Cell,
Vol. 14, 501-509,
July
1978. Copyright
0 1978 by MIT
The Isolation of Vascular with Altered Cell Surface Properties Bruce R. Zetter, Lorin K. Johnson, Mark A. Shuman and Denis Gospodarowicz Endocrine Research Division Cancer Research Institute and Department of Medicine University of California Medical Center San Francisco, California 94143
Summary The vascular endothelium consists of a single layer of flattened, contact-inhibited cells that line the inner surface of blood vessels. One important characteristic of these cells is that circulating blood cells should not adhere or become activated by contact with the endothelial cell surface that is exposed to the bloodstream. This nonthrombogenic surface is a characteristic not only of the endothelium in vivo, but also of cultured endothelial cells such as the clonal line of adult bovine aortic endothelium used in this study. Using the mutagen 2-chloroacetaldehyde, we have been able to isolate cell lines of endothelial origin which have stable alterations in morphology and in the ability to bind platelets to the upper cell surface. Other cell surface properties that are altered in these cells include the distribution of a major cell surface glycoprotein (LETS, fibronectin), the response to incubation with a multivalent lectin (concanavalin A) and the distribution of iodinatable cell surface proteins. The binding of platelets to the endothelial cell variants can be quantified by radioisotope labeling of the platelets and measurement of the amount of label on cell-associated platelets. When platelets were incubated with “C-serotonin prior to incubation with the variant endothelial cells, it was found that the binding of platelets to these cells was not accompanied by release of serotonin from platelet granules. This is in contrast to the release reported when platelets are allowed to adhere to subendothelial vessel components, and demonstrates that adhesion to endothelial surfaces is dissociable from the platelet release reaction. The use of such endothelial cell variants should allow elucidation of the biochemical properties of the normal endothelial cell surface which render it nonthrombogenic. Introduction The vascular endothelium forms the lining of the inner surface of blood vessels and consists of cells that have a specialized morphology and function (French 1971). These endothelial cells form a contact-inhibited monolayer of highly flattened cells that present an inert nonthrombogenic surface to
Endothelial Cell Lines and Platelet-Binding the bloodstream and thus ensure that circulating blood cells do not adhere to the walls of blood vessels (Spaet and Stemmerman 1972; Spaet 1977). These properties of the endothelial cell layer are critical for the normal functioning of the blood vessels, and it has been proposed that alterations in the endothelial cell layer could lead to an increased incidence of thrombosis, as well as to platelet-vessel interactions that might have a role in the early stages of atherogenesis (Ross et al., 1977). This report describes the isolation of endothelial cell lines which exhibit stable alterations in cell morphology and have lost the contact inhibition of growth and nonthrombogenic surface characteristic of normal endothelial cells. These cell lines, isolated after treatment with the mutagen 2-chloroacetaldehyde (Huberman, Bartsch and Sachs, 1975; McCann et al., 1975; Malaveille et al., 1975; Elmore et 1976), display cell surface properties that differ from the parental endothelial cell line in several significant ways, including the alteration of cell-cell interactions, membrane mobility, distribution of a large cell surface glycoprotein and adhesiveness to circulating blood cells. These cells therefore provide an excellent model system for the elucidation of the nonthrombogenic nature of the endothelial cell layer. Results Following treatment of cultured vascular endothelial cells with the mutagen 2-chloroacetaldehyde, we observed that colonies of cells arose which had lost the characteristic morphology and contactinhibited growth pattern of normal endothelial cells. After sequential selection for cells capable of overgrowth as described in Experimental Procedures, we have been able to establish long-term cell lines which maintain certain alterations in morphology, cell-cell interactions and cell surface properties. To evaluate properly drug-induced changes in cell type, it is necessary to start with a homogeneous parental cell population. We have therefore chosen the previously described clonal line of adult bovine aortic endothelial cells (ABAE) (Gospodarowicz et al., 1976; Gospodarowicz, Moran and Braun, 1977) for use as the parental stock. These cells display the following characteristics of normal endothelial cells: contact inhibition of growth, a nonthrombogenic surface, production of factor VIII antigen (Jaffe, Hoyer and Nachman, 1973), and a dependence on the fibroblast growth factor (FGF) (Gospodarowicz 1974, 1975) for growth and survival in culture. This report describes the characteristic of one stably altered cell line isolated after
Cell 502
treatment with 2-chloroacetaldehyde that has been altered in each of the above properties. The variant cell line (ABAE-CA,) has now been cultivated inthe laboratory for more than one year. As shown in Table 1, the cells are responsive to the mitogenic activity of FGF and can be grown from low density (320 cells per cm*) in medium containing 10% calf serum and 100 rig/ml FGF. No significant differences have been found between the parental and variant cells with respect to serum dependence or to the growth response to FGF. Whereas the parental cells will maintain their normal monolayered morphology only when cultured with FGF, the CA, variants maintain their altered morphology in either the presence or absence of FGF. In either case, these cells are larger and somewhat more elongated than the parental cells and fail to grow as an ordered monolayer, but rather show extensive cytoplasmic overlapping (Figure 1). This altered morphology has been maintained continuously for more than 70 generations, and no cultures have yet reverted to the normal contact-inhibited morphology. Table
1. Growth
of Normal
(ABAE)
and Variant
(ABAE-CA,)
To determine whether the variant cells had lost any of the specific characteristics of endothelial cells other than morphology, we tested both the parental and variant cells for the presence of the factor VIII antigen. Figure 2 demonstrates that although the parental cells express this antigen, the CA, variant cells do not retain this ability. Since the parental cell line was isolated as a single cell clone, it is not possible that the variant represents a contaminating nonendothelial cell type isolated by the selection procedure, but must rather be an endothelial derived cell line that has lost certain of the differentiated functions of normal endothelial cells. Since cell-cell interactions such as those which regulate contact inhibition of growth must be mediated at the cell surface, we have examined the normal and variant cell lines for differences in the distribution of cell surface proteins. Following lactoperoxidase-catalyzed iodination with 13’1, cell surface proteins from the normal or variant endothelial cells were subjected to slab gel electrophoresis and autoradiography. As shown in Figure 3,
Endothelial
Cells
and Absence
of FGF
ABAE-CA1
ABAE Serum
in the Presence
Concentration
w 0.25 2.50 10.0
-FGF
+FGF
6.6 + 0.12=
-FGF
7.6 + 0.31
7.2 + 0.36
6.8 + 0.34
11.4 + 0.11
40.5
+ 1.80
9.1 + 0.54
34.9 + 0.35
17.6 + 0.17
106.0
+ 5.30
15.3 + 0.78
92.0 + 2.84
a Cell number x IO-’ + standard error. 2 x 10’ cells were seeded in 8 cm tissue culture dishes in medium containing 10% calf serum. After fresh medium containing the indicated serum concentration with or without 100 nglml FGF. Triplicate
Figure 1. Morphology (Magnification 150x)
+FGF
of (A) Normal
Vascular
Endothelial
Cells
and
(9)
2-Chloroacetaldehyde-Induced
18 hr, the medium was replaced with cultures were counted after 10 days.
Variant
Cell
Line
(ABAE-CA,)
Endothelial 503
Cell Surface
Variants
when cultures were grown to confluence and maintained in culture for an additional 7 days, the distribution of cell surface proteins in the two cell types differed significantly. In comparison with the parental cell line, the CA, variants have decreased concentrations of four proteins with molecular weights of 220,000, 177,000, 150,000 and 26,000 daltons, respectively. In addition, the variant cells have increased concentrations of two high molecular weight proteins (400,000 and 285,000 daltons). With the exception of the 26,000 dalton protein, these patterns do not reflect cell surface differences between growing and resting cells (I. Vlodavsky, L. K. Johnson and D. Gospodarowicz, unpublished results). One particular protein which is prominent on the surface of many cell types is a high molecular weight (220,000 daltons) glycoprotein termed fibro-
400K 285K 220K+ 177K+ 15OK-,
26K+
Figure 2. Determination of Factor VIII Normal and Variant Endothelial Cells
Antigen
in Cultures
of
(A) Phase-contrast micrograph of the variant ABAE-CA, cells (mangification 400x). (8) Fluorescence micrograph of the same field shown in .(A). No factor VIII antigen is detectable in the variant cells. (C) Fluorescence micrograph of parental ABAE endothelial cells showing abundant intracellular factor VII antigen.
Figure 3. Distribution of lodinatable Normal and CA, Variant Endothelial
Cell Cells
Surface
Proteins
from
Confluent cultures of (A) normal and (8) CA, variant endothelial cells were labeled by lactoperoxidase-catalyzed iodination with ‘3’l and subjected to electrophoresis on 515% exponential gradient SDS-polyacrylamide slab gels.
Cell 504
nectin or LETS protein (for a review, see Hynes 1976; Vaheri et al., 1976). This protein has been implicated as a major factor in cell substratum adhesion (Yamada, Yamada and Pastan, 1976; Ali et al., 1977) and in the maintenance of cell-cell contacts (Yamada, Yamada and Pastan, 1975; Chen, Gallimore and McDougall, 1976; Zetter, Chen and Buchanan, 1976). At low cell densitities, relatively small amounts of this protein can be detected after iodination of either cell type (not shown). When confluent cultures were tested, however, significantly greater amounts were expressed by the parental cells (see Figure 3, band at 220,000). To determine the distribution of the relatively smaller amounts of LETS protein on the surface of cells in sparse culture, we have used a highly sensitive immunofluorescent staining technique (Ruoslahti et al., 1973). As shown in Figure 4, there is a striking difference between the LETS protein distribution on the two cell types. As demonstrated by Birdwell, Gospodarowicz and Nicolson (1978), the contact-inhibited parental cells deposit LETS only onto the plastic dish and not onto the surface of the cells themselves. The importance of cell-cell contacts for the depositing of LETS is evident in subconfluent cultures of these cells, since there is no evidence of LETS protein on isolated cells but only on cells in contact with others. In contrast, the variant cells that are capable of overgrowth can anchor the protein to their surfaces in a pattern corresponding to the distribution of stress fibers visible under phase microscopy. Here, totally isolated cells anchor LETS to their surface. The difference between the parental and variant
Figure
4. Distribution
of LETS
Protein
Subconfluent normal endothelial cells their cell surfaces even when completely
on Normal
and Variant
Endothelial
cell lines in the distribution of LETS would indicate that the CA, cells have lost the polarity between the upper and lower cell surfaces that is so important for the normal functioning of the endothelium (Birdwell et al., 1978; Gospodarowicz et al., 1978). This is especially evident in the distribution of cell surface LETS protein on the well spread variant cell shown in Figure 5. Here, the LETS protein is located on surface-associated fibers that enclose the cell and describe its three-dimensional appearance. Alterations in the cell surface composition of a given cell type will often be reflected by a change in the mobility of receptors localized in the cell membrane. When FITC-conjugated concanavalin A (ConA) was incubated with suspensions of the parental aortic endothelial cell, the fluorescence was found to be localized in discrete areas (caps) on the cell surface. Capping was observed on 72% of the ABAE cells. On the other hand, when the same experiment was performed with the CA, cells, the fluorescence was distributed more evenly over the entire cell surface (Figure 6), and caps were seen on only 6% of all cells counted. This result implies that membrane mobility is greater in the normal cells than in the altered line. One of the most intriguing aspects of endothelial cell biology is the nonthrombogenic nature of the endothelial cell surface exposed to the bloodstream. Circulating platelets will not interact with a vessel wall unless it has been in some way injured or altered as in inflammation, wounding or the early stages of atherosclerosis. We have therefore examined the ability of the parental and variant cells to bind washed human platelets by incubating
Cells
(A) deposit LETS protein in areas of cell-cell contact, isolated from other cells (magnification 400x).
whereas
CA, variants
(B) have
deposits
on
Endothelial 505
Figure
Cell Surface
5. Distribution
Variants
of LETS Protein
on CA, Cell
The disruption of the normal bipolarity of the endothelial of this well spread variant cell (magnification 630x).
Figure
6. Distribution
of Fluorescein-Conjugated
ConA
cell surface
on Normal
is reflected
and Variant
in the deposits
Endothelial
of LETS on fibers
the surface
Cells
Cell suspensions were incubated for 15 min at 37°C with 100 pglml fluorescein-conjugated ConA, fluorescence microscopy. Capping of ConA receptors is observed on the normal endothelial distributed more randomly on the surface of the variant cell (B) (magnification 630x).
cultures of each cell type with 2 x lOa fresh platelets for 30 min at 37°C. After washing the cultures 10 times in medium containing 0.25% BSA, it was found that only the variant cells have platelets bound tightly to their upper surfaces (Figure 7). The platelets used for these studies were
surrounding
washed with PBS and observed under cell (A), whereas the fluorescence is
normal by the criteria of morphology (discoidal) and the ability to take up 14C-serotonin and to secrete it in response to specific stimuli such as thrombin or collagen. It has been previously demonstrated that when large blood vessels are denuded of their endothe-
Cell 506
hum by mechanical or hydrolytic treatment, platelets will adhere to the newly exposed subendothelial surface. When this occurs, the platelets change shape, release materials from cytoplasmic granules and coalesce into large aggregates (for review, see Weiss et al., 1977). As shown in Figure 7, however, the platelets adhering to the CA, cells attach singly and do not form aggregates. To determine whether the attached platelets undergo the platelet-release reaction, we preincubated the platelets with 14Cserotonin and then incubated the labeled platelets with normal and altered endothelial cells. After 30 min at 37”C, the culture medium containing unattached platelets and any serotonin released during binding was removed, and the platelets were col-
Figure
7. Adherence
2 x 10’ platelets washed 10 times Table
2. Binding
of Platelets
in 1 ml culture and observed of Platelets
to Normal
to Normal
Endothelial
Cells
0.25% BSA were incubated microscopy (magnification
and CA, Variant
Endothelial
ABAE
650
ABAE-CA, ABAE-CA1
+ 0.5 pg/ml
Trypsin
ABAE-CA,
+ Anti-LETS
Antiserum
(+)
with normal 150x).
(A) and variant
CA, (B) cells for 30 min at 37”C,
Cells
Cell-Bound Platelets (cpm “C-Serotonin)
Cell Type
No Cells
and Variant
medium containing under phase-contrast
lected by centrifugation. The cells and attached platelets were washed 10 times with phosphatebuffered saline and then hydrolyzed in 0.2 N NaOH. The cells, platelets and platelet-free supernatant were counted separately to determine the distribution of the “C-serotonin. As shown in Table 2, normal (ABAE) endothelial cells bind very few platelets (0.5 platelets per cell), whereas the altered CA, cells bind platelets over their entire upper cell surface (12-15 platelets per cell). In neither case is any serotonin release seen beyond that spontaneously released by platelets incubated at 37°C for 30 min in the absence of cells. The capability of the platelets to release 14C-serotonin under appropriate conditions can be demonstrated by adding
Released
“C-Serotonin
(cm 5670
11,760
5666
9,306
5026
11,560
5692
-
5653*
2 x lOa platelets (79,270 cpm) were incubated with confluent cell cultures for 30 min at 3PC. The binding of platelets to cells and the release of “C-serotonin from platelets were determined as described in Experimental Procedures. The amount of serotonin released by the platelets was in all cases equivalent to that spontaneously released by platelets during the time course of the experiment (*). Treatment of 2 x lo* platelets with 10 units par ml human thrombin caused release of 63,760 cpm of ‘.C-serotonin. (+) Cells were preincubated for 1 hr with serial dilutions of the anti-LETS antiserum (1:2 to 1:512) with no effect on platelet binding or release.
Endothelial
Cell Surface
Variants
507
thrombin (1 unit per ml) to the dish containing the cells and platelets. Under these conditions, 7090% of the counts will be released into the culture medium. Discussion The vascular endothelial cells that line the inner surfaces of blood vessels have several interesting properties that are amenable to study in tissue culture, and in recent years, considerable progress has been made in propagating cultured cell lines that maintain the properties characteristic of the vascular endothelium in vivo. Most of these cell lines demonstrate contact inhibition of growth and have been found to produce factor VIII antigen, prostaglandin 12, Weibel-Palade bodies and angiotensin-converting factor (for review, see Gimbrone, 1976). One of the most intriguing aspects of endothelial cells is the bifunctional nature of their cell surfaces. One side of these cells is attached to a basement membrane and functions to keep the cells in a flattened configuration tightly adherent to the vessel wall. This flattened monolayer configuration serves to prevent the cells from occluding the vessel lumen through which the blood flows. Tight adherence to the vessel wall is also essential in face of the large shear force to which these cells are subjected. On the other hand, the side of the endothelial cells that is exposed to the flowing blood stream must present a surface that is relatively inert and resists adhesion by circulating blood cells such as platelets and leukocytes. This bipolarity of the endothelial cell surface is reflected in the distribution of the LETS protein, which has been shown to be deposited by normal endothelial cells only on the side at which the cells adhere to a substratum and not on the surface exposed to the culture fluid (Birdwell et al., 1978). Since this protein has been implicated in cell substratum adhesiveness (Yamada et al., 1976; Ali et al., 1977), it is conceivable that it has a role in maintaining the tight contact between the endothelium and the basal lamina to which it is attached. To study the cell surface biology of vascular endothelial cells more effectively, it would be advantageous to develop mutant cell lines having lesions in specific cell surface properties. Although one virally transformed endothelial cell line has been described that possesses altered growth properties and lacks some of the differentiated properties of normal endothelial cells (Gimbrone and Fareed 1976), no variant cell lines have yet been isolated which are altered with respect to the ability to interact with circulating blood cells. Through the use of a mutagen, 2-chloroacetalde-
hyde, we have been able to isolate endothelial cell lines with stable alterations that result in a loss of the thrombogenic cell surface. The altered cell line (ABAE-CA,) described in this report was isolated on the basis of its ability to undergo extensive cellular overlapping in dense cultures, unlike the normal endothelial cells which always form a flattened monolayer. The morphology of these altered cells has remained constant for more than 70 population doublings in vitro, and in no case have the cells reverted to the parental morphology. It is important to note that the parental cell line used to isolate this variant was originated as a single cell clone and therefore contained no contaminating cell types that could have been isolated in the selection procedure. Since every cell in the parental stock is an endothelial cell, the variants must be of endothelial derivation. When iodinated cell surface proteins from the two cell types are compared on SDS-polyacrylamide slab gel electrophoresis, the gel patterns are basically similar, but several specific differences can be noted. In comparison with the parental cell line, the CA, variants have an increased concentration of two high molecular weight proteins (400,000 and 285,000 daltons) and a decrease in the amount of at least four other bands having apparent molecular weights of 220,000, 177,000, 150,000 and 26,000 daltons. That these cell surface changes are reflected in altered surface properties has been shown in three ways. First, it was shown that whereas the parental cells can mobilize receptors for ConA to form caps, the altered cells display a more uniform distribution of ConA over the entire cell surface. This would suggest that the membrane fluidity of the CA, cells is restricted in comparison to that of the normal cells. Second, when the expression of a high molecular weight cell. surface glycoprotein (LETS, fibronectin) was compared by immunofluorescent staining of the two cell types, the distribution of the protein was found to be markedly different. In contrast to the normal cells that deposit LETS only onto the surface attached to the substratum, the CA, cells deposit the protein on both the upper and lower cell surface. One might infer that the bipolar nature of the endothelial cell surface on these cells has been disrupted. Finally, only the altered cells possess the ability to bind platelets, whereas the normal cells present a nonthrombogenic surface similar to that which is characteristic of the vascular endothelium in vivo. Although there has been considerable study of the interaction of platelets with the subendothelial matrix of blood vessels (for a review, see Weiss et al., 1977), the properties of the endothelial cell surface that render it nonthrombogenic are rela-
Cdl 508
tively unexplored. We believe that the CA, cell line should provide a valuable model for investigating the interactions between the inner surfaces of blood vessels and the circulating blood cells. In this regard, it is of particular interest that the platelets which firmly attach to the surface of the CA, cells do not aggregate or release the contents of cytoplasmic granules as they do when they attach to the subendothelial matrix (Weiss et al., 1977). It remains to be determined whether the long-term contact of platelets with endothelial cells has deleterious effects for either cell type. Although it would be tempting to correlate the altered distribution of LETS protein in the CA, cells with their ability to bind platelets, two pieces of evidence indicate that this is not the case. First, incubation of CA, cells with a concentration of trypsin sufficient to remove all surface-associated LETS (as determined by immunofluorescent staining) did not reduce the binding of platelets to the cells. Second, preincubation of the CA, cells with antiserum against the LETS protein also failed to reduce the number of platelets bound to the cells. The altered distribution of LETS may, however, reflect a more general cell surface modification that is in itself responsible for the interactions with platelets. The ability of 2-chloroacetaldehyde to cause stable changes in the properties of vascular endothelial cells is of particular interest in view of the finding that this compound is a metabolite of the carcinogen vinyl chloride (Barbin et al., 1975; Kappus et al., 1976). One of the special properties of this carcinogen is the ability to induce endothelial cell tumors (malignant hemangioendotheliomas) (Creech and Johnson, 1974; Maltoni and Lefemine 1974; Stewart ‘1976; Infante, Wagoner and Waxweiler, 1976), and although the data presented here do not provide direct evidence that 2-chloroacetaldehyde is responsible for vinyl chloride carcinogenesis, they do demonstrate an interesting ability on the part of a carcinogenic metabolite to affect the target cell of the carcinogen. The variant cell line described here shares some features with the virally transformed endothelial cell line isolated by Gimbrone and Fareed (1976), such as the loss of contact inhibition and the cessation of factor VIII antigen production as revealed by indirect immunofluorescence. The cells do, however, retain some properties of normal endothelial cells-for example, the ability to respond to the mitogenic activity of FGF and a requirement for normal concentrations (5-10%) of calf serum in the growth medium. In this respect, the cells are similar to revertants of transformed cells that have lost some, but not all, of the dissociable parameters of transformation (Vogel and
Pollack 1973; Pollack and Risser 1974). It is possible that not all of the cell lines isolated following 2chloroacetaldehyde treatment will share all of the same properties. To date, however, each has demonstrated the ability to overgrow, as well as the ability to bind platelets. The altered endothelial cells described here should have several uses for the further study of vascular cell biology. Further biochemical elucidation of the cell surface differences between these cells and the normal endothelium may help to elucidate the loss of contact inhibition in one of the few cell types to display this kind of control in vivo. In addition, the ability of these cells to bind platelets provides the first model system for studying the nonthrombogenic properties of the vascular endothelium. Such cells could conceivably be of use as an assay for pathological conditions in which the adhesive properties of platelets have been altered. Experimental
Procedures
Isolation of Variant Endothelial Cell Lines Cultures of the parental endothelial cells (ABAE) were grown to subconfluence in 6 cm Falcon tissue culture dishes in Dulbecco’s modified Eagle’s medium with 10% calf serum and 100 rig/ml FGF, and then treated for 48 hr with 5-20 PM 2-chloroacetaldehyde. The cultures were than washed twice and maintained in fresh medium containing serum only. Following this treatment, the cells grew to a confluent monolayer, and in certain areas, colonies of overlapping cells appeared. It was subsequently found that the cells in these colonies were more rapidly detached by trypsinization than the cells that were attached directly to the plate and could therefore be selectively isolated by short treatment (1 min) with 0.25% trypsin in phosphate-buffered saline (PBS). The subcultures were grown to confluence and passaged repeatedly until cultures were obtained in which all the cells were capable of overlapping. One such cell line which is described in this report has been designated “ABAE-CA,.” lmmunofluorescent Staining Cultures to be assayed for the presence of LETS protein were grown on 12 mm glass coverslips and fixed in 3.7% formaldehyde. Indirect immunofluorescence was performed according to the procedure described by Chen et al. (1976) using antiserum provided by Dr. L. B. Chen. The properties of this monospecific antiserum, originally prepared against the antigenically identical cold-insoluble globulin (GIG) by Mosseson and Umfleet (1970), have been described by Chen et al. (1976) and by Burridge (1976). Cultures to be assayed for the presence of factor VIII antigen were treated as described by Jaffe (1977), using antiserum against bovine factor VIII antigen provided by Dr. J. Brown. The antiserum to LETS protein was used at a dilution of 1:80, and the antiserum to factor VIII antigen was used at a dilution of 1:200. Fluorescence was observed using a Leitz Orthoplan microscope under epiillumination and photographed with Kodak Tri-X film using a Leitz Orthomat camera. In all cases where positive fluorescence was observed, the specificity of the staining was determined by control experiments in which nonimmune rabbit serum was used in place of the anti-CIG antiserum. Treatment of Cells with Fluorescein-Conjugated ConA Cells were removed from the culture dish with 0.03% EDTA, centrifuged and resuspended in PBS. They were then incubated
Endothelial
Cell Surface
Variants
509
for 15 min at 37°C with 100 pg/ml FITC-conjugated ConA. Following this incubation, the cells were washed with PBS, pipetted to dissociated aggregates and placed on glass microscope slides for observation by fluorescent microscopy. Lactoperoxidase-Catalyzed lodination and Slab Gel Electrophoresis of Cell Surface Proteins Monolayer cultures of ABAE and ABAE-CA, were iodinated with 13’1 according to the method of Teng and Chen (1976). The cells were subsequently solubilized in sample buffer containing 2 mM PMSF and 1% 2-mercaptoethanol, and subjected to SDS-poly acrylamide gel electrophoresis on gels prepared with a 5-15% exponential gradient of acrylamide. Autoradiograms of dried gels were made using Kodak NS-2T X-ray film and developed with Kodak D-19 developer. Binding of Platelets to Cultured Endothelial Cells Platelets were prepared from 60-120 ml human whole blood according to the procedure described by Tollefsen, Feagler and Majerus (1976). To observe platelet binding, 2 x lo* platelets were added to confluent cultures of cells in 35 mm culture dishes containing 1 ml of Dulbecco’s modified Eagle’s medium with 0.25% bovine serum albumin (BSA) and incubated for 30 min at 37°C in a humidifed CO, incubator. Following incubation, the cultures were washed 10 times with the same medium and viewed under phase microscopy. For quantification of platelet binding and serotonin release from platelet granules, the platelets were preincubated for 30 min with “C-serotonin (0.2 &i/ml). After incubation of the labeled platelets with the cells, the culture medium was removed and the platelets were collected by centrifugation. The platelets were then hydrolyzed in 0.2 M NaOH, as were the cells with bound platelets remaining on the culture dish. The three fractions-platelets, cells and the platelet-free supernatant -were then counted separately in Bray’s solution using a Beckman LS 8000 scintillation counter. Acknowledgments
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Yamada, K. M., Yamada, S. S. and Acad. Sci. USA 72, 3158-3162.
Pastan,
I. (1975).
Proc.
Nat.
Yamada, K. M., Yamada, S. S. and Pastan, Acad. Sci. USA 73, 1217-1221.
I. (1976).
Proc.
Nat.
Zetter, 412.
Sci. USA 73, 4457-4461.
Elmore, J. D.. Wong. J. L., Laumbach, A. D. and (1976). Biochim. Biophys. Acta 442, 405-419. Gimbrone,
Cell 71,
A., Jacquignon, P., Malaveille, H. (1975). Biochim. Biophys.
Birdwell, C. R., Gospodarowicz, Nat. Acad. Sci. USA, in press.
York:
Vaheri, A., Ruoslahti, E., Linder, E., Wartiovaara, J., Keski-Oja, J.. Kuusela, P. and Saksela, 0. (1976). J. Supramol. Structure 4, 63-70.
April 3, 1978
R. and Hynes,
ed. (New
M. A., Jr. and Fareed,
Gospodarowicz,
Vogel,
Burridge,
3, T. H. Spaet.
Ruoslahti, Biochim.
We wish to thank Dr. L. B. Chen for a generous gift of antiserum to LETS protein (CIG); M. Botney for the preparation of washed human platelets and “C-serotonin-labeled human platelets; and Dr. J. Brown for antiserum to bovine factor VIII antigen. This work was supported by a grant from the NIH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
bosis,
Gimbrone.
B. R., Chen,
L. B. and Buchanan,
J. M. (1976).
V.
Cell 7, 407-
Vol. 14, 501-509,
July
1978. Copyright
0 1978 by MIT
The Isolation of Vascular with Altered Cell Surface Properties Bruce R. Zetter, Lorin K. Johnson, Mark A. Shuman and Denis Gospodarowicz Endocrine Research Division Cancer Research Institute and Department of Medicine University of California Medical Center San Francisco, California 94143
Summary The vascular endothelium consists of a single layer of flattened, contact-inhibited cells that line the inner surface of blood vessels. One important characteristic of these cells is that circulating blood cells should not adhere or become activated by contact with the endothelial cell surface that is exposed to the bloodstream. This nonthrombogenic surface is a characteristic not only of the endothelium in vivo, but also of cultured endothelial cells such as the clonal line of adult bovine aortic endothelium used in this study. Using the mutagen 2-chloroacetaldehyde, we have been able to isolate cell lines of endothelial origin which have stable alterations in morphology and in the ability to bind platelets to the upper cell surface. Other cell surface properties that are altered in these cells include the distribution of a major cell surface glycoprotein (LETS, fibronectin), the response to incubation with a multivalent lectin (concanavalin A) and the distribution of iodinatable cell surface proteins. The binding of platelets to the endothelial cell variants can be quantified by radioisotope labeling of the platelets and measurement of the amount of label on cell-associated platelets. When platelets were incubated with “C-serotonin prior to incubation with the variant endothelial cells, it was found that the binding of platelets to these cells was not accompanied by release of serotonin from platelet granules. This is in contrast to the release reported when platelets are allowed to adhere to subendothelial vessel components, and demonstrates that adhesion to endothelial surfaces is dissociable from the platelet release reaction. The use of such endothelial cell variants should allow elucidation of the biochemical properties of the normal endothelial cell surface which render it nonthrombogenic. Introduction The vascular endothelium forms the lining of the inner surface of blood vessels and consists of cells that have a specialized morphology and function (French 1971). These endothelial cells form a contact-inhibited monolayer of highly flattened cells that present an inert nonthrombogenic surface to
Endothelial Cell Lines and Platelet-Binding the bloodstream and thus ensure that circulating blood cells do not adhere to the walls of blood vessels (Spaet and Stemmerman 1972; Spaet 1977). These properties of the endothelial cell layer are critical for the normal functioning of the blood vessels, and it has been proposed that alterations in the endothelial cell layer could lead to an increased incidence of thrombosis, as well as to platelet-vessel interactions that might have a role in the early stages of atherogenesis (Ross et al., 1977). This report describes the isolation of endothelial cell lines which exhibit stable alterations in cell morphology and have lost the contact inhibition of growth and nonthrombogenic surface characteristic of normal endothelial cells. These cell lines, isolated after treatment with the mutagen 2-chloroacetaldehyde (Huberman, Bartsch and Sachs, 1975; McCann et al., 1975; Malaveille et al., 1975; Elmore et 1976), display cell surface properties that differ from the parental endothelial cell line in several significant ways, including the alteration of cell-cell interactions, membrane mobility, distribution of a large cell surface glycoprotein and adhesiveness to circulating blood cells. These cells therefore provide an excellent model system for the elucidation of the nonthrombogenic nature of the endothelial cell layer. Results Following treatment of cultured vascular endothelial cells with the mutagen 2-chloroacetaldehyde, we observed that colonies of cells arose which had lost the characteristic morphology and contactinhibited growth pattern of normal endothelial cells. After sequential selection for cells capable of overgrowth as described in Experimental Procedures, we have been able to establish long-term cell lines which maintain certain alterations in morphology, cell-cell interactions and cell surface properties. To evaluate properly drug-induced changes in cell type, it is necessary to start with a homogeneous parental cell population. We have therefore chosen the previously described clonal line of adult bovine aortic endothelial cells (ABAE) (Gospodarowicz et al., 1976; Gospodarowicz, Moran and Braun, 1977) for use as the parental stock. These cells display the following characteristics of normal endothelial cells: contact inhibition of growth, a nonthrombogenic surface, production of factor VIII antigen (Jaffe, Hoyer and Nachman, 1973), and a dependence on the fibroblast growth factor (FGF) (Gospodarowicz 1974, 1975) for growth and survival in culture. This report describes the characteristic of one stably altered cell line isolated after
Cell 502
treatment with 2-chloroacetaldehyde that has been altered in each of the above properties. The variant cell line (ABAE-CA,) has now been cultivated inthe laboratory for more than one year. As shown in Table 1, the cells are responsive to the mitogenic activity of FGF and can be grown from low density (320 cells per cm*) in medium containing 10% calf serum and 100 rig/ml FGF. No significant differences have been found between the parental and variant cells with respect to serum dependence or to the growth response to FGF. Whereas the parental cells will maintain their normal monolayered morphology only when cultured with FGF, the CA, variants maintain their altered morphology in either the presence or absence of FGF. In either case, these cells are larger and somewhat more elongated than the parental cells and fail to grow as an ordered monolayer, but rather show extensive cytoplasmic overlapping (Figure 1). This altered morphology has been maintained continuously for more than 70 generations, and no cultures have yet reverted to the normal contact-inhibited morphology. Table
1. Growth
of Normal
(ABAE)
and Variant
(ABAE-CA,)
To determine whether the variant cells had lost any of the specific characteristics of endothelial cells other than morphology, we tested both the parental and variant cells for the presence of the factor VIII antigen. Figure 2 demonstrates that although the parental cells express this antigen, the CA, variant cells do not retain this ability. Since the parental cell line was isolated as a single cell clone, it is not possible that the variant represents a contaminating nonendothelial cell type isolated by the selection procedure, but must rather be an endothelial derived cell line that has lost certain of the differentiated functions of normal endothelial cells. Since cell-cell interactions such as those which regulate contact inhibition of growth must be mediated at the cell surface, we have examined the normal and variant cell lines for differences in the distribution of cell surface proteins. Following lactoperoxidase-catalyzed iodination with 13’1, cell surface proteins from the normal or variant endothelial cells were subjected to slab gel electrophoresis and autoradiography. As shown in Figure 3,
Endothelial
Cells
and Absence
of FGF
ABAE-CA1
ABAE Serum
in the Presence
Concentration
w 0.25 2.50 10.0
-FGF
+FGF
6.6 + 0.12=
-FGF
7.6 + 0.31
7.2 + 0.36
6.8 + 0.34
11.4 + 0.11
40.5
+ 1.80
9.1 + 0.54
34.9 + 0.35
17.6 + 0.17
106.0
+ 5.30
15.3 + 0.78
92.0 + 2.84
a Cell number x IO-’ + standard error. 2 x 10’ cells were seeded in 8 cm tissue culture dishes in medium containing 10% calf serum. After fresh medium containing the indicated serum concentration with or without 100 nglml FGF. Triplicate
Figure 1. Morphology (Magnification 150x)
+FGF
of (A) Normal
Vascular
Endothelial
Cells
and
(9)
2-Chloroacetaldehyde-Induced
18 hr, the medium was replaced with cultures were counted after 10 days.
Variant
Cell
Line
(ABAE-CA,)
Endothelial 503
Cell Surface
Variants
when cultures were grown to confluence and maintained in culture for an additional 7 days, the distribution of cell surface proteins in the two cell types differed significantly. In comparison with the parental cell line, the CA, variants have decreased concentrations of four proteins with molecular weights of 220,000, 177,000, 150,000 and 26,000 daltons, respectively. In addition, the variant cells have increased concentrations of two high molecular weight proteins (400,000 and 285,000 daltons). With the exception of the 26,000 dalton protein, these patterns do not reflect cell surface differences between growing and resting cells (I. Vlodavsky, L. K. Johnson and D. Gospodarowicz, unpublished results). One particular protein which is prominent on the surface of many cell types is a high molecular weight (220,000 daltons) glycoprotein termed fibro-
400K 285K 220K+ 177K+ 15OK-,
26K+
Figure 2. Determination of Factor VIII Normal and Variant Endothelial Cells
Antigen
in Cultures
of
(A) Phase-contrast micrograph of the variant ABAE-CA, cells (mangification 400x). (8) Fluorescence micrograph of the same field shown in .(A). No factor VIII antigen is detectable in the variant cells. (C) Fluorescence micrograph of parental ABAE endothelial cells showing abundant intracellular factor VII antigen.
Figure 3. Distribution of lodinatable Normal and CA, Variant Endothelial
Cell Cells
Surface
Proteins
from
Confluent cultures of (A) normal and (8) CA, variant endothelial cells were labeled by lactoperoxidase-catalyzed iodination with ‘3’l and subjected to electrophoresis on 515% exponential gradient SDS-polyacrylamide slab gels.
Cell 504
nectin or LETS protein (for a review, see Hynes 1976; Vaheri et al., 1976). This protein has been implicated as a major factor in cell substratum adhesion (Yamada, Yamada and Pastan, 1976; Ali et al., 1977) and in the maintenance of cell-cell contacts (Yamada, Yamada and Pastan, 1975; Chen, Gallimore and McDougall, 1976; Zetter, Chen and Buchanan, 1976). At low cell densitities, relatively small amounts of this protein can be detected after iodination of either cell type (not shown). When confluent cultures were tested, however, significantly greater amounts were expressed by the parental cells (see Figure 3, band at 220,000). To determine the distribution of the relatively smaller amounts of LETS protein on the surface of cells in sparse culture, we have used a highly sensitive immunofluorescent staining technique (Ruoslahti et al., 1973). As shown in Figure 4, there is a striking difference between the LETS protein distribution on the two cell types. As demonstrated by Birdwell, Gospodarowicz and Nicolson (1978), the contact-inhibited parental cells deposit LETS only onto the plastic dish and not onto the surface of the cells themselves. The importance of cell-cell contacts for the depositing of LETS is evident in subconfluent cultures of these cells, since there is no evidence of LETS protein on isolated cells but only on cells in contact with others. In contrast, the variant cells that are capable of overgrowth can anchor the protein to their surfaces in a pattern corresponding to the distribution of stress fibers visible under phase microscopy. Here, totally isolated cells anchor LETS to their surface. The difference between the parental and variant
Figure
4. Distribution
of LETS
Protein
Subconfluent normal endothelial cells their cell surfaces even when completely
on Normal
and Variant
Endothelial
cell lines in the distribution of LETS would indicate that the CA, cells have lost the polarity between the upper and lower cell surfaces that is so important for the normal functioning of the endothelium (Birdwell et al., 1978; Gospodarowicz et al., 1978). This is especially evident in the distribution of cell surface LETS protein on the well spread variant cell shown in Figure 5. Here, the LETS protein is located on surface-associated fibers that enclose the cell and describe its three-dimensional appearance. Alterations in the cell surface composition of a given cell type will often be reflected by a change in the mobility of receptors localized in the cell membrane. When FITC-conjugated concanavalin A (ConA) was incubated with suspensions of the parental aortic endothelial cell, the fluorescence was found to be localized in discrete areas (caps) on the cell surface. Capping was observed on 72% of the ABAE cells. On the other hand, when the same experiment was performed with the CA, cells, the fluorescence was distributed more evenly over the entire cell surface (Figure 6), and caps were seen on only 6% of all cells counted. This result implies that membrane mobility is greater in the normal cells than in the altered line. One of the most intriguing aspects of endothelial cell biology is the nonthrombogenic nature of the endothelial cell surface exposed to the bloodstream. Circulating platelets will not interact with a vessel wall unless it has been in some way injured or altered as in inflammation, wounding or the early stages of atherosclerosis. We have therefore examined the ability of the parental and variant cells to bind washed human platelets by incubating
Cells
(A) deposit LETS protein in areas of cell-cell contact, isolated from other cells (magnification 400x).
whereas
CA, variants
(B) have
deposits
on
Endothelial 505
Figure
Cell Surface
5. Distribution
Variants
of LETS Protein
on CA, Cell
The disruption of the normal bipolarity of the endothelial of this well spread variant cell (magnification 630x).
Figure
6. Distribution
of Fluorescein-Conjugated
ConA
cell surface
on Normal
is reflected
and Variant
in the deposits
Endothelial
of LETS on fibers
the surface
Cells
Cell suspensions were incubated for 15 min at 37°C with 100 pglml fluorescein-conjugated ConA, fluorescence microscopy. Capping of ConA receptors is observed on the normal endothelial distributed more randomly on the surface of the variant cell (B) (magnification 630x).
cultures of each cell type with 2 x lOa fresh platelets for 30 min at 37°C. After washing the cultures 10 times in medium containing 0.25% BSA, it was found that only the variant cells have platelets bound tightly to their upper surfaces (Figure 7). The platelets used for these studies were
surrounding
washed with PBS and observed under cell (A), whereas the fluorescence is
normal by the criteria of morphology (discoidal) and the ability to take up 14C-serotonin and to secrete it in response to specific stimuli such as thrombin or collagen. It has been previously demonstrated that when large blood vessels are denuded of their endothe-
Cell 506
hum by mechanical or hydrolytic treatment, platelets will adhere to the newly exposed subendothelial surface. When this occurs, the platelets change shape, release materials from cytoplasmic granules and coalesce into large aggregates (for review, see Weiss et al., 1977). As shown in Figure 7, however, the platelets adhering to the CA, cells attach singly and do not form aggregates. To determine whether the attached platelets undergo the platelet-release reaction, we preincubated the platelets with 14Cserotonin and then incubated the labeled platelets with normal and altered endothelial cells. After 30 min at 37”C, the culture medium containing unattached platelets and any serotonin released during binding was removed, and the platelets were col-
Figure
7. Adherence
2 x 10’ platelets washed 10 times Table
2. Binding
of Platelets
in 1 ml culture and observed of Platelets
to Normal
to Normal
Endothelial
Cells
0.25% BSA were incubated microscopy (magnification
and CA, Variant
Endothelial
ABAE
650
ABAE-CA, ABAE-CA1
+ 0.5 pg/ml
Trypsin
ABAE-CA,
+ Anti-LETS
Antiserum
(+)
with normal 150x).
(A) and variant
CA, (B) cells for 30 min at 37”C,
Cells
Cell-Bound Platelets (cpm “C-Serotonin)
Cell Type
No Cells
and Variant
medium containing under phase-contrast
lected by centrifugation. The cells and attached platelets were washed 10 times with phosphatebuffered saline and then hydrolyzed in 0.2 N NaOH. The cells, platelets and platelet-free supernatant were counted separately to determine the distribution of the “C-serotonin. As shown in Table 2, normal (ABAE) endothelial cells bind very few platelets (0.5 platelets per cell), whereas the altered CA, cells bind platelets over their entire upper cell surface (12-15 platelets per cell). In neither case is any serotonin release seen beyond that spontaneously released by platelets incubated at 37°C for 30 min in the absence of cells. The capability of the platelets to release 14C-serotonin under appropriate conditions can be demonstrated by adding
Released
“C-Serotonin
(cm 5670
11,760
5666
9,306
5026
11,560
5692
-
5653*
2 x lOa platelets (79,270 cpm) were incubated with confluent cell cultures for 30 min at 3PC. The binding of platelets to cells and the release of “C-serotonin from platelets were determined as described in Experimental Procedures. The amount of serotonin released by the platelets was in all cases equivalent to that spontaneously released by platelets during the time course of the experiment (*). Treatment of 2 x lo* platelets with 10 units par ml human thrombin caused release of 63,760 cpm of ‘.C-serotonin. (+) Cells were preincubated for 1 hr with serial dilutions of the anti-LETS antiserum (1:2 to 1:512) with no effect on platelet binding or release.
Endothelial
Cell Surface
Variants
507
thrombin (1 unit per ml) to the dish containing the cells and platelets. Under these conditions, 7090% of the counts will be released into the culture medium. Discussion The vascular endothelial cells that line the inner surfaces of blood vessels have several interesting properties that are amenable to study in tissue culture, and in recent years, considerable progress has been made in propagating cultured cell lines that maintain the properties characteristic of the vascular endothelium in vivo. Most of these cell lines demonstrate contact inhibition of growth and have been found to produce factor VIII antigen, prostaglandin 12, Weibel-Palade bodies and angiotensin-converting factor (for review, see Gimbrone, 1976). One of the most intriguing aspects of endothelial cells is the bifunctional nature of their cell surfaces. One side of these cells is attached to a basement membrane and functions to keep the cells in a flattened configuration tightly adherent to the vessel wall. This flattened monolayer configuration serves to prevent the cells from occluding the vessel lumen through which the blood flows. Tight adherence to the vessel wall is also essential in face of the large shear force to which these cells are subjected. On the other hand, the side of the endothelial cells that is exposed to the flowing blood stream must present a surface that is relatively inert and resists adhesion by circulating blood cells such as platelets and leukocytes. This bipolarity of the endothelial cell surface is reflected in the distribution of the LETS protein, which has been shown to be deposited by normal endothelial cells only on the side at which the cells adhere to a substratum and not on the surface exposed to the culture fluid (Birdwell et al., 1978). Since this protein has been implicated in cell substratum adhesiveness (Yamada et al., 1976; Ali et al., 1977), it is conceivable that it has a role in maintaining the tight contact between the endothelium and the basal lamina to which it is attached. To study the cell surface biology of vascular endothelial cells more effectively, it would be advantageous to develop mutant cell lines having lesions in specific cell surface properties. Although one virally transformed endothelial cell line has been described that possesses altered growth properties and lacks some of the differentiated properties of normal endothelial cells (Gimbrone and Fareed 1976), no variant cell lines have yet been isolated which are altered with respect to the ability to interact with circulating blood cells. Through the use of a mutagen, 2-chloroacetalde-
hyde, we have been able to isolate endothelial cell lines with stable alterations that result in a loss of the thrombogenic cell surface. The altered cell line (ABAE-CA,) described in this report was isolated on the basis of its ability to undergo extensive cellular overlapping in dense cultures, unlike the normal endothelial cells which always form a flattened monolayer. The morphology of these altered cells has remained constant for more than 70 population doublings in vitro, and in no case have the cells reverted to the parental morphology. It is important to note that the parental cell line used to isolate this variant was originated as a single cell clone and therefore contained no contaminating cell types that could have been isolated in the selection procedure. Since every cell in the parental stock is an endothelial cell, the variants must be of endothelial derivation. When iodinated cell surface proteins from the two cell types are compared on SDS-polyacrylamide slab gel electrophoresis, the gel patterns are basically similar, but several specific differences can be noted. In comparison with the parental cell line, the CA, variants have an increased concentration of two high molecular weight proteins (400,000 and 285,000 daltons) and a decrease in the amount of at least four other bands having apparent molecular weights of 220,000, 177,000, 150,000 and 26,000 daltons. That these cell surface changes are reflected in altered surface properties has been shown in three ways. First, it was shown that whereas the parental cells can mobilize receptors for ConA to form caps, the altered cells display a more uniform distribution of ConA over the entire cell surface. This would suggest that the membrane fluidity of the CA, cells is restricted in comparison to that of the normal cells. Second, when the expression of a high molecular weight cell. surface glycoprotein (LETS, fibronectin) was compared by immunofluorescent staining of the two cell types, the distribution of the protein was found to be markedly different. In contrast to the normal cells that deposit LETS only onto the surface attached to the substratum, the CA, cells deposit the protein on both the upper and lower cell surface. One might infer that the bipolar nature of the endothelial cell surface on these cells has been disrupted. Finally, only the altered cells possess the ability to bind platelets, whereas the normal cells present a nonthrombogenic surface similar to that which is characteristic of the vascular endothelium in vivo. Although there has been considerable study of the interaction of platelets with the subendothelial matrix of blood vessels (for a review, see Weiss et al., 1977), the properties of the endothelial cell surface that render it nonthrombogenic are rela-
Cdl 508
tively unexplored. We believe that the CA, cell line should provide a valuable model for investigating the interactions between the inner surfaces of blood vessels and the circulating blood cells. In this regard, it is of particular interest that the platelets which firmly attach to the surface of the CA, cells do not aggregate or release the contents of cytoplasmic granules as they do when they attach to the subendothelial matrix (Weiss et al., 1977). It remains to be determined whether the long-term contact of platelets with endothelial cells has deleterious effects for either cell type. Although it would be tempting to correlate the altered distribution of LETS protein in the CA, cells with their ability to bind platelets, two pieces of evidence indicate that this is not the case. First, incubation of CA, cells with a concentration of trypsin sufficient to remove all surface-associated LETS (as determined by immunofluorescent staining) did not reduce the binding of platelets to the cells. Second, preincubation of the CA, cells with antiserum against the LETS protein also failed to reduce the number of platelets bound to the cells. The altered distribution of LETS may, however, reflect a more general cell surface modification that is in itself responsible for the interactions with platelets. The ability of 2-chloroacetaldehyde to cause stable changes in the properties of vascular endothelial cells is of particular interest in view of the finding that this compound is a metabolite of the carcinogen vinyl chloride (Barbin et al., 1975; Kappus et al., 1976). One of the special properties of this carcinogen is the ability to induce endothelial cell tumors (malignant hemangioendotheliomas) (Creech and Johnson, 1974; Maltoni and Lefemine 1974; Stewart ‘1976; Infante, Wagoner and Waxweiler, 1976), and although the data presented here do not provide direct evidence that 2-chloroacetaldehyde is responsible for vinyl chloride carcinogenesis, they do demonstrate an interesting ability on the part of a carcinogenic metabolite to affect the target cell of the carcinogen. The variant cell line described here shares some features with the virally transformed endothelial cell line isolated by Gimbrone and Fareed (1976), such as the loss of contact inhibition and the cessation of factor VIII antigen production as revealed by indirect immunofluorescence. The cells do, however, retain some properties of normal endothelial cells-for example, the ability to respond to the mitogenic activity of FGF and a requirement for normal concentrations (5-10%) of calf serum in the growth medium. In this respect, the cells are similar to revertants of transformed cells that have lost some, but not all, of the dissociable parameters of transformation (Vogel and
Pollack 1973; Pollack and Risser 1974). It is possible that not all of the cell lines isolated following 2chloroacetaldehyde treatment will share all of the same properties. To date, however, each has demonstrated the ability to overgrow, as well as the ability to bind platelets. The altered endothelial cells described here should have several uses for the further study of vascular cell biology. Further biochemical elucidation of the cell surface differences between these cells and the normal endothelium may help to elucidate the loss of contact inhibition in one of the few cell types to display this kind of control in vivo. In addition, the ability of these cells to bind platelets provides the first model system for studying the nonthrombogenic properties of the vascular endothelium. Such cells could conceivably be of use as an assay for pathological conditions in which the adhesive properties of platelets have been altered. Experimental
Procedures
Isolation of Variant Endothelial Cell Lines Cultures of the parental endothelial cells (ABAE) were grown to subconfluence in 6 cm Falcon tissue culture dishes in Dulbecco’s modified Eagle’s medium with 10% calf serum and 100 rig/ml FGF, and then treated for 48 hr with 5-20 PM 2-chloroacetaldehyde. The cultures were than washed twice and maintained in fresh medium containing serum only. Following this treatment, the cells grew to a confluent monolayer, and in certain areas, colonies of overlapping cells appeared. It was subsequently found that the cells in these colonies were more rapidly detached by trypsinization than the cells that were attached directly to the plate and could therefore be selectively isolated by short treatment (1 min) with 0.25% trypsin in phosphate-buffered saline (PBS). The subcultures were grown to confluence and passaged repeatedly until cultures were obtained in which all the cells were capable of overlapping. One such cell line which is described in this report has been designated “ABAE-CA,.” lmmunofluorescent Staining Cultures to be assayed for the presence of LETS protein were grown on 12 mm glass coverslips and fixed in 3.7% formaldehyde. Indirect immunofluorescence was performed according to the procedure described by Chen et al. (1976) using antiserum provided by Dr. L. B. Chen. The properties of this monospecific antiserum, originally prepared against the antigenically identical cold-insoluble globulin (GIG) by Mosseson and Umfleet (1970), have been described by Chen et al. (1976) and by Burridge (1976). Cultures to be assayed for the presence of factor VIII antigen were treated as described by Jaffe (1977), using antiserum against bovine factor VIII antigen provided by Dr. J. Brown. The antiserum to LETS protein was used at a dilution of 1:80, and the antiserum to factor VIII antigen was used at a dilution of 1:200. Fluorescence was observed using a Leitz Orthoplan microscope under epiillumination and photographed with Kodak Tri-X film using a Leitz Orthomat camera. In all cases where positive fluorescence was observed, the specificity of the staining was determined by control experiments in which nonimmune rabbit serum was used in place of the anti-CIG antiserum. Treatment of Cells with Fluorescein-Conjugated ConA Cells were removed from the culture dish with 0.03% EDTA, centrifuged and resuspended in PBS. They were then incubated
Endothelial
Cell Surface
Variants
509
for 15 min at 37°C with 100 pg/ml FITC-conjugated ConA. Following this incubation, the cells were washed with PBS, pipetted to dissociated aggregates and placed on glass microscope slides for observation by fluorescent microscopy. Lactoperoxidase-Catalyzed lodination and Slab Gel Electrophoresis of Cell Surface Proteins Monolayer cultures of ABAE and ABAE-CA, were iodinated with 13’1 according to the method of Teng and Chen (1976). The cells were subsequently solubilized in sample buffer containing 2 mM PMSF and 1% 2-mercaptoethanol, and subjected to SDS-poly acrylamide gel electrophoresis on gels prepared with a 5-15% exponential gradient of acrylamide. Autoradiograms of dried gels were made using Kodak NS-2T X-ray film and developed with Kodak D-19 developer. Binding of Platelets to Cultured Endothelial Cells Platelets were prepared from 60-120 ml human whole blood according to the procedure described by Tollefsen, Feagler and Majerus (1976). To observe platelet binding, 2 x lo* platelets were added to confluent cultures of cells in 35 mm culture dishes containing 1 ml of Dulbecco’s modified Eagle’s medium with 0.25% bovine serum albumin (BSA) and incubated for 30 min at 37°C in a humidifed CO, incubator. Following incubation, the cultures were washed 10 times with the same medium and viewed under phase microscopy. For quantification of platelet binding and serotonin release from platelet granules, the platelets were preincubated for 30 min with “C-serotonin (0.2 &i/ml). After incubation of the labeled platelets with the cells, the culture medium was removed and the platelets were collected by centrifugation. The platelets were then hydrolyzed in 0.2 M NaOH, as were the cells with bound platelets remaining on the culture dish. The three fractions-platelets, cells and the platelet-free supernatant -were then counted separately in Bray’s solution using a Beckman LS 8000 scintillation counter. Acknowledgments
February
17, 1978;
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