and collagen type I and III substrata were 6.27 x 103, 1.80 x 103, and 1.68 x lo3 dpm/104 cells, respectively (*P < 0.001, plastic vs collagen type I or III). However, on the third and fifth days of culture, the mean values of [3H]thymidine incorporation of cells on plastic decreased to 0.72 x lo3 and 1.36 x lo3 dpm/104 cells, respectively. In contrast, uptake of [3H]thymidine of cells grown on collagen type I and III gels increased on the third and fifth days of culture. Hence, cells grown on collagen gels incorporated significantly more [3H]thymidine into DNA on the third and fifth days than did cells grown on plastic (**P < 0.001, ***P < 0.002, ****p < 0.02). 4. Time Course of Formation Collagen Substrata
of Actin Filament
on Glass and
Table I shows the frequency distribution of cells grown on each substrate and the developing actin filaments. Many cells in all groups had few bundles of actin filaments after 3 hr. Cells in all groups began to form actin filaments after 6 hr, and the mean value of frequency of cells with actin filaments was approximately 18 to 36%. More than 50% of cells had filaments after 12 hr, and cells on collagen type III had significantly less filaments than did the other groups (*P < 0.05). The majority of cells in all groups showed formation of actin filaments after 24 hr, but the mean value of frequency of cells forming actin filaments was significantly higher in the glass group than in the type I and III groups (**P < 0.01). 5. Actin Filament
Morphology
In the smooth muscle cells there was no formation of actin filaments during the first 3 hr of plating on any substrate. The well-spread cells cultured on glass $$J:plastic z:collagen n:collagen
l-3
I III
3-5
Days in culture FIG. 5. Doubling times of smooth muscle cells were affected over the time course of the culture. The data were expressed as the mean values of two experiments done in triplicate.
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SAKATA,
KAWAMUBA,
AND
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•j : plastic q : collagen1 0
: collagen
1
III
5
Days &Mm? FIG. 6. Effects of collagen gels on DNA synthesis of smooth muscle cells were significantly varied in the time course of the culture. The mean value of incorporation of [‘Hlthymidine in smooth muscle cells of the plastic group was significantly higher than those of collagen type I and III groups on the first day of culture (*P < O.OOl), while the former was significantly lower than the latter on the third and fifth days of culture (**P i 0.001, ***P < 0.002, ****P < 0.02).
coverslips for 12 hr had a continuous staining of linear structures which ran in various directions within the cytoplasm (Fig. 7). Smooth muscle cells pretreated with 50x excess of unlabeled phalloidin and then stained with NBD-phallacidin stained weakly. The elongated cells grown on collagen substrata for 12 hr were mostly in straight bundles parallel to the major axis of cells and ran from end to end (Fig. 8). The very elongated cells with processes containing actin filaments between other cells were more frequently found on the collagen substrates, compared to those on the glass (Fig. 9). DISCUSSION In the present study, cultured smooth muscle cells derived from explants of abdominal aortic media were used. Enzyme-dispersed contractile smooth muscle cells undergo a spontaneous change in phenotype to the synthetic state after 6 to Frequency Distribution Duration W 3 6 12 24
TABLE I of Smooth Muscle Cells Developing Distinct Actin Filaments during 24 hf’
Grade + ++ + ++ + ++ + ++
Glass 96.0 + 2.4 4.0 ? 2.4 Ok0 64.2 +- 14 32.2 2 10 3.6 k 4.2 32.5 i 6.1 62.2 k 7.0 5.3 2 4.1 14.2 -+ 4.6** 74.0 t 7.4 11.8 + 6.2
Collagen I
Collagen III
92.8 + 7.2 f olro 74.7 + 23.0 + 2.3 f 38.3 f 57.0 2 4.7 + 33.8 f 64.0 2 2.2 2
98.8 + 1.2 2 020 82.2 _’ 17.0 ” 0.8 + 49.5 k 48.2 f 2.3 + 26.0 f 72.5 f 1.5 f
4.6 4.6 8.5 7.3 2.6 5.2 7.4 2.8 9.0 8.7 1.6
1.2 1.2 5.2 4.4 1.3 5.6* 6.9 2.1 5.8 5.0 1.6
’ Data (%) are mean values f SD. n = 6. *P < 0.05 vs glass and collagen type I. **P < 0.01 vs collagen types I and III.
EFFECTS
OF
COLLAGEN
ON
DIFFERENTIATION
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FIG. 7. Smooth muscle cells grown on a coverslip for 12 hr. Note bundles of actin filaments traveling from end to end. FIG. 8. Smooth muscle cells grown on collagen type I gel for 12 hr. Note bundles of actin filaments running parallel to the direction of the major axis of the elongated cytoplasm.
8 days in primary culture. Culture conditions and number of cumulative population doublings required to reach confluency relate to whether they spontaneously return to the contractile state (Charnley-Campbell and Campbell, 1981; Campbell and Campbell, 1985). On the other hand, smooth muscle cells can divide without loss of smooth muscle myosin, which is an important marker protein of differentiated contractile smooth muscle cells. Cells which lose smooth muscle myosin may regain it in the postconfluent state (Hammerle ef al., 1988). We have recently shown that smooth muscle cells derived from explants of coronary arterial media have a significantly higher frequency of desmin positivity in the conlluent state than in the logarithmic growth state, desmin being an important protein of intermediate filaments of muscle cells (unpublished data). Moreover, it has been shown that type I collagen increased the number of cytoskeletal filaments and suppressed DNA synthetic activity in smooth muscle cells derived from explants of the thoracic aortas of rabbits (Yoshida et al., 1988). Subcultured smooth muscle cells derived from explant outgrowth are also considered to be able to undergo phenotypic modulation, to a certain degree. Intercellular matrices, including collagens, play an important role in the control of cell proliferation, in vivo and in vitro. There are, however, conflicting reports concerning the effect of collagen substrata, one being that they promote cell growth (Ehrmann and Gey, 1956), the other that they suppress it (Yoshizato et al., 1985). In our study, porcine smooth muscle cells were grown on plastic and
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FIG. 9. Smooth muscle cells grown on collagen type I gel for 12 hr. Cytoplasmic processes containing actin filaments are present between the adjacent cells.
collagen type I and III substrata. Growth rates, estimated by doubling time, and incorporation of [3H]thymidine into DNA of cells were suppressed on the collagen gels on the first day of culture, compared with the cells grown on the plastic substrate. Cells on the collagen gels had a shorter doubling time and a greater incorporation of [3H]thymidine between the third and the fifth days of culture than did cells grown on the plastic substrate. Moreover, differences in cell number between plastic and collagen type I or III groups decreased with the continuation of cultivation. These results suggest that the growth rate of the smooth muscle cells is suppressed in the early stage of culture, followed by a gradual proliferation when the cells are cultivated under conditions which more closely resemble the in vivo environment, e.g., collagen gel substrate. Herman and Castellot (1987) reported that collagen matrices did not influence the doubling time and saturation density of a cultured smooth muscle cell. The saturation density of cultured smooth muscle cells is considered to be unaffected by collagen substrata, because differences in cell number between plastic and collagen groups decreased during the culture period. There is, however, a discrepancy between our data and those of Herman and Castellot (1987) with regard to the doubling time. In their study, the doubling time was examined only between the second and the fourth days of culture, while we observed it from the first to the third days and also from the third to the fifth days of culture. Thus, we obtained good evidence that the effect of collagen matrices on the doubling time of cells varies with the period of culture.
EFFECTS
OF COLLAGEN
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DIFFERENTIATION
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This result is consistent with the finding concerning the incorporation of [3H]thymidine into DNA. Smooth muscle cells cultivated on the type III collagen gel aligned in the hills and valleys arrangement on the first day of culture and this pattern was preserved throughout the culture period. In contrast, cells on the plastic substrate did not show the hills and valleys alignment until after 5 days of culture. It is known that collagen matrices influence the function and differentiation of cells (Lark and Wight, 1986; Gibson et al., 1984). However, there are few reports that the hills and valleys alignment, a characteristic of cultured smooth muscle cells, occurred on the first day of culture on the collagen type III gel. Our observations suggest that collagen matrices, especially type III collagen, may play an important role in the expression of differentiation of cultured smooth muscle cells. Moreover this finding is in accord with the fact that type III collagen exists predominantly in the media of an artery (McCullagh er al., 1979). The actin filament is the most important part of the cytoskeleton. Stress fibers that are prominent components of the cytoskeleton in cultured cells are closely linked to the maintenance of cell shape and adhesiveness to the substrates. Farsi and Aubin (1984) noted prominent bundles of microfilaments in very elongated fibroblasts cultured within collagen gels. We found that smooth muscle cells grown on collagen gels were more elongated and had exclusively actin filaments traveling in parallel to the major axis of their cytoplasm, some of which had cell processes containing the actin filaments between adjacent smooth muscle cells. These observations are interpreted to mean that the mode of actin filaments is related to the shape and arrangement of smooth muscle cells grown on the collagen gels. Smooth muscle cells, when grown on a glass substrate, began to form actin filaments after 6 hr, and the majority of the cells had adequate actin filaments after 24 hr. In contrast, the formation of actin filaments was delayed in the smooth muscle cells grown on collagen gels. In other studies (Willingham et al., 1977; Singhal and Hays, 1988), the rate of formation of actin filaments was shown to relate to the adhesiveness to the substrata. Since cell numbers on the first day of culture varied in the various substrata including the plastic, glass, and collagen types I and III, there is the possibility that the development of actin filaments is related to the adherence of cells and quality of the substrate. However, the collagen substrate has been also reported to promote the attachment of smooth muscle cells (Bjorkerud, 1985). The relation between the adhesive properties of collagen matrices and the development of actin filaments remains to be elucidated. Ultrastructurally, the development of microfilaments of cells grown on collagen gels was similar to that of cells on the plastic substrate after 1 and 3 days of culture. On the other hand, the formation of F-actin was more delayed in the former, as determined using NBD-phallacidin staining which is specific for Factin. Microfilaments contain other proteins including myosin, tropomyosin, and a-actinin (De Robertis and De Robertis, 1987). Therefore, these findings suggest that the collagen substrata may not affect the quantity of microfilament bundles but do change the quality of components such as the content of actin. The present study demonstrated that the rate of proliferation, the shape and arrangement, and the development of actin filaments of smooth muscle cells
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grown on collagen gels differed from those observed on a plastic substrate. In addition, in cultured smooth muscle cells, the suppression of proliferation and expression of differentiation in the early stages of culture on the collagen matrix with a more physiological environment than plastic substrate, and the effects of collagen matrix on the cell differentiation, might vary with the species of collagen. ACKNOWLEDGMENTS We thank M. Ohara for comments and T. Sato for technical assistance. Part of this work was supported by funds from the Central Research Institute of Fukuoka University.
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MOLECULAR
PATHOLOGY
52, 179-191 (1990)
Effects of Collagen Matrix on Proliferation and Differentiation of Vascular Smooth Muscle Cells in Vitro NORIYUKI SAKATA, KOHJI KAWAMURA,AND Department
of
Pathology, School
of
SHIGEO TAKEBAYASHI
Medicine, Fukuoka University, Fukuoka, Japan
Received August 29, 1989, and in revised
form
December 18, 1989
In an attempt to better define the relationship between collagen matrices and vascular smooth muscle cells in vitro, proliferation of smooth muscle cells was observed in the early stages of culture. Cells spread on collagen gels had a longer doubling time and less incorporation of r3H]thymidine into DNA on the fust day of culture than did cells grown on a plastic substrate. Cells on collagen gels were more elongated than were those on the plastic substrate and showed a “hills and valleys” arrangement from the first day in culture on the collagen type III gel. All cells were identified as smooth muscle having definite microfilaments, dense bodies, and pinocytotic vesicles. They had a distinct actin filament running from end to end when labeled with nitrobenzoxadiazole-phallacidin. Cells on the collagen gels had a larger number of actin filaments traveling parallel to the direction of the major axis of their cytoplasm than did those on the glass substrate. Therefore, cultured smooth muscle cells in the more physiological environment for cells in vitro, i.e., on collagen gels, show a suppression of cellular proliferation and an enhancement of differentiation in the early stages of culture. The effects of collagens on the differentiation of cells vary with the collagen phenotype. 8 I’BO Academic Press. Inc.
INTRODUCTION Vascular smooth muscle cells seem to interact with extracellular matrices such as various phenotypes of collagens, glycosaminoglycans, and glycoproteins (Gospodarowicz et al., 1980; Schor et al., 1983; Herman, 1987). While collagen constituents are the major components of extracellular substances in arteries, the precise collagen-cell interaction remains obscure. Type I and III collagens are present in the arterial wall, the former being predominant in atherosclerotic lesions and the latter in the media (McCullagh et al., 1979). On the other hand, in the early stages of intimal thickening, type III collagen predominates and is the most important of the collagen species in atherogenesis (Gay and Balleisen, 1977). According to current studies, type I collagen is the predominant species in both atherosclerotic lesions and media and there is little change of the type III/type I ratio between them. Moreover, type V collagen is increased in the atherosclerotic plaques, compared to findings in the media and adventitia (Ooshima, 1981; Morton and Barnes, 1982). We examined the influence of type I and III collagens on the proliferation and differentiation of vascular smooth muscle cells, in vitro, and our findings are reported herein. MATERIALS
AND METHODS
1. Cell Culture Cultured smooth muscle cells were derived from explants isolated from the abdominal aortic media of a 6-month-old pig, under aseptic conditions. All cells were cultured at 37°C in a humidified, 5% CO, and 95% air atmosphere. Cultivation was done in Dulbecco’s modified Eagle’s medium (DME, GIBCO) containing 179 0014-4800/90 $3.00 Copyri& 0 1990 by Academic Press, Inc. AU ligbfs of reproduction in any form reserved.
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10% fetal calf serum and antibiotics, 100 k/ml penicillin (GIBCO) and 100 t&ml streptomycin (GIBCO), adjusted to pH 7.4. The cells were dispersed using 0.05% trypsina.53 m&f EDTA (GIBCO) and were subcultured into 75cm* tissue culture flasks (Falcon). Cultured cells between the third and seventh population doubling were used for the experiments. 2. Preparation
of Collagen
Gels
Type I and III collagens (Koken Co., Japan) were prepared from calf dermis and placenta, respectively. Briefly, tissues were digested by 0.5 M acetic acid containing pepsin. Collagens were extracted with incubation for 24 hr at 4°C. The isolation of type I and III collagens was made by the differential salt precipitation method described by Ooshima (1981). Purity of the type I and III collagens was checked by 5% SDS-PAGE according to Laemmli (1970). The samples for electrophoresis were redissolved in the sample buffer, which contained the final concentrations 62.5 mM Tris-HCL buffer (pH 6.8), 10% glycerol, 2% SDS, and 5% 2-mercaptoethanol. The proteins were dissociated by immersing the samples for 3 min in boiling water. Electrophoresis was performed with a constant current of 25 mA using a 3% stacking gel (pH 6.8) and a 5% separation gel (PH 8.9). After staining by Coomassie brilliant blue, the density of the band was determined by densitometry using a Dual-Wavelength TLC Scanner (CS-900, Shimadzu Co., Japan) and Chromatopack C-RIB (Shimadzu Co.). The SDS-PAGE pattern of collagen preparation is presented in Fig. 1. Several protein bands corresponded mainly to CX-,B-, and y-chains. The purity of collagen preparations exceeded 83% with densitometry. Type I and III collagens were prepared in 0.2% solutions by rapidly mixing 2 vol of 0.3% pepsin solubilized type I and III collagen solutions (Koken Co.) with 1 vol of 3x concentrated DME-12.5 mM Hepes (PH 7.4), on ice; 0.5 ml of 0.2% collagen solution was added to each dish (35 mm, Falcon); and the preparation was incubated at 37°C for 2 hr for gelation of the collagen. The polymerized collagen gels were then equilibrated overnight with complete media. 3. Growth
Curve
Cell monolayers were prepared by plating 2-ml aliquots of a cell suspension of smooth muscle cells (5 x IO4 cells/ml) onto plastic (plastic group) and type I (type I group) and type III (type III group) collagen gel substrata. The cultured cells were maintained in DME-12.5 mM Hepes (pH 7.4) containing 10% FCS and antibiotics for 9 days and the medium was changed every third day. After 1, 3, 5, 7, and 9 days in culture, the cells were observed under a phase contrast microscope (Nikon, TMD, Japan) and then washed twice with phosphate-buffered saline (PBS). Cells grown on plastic substrata were trypsinized with 0.05% trypsin0.53 mM EDTA at 37°C for 15 min. Cells grown on collagen gels were removed from dishes by incubation with 0.2% collagenase (Worthington Biochemical Corp. )-I mM CaCl, at 37°C for 20 min. The suspension was then pipetted to disperse the cells and a counting chamber was used for enumeration. After 24 hr of cultivation, cells on the plastic, glass, and collagen type I and type III substrates were detached and counted by the above-mentioned methods, to estimate plating efficiency. of DNA Synthesis Cultured smooth muscle cells were incubated in the media containing
4. Assay
2 t~Ci/ml
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FIG. 1. SDS-PAGE (5%) patterns of type I and III collagen solutions. The gel was stained with Coomassie brilliant blue R-250. Molecular weight standards (lane 3) include a,-macroglobulin (170 kDa) and phosphorylase b (97 kDa). Both type I (lane 2) and III (lane 1) collagen solutions consisted mainly of protein bands that corresponded to a-chain, P-chain, and y-chain. F, front. T, top.
[3H]thymidine at 37°C for 2 hr on the first, third, and fifth days of culture. The media were removed and the cell sheet was rinsed three times with cold PBS containing 50 p,M thymidine (Wako Chemical Co., Japan). Cultured smooth muscle cells were harvested by incubation with 0.05% trypsin-0.53 mM EDTA-50 @4 thymidine in PBS in the case of uncoated plastic dishes or 0.2% collagenase-1 m&f CaClz50 l.~A4thymidine in PBS in the case of the collagen-coated dishes. A cell suspension was pipetted and a sample was taken for cell counting. Cell pellets, obtained by centrifugation at 1000 rpm for 10 min, were suspended in 1 ml of distilled water and then disrupted for 20 set at 0°C with a sonicator (Model W-10) tuned to 5, at the maximum power. One milliliter of 10% TCA was added to the sonicated cell suspension, and a TCA-precipitate was collected by centrifugation at 3000 t-pm for 15 min and then washed three times with 2 ml of cold 5% TCA. Precipitates were suspended in 1 ml of cold 10% TCA, incubated at 95°C for 15 min, and then placed on ice for 30 min. After centrifugation at 3000 rpm for 15 min, 0.1 ml of the hot TCA-soluble fraction was added to 6 ml of a scintillator (ACS II, Amersham, Canada). The amount of the radioactivity was determined with a liquid scintillation counter. A value was expressed as dpm per lo4 cells during 1 hr. 5. Electron Microscopic
Observation
After 1 and 3 days in culture,
the smooth muscle cells were fixed in 1.4%
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glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4) for 2 hr at 4”C, rinsed three times in the same buffer at 4°C and postfixed in 1% osmium tetroxide in the same buffer at 4°C for 2 hr. They were then dehydrated in a graded series of ethanols and embedded in Epon 812. Ultrathin sections were double-stained with uranyl acetate and lead citrate. All sections were observed under a JEM-1OOCX electron microscope. 6. Observation
of Actin Filaments
To visualize actin filaments, we used fluorophore nitrobenzoxadiazole (NBDb phallacidin (Molecular Probes), a specific probe for F-actin (Barak et al., 1980). A collagen-coated or uncoated coverslip (18 x 18 mm) was put in each dish (35 mm). Cells were inoculated onto the glass or collagen-coated coverslip in a dish at an initial plating density of 2 X 104/dish and cultivated with 2 ml of DME-12.5 mM Hepes containing 10% FCS and antibiotics. After 3,6, 12, and 24 hr in culture, the cultured cells were fixed in 4% paraformaldehyde in PBS at room temperature for 10 min. After rinsing twice with PBS at 4°C they were extracted with acetone at - 20°C for 5 min and then air-dried. Cells were incubated with NBD-phallacidin (1.6 x lop6 M) for 45 min at room temperature in a dark moist chamber, followed by washing twice in PBS for 3 min each time. Another coverslip was pretreated with a 50-fold excess of unlabeled phallacidin and then stained with NBDphallacidin, under identical conditions. The latter coverslip was considered to be a negative control. Cells were mounted in glycerol and viewed through a fluorescence microscope (Olympus, Japan). The frequency of cells with the distinct actin filaments in the cytoplasm was compared among the glass and collagen I and III groups. 7. Statistical
Analysis
Data were expressed as the means + SD. A statistical analysis was carried out by unpaired Student’s t test and a value of P < 0.05 was considered to be statistically significant. RESULTS 1. Phase Contrast Microscopy Figure 2 shows the phase contrast microscopic views of cultured smooth muscle cells on the first and ninth days of culture. Cells on the collagen gels were more elongated than those on plastic subtrata, and in particular the bipolar elongation was considerable on the type III collagen gel (Fig. 2F). Cells on the type III collagen gel aligned in the “hills and valleys” arrangement on the first day of culture, a characteristic of the cultured smooth muscle cell (Fig. 2C), and maintained this arrangement during the culture (Fig. 2F). Cells on type I collagen gel showed a similar structure on the first and third days of culture, but not as clearly as that on the type III collagen gel (Fig. 2B). In contrast, cells on plastic substrata did not show the hills and valleys alignment on the first and third days of culture (Fig. 2A). After 7 days, all the cells had the hills and valleys arrangement (Figs. 2D-2F). 2. Electron Microscopic Electron
microscopic
Observation observations
of the smooth muscle cells cultured
for 1
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FIG. 2. Phase contrast micrographs of cultured smooth muscle cells. (A-C) On the fust day. (D-F) On the ninth day. (A,D) Plastic group. (B,E) Type I group. (C,F) Type III group. x24.
and 3 days are shown in Fig. 3. Smooth muscle cells on both collagen gel and plastic substrata had a rough endoplasmic reticulum (rER), a Golgi apparatus, and polysomes, in addition to discrete microtilament bundles, dense patches, and pinocytotic vesicles beneath the adjacent plasma membrane. The cells were connected through junctional formations. Smooth muscle cells on collagen gels were more hypertrophic and enriched with rER, polysomes, and glycogens than were those grown on the plastic substratum (Fig. 3). In addition, numerous vacuoles containing residual body-like substances were observed within the cytoplasm of cells cultured on the collagen gels (Fig. 3). There was no definitive difference in the development of microfilament bundles between cells on the collagen gels and plastic substrata on first and third days of culture. 3. Cell Growth To test for the effect of collagen matrices on smooth muscle cell proliferation, cell number and incorporation of [3H]thymidine of each group were determined
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FIG. 3. Smooth muscle cells grown on collagen type I gel for 1 day. Cells show a hypertrophic cytoplasm rich in endoplasmic reticulum, polysomes, and lysosome-like vacuoles (L). Microfilament bundles (MF) are also present beneath the plasma membrane. X 11,120.
during culture. Figure 4 shows the growth curve of each group. The cell number of the plastic group was much greater than those of type I and III groups during the culture period (*P < 0.001, **P < 0.02 vs type III on the first day). However, the difference between plastic and collagen groups diminished during the process of the culture. There was no difference in cell number between type I and III groups, except for the first day of culture. The plating efficiencies were affected by the substrata; that is, the mean values of relative plating efficiencies of the glass and collagens type I and type III to the plastic substrate were 85.2,69.7 and 83.8%, respectively. The population doubling time of smooth muscle cells of the plastic group was shorter (32.6 hr) from the first to the third day of culture than those of the type I (56.7 hr) and III (64.1 hr) groups, which was longer (80.2 hr)
0:
plastic
A: collagen
I
O:collagen
III
FIG. 4. Effect of collagen gels on growth of cultured smooth muscle cells. There was a statistically significant difference in counts of cells grown on plastic and type I or III groups during the experiment. The data are mean values + SD of two separate experiments done in triplicate. *P < 0.001, **P < 0.02, plastic group vs type III group.
EFFECTS OF COLLAGEN
185
ON DIFFERENTIATION
from the third to the fifth day of culture than those of types I (45.2 hr) and III (49.8 hr) (Fig. 5). In contrast, the doubling time of cells grown on collagen type I and III gels was shorter from the third to the fifth day rather than from the first to the third day of culture. The incorporation of [3H]thymidine into DNA was compared between the cells on the plastic and the collagen gel-coated dishes (Fig. 6). DNA synthetic activity of cells on both type I and III collagen gels was remarkably suppressed on the first day of culture, compared to that on the plastics (Fig. 6). Namely, the mean values of [3H]thymidine incorporation of cells on plastic and collagen type I and III substrata were 6.27 x 103, 1.80 x 103, and 1.68 x lo3 dpm/104 cells, respectively (*P < 0.001, plastic vs collagen type I or III). However, on the third and fifth days of culture, the mean values of [3H]thymidine incorporation of cells on plastic decreased to 0.72 x lo3 and 1.36 x lo3 dpm/104 cells, respectively. In contrast, uptake of [3H]thymidine of cells grown on collagen type I and III gels increased on the third and fifth days of culture. Hence, cells grown on collagen gels incorporated significantly more [3H]thymidine into DNA on the third and fifth days than did cells grown on plastic (**P < 0.001, ***P < 0.002, ****p < 0.02). 4. Time Course of Formation Collagen Substrata
of Actin Filament
on Glass and
Table I shows the frequency distribution of cells grown on each substrate and the developing actin filaments. Many cells in all groups had few bundles of actin filaments after 3 hr. Cells in all groups began to form actin filaments after 6 hr, and the mean value of frequency of cells with actin filaments was approximately 18 to 36%. More than 50% of cells had filaments after 12 hr, and cells on collagen type III had significantly less filaments than did the other groups (*P < 0.05). The majority of cells in all groups showed formation of actin filaments after 24 hr, but the mean value of frequency of cells forming actin filaments was significantly higher in the glass group than in the type I and III groups (**P < 0.01). 5. Actin Filament
Morphology
In the smooth muscle cells there was no formation of actin filaments during the first 3 hr of plating on any substrate. The well-spread cells cultured on glass $$J:plastic z:collagen n:collagen
l-3
I III
3-5
Days in culture FIG. 5. Doubling times of smooth muscle cells were affected over the time course of the culture. The data were expressed as the mean values of two experiments done in triplicate.
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•j : plastic q : collagen1 0
: collagen
1
III
5
Days &Mm? FIG. 6. Effects of collagen gels on DNA synthesis of smooth muscle cells were significantly varied in the time course of the culture. The mean value of incorporation of [‘Hlthymidine in smooth muscle cells of the plastic group was significantly higher than those of collagen type I and III groups on the first day of culture (*P < O.OOl), while the former was significantly lower than the latter on the third and fifth days of culture (**P i 0.001, ***P < 0.002, ****P < 0.02).
coverslips for 12 hr had a continuous staining of linear structures which ran in various directions within the cytoplasm (Fig. 7). Smooth muscle cells pretreated with 50x excess of unlabeled phalloidin and then stained with NBD-phallacidin stained weakly. The elongated cells grown on collagen substrata for 12 hr were mostly in straight bundles parallel to the major axis of cells and ran from end to end (Fig. 8). The very elongated cells with processes containing actin filaments between other cells were more frequently found on the collagen substrates, compared to those on the glass (Fig. 9). DISCUSSION In the present study, cultured smooth muscle cells derived from explants of abdominal aortic media were used. Enzyme-dispersed contractile smooth muscle cells undergo a spontaneous change in phenotype to the synthetic state after 6 to Frequency Distribution Duration W 3 6 12 24
TABLE I of Smooth Muscle Cells Developing Distinct Actin Filaments during 24 hf’
Grade + ++ + ++ + ++ + ++
Glass 96.0 + 2.4 4.0 ? 2.4 Ok0 64.2 +- 14 32.2 2 10 3.6 k 4.2 32.5 i 6.1 62.2 k 7.0 5.3 2 4.1 14.2 -+ 4.6** 74.0 t 7.4 11.8 + 6.2
Collagen I
Collagen III
92.8 + 7.2 f olro 74.7 + 23.0 + 2.3 f 38.3 f 57.0 2 4.7 + 33.8 f 64.0 2 2.2 2
98.8 + 1.2 2 020 82.2 _’ 17.0 ” 0.8 + 49.5 k 48.2 f 2.3 + 26.0 f 72.5 f 1.5 f
4.6 4.6 8.5 7.3 2.6 5.2 7.4 2.8 9.0 8.7 1.6
1.2 1.2 5.2 4.4 1.3 5.6* 6.9 2.1 5.8 5.0 1.6
’ Data (%) are mean values f SD. n = 6. *P < 0.05 vs glass and collagen type I. **P < 0.01 vs collagen types I and III.
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OF
COLLAGEN
ON
DIFFERENTIATION
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FIG. 7. Smooth muscle cells grown on a coverslip for 12 hr. Note bundles of actin filaments traveling from end to end. FIG. 8. Smooth muscle cells grown on collagen type I gel for 12 hr. Note bundles of actin filaments running parallel to the direction of the major axis of the elongated cytoplasm.
8 days in primary culture. Culture conditions and number of cumulative population doublings required to reach confluency relate to whether they spontaneously return to the contractile state (Charnley-Campbell and Campbell, 1981; Campbell and Campbell, 1985). On the other hand, smooth muscle cells can divide without loss of smooth muscle myosin, which is an important marker protein of differentiated contractile smooth muscle cells. Cells which lose smooth muscle myosin may regain it in the postconfluent state (Hammerle ef al., 1988). We have recently shown that smooth muscle cells derived from explants of coronary arterial media have a significantly higher frequency of desmin positivity in the conlluent state than in the logarithmic growth state, desmin being an important protein of intermediate filaments of muscle cells (unpublished data). Moreover, it has been shown that type I collagen increased the number of cytoskeletal filaments and suppressed DNA synthetic activity in smooth muscle cells derived from explants of the thoracic aortas of rabbits (Yoshida et al., 1988). Subcultured smooth muscle cells derived from explant outgrowth are also considered to be able to undergo phenotypic modulation, to a certain degree. Intercellular matrices, including collagens, play an important role in the control of cell proliferation, in vivo and in vitro. There are, however, conflicting reports concerning the effect of collagen substrata, one being that they promote cell growth (Ehrmann and Gey, 1956), the other that they suppress it (Yoshizato et al., 1985). In our study, porcine smooth muscle cells were grown on plastic and
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FIG. 9. Smooth muscle cells grown on collagen type I gel for 12 hr. Cytoplasmic processes containing actin filaments are present between the adjacent cells.
collagen type I and III substrata. Growth rates, estimated by doubling time, and incorporation of [3H]thymidine into DNA of cells were suppressed on the collagen gels on the first day of culture, compared with the cells grown on the plastic substrate. Cells on the collagen gels had a shorter doubling time and a greater incorporation of [3H]thymidine between the third and the fifth days of culture than did cells grown on the plastic substrate. Moreover, differences in cell number between plastic and collagen type I or III groups decreased with the continuation of cultivation. These results suggest that the growth rate of the smooth muscle cells is suppressed in the early stage of culture, followed by a gradual proliferation when the cells are cultivated under conditions which more closely resemble the in vivo environment, e.g., collagen gel substrate. Herman and Castellot (1987) reported that collagen matrices did not influence the doubling time and saturation density of a cultured smooth muscle cell. The saturation density of cultured smooth muscle cells is considered to be unaffected by collagen substrata, because differences in cell number between plastic and collagen groups decreased during the culture period. There is, however, a discrepancy between our data and those of Herman and Castellot (1987) with regard to the doubling time. In their study, the doubling time was examined only between the second and the fourth days of culture, while we observed it from the first to the third days and also from the third to the fifth days of culture. Thus, we obtained good evidence that the effect of collagen matrices on the doubling time of cells varies with the period of culture.
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This result is consistent with the finding concerning the incorporation of [3H]thymidine into DNA. Smooth muscle cells cultivated on the type III collagen gel aligned in the hills and valleys arrangement on the first day of culture and this pattern was preserved throughout the culture period. In contrast, cells on the plastic substrate did not show the hills and valleys alignment until after 5 days of culture. It is known that collagen matrices influence the function and differentiation of cells (Lark and Wight, 1986; Gibson et al., 1984). However, there are few reports that the hills and valleys alignment, a characteristic of cultured smooth muscle cells, occurred on the first day of culture on the collagen type III gel. Our observations suggest that collagen matrices, especially type III collagen, may play an important role in the expression of differentiation of cultured smooth muscle cells. Moreover this finding is in accord with the fact that type III collagen exists predominantly in the media of an artery (McCullagh er al., 1979). The actin filament is the most important part of the cytoskeleton. Stress fibers that are prominent components of the cytoskeleton in cultured cells are closely linked to the maintenance of cell shape and adhesiveness to the substrates. Farsi and Aubin (1984) noted prominent bundles of microfilaments in very elongated fibroblasts cultured within collagen gels. We found that smooth muscle cells grown on collagen gels were more elongated and had exclusively actin filaments traveling in parallel to the major axis of their cytoplasm, some of which had cell processes containing the actin filaments between adjacent smooth muscle cells. These observations are interpreted to mean that the mode of actin filaments is related to the shape and arrangement of smooth muscle cells grown on the collagen gels. Smooth muscle cells, when grown on a glass substrate, began to form actin filaments after 6 hr, and the majority of the cells had adequate actin filaments after 24 hr. In contrast, the formation of actin filaments was delayed in the smooth muscle cells grown on collagen gels. In other studies (Willingham et al., 1977; Singhal and Hays, 1988), the rate of formation of actin filaments was shown to relate to the adhesiveness to the substrata. Since cell numbers on the first day of culture varied in the various substrata including the plastic, glass, and collagen types I and III, there is the possibility that the development of actin filaments is related to the adherence of cells and quality of the substrate. However, the collagen substrate has been also reported to promote the attachment of smooth muscle cells (Bjorkerud, 1985). The relation between the adhesive properties of collagen matrices and the development of actin filaments remains to be elucidated. Ultrastructurally, the development of microfilaments of cells grown on collagen gels was similar to that of cells on the plastic substrate after 1 and 3 days of culture. On the other hand, the formation of F-actin was more delayed in the former, as determined using NBD-phallacidin staining which is specific for Factin. Microfilaments contain other proteins including myosin, tropomyosin, and a-actinin (De Robertis and De Robertis, 1987). Therefore, these findings suggest that the collagen substrata may not affect the quantity of microfilament bundles but do change the quality of components such as the content of actin. The present study demonstrated that the rate of proliferation, the shape and arrangement, and the development of actin filaments of smooth muscle cells
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grown on collagen gels differed from those observed on a plastic substrate. In addition, in cultured smooth muscle cells, the suppression of proliferation and expression of differentiation in the early stages of culture on the collagen matrix with a more physiological environment than plastic substrate, and the effects of collagen matrix on the cell differentiation, might vary with the species of collagen. ACKNOWLEDGMENTS We thank M. Ohara for comments and T. Sato for technical assistance. Part of this work was supported by funds from the Central Research Institute of Fukuoka University.
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