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Disassembly of F-actin filaments in human endothelial cells cultured on type V collagen
EXPERIMENTAL
CELL
RESEARCH
201,55-63
(1992)
Disassembly of F-Actin Filaments in Human Endothelial Cells Cultured on Type V Collagen KIYOTAKA YAMAMOTO,*,’
MARI YAMAMOTO,*~~ AND TETSUO NOUMURA~
*Department of Cell Biology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Ztabashi-ku, Tokyo 173, Japan; and tDepartment of Regulation Biology, Faculty of Science, Saitama University, 255 Shimo-ookubo, Urawa 338, Japan
such vascular diseases as thrombosis and atherosclerosis, since the subendothelium can initiate platelet adhesion and aggregation [ 11. Endothelial cells in culture form a contact-inhibited monolayer [2], synthesize several compounds that are integral to hemostasis, including prostacyclin, heparin-like glycosaminoglycans, plasminogen activator, thrombin, and factor VIII antigen [3], and have been widely used as a model system for studying the mechanisms of vascular diseases [4-71. The extracellular matrix (ECM) which anchors the endothelial cell layer plays an integral role not only in determining endothelial cell polarity, orientation, and morphology, but also in regulating endothelial cell proliferation [8, 91 by releasing biologically active basic fibroblast growth factor [lo, 111. Folkman and Moscona [12] found that the proliferation of endothelial cells is correlated very closely with cell shape. Schor et al. [13] suggested that collagen-containing substrata influence the response of capillary endothelium to tumor angiogenie factor. Collagens in the blood vessel walls serve as key components for maintaining structural integrity and for mediating thrombosis through their interaction with platelets [14]. Proportional changes in type I and type III collagens [15-171 and increases in type V collagen [16, 181 within advanced atherosclerotic plaques have been reported. Type V collagen is an interstitial fibrillar collagen rather than a basement membrane collagen, with a tissue pattern completely different from that of collagen types I, III, and VI or fibronectin [19]. Fukuda et al. [20] found that type V collagen remarkably inhibits the proliferation of human umbilical vein endothelial cells but not that of vascular smooth muscle cells and that it does not affect cell attachment but rather seems to promote cell detachment. The detachment of endothelial cells from the subendothelium and the attachment of platelets and leukocytes to the normally inaccessible subendothelial matrix can lead to thrombosis and atherosclerosis [ 11. Therefore, understanding the factors that control the adhesion of endothelial cells to the substratum is important in a wide variety of vascular disease states. Many cells, including fibroblasts, smooth muscle cells, and endothelial cells, develop focal
We examined the inhibitory activity of type V collagen on cell attachment and cell growth and the role of stress fibers and & integrin in cultured human endothelial cells. Human endothelial cells cultured on type V collagen attached temporarily to the substrate and formed stress fibers. However, the cells failed to proliferate and gradually detached from the substrate. After 24 h, the cells on type V collagen lacked discernible stress fibers (F-actin filaments) and exhibited dots in small aggregates of F-actin. In addition, the cells expressed little or no proteins as focal adhesions, including vinculin and j3i integrin. In contrast, the cells on fibronectin and type I collagen formed complete F-actin filaments, exhibited sufficient vinculin and 8, integrin, and grew logarithmically from 2 days. On the other hand, human smooth muscle cells formed complete Factin filaments, revealed typical focal adhesions, and started to proliferate rapidly after 24 h on type V collagen as well as on fibronectin and type I collagen. Thus, the disassembly of F-actin filaments was observed as a specific phenomenon in human endothelial cells cultured on type V collagen. Moreover, the F-actin filaments disappeared from endothelial cells treated with cytochalasin D after 24 h and the cells detached from fibronectin and type I collagen with time, a result consistent with the observations on type V collagen. Accordingly, the disassembly of F-actin filaments in focal adhesions may result in the detachment of endothelial cells from type V collagen. 0 1992 Academic PWS, 1~.
INTRODUCTION Vascular endothelial cells in uiuo are disposed as a contact-inhibited monolayer on the subendothelium and are directly involved in the maintenance of nonthrombogenic surfaces, as a permeability barrier between blood and subendothelial matrix, and in intimal repair following vascular endothelial injury. Loss of endothelial cells and the subsequent exposure of the subendothelium is an important step of the development of ’ To whom reprint
requests should be addressed. 55
All
Copyright 0 1992 rights of reproduction
0014-4827/92 $5.00 by Academic Press, Inc. in any form reserved.
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Coating of culture dishes. Type I and type V collagens (3 mg/ml; Koken, Tokyo) were applied to dishes and air-dried at room temperature. Human plasma fibronectin (HFN, Boehringer-Mannheim) was applied to dishes at a concentration of 20 pg/ml in MCDB 107 or MEM and left for 45 min at room temperature. Before use, these coated dishes were washed twice with each medium. Cellgrowth. HUVE-9 cells (2 X 10’ldish) were cultured in MCDB 107 containing 1% hi-FBS, 0.2% bovine serum albumin (BSA), ECGF (100 rg/ml), recombinant epidermal growth factor (EGF, 10 rig/ml), recombinant basic fibroblast growth factor (b-FGF, 10 rig/ml), recombinant insulin-like growth factor-I (IGF-I, 20 rig/ml), and transferrin (5 pg/ml) (EC medium) in 16.mm Falcon multiple dishes (3047) coated with type I or type V collagen, or HFN at 37°C. HAS-2 cells (2 X lO’/dish) were cultured in MEM containing 1% FBS, 0.2% BSA, recombinant platelet-derived growth factor (PDGF, C-sis, 10 rig/ml), EGF (10 rig/ml), b-FGF (10 rig/ml), IGF-I (20 rig/ml), and transferrin (5 pg/ml) (SMC medium) in 16-mm Falcon dishes coated with type I or type V collagen, or HFN at 37°C. All growth factors were purchased from Boehringer-Mannheim except for PDGF (Amersham). Endothelial cells (2 X Incorporation of BrdU into cellular DNA. 104/dish) were cultured in 16-mm Falcon plastic dishes coated with type I or type V collagen, or HFN in 0.5 ml of EC medium at 37°C. The growth medium was replaced with fresh medium containing a 1:lOOO dilution of 10 mM BrdU and the cells were incubated for 24 h. The cells were washed twice with phosphate-buffered saline (PBS), fixed in ethanol containing 0.1% hydrogen peroxide for 30 min, then washed again in PBS. BrdU incorporation into cellular DNA was
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FIG. 1. Cell growth of both human vascular endothelial cells (a) and smooth muscle cells (b). Cells (2 X 10’) were cultured in medium containing 1% FBS and growth factors (EC or SMC medium) in human plasma fibronectin (HFN)- (O), type I collagen- (0), or type V collagen-coated dishes (A) as described under Materials and Methods. The medium was renewed every 2 or 3 days, and cell numbers were determined with a Coulter Counter. Each point represents the mean of the triplicate cultures. 0, cell growth of endothelial cells on plastic dishes.
adhesions when plated onto appropriate substrates [2124]. The formation of focal adhesions is preceded by a structural precursor consisting of a microspike or bundle of actin filaments oriented radially within the leading edge of a cell [25-271. In this paper, we describe that the assembly and the disassembly of F-actin filaments in focal adhesions after 24 h of culture are related closely to cell attachment and detachment as well as the control of the cell proliferation.
MATERIALS
AND
METHODS
Cell culture. Human umbilical vein endothelial cells (HUVE-9) and human arterial smooth muscle cells (HAS-2) have been described [28]. HUVE-9 cells were cultured in MCDB 107 containing 20% heatinactivated fetal bovine serum (hi-FBS, HyClone Laboratories Inc., lot No. 1111654) and 150 pg of endothelial cell growth factor (ECGF, Culture grade, Boehringer-Mannheim) per milliliter in type I collagen-coated dishes (Corning) at 37°C under humidified 5% CO,-95% air. The growth medium was renewed every 2 or 3 days, and the cells were subcultured at a 1:4 split ratio. The HUVE-9 cell strain had a limited life span of about 50 population doubling levels (PDL) under these culture conditions. In this study, we used the cells at 20-30 PDL. HAS-2 cells were cultured in Eagle’s minimum essential medium (MEM) containing 15% FBS in plastic dishes in the CO, incubator and subcultured at a 1:4 split ratio. The HAS-2 cell strain had a limited life span of about 29 PDL under these conditions and was used at 9-13 PDL in this study. The cells were counted with a hemocytometer or Model ZBI Coulter Counter (Coulter Electronics Inc.). The cells were judged mycoplasma-free by the method described previously [28].
FIG. 2. Morphological characteristics of cells grown on fibronectin, type I collagen, and type V collagen. Cells were cultured in EC or SMC medium for 7 days as described under Materials and Methods. Human endothelial cells reached near confluence on HFN (a) and type I collagen (b), but were detached from type V collagen and remained slightly so (c). Human smooth muscle cells reached near confluence on type V collagen (f) as well as on HFN (d) and type I collagen (e). Bar, 100 pm.
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25
20 “E E 15 Pi 6 =07 10
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HFN FIG. 3. BrdU incorporation into cellular DNA of human endothelial cells. Cells (2 X lO*/dish) were cultured in EC medium containing BrdU, fixed, and stained with monoclonal anti-BrdU antibody as described under Materials and Methods. BrdU incorporation into cellular DNA after 24 h (Cl) and 48 h (m) of culture was measured in a constant area (0.321 mm’), and the spread cells after 24 h (0) and 48 h @I) were also counted in the same area. Each point represents the mean of triplicate determinations with the standard deviation indicated as a vertical line.
measured using an immunoperoxidase technique (cell proliferation kit; Amersham) as described previously [29]. The percentage of labeled nuclei was determined by counting more than 200 cells in each experiment. Fluorescence assay. F-actin filaments were stained according to the method of Hall et al. [30]. Endothelial cells and smooth muscle cells (2 X 104) were cultured on Lab-tek chamber glass slides (Nunc, 4804) coated with type I or type V collagen, or HFN in EC medium and SMC medium, respectively, at 37°C. After 24 h, when cell spreading reached a maximum on type I collagen and HFN, the cells were fixed with 3.7% (v/v) paraformaldehyde in PBS for 20 min at room temperature and permeabilized using 1 ml of 0.2% (v/v) Triton X-100 in PBS for 5 min at room temperature. After washing with PBS, the cells were stained with rhodamine phalloidin (2.0 units/ml, Molecular Probes, Inc.) for 1 hat room temperature. Chamber slides were extensively rinsed with PBS, mounted in 50% glycerol-50% PBS, and examined with an Olympus microscope equipped with epifluorescence illumination. The relative fluorescence intensity of cells was measured with a Model OSP-1 Olympus spectrophotometer. For immunocytochemical detection of vinculin and pi integrin, cells fixed as described above were incubated with a 1:50 dilution of monoclonal anti-vinculin antibody (Amersham) and anti-& integrin antibody (Immunotech, Marseille) for 45 min at room temperature, washed, and exposed to a 1:200 dilution of rhodamine-conjugated rabbit anti-mouse IgG (Cappel Products), as described previously [28, 291. Confocal micrographs were taken using a Bio-Rad MRCBOO laser scanning focal imaging system connected to a Nikon X2F-EFD2 microscope.
RESULTS Cell Proliferation on Dishes Coated with Type I Collagen, Type V Collagen, and HFN Human umbilical vein endothelial cells (HUVE-9) attached to the substrate within 0.5-l h after seeding,
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when they were cultured in type I collagen- or HFNcoated dishes. Cell attachment on HFN and type I collagen after 24 h incubation were 75.1 f 5.62 and 78.4 + 4.21%, respectively. Growth curves showed that the cells on HFN-coated dishes and type I collagen-coated dishes started to proliferate on the second day of culture and thereafter grew logarithmically for up to 5 days of culture (Fig. la). The growth curves were similar to those in MCDB 107 containing 20% hi-FBS and 150 pg of ECGF per milliliter on HFN and type I collagen (data not shown). In contrast, the cells cultured on type V collagen temporarily adhered and spread out as well as on HFN and type I collagen, but gradually detached from the substrate. The cell attachment after 24 h was 41.2 + 7.21%. The cells were rounded, detached from type V collagen-coated dishes, and remained slightly so on the seventh day of culture (Figs. la and 2c), when the cell densities reached confluence on HFN and type I collagen (Figs. 2a and 2b). These results under serum deficient conditions (1% FBS) corresponded with the findings of Fukuda et al., which were in MCDB 107 containing a higher concentration of serum [20]. The cells hardly grew on plastic dishes under these conditions but never detached from them (Fig. la). On the other hand, human smooth muscle cells (HAS-2) adhered and spread well on the three substrates. The cells started to proliferate rapidly after 24 h of culture and reached near confluence even on type V collagen-coated dishes after 7 days of culture (Figs. lb and 2f) as well as on HFN- and type I collagen-coated dishes (Figs. lb, 2d, and 2e). These results obtained under conditions that excluded the effect of serum fibro-
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FIG. 4. Growth curve of the cells detached from type V collagen. Human endothelial cells (2 X 104/dish) were cultured in EC medium on type V collagen (a). The detached cells into the medium during 12-36 h of culture were collected and recultured in EC medium on type I collagen (0) and type V collagen (A). Each point represents the mean of triplicate determinations. No differences in growth characteristics between the original cells and detached cells were observed. 0, cells on type I collagen as a control.
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FIG. 5. Localization of F-actin filaments with rhodamine phalloidin in human endothelial cells. Cells (2 X 10’) were cultured in EC medium for 24 h on fibronectin, type I collagen, and type V collagen, fixed with 3.7% paraformaldehyde, and stained with rhodamine phalloidin as described under Materials and Methods. The cells formed complete F-actin filaments both on HFN (a) and type I collagen (b), but not on type V collagen (c). Bar, 25 pm.
nectin within reasonable limits revealed that type V collagen selectively inhibited the growth of human endothelial cells. Figure 3 shows BrdU incorporation into cellular DNA of the endothelial cells after 24 and 48 h of culture. BrdU incorporation into cellular DNA was not observed on any substratum when cultures were incubated for 24 h in medium containing BrdU. After 48 h of culture, the BrdU incorporation into the cellular DNA of spread cells was observed on HFN (12.9 + 9.60%) and type I collagen (14.5 + 11.4%), but not on type V collagen. These results indicated that the endothelial cells which detached from type V collagen within 2 days were resting in G, phase and were not mitotic cells. The endothelial cells in the G, phase that were floating in the medium between 12 and 36 h of culture on type V collagen were then collected and again cultured on type I collagen-coated dishes. These detached cells were reattached, spread, and started to proliferate. No
significant difference in the rate of adhesion and proliferation between the detached cells from type V collagen and the cells cultured originally on type I collagen was observed (Fig. 4). These results indicated that the inhibition of the cell growth on type V collagen was not caused by cell damage, but was rather attributable to some factors that promote cell detachment, and that the endothelial cell detachment was determined by the origin of the extracellular matrix. Reorganization
of F-Actin
Filaments
We examined the reorganization of F-actin filaments of the cells in G, phase under serum deficient conditions. HUVE-9 cells cultured on type V collagen formed stress fibers composed of F-actin filaments after 1 h of culture, but eliminated the actin filaments after 24 h, generating focal pools of F-actin in the cytoplasm (Fig. k), though complete F-actin filaments were observed
FIG. 6. Localization of F-actin filaments in human smooth muscle cells. Cells (2 X 104) were cultured in SMC medium for 24 h and stained with rhodamine phalloidin. Stress fibers in the cytoplasm of the cells on type V collagen (c) as well as on HFN (a) and type I collagen (b) were clearly observed. Bar, 25 Wm.
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specific protein of focal adhesions, and & integrin, an integral membrane component identified in focal adhesions, was examined using a laser scanning confocal imaging system. In human endothelial cells cultured for 24 h on type V collagen, ,L3,integrin was not detected though it was present in all the cells on HFN and type I collagen (Fig. 8). The localization of vinculin in the cells
Type V
FIG. ‘7. Fluorescence intensity of F-actin filaments stained with rhodamine phalloidin. Cells were stained with rhodamine phalloidin after 24 h of culture, and the relative fluorescence intensity per cell was measured with a Model OSP-1 spectrophotometer (Olympus). Each column represents the mean and standard deviation of three experiments, determined by counting 50 cells in each experiment.
throughout the cytoplasm of all the cells on both HFN and type I collagen (Figs. 5a and 5b). Despite the absence of the stress fibers, however, these cells demonstrated effective cytoplasmic spreading over type V collagen, presumably by other cytoskeletal networks. In contrast, the cells that detached from type V collagen restored the F-actin filaments when they were recultured on HFN or type I collagen (data not shown). On the other hand, the smooth muscle cells cultured for 24 h on type V collagen as well as on HFN and type I collagen reorganized extensive arrays of stress fibers (Fig. 6). Using microscopic fluorometry, the fluorescence intensity of rhodamine phalloidin per cell was then measured (Fig. 7). In HUVE-9 cultured on HFN and type I collagen, the relative levels of F-actin filaments were higher, namely, 112 f 18.6 and 136 f 28.3, respectively, while on type V collagen, the values were very low (8.11 -t 7.03). On the other hand, HAS-2 cells indicated similarly high relative contents on type V collagen as well as on HFN and type I collagen (114 f 30.5,120 f 41.4, and 122 f 40.1, respectively). The disassembly of F-actin filaments in the endothelial cells cultured for 24 h on type V collagen may have resulted in detaching of endothelial cells from the substrate. Formation of Focal Adhesions and Expression of p, Integrin Focal adhesions are clearly important for anchoring stress fibers to the plasma membrane, but they probably also regulate the assembly and disassembly of the attached actin filaments [31]. The presence of vinculin, a
FIG. 8. Localization of pi integrin in human endothelial cells. Cells were cultured in EC medium, fixed with 3.7% paraformaldehyde, stained with monoclonal anti-b, integrin antibody, then visualized by indirect immunofluorescence as described under Materials and Methods. The & integrin was concentrated on HFN (a) and type I collagen (b), but not on type V collagen (c). Bars, 25 pm.
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FIG. 9. Fluorescence intensity of vinculin and & integrin in human endothelial cells. Cells were cultured in EC medium, stained with monoclonal antibodies against vinculin and & integrin, and visualized by indirect immunofluorescence. Relative fluorescence intensity per cell was measured with a MRC-600 laser scanning focal imaging system (Bio-Rad). Each column represents the mean and standard deviation of three experiments that were determined by counting 50 cells in each experiment. The cells on type V collagen failed to develop focal adhesions and to concentrate pi integrin in adhesion regions because the cells were little stained by these two antibodies.
was almost the same as that of pi integrin (data not shown), as described previously [24,32,33]. As shown in Fig. 9, the relative levels per cell were higher on HFN and type I collagen, and very low value on type V collagen. After 24 h of culture thus, human vascular smooth muscle cells reorganized F-actin filaments and revealed focal adhesions regardless of differences in substrates, whereas human vascular endothelial cells failed to develop F-actin filaments and to concentrate vinculin and p, integrin, and revealed abnormal adhesions or close contacts on type V collagen. Consequently, the endothelial cells gradually detached from type V collagen and did not subsequently proliferate. Effects of Cytochalasin
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cytoplasm of the cells both on HFN and type I collagen (Figs. llc and lld), though the cells without cytochalasin D formed extensive F-actin filaments (Fig. lla and lib). As shown in Fig. 12, the relative fluorescence intensity of the cells treated with cytochalasin D revealed far lower values (HFN, 9.03 f 8.01; type I collagen, 26.1 + 18.0) than controls without cytochalasin D (HFN, 112 + 16.4; type I collagen, 132 + 30.3) and these levels were similar to that (8.11 f 7.03) on type V collagen. Thus, the cytochalasin D-treated cells, which effectively spread over the substrates at 24 h, revealed close contact or abnormal adhesions, and thereafter detached from the substrates, as observed on type V collagen. Consequently, it is considered that the distribution patterns of F-actin filaments after 24 h of culture determine the subsequent events (cell growth or cell detachment). The difference in the rearrangement of F-actin filaments in the G, phase and in the growth pattern between endothelial cells and smooth muscle cells on type V collagen are of interest in view of the development of vascular diseases. DISCUSSION
Cellular adhesion to and detachment from the substratum involves interaction between constituents of the cell surface and the extracellular matrix [35, 361. Several investigators have examined endothelial cell detachment in vitro under conditions that led to cell lysis and killing [37-391. Gordon et al. [40] have reported that the detachment of human endothelial cells by detaching agents is controlled by the extracellular
I
D
Cytochalasin disrupts actin fibers but microtubules remain intact [34]. Human endothelial cells were cultured on type I collagen and HFN in EC medium containing 5 pg/ml cytochalasin D. The cell growth differed extremely with or without cytochalasin D. The cytochalasin D treatment gradually induced cell detachment from the substrate after 24 h of culture, and the growth profile resembled that of the cells on type V collagen (Fig. 10). After 24 h of culture, furthermore, the F-actin filament fibers of the cells were completely disrupted by cytochalasin D and condensed into focal pools in the
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FIG. 10. Effect of cytochalasin D on the cell growth of human endothelial cells. Cells (2 X 104/dish) were cultured in EC medium containing 5 pg cytochalasin D per milliliter on HFN and type I collagen. Each point represents the mean of triplicate determinations. The cells treated with cytochalasin D gradually detached from HFN (m) and type I collagen (O), though the cells without cytochalasin D rapidly grew on HFN (Cl) and type I collagen (0).
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FIG. 11. Effect of cytochalasin D on localization of F-actin filaments in human endothelial cells. Cells were cultured with cytochalasin D and stained with rhodamine phalloidin after 24 h of culture. The cells treated with cytochalasin D did not show F-actin filaments on HFN (c) and type I collagen (d), though control cells without cytochalasin D formed complete F-actin filaments on HFN (a) and type I collagen (h). Bar, 25 pm.
matrices and not by the cells themselves. We showed in the present study that type V collagen selectively inhibited human endothelial cell proliferation, probably promoting cell detachment by the disassemble of F-actin filaments, and that the endothelial cells detached in the G, phase from type V collagen reattached, spread, reorganized the filaments, and started to proliferate when recultured on type I collagen. The growth characteris-
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FIG. 12. Fluorescence intensity of F-actin filaments of cells treated with cytochalasin D. Cells were cultured and stained with rhodamine phalloidin, and the relative fluorescence intensity per cell was measured as described in the legend to Fig. 7.
tics of the detached cells were essentially the same as those of the cells that were originally cultured on HFN and type I collagen. The phenomenon that the detached cells reattached and continued to proliferate resembles that reported by Gordon et al. [40], using cells detached by specific agents. The growth inhibition of the endothelial cells on type V collagen was not caused by cell damage and is attributed to cell detachment determined by the extracellular matrices. Human endothelial cells, even though the cells could adhere to and spread over type V collagen, disassembled F-actin stress fibers after 24 h of culture and thereafter gradually detached. In contrast, the endothelial cells on HFN and type I collagen and human smooth muscle cells on all three substrates formed complete F-actin filaments after 24 h of culture and started to grow rapidly. Also the disruption of the actin fibers by cytochalasin D inhibited the growth and induced the detachment of the human endothelial cells on HFN or type I collagen. In this way, the assembly and disassembly of F-actin filaments appear to exactly reflect the subsequent growth on the substrate and detachment of cells from the substrate. Cells can adhere to substrates by both focal and close contacts, as classified by Izzard and Lochner [ 221. Many cells, including fibroblasts, smooth muscle cells, and endothelial cells, form focal adhesions when plated onto appropriate substrates [21-241. In contrast, migrating cells initially reveal close contacts rather than focal adhesions and lack stress fibers. With time, the cells de-
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velop focal adhesions and stress fibers [41]. In the focal adhesion regions, the assembly and the disassembly of actin filament stress fibers occurs [42]. In this manner, focal adhesions serve as sites not only to anchor stress fibers to the plasma membrane but also to nucleate actin polymerization. Moreover, they probably regulate the assembly and the disassembly of the attached actin filaments [31]. When cells were plated onto naked glass coverslips in the absence of serum, or on glass coated with nonspecific proteins, such as BSA, normal fibroblasts adhere poorly and fail to develop focal adhesions [43], and transformed cells reveal abnormal rosette adhesions or podosomes [44]. The addition of fibronectin to transformed cells temporarily restored stress fibers [35,45] and the disruption of the actin cytoskeleton resulted in a loss of fibronectin from the cell surface [46, 471. These results indicate the existence of a transmembrane link between the ECM and the cytoskeleton. Perhaps related to this lack of requirement for an ECM, integrin is not usually concentrated within these abnormal adhesions [48, 491. Carter et al. [50] reported that the relocation of integrin in neonatal human foreskin keratinocytes may result in detachment of cells from the membrane basement zone. In the present study, human vascular endothelial cells temporarily attached to type V collagen and assembled F-actin filaments. However, the cells disassembled actin filaments after 24 h on type V collagen, failed to develop focal adhesions and to concentrate /3,-integrin in adhesion regions, and detached from type V collagen, though focal adhesions were found and & integrin was concentrated on HFN and type I collagen. In addition, the cells that detached from type V collagen immediately restored F-actin stress fibers when transferred to HFN or type I collagen. Taken together, these results suggest that the ECM-integrin-actin filament system was reversed by the ECM and that the relocation of integrin following the disassembly of the actin filaments may occur and result in detachment of the endothelial cells on type V collagen. The simplest model of an ECM-integrin-cytoskeleton system is that binding of extracellular ligand produces a conformational change in the integrin receptor, which favors interaction with cytoplasmic (cytoskeletal?) components and that both external and internal interactions are necessary for stable assembly of integrins into focal contacts 1511. Finding integrins concentrated at adhesion sites has drawn attention to these ECM receptors as critical elements in the transmembrane link between the ECM and the cytoskeleton. The interaction between rabbit aortic SMC and type V collagen was originally shown to be mediated by a cell-surface type V collagen receptor. The binding system consists of integral membrane glycoproteins (80,000 and 50,000) which act alone or in combination with a surface glycolipid in type V collagen attachment [52]. Taken
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together, human endothelial cells may lack these type V collagen receptor components on the cell surface, may use other molecules as type V collagen receptor(s), but may relocate them with time. Additionally, not only ECM receptors but also ECM itself may serve as a regulator in focal adhesions. We thank Drs. K. Okumura and M. Kihara, National Institute of Health, Tokyo, and Dr. M. Takeuchi, Institute for Fermentation, Osaka, for mycoplasma testing of the cultures. We thank Miss H. Inoue, Japan Bio-Rad Laboratories Co., for confocal microscope operation. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan.
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27. Rinnerthaler, G., Geiger, B., and Small, J. V. (1988) J. Cell Biol. 106,747-760. 28. Yamamoto, K., Yamamoto, M., Ishi-i, K., Eitoku, H., Ikuta, S., Ishi-i, T., Tomonaga, M., and Ooyama, T. (1990) Artery 17, 213-232.
111,1233-1243. 33. Cheng, Y.-F., Clyman, R. I., Enenstein, 34. 35. 36. 37. 38. 39.
J., Waleh, N., Pytela, R., and Kramer, R. H. (1991) Exp. Cell Res. 194,69-77. Van Gansen, P., Siebertz, B., Capone, B., and Malherbe, L. (1984) Biol. Cell 52, 161-174. Yamada, K. M., Yamada, S. S., and Pastan, I. (1976) Proc. N&l. Acad. Sci. USA 73,1217-1221. Grinnell, F. (1978) Int. Reu. Cytol. 53, 65-144. Henriksen, T., Evensen, S. A., and Carlander, B. (1979) &and. J. Clin. Lab. Invest. 39, 361-368. Wall, R. T., Harlan, J. M., Harker, L. A., and Striker, G. E. (1980) Thromb. Res. 18, 113-121. Klein-Soyer, C., Beretz, A., Millon-Collard, R., Abecassis, J., and Cazenave, J.-P. (1986) Thromb. Haemost. 56, 232-235.
Received December
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40. Gordon, P. B., Levitt, M. A., Jenkins, C. S. P., and Hatcher, V. B. (1984) J. Cell. Physiol. 121, 467-475. 41. Couchman, J. R., Rees, D. A., Green, M. R., and Smith, C. G. (1982) J. Cell Biol. 93, 402-410. 42. Wang, E., Yin, H. L., Krueger, J. G., Caliguiri, L. A., and Tamm,
29.
Yamamoto, M., Fujita, K., Shinkai, T., Yamamoto, K., and Noumura, T. (1992) Exp. Cell Res. 198,43-51. 30. Hall, M. D., Flickinger, K. S., Cutolo, M., Zardi, L., and Culp, L. A. (1988) Exp. Cell Res. 179, 115-136. 31. Burridge, K., Fath, K., Kelly, T., Nuckolls, G., and Turner, C. (1988) Annu. Rev. Cell Biol. 4, 487-525. 32. Kramer, R. H., Cheng, Y.-F., and Clyman, R. (1990) J. Cell Biol.
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I. (1984) J. Cell Biol. 98, 761-771.
43. Grinnell, F., and Feld, M. K. (1979) Cell 17, 117-129. 44. Tarone, G., Cirillo, D., Giancotti, F. G., Comoglio, P. M., and Marchisio, P. C. (1985) Exp. Cell Res. 159, 141-157. 45. Willingham, M. C., Yamada, K. M., Yamada, S. S., Pouyssegur, J., and Pastan, I. (1977) Cell 10, 375-380. 46. Ali, I. U., and Hynes, R. 0. (1977) Biochim. Biophys. Actu 471, 16-24. 47. Kurkinen, M., Wartiovaara, J., and Vaheri, A. (1978) Exp. Cell Res. 111,127-137. 48. Chen, W.-T., Wang, J., Hasegawa, T., Yamada, S. S., and Yamada, K. M. (1986) J. Cell Biol. 103, 1649-1661. 49.
Giancotti, F. G., Comoglio, CellRes. 163,47-62.
50. Carter, (199O)J.
P. M., and Tarone,
W. G., Wayner, E. A., Bouchard, CellBiol. 110,1387-1404.
51. Hynes, R. 0. (1990) in Fibronectin 248, Springer-Verlag,
G. (1986) Exp.
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52. Leushner, J. R. A., and Haust, M. D. (1985) Can. J. Biochem. Cell Biol. 63, 1176-1182.
CELL
RESEARCH
201,55-63
(1992)
Disassembly of F-Actin Filaments in Human Endothelial Cells Cultured on Type V Collagen KIYOTAKA YAMAMOTO,*,’
MARI YAMAMOTO,*~~ AND TETSUO NOUMURA~
*Department of Cell Biology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Ztabashi-ku, Tokyo 173, Japan; and tDepartment of Regulation Biology, Faculty of Science, Saitama University, 255 Shimo-ookubo, Urawa 338, Japan
such vascular diseases as thrombosis and atherosclerosis, since the subendothelium can initiate platelet adhesion and aggregation [ 11. Endothelial cells in culture form a contact-inhibited monolayer [2], synthesize several compounds that are integral to hemostasis, including prostacyclin, heparin-like glycosaminoglycans, plasminogen activator, thrombin, and factor VIII antigen [3], and have been widely used as a model system for studying the mechanisms of vascular diseases [4-71. The extracellular matrix (ECM) which anchors the endothelial cell layer plays an integral role not only in determining endothelial cell polarity, orientation, and morphology, but also in regulating endothelial cell proliferation [8, 91 by releasing biologically active basic fibroblast growth factor [lo, 111. Folkman and Moscona [12] found that the proliferation of endothelial cells is correlated very closely with cell shape. Schor et al. [13] suggested that collagen-containing substrata influence the response of capillary endothelium to tumor angiogenie factor. Collagens in the blood vessel walls serve as key components for maintaining structural integrity and for mediating thrombosis through their interaction with platelets [14]. Proportional changes in type I and type III collagens [15-171 and increases in type V collagen [16, 181 within advanced atherosclerotic plaques have been reported. Type V collagen is an interstitial fibrillar collagen rather than a basement membrane collagen, with a tissue pattern completely different from that of collagen types I, III, and VI or fibronectin [19]. Fukuda et al. [20] found that type V collagen remarkably inhibits the proliferation of human umbilical vein endothelial cells but not that of vascular smooth muscle cells and that it does not affect cell attachment but rather seems to promote cell detachment. The detachment of endothelial cells from the subendothelium and the attachment of platelets and leukocytes to the normally inaccessible subendothelial matrix can lead to thrombosis and atherosclerosis [ 11. Therefore, understanding the factors that control the adhesion of endothelial cells to the substratum is important in a wide variety of vascular disease states. Many cells, including fibroblasts, smooth muscle cells, and endothelial cells, develop focal
We examined the inhibitory activity of type V collagen on cell attachment and cell growth and the role of stress fibers and & integrin in cultured human endothelial cells. Human endothelial cells cultured on type V collagen attached temporarily to the substrate and formed stress fibers. However, the cells failed to proliferate and gradually detached from the substrate. After 24 h, the cells on type V collagen lacked discernible stress fibers (F-actin filaments) and exhibited dots in small aggregates of F-actin. In addition, the cells expressed little or no proteins as focal adhesions, including vinculin and j3i integrin. In contrast, the cells on fibronectin and type I collagen formed complete F-actin filaments, exhibited sufficient vinculin and 8, integrin, and grew logarithmically from 2 days. On the other hand, human smooth muscle cells formed complete Factin filaments, revealed typical focal adhesions, and started to proliferate rapidly after 24 h on type V collagen as well as on fibronectin and type I collagen. Thus, the disassembly of F-actin filaments was observed as a specific phenomenon in human endothelial cells cultured on type V collagen. Moreover, the F-actin filaments disappeared from endothelial cells treated with cytochalasin D after 24 h and the cells detached from fibronectin and type I collagen with time, a result consistent with the observations on type V collagen. Accordingly, the disassembly of F-actin filaments in focal adhesions may result in the detachment of endothelial cells from type V collagen. 0 1992 Academic PWS, 1~.
INTRODUCTION Vascular endothelial cells in uiuo are disposed as a contact-inhibited monolayer on the subendothelium and are directly involved in the maintenance of nonthrombogenic surfaces, as a permeability barrier between blood and subendothelial matrix, and in intimal repair following vascular endothelial injury. Loss of endothelial cells and the subsequent exposure of the subendothelium is an important step of the development of ’ To whom reprint
requests should be addressed. 55
All
Copyright 0 1992 rights of reproduction
0014-4827/92 $5.00 by Academic Press, Inc. in any form reserved.
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YAMAMOTO,
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Coating of culture dishes. Type I and type V collagens (3 mg/ml; Koken, Tokyo) were applied to dishes and air-dried at room temperature. Human plasma fibronectin (HFN, Boehringer-Mannheim) was applied to dishes at a concentration of 20 pg/ml in MCDB 107 or MEM and left for 45 min at room temperature. Before use, these coated dishes were washed twice with each medium. Cellgrowth. HUVE-9 cells (2 X 10’ldish) were cultured in MCDB 107 containing 1% hi-FBS, 0.2% bovine serum albumin (BSA), ECGF (100 rg/ml), recombinant epidermal growth factor (EGF, 10 rig/ml), recombinant basic fibroblast growth factor (b-FGF, 10 rig/ml), recombinant insulin-like growth factor-I (IGF-I, 20 rig/ml), and transferrin (5 pg/ml) (EC medium) in 16.mm Falcon multiple dishes (3047) coated with type I or type V collagen, or HFN at 37°C. HAS-2 cells (2 X lO’/dish) were cultured in MEM containing 1% FBS, 0.2% BSA, recombinant platelet-derived growth factor (PDGF, C-sis, 10 rig/ml), EGF (10 rig/ml), b-FGF (10 rig/ml), IGF-I (20 rig/ml), and transferrin (5 pg/ml) (SMC medium) in 16-mm Falcon dishes coated with type I or type V collagen, or HFN at 37°C. All growth factors were purchased from Boehringer-Mannheim except for PDGF (Amersham). Endothelial cells (2 X Incorporation of BrdU into cellular DNA. 104/dish) were cultured in 16-mm Falcon plastic dishes coated with type I or type V collagen, or HFN in 0.5 ml of EC medium at 37°C. The growth medium was replaced with fresh medium containing a 1:lOOO dilution of 10 mM BrdU and the cells were incubated for 24 h. The cells were washed twice with phosphate-buffered saline (PBS), fixed in ethanol containing 0.1% hydrogen peroxide for 30 min, then washed again in PBS. BrdU incorporation into cellular DNA was
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FIG. 1. Cell growth of both human vascular endothelial cells (a) and smooth muscle cells (b). Cells (2 X 10’) were cultured in medium containing 1% FBS and growth factors (EC or SMC medium) in human plasma fibronectin (HFN)- (O), type I collagen- (0), or type V collagen-coated dishes (A) as described under Materials and Methods. The medium was renewed every 2 or 3 days, and cell numbers were determined with a Coulter Counter. Each point represents the mean of the triplicate cultures. 0, cell growth of endothelial cells on plastic dishes.
adhesions when plated onto appropriate substrates [2124]. The formation of focal adhesions is preceded by a structural precursor consisting of a microspike or bundle of actin filaments oriented radially within the leading edge of a cell [25-271. In this paper, we describe that the assembly and the disassembly of F-actin filaments in focal adhesions after 24 h of culture are related closely to cell attachment and detachment as well as the control of the cell proliferation.
MATERIALS
AND
METHODS
Cell culture. Human umbilical vein endothelial cells (HUVE-9) and human arterial smooth muscle cells (HAS-2) have been described [28]. HUVE-9 cells were cultured in MCDB 107 containing 20% heatinactivated fetal bovine serum (hi-FBS, HyClone Laboratories Inc., lot No. 1111654) and 150 pg of endothelial cell growth factor (ECGF, Culture grade, Boehringer-Mannheim) per milliliter in type I collagen-coated dishes (Corning) at 37°C under humidified 5% CO,-95% air. The growth medium was renewed every 2 or 3 days, and the cells were subcultured at a 1:4 split ratio. The HUVE-9 cell strain had a limited life span of about 50 population doubling levels (PDL) under these culture conditions. In this study, we used the cells at 20-30 PDL. HAS-2 cells were cultured in Eagle’s minimum essential medium (MEM) containing 15% FBS in plastic dishes in the CO, incubator and subcultured at a 1:4 split ratio. The HAS-2 cell strain had a limited life span of about 29 PDL under these conditions and was used at 9-13 PDL in this study. The cells were counted with a hemocytometer or Model ZBI Coulter Counter (Coulter Electronics Inc.). The cells were judged mycoplasma-free by the method described previously [28].
FIG. 2. Morphological characteristics of cells grown on fibronectin, type I collagen, and type V collagen. Cells were cultured in EC or SMC medium for 7 days as described under Materials and Methods. Human endothelial cells reached near confluence on HFN (a) and type I collagen (b), but were detached from type V collagen and remained slightly so (c). Human smooth muscle cells reached near confluence on type V collagen (f) as well as on HFN (d) and type I collagen (e). Bar, 100 pm.
DISASSEMBLY
OF F-ACTIN
FILAMENTS
25
20 “E E 15 Pi 6 =07 10
t
s 5
HFN FIG. 3. BrdU incorporation into cellular DNA of human endothelial cells. Cells (2 X lO*/dish) were cultured in EC medium containing BrdU, fixed, and stained with monoclonal anti-BrdU antibody as described under Materials and Methods. BrdU incorporation into cellular DNA after 24 h (Cl) and 48 h (m) of culture was measured in a constant area (0.321 mm’), and the spread cells after 24 h (0) and 48 h @I) were also counted in the same area. Each point represents the mean of triplicate determinations with the standard deviation indicated as a vertical line.
measured using an immunoperoxidase technique (cell proliferation kit; Amersham) as described previously [29]. The percentage of labeled nuclei was determined by counting more than 200 cells in each experiment. Fluorescence assay. F-actin filaments were stained according to the method of Hall et al. [30]. Endothelial cells and smooth muscle cells (2 X 104) were cultured on Lab-tek chamber glass slides (Nunc, 4804) coated with type I or type V collagen, or HFN in EC medium and SMC medium, respectively, at 37°C. After 24 h, when cell spreading reached a maximum on type I collagen and HFN, the cells were fixed with 3.7% (v/v) paraformaldehyde in PBS for 20 min at room temperature and permeabilized using 1 ml of 0.2% (v/v) Triton X-100 in PBS for 5 min at room temperature. After washing with PBS, the cells were stained with rhodamine phalloidin (2.0 units/ml, Molecular Probes, Inc.) for 1 hat room temperature. Chamber slides were extensively rinsed with PBS, mounted in 50% glycerol-50% PBS, and examined with an Olympus microscope equipped with epifluorescence illumination. The relative fluorescence intensity of cells was measured with a Model OSP-1 Olympus spectrophotometer. For immunocytochemical detection of vinculin and pi integrin, cells fixed as described above were incubated with a 1:50 dilution of monoclonal anti-vinculin antibody (Amersham) and anti-& integrin antibody (Immunotech, Marseille) for 45 min at room temperature, washed, and exposed to a 1:200 dilution of rhodamine-conjugated rabbit anti-mouse IgG (Cappel Products), as described previously [28, 291. Confocal micrographs were taken using a Bio-Rad MRCBOO laser scanning focal imaging system connected to a Nikon X2F-EFD2 microscope.
RESULTS Cell Proliferation on Dishes Coated with Type I Collagen, Type V Collagen, and HFN Human umbilical vein endothelial cells (HUVE-9) attached to the substrate within 0.5-l h after seeding,
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when they were cultured in type I collagen- or HFNcoated dishes. Cell attachment on HFN and type I collagen after 24 h incubation were 75.1 f 5.62 and 78.4 + 4.21%, respectively. Growth curves showed that the cells on HFN-coated dishes and type I collagen-coated dishes started to proliferate on the second day of culture and thereafter grew logarithmically for up to 5 days of culture (Fig. la). The growth curves were similar to those in MCDB 107 containing 20% hi-FBS and 150 pg of ECGF per milliliter on HFN and type I collagen (data not shown). In contrast, the cells cultured on type V collagen temporarily adhered and spread out as well as on HFN and type I collagen, but gradually detached from the substrate. The cell attachment after 24 h was 41.2 + 7.21%. The cells were rounded, detached from type V collagen-coated dishes, and remained slightly so on the seventh day of culture (Figs. la and 2c), when the cell densities reached confluence on HFN and type I collagen (Figs. 2a and 2b). These results under serum deficient conditions (1% FBS) corresponded with the findings of Fukuda et al., which were in MCDB 107 containing a higher concentration of serum [20]. The cells hardly grew on plastic dishes under these conditions but never detached from them (Fig. la). On the other hand, human smooth muscle cells (HAS-2) adhered and spread well on the three substrates. The cells started to proliferate rapidly after 24 h of culture and reached near confluence even on type V collagen-coated dishes after 7 days of culture (Figs. lb and 2f) as well as on HFN- and type I collagen-coated dishes (Figs. lb, 2d, and 2e). These results obtained under conditions that excluded the effect of serum fibro-
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FIG. 4. Growth curve of the cells detached from type V collagen. Human endothelial cells (2 X 104/dish) were cultured in EC medium on type V collagen (a). The detached cells into the medium during 12-36 h of culture were collected and recultured in EC medium on type I collagen (0) and type V collagen (A). Each point represents the mean of triplicate determinations. No differences in growth characteristics between the original cells and detached cells were observed. 0, cells on type I collagen as a control.
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FIG. 5. Localization of F-actin filaments with rhodamine phalloidin in human endothelial cells. Cells (2 X 10’) were cultured in EC medium for 24 h on fibronectin, type I collagen, and type V collagen, fixed with 3.7% paraformaldehyde, and stained with rhodamine phalloidin as described under Materials and Methods. The cells formed complete F-actin filaments both on HFN (a) and type I collagen (b), but not on type V collagen (c). Bar, 25 pm.
nectin within reasonable limits revealed that type V collagen selectively inhibited the growth of human endothelial cells. Figure 3 shows BrdU incorporation into cellular DNA of the endothelial cells after 24 and 48 h of culture. BrdU incorporation into cellular DNA was not observed on any substratum when cultures were incubated for 24 h in medium containing BrdU. After 48 h of culture, the BrdU incorporation into the cellular DNA of spread cells was observed on HFN (12.9 + 9.60%) and type I collagen (14.5 + 11.4%), but not on type V collagen. These results indicated that the endothelial cells which detached from type V collagen within 2 days were resting in G, phase and were not mitotic cells. The endothelial cells in the G, phase that were floating in the medium between 12 and 36 h of culture on type V collagen were then collected and again cultured on type I collagen-coated dishes. These detached cells were reattached, spread, and started to proliferate. No
significant difference in the rate of adhesion and proliferation between the detached cells from type V collagen and the cells cultured originally on type I collagen was observed (Fig. 4). These results indicated that the inhibition of the cell growth on type V collagen was not caused by cell damage, but was rather attributable to some factors that promote cell detachment, and that the endothelial cell detachment was determined by the origin of the extracellular matrix. Reorganization
of F-Actin
Filaments
We examined the reorganization of F-actin filaments of the cells in G, phase under serum deficient conditions. HUVE-9 cells cultured on type V collagen formed stress fibers composed of F-actin filaments after 1 h of culture, but eliminated the actin filaments after 24 h, generating focal pools of F-actin in the cytoplasm (Fig. k), though complete F-actin filaments were observed
FIG. 6. Localization of F-actin filaments in human smooth muscle cells. Cells (2 X 104) were cultured in SMC medium for 24 h and stained with rhodamine phalloidin. Stress fibers in the cytoplasm of the cells on type V collagen (c) as well as on HFN (a) and type I collagen (b) were clearly observed. Bar, 25 Wm.
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Cl SMC
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specific protein of focal adhesions, and & integrin, an integral membrane component identified in focal adhesions, was examined using a laser scanning confocal imaging system. In human endothelial cells cultured for 24 h on type V collagen, ,L3,integrin was not detected though it was present in all the cells on HFN and type I collagen (Fig. 8). The localization of vinculin in the cells
Type V
FIG. ‘7. Fluorescence intensity of F-actin filaments stained with rhodamine phalloidin. Cells were stained with rhodamine phalloidin after 24 h of culture, and the relative fluorescence intensity per cell was measured with a Model OSP-1 spectrophotometer (Olympus). Each column represents the mean and standard deviation of three experiments, determined by counting 50 cells in each experiment.
throughout the cytoplasm of all the cells on both HFN and type I collagen (Figs. 5a and 5b). Despite the absence of the stress fibers, however, these cells demonstrated effective cytoplasmic spreading over type V collagen, presumably by other cytoskeletal networks. In contrast, the cells that detached from type V collagen restored the F-actin filaments when they were recultured on HFN or type I collagen (data not shown). On the other hand, the smooth muscle cells cultured for 24 h on type V collagen as well as on HFN and type I collagen reorganized extensive arrays of stress fibers (Fig. 6). Using microscopic fluorometry, the fluorescence intensity of rhodamine phalloidin per cell was then measured (Fig. 7). In HUVE-9 cultured on HFN and type I collagen, the relative levels of F-actin filaments were higher, namely, 112 f 18.6 and 136 f 28.3, respectively, while on type V collagen, the values were very low (8.11 -t 7.03). On the other hand, HAS-2 cells indicated similarly high relative contents on type V collagen as well as on HFN and type I collagen (114 f 30.5,120 f 41.4, and 122 f 40.1, respectively). The disassembly of F-actin filaments in the endothelial cells cultured for 24 h on type V collagen may have resulted in detaching of endothelial cells from the substrate. Formation of Focal Adhesions and Expression of p, Integrin Focal adhesions are clearly important for anchoring stress fibers to the plasma membrane, but they probably also regulate the assembly and disassembly of the attached actin filaments [31]. The presence of vinculin, a
FIG. 8. Localization of pi integrin in human endothelial cells. Cells were cultured in EC medium, fixed with 3.7% paraformaldehyde, stained with monoclonal anti-b, integrin antibody, then visualized by indirect immunofluorescence as described under Materials and Methods. The & integrin was concentrated on HFN (a) and type I collagen (b), but not on type V collagen (c). Bars, 25 pm.
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1 HFN
YAMAMOTO,
Vinculin 61 integrin
-I
% TYPO 1
Type V
FIG. 9. Fluorescence intensity of vinculin and & integrin in human endothelial cells. Cells were cultured in EC medium, stained with monoclonal antibodies against vinculin and & integrin, and visualized by indirect immunofluorescence. Relative fluorescence intensity per cell was measured with a MRC-600 laser scanning focal imaging system (Bio-Rad). Each column represents the mean and standard deviation of three experiments that were determined by counting 50 cells in each experiment. The cells on type V collagen failed to develop focal adhesions and to concentrate pi integrin in adhesion regions because the cells were little stained by these two antibodies.
was almost the same as that of pi integrin (data not shown), as described previously [24,32,33]. As shown in Fig. 9, the relative levels per cell were higher on HFN and type I collagen, and very low value on type V collagen. After 24 h of culture thus, human vascular smooth muscle cells reorganized F-actin filaments and revealed focal adhesions regardless of differences in substrates, whereas human vascular endothelial cells failed to develop F-actin filaments and to concentrate vinculin and p, integrin, and revealed abnormal adhesions or close contacts on type V collagen. Consequently, the endothelial cells gradually detached from type V collagen and did not subsequently proliferate. Effects of Cytochalasin
AND
NOUMURA
cytoplasm of the cells both on HFN and type I collagen (Figs. llc and lld), though the cells without cytochalasin D formed extensive F-actin filaments (Fig. lla and lib). As shown in Fig. 12, the relative fluorescence intensity of the cells treated with cytochalasin D revealed far lower values (HFN, 9.03 f 8.01; type I collagen, 26.1 + 18.0) than controls without cytochalasin D (HFN, 112 + 16.4; type I collagen, 132 + 30.3) and these levels were similar to that (8.11 f 7.03) on type V collagen. Thus, the cytochalasin D-treated cells, which effectively spread over the substrates at 24 h, revealed close contact or abnormal adhesions, and thereafter detached from the substrates, as observed on type V collagen. Consequently, it is considered that the distribution patterns of F-actin filaments after 24 h of culture determine the subsequent events (cell growth or cell detachment). The difference in the rearrangement of F-actin filaments in the G, phase and in the growth pattern between endothelial cells and smooth muscle cells on type V collagen are of interest in view of the development of vascular diseases. DISCUSSION
Cellular adhesion to and detachment from the substratum involves interaction between constituents of the cell surface and the extracellular matrix [35, 361. Several investigators have examined endothelial cell detachment in vitro under conditions that led to cell lysis and killing [37-391. Gordon et al. [40] have reported that the detachment of human endothelial cells by detaching agents is controlled by the extracellular
I
D
Cytochalasin disrupts actin fibers but microtubules remain intact [34]. Human endothelial cells were cultured on type I collagen and HFN in EC medium containing 5 pg/ml cytochalasin D. The cell growth differed extremely with or without cytochalasin D. The cytochalasin D treatment gradually induced cell detachment from the substrate after 24 h of culture, and the growth profile resembled that of the cells on type V collagen (Fig. 10). After 24 h of culture, furthermore, the F-actin filament fibers of the cells were completely disrupted by cytochalasin D and condensed into focal pools in the
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FIG. 10. Effect of cytochalasin D on the cell growth of human endothelial cells. Cells (2 X 104/dish) were cultured in EC medium containing 5 pg cytochalasin D per milliliter on HFN and type I collagen. Each point represents the mean of triplicate determinations. The cells treated with cytochalasin D gradually detached from HFN (m) and type I collagen (O), though the cells without cytochalasin D rapidly grew on HFN (Cl) and type I collagen (0).
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61
FIG. 11. Effect of cytochalasin D on localization of F-actin filaments in human endothelial cells. Cells were cultured with cytochalasin D and stained with rhodamine phalloidin after 24 h of culture. The cells treated with cytochalasin D did not show F-actin filaments on HFN (c) and type I collagen (d), though control cells without cytochalasin D formed complete F-actin filaments on HFN (a) and type I collagen (h). Bar, 25 pm.
matrices and not by the cells themselves. We showed in the present study that type V collagen selectively inhibited human endothelial cell proliferation, probably promoting cell detachment by the disassemble of F-actin filaments, and that the endothelial cells detached in the G, phase from type V collagen reattached, spread, reorganized the filaments, and started to proliferate when recultured on type I collagen. The growth characteris-
IJ H
Control Cytochalasin
T A-
D
r
-I
HFN
Type 1
FIG. 12. Fluorescence intensity of F-actin filaments of cells treated with cytochalasin D. Cells were cultured and stained with rhodamine phalloidin, and the relative fluorescence intensity per cell was measured as described in the legend to Fig. 7.
tics of the detached cells were essentially the same as those of the cells that were originally cultured on HFN and type I collagen. The phenomenon that the detached cells reattached and continued to proliferate resembles that reported by Gordon et al. [40], using cells detached by specific agents. The growth inhibition of the endothelial cells on type V collagen was not caused by cell damage and is attributed to cell detachment determined by the extracellular matrices. Human endothelial cells, even though the cells could adhere to and spread over type V collagen, disassembled F-actin stress fibers after 24 h of culture and thereafter gradually detached. In contrast, the endothelial cells on HFN and type I collagen and human smooth muscle cells on all three substrates formed complete F-actin filaments after 24 h of culture and started to grow rapidly. Also the disruption of the actin fibers by cytochalasin D inhibited the growth and induced the detachment of the human endothelial cells on HFN or type I collagen. In this way, the assembly and disassembly of F-actin filaments appear to exactly reflect the subsequent growth on the substrate and detachment of cells from the substrate. Cells can adhere to substrates by both focal and close contacts, as classified by Izzard and Lochner [ 221. Many cells, including fibroblasts, smooth muscle cells, and endothelial cells, form focal adhesions when plated onto appropriate substrates [21-241. In contrast, migrating cells initially reveal close contacts rather than focal adhesions and lack stress fibers. With time, the cells de-
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velop focal adhesions and stress fibers [41]. In the focal adhesion regions, the assembly and the disassembly of actin filament stress fibers occurs [42]. In this manner, focal adhesions serve as sites not only to anchor stress fibers to the plasma membrane but also to nucleate actin polymerization. Moreover, they probably regulate the assembly and the disassembly of the attached actin filaments [31]. When cells were plated onto naked glass coverslips in the absence of serum, or on glass coated with nonspecific proteins, such as BSA, normal fibroblasts adhere poorly and fail to develop focal adhesions [43], and transformed cells reveal abnormal rosette adhesions or podosomes [44]. The addition of fibronectin to transformed cells temporarily restored stress fibers [35,45] and the disruption of the actin cytoskeleton resulted in a loss of fibronectin from the cell surface [46, 471. These results indicate the existence of a transmembrane link between the ECM and the cytoskeleton. Perhaps related to this lack of requirement for an ECM, integrin is not usually concentrated within these abnormal adhesions [48, 491. Carter et al. [50] reported that the relocation of integrin in neonatal human foreskin keratinocytes may result in detachment of cells from the membrane basement zone. In the present study, human vascular endothelial cells temporarily attached to type V collagen and assembled F-actin filaments. However, the cells disassembled actin filaments after 24 h on type V collagen, failed to develop focal adhesions and to concentrate /3,-integrin in adhesion regions, and detached from type V collagen, though focal adhesions were found and & integrin was concentrated on HFN and type I collagen. In addition, the cells that detached from type V collagen immediately restored F-actin stress fibers when transferred to HFN or type I collagen. Taken together, these results suggest that the ECM-integrin-actin filament system was reversed by the ECM and that the relocation of integrin following the disassembly of the actin filaments may occur and result in detachment of the endothelial cells on type V collagen. The simplest model of an ECM-integrin-cytoskeleton system is that binding of extracellular ligand produces a conformational change in the integrin receptor, which favors interaction with cytoplasmic (cytoskeletal?) components and that both external and internal interactions are necessary for stable assembly of integrins into focal contacts 1511. Finding integrins concentrated at adhesion sites has drawn attention to these ECM receptors as critical elements in the transmembrane link between the ECM and the cytoskeleton. The interaction between rabbit aortic SMC and type V collagen was originally shown to be mediated by a cell-surface type V collagen receptor. The binding system consists of integral membrane glycoproteins (80,000 and 50,000) which act alone or in combination with a surface glycolipid in type V collagen attachment [52]. Taken
AND
NOUMURA
together, human endothelial cells may lack these type V collagen receptor components on the cell surface, may use other molecules as type V collagen receptor(s), but may relocate them with time. Additionally, not only ECM receptors but also ECM itself may serve as a regulator in focal adhesions. We thank Drs. K. Okumura and M. Kihara, National Institute of Health, Tokyo, and Dr. M. Takeuchi, Institute for Fermentation, Osaka, for mycoplasma testing of the cultures. We thank Miss H. Inoue, Japan Bio-Rad Laboratories Co., for confocal microscope operation. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan.
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