Distribution and movement of anionic cell surface sites in cultured human vascular endothelial cells

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Atherosclerosis, 32 (1979) 69-80 @ Elsevier/North-Holland Scientific

Publishers,

Ltd.

DISTRIBUTION IN CULTURED

AND MOVEMENT OF ANIONIC CELL SURFACE HUMAN VASCULAR ENDOTHELIAL CELLS

PETER

MICHAEL

PELIKAN,

A. GIMBRONE,

Jr. and RAMZI

SITES

S. COTRAN

Department of Pathology, Peter Bent Brigham Hospital and Harvard Medical School, Boston, MA 02115 (U.S.A.) (Received (Revised, (Accepted

21 June, 1978) received 5 September, 1978) 11 September, 1978)

Summary

Cell surface anionic sites on primary and transformed cultures of human vascular endothelium were studied using cationized ferritin (CF) as an ultrastructural marker. The native distribution of anionic sites on the upper (free) surfaces of cells, fixed in situ with glutaraldehyde, was uniform. Binding of the polycationic ligand, CF, in unfixed cells induced redistribution of anionic sites. Rapid formation of discrete patches of CF particles was followed by reappearance of binding between patches, movement of surface-bound CF into intercellular clefts, and endocytosis, over the next 30 min. Aldehyde-fixed cells, detached from the culture surface, bound CF on both upper and lower surfaces. The distribution and mobility of negatively charged membrane components in vascular endothelium may have relevance for thrombosis, atherogenesis, and vascular permeability. Key words:

Anionic sites -Blood vessels - Cationized ferritin - Cell culture - Cell membrane - Electron microscopy - Endothelium - Viral transformation

Introduction

The luminal surface of vascular endothelium is a biologically active interface which participates in many physiological and pathological processes. In vivo and in vitro studies of endothelial cells have demonstrated membrane-assoThese studies of an Established

were supported by NIH grants HL-08251 and HL-20054. Investigator award from the American Heart Association.

Address correspondence to: Dr. Ramzi S. Cotran. Department Hospital, 721 Huntington Ave. Boston, MA 02115, U.S.A.

Dr. Gimbrone

of Pathology.

Peter

is the recipient Bent

Brigham

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ciated lectin binding sites [l]; specific receptors for low density lipoproteins [ 2,3], insulin [4] and vasoactive hormones [ 51; histocompatibility [6,7] and blood group antigens [8]; proteolytic activities and inhibitors [9,10]; and enzymes important in lipid [ll] and vasoactive peptide metabolism [12]. In addition to these functionally defined components, the endothelial cell surface contains heparin-like sulfated mucopolysaccharides [ 131, as well ‘as neuraminidase-sensitive and -resistant anionic groups [14], and bears a negative charge at physiological pH and ionic strength [15]. It has been suggested that this negative surface charge might contribute, by electrostatic repulsion, to the nonreactivity of the normal endothelial lining with circulating blood cells and platelets [16]. Recent studies on the glomerular capillary wall also indicate that structural poly anionic elements can influence the permeability to charged plasma macromolecules [ 171. Thus, endothelial cell surface properties, such as the distribution and movement of anionic membrane components, may be relevant to the normal function of the blood vessel wall and its involvement in disease processes such as thrombosis, inflammation and atherosclerosis. Negatively charged sites on cell surfaces can be visualized by electron microscopy, using a positively charged, electron-dense label such as cationized ferritin [15]. When applied following aldehyde fixation, cationized ferritin electrostatically “stains” surface anionic groups in their native distribution; however, exposure of a living cell to this polyvalent ligand can induce redistribution of anionic membrane components [ 121, in a manner similar to that observed with lectin and immunoglobulin binding in lymphocytes [ 191. Using both approaches, Danon et al. have studied anionic sites on luminal endothelial surfaces and subendothelial connective tissues of guinea pig and rabbit blood vessels [ 14,201. In our laboratory, we have selectively isolated and cultured human vascular endothelial cells, which retain differentiated structural and functional features [21,22], and also have transformed these cells with SV40 viral DNA [23]. Such cultures lend themselves to controlled experimental manipulation. In the studies reported here, we have utilized cationized ferritin to visualize native and polycation-induced patterns of distribution of surface anionic sites in primary and SV40-transformed human endothelial cells in culture. Materials and Methods Vascular endothelial cultures Primary cells. Human endothelial cells were harvested, essentially free of contamination with other cell types, by limited collagenase digestion of the intimal surface of normal, term umbilical cord veins, as previously described [21]. Cells from 2-5 vessel segments were pooled in Medium 199 (Microbiological Associates, Bethesda, MD), supplemented with 25 mM HEPES buffer (N-2-hydroxyethyl-piperazine-N’-2 ethanesulfonic acid) at pH 7.4, 20% heatinactivated (55”C, 30 min) fetal calf serum (Microbiological Associates), 60 pg/ ml penicillin and 120 E.cg/mlstreptomycin. Aliquots of 0.5-1.0 X lo5 cells were plated in 16-mm diameter plastic culture wells (Cluster-24, COSTAR, Cambridge, MA) and maintained in a humidified atmosphere at 37”C, with daily medium changes. These primary cultures were used 3-5 days after plating,

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when partially confluent monolayers of polygonal cells had formed. Transformed cells. Cells from a line of SV40-transformed human endothelium (SVHEC-F), originally derived by SV40-DNA transvection of primary umbilical vein cultures [23], were studied in subculture passages 5-12. Aliquots of 0.2-0.5 X 10’ cells were plated, as described above, in supplemented Medium 199 with 10% heat-inactivated fetal calf serum. These cultures were used after 3-5 days, when multilayered arrays of pleomorphic cells were present. Reagents Cationized ferritin (CF) was purchased from Miles-Yeda (Lot No. CF-10; Rehovot, Israel), and had an isoelectric point of 8.8-10.9, as determined by isoelectric focusing. Native (anionic) horse spleen ferritin (NF), isoelectric point of 4.4-4.8, was purchased from Sigma Chemical Co. (St. Louis, MO). Stock solutions of CF (11.5 mg/ml) and NF (250 mg/ml) were diluted immediately before use with Dulbecco’s phosphate-buffered saline (DPBS), pH 7.3-7.4 (Microbiological Associates). General procedures Prior to incubation with ferritin, all cultures were washed 5 times with DPBS at 37°C. In pilot studies, this washing procedure yielded optimal CF binding. Primary and transformed cells were incubated at 37°C with CF or NF, at final concentrations of 0.08, 0.16 and 0.32 mg/ml DPBS, for time intervals ranging from 15 set to 30 min, either before or after fixation in 2.5% glutaraldehyde-0.1 M cacodylate buffer (pH 7.4) for 30 min at room temperature. In unfixed cells, incubation with ferritin was terminated, at specified intervals, by 5 changes of 2.5% glutamldehyde in DPBS. In fixed cells, incubation was terminated with 5 changes of DPBS alone. To determine whether CF might be interacting with free (unreacted) aldehyde groups on pre-fixed cell surfaces, 0.1 M glycine rinses were used, as described by Skutelsky and Danon [ 201. This quenching procedure did not affect the pattern of CF binding. As the final step in all experiments, cells were fixed at room temperature for an additional 30 min in 2.5% glutaraldehyde-O.1 M cacodylate buffer. Each culture then was post-fixed in 2% osmium tetroxide, stained en bloc with uranyl acetate, dehydrated through graded ethanol solutions, and embedded in Epon at 55°C for 12 h as previously described [ 211. This procedure yielded a semi-hard cast which could be peeled from the culture wells and further polymerized at 60” C for 48 h. Portions of each cast were then remounted and sections cut perpendicular to the plane of the culture. These were examined unstained, or stained (uranyl acetate), with a Philips EM-201 electron microscope. Results Distribution of anionic sites on aldehyde-fixed cells To determine the native distribution of anionic sites on the unattached (upper) surfaces of primary and transformed endothelial cells, culture monolayers were fixed with 2.5% glutaraldehyde for 30 min prior to exposure to CF.

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lb

Fig. 1. a: Human endothelial cells in primary culture. fixed in situ with 2.5% glutaraldehyde for 30 min prior to exposure to 0.08 mg CF/ml phosphate-buffered saline for 15 sec. CF particles are distributed singly and in small groups along the free cell surface. X50.000. b: Cultured endothelial cell. fixed as above, and incubated with 0.16 mg CF/ml PBS for one minute. Note uniform linear pattern of staining and increased density compared to a. X60.000.

Under these conditions, CF binding occurred in a linear pattern along the cell surface. The amount of surface staining was proportional to CF concentration (0.08-0.32 mg/ml) and the duration of incubation (15 see-3 min). Surfacebound CF was usually embedded in an amorphous, electron-dense material. Brief incubations (0.08 mg CF/ml for 15 set) resulted in a distribution of CF as either isolated molecules, or small groups of adjacent molecules (Fig. la). With higher concentrations or longer incubations, CF particles were more closely packed, sometimes several layers deep, uniformly along the cell surface (Fig. lb). CF particles occasionally were seen in the mouth, and rarely in the center, of flask-shaped surface vesicles. Most surface and intracytoplasmic vesicles, however, were free of ferritin. Ferritin was not present in areas of intercellular contacts or within intercellular clefts. In some experiments, prefixation also was performed with 0.2% glutaraldehyde for 10 min, as described by Grinnell et al. [ 241. Following exposure to 0.08 mg CF/ml for 15 set, CF appeared in discrete patches, consisting of lo40 particles, separated by bare areas of cell surface. Since such patches were not observed in cells which had been fixed with a higher concentration of glutaraldehyde, it is likely that this pattern represented rearrangement of anionic sites induced by binding of the polycationic ligand in inadequately fixed cells (see next section).

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No consistent differences in the pattern of CF binding between prefixed primary and transformed cells were noted. In addition, no binding was observed in prefixed primary endothelial cell cultures exposed to native (anionic) horse spleen ferritin (0.16 mg/ml) for 3 min.

Distribution of anionic sites on live cells exposed to polycation To determine changes in surface anionic charge distribution induced by binding of polyvalent CF, unfixed primary and transformed cultures were washed, incubated live with CF (0.16 mg/ml) for varying time intervals at 37°C and then immediately fixed in situ and prepared for electron microscopy. When live cultures were exposed to CF for as short a period as 15 set (Fig. 2a), we observed iarge, discrete patches of ferritin particles at intervals along the cell surface, in striking contrast to the uniform distribution of CF on adequately fixed cells (cf. Fig. 1). In the broad interpatch areas, only a small fraction of the surface was stained by CF in the form of isolated molecules or small clusters. Exposure to CF for one minute resulted in a similar distribution, with larger patches and less binding in the interpatch areas on some cells (Fig. 2b). In cultures incubated for 5 min, surface-bound CF was still distributed in patches of comparable size to those seen at one minute. However, in certain cells, scattered CF binding was apparent between patches (Fig. 2~). Longer incubations resulted in a progressive increase in binding to the interpatch areas, as well as a modest increase in patch size (Fig. 3a). This trend eventually produced a uniform distribution of CF along the cell surface (Fig. 3b) similar to that seen in prefixed cultures (cf. Fig. lb). In addition, in live cultures, CF also was observed in intracellular vesicles (Fig. 3) and in intercellular clefts (Fig. 4), configurations not seen in prefixed cultures. In certain transformed cells, the free surface in the vicinity of intercellular clefts showed noticably less CF binding (Fig. 4). The above observations suggested that binding of polycation to cell surface anionic moieties induced their lateral migration, thus forming the observed patches of CF and transiently leaving the interpatch areas free of anionic sites. In cultures exposed for only 15 set, the modest amount of CF binding in the interpatch areas was thought to represent anionic sites which had not yet completed their lateral migration. By one minute, patch formation was completed, as evidenced by the decrease in interpatch binding. Subsequently, however, Cl? binding reappeared in the previously bare interpatch areas. In order to exclude the possibility that this phenomenon was due to staining of less accessible or lower affinity anionic sites, which might require prolonged incubation to be visualized, the following experiment was done. Live primary cultures were exposed to CF (0.16 mg/ml) for 3 min, followed by incubation in CF-free PBS for 5 and 30 min. They were then fixed and additionally stained with CF (0.16 mg/ml) for 3 min. The distribution of CF binding in these experiments was similar to that described above in live cells continuously exposed to Cl? for 5 or 30 min. Therefore, binding in the interpatch areas was not due to staining of sites requiring longer time to bind CF. Rather, this phenomenon is consistent with the repopulation of the interpatch areas with anionic sites. The latter could represent nascent surface moieties, newly formed or exposed, or the

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2a

2b

2c

Fig. 2. Live endothelial cell cultures were incubated continuously with 0.16 mg CF/ml PBS at 37’C for the specified intervals and then fixed. o: 15 seconds: CF is distributed in large. discrete patches along the cell surface, in contrast to the uniform, linear staining observed in prefixed cells (cf. Fig. 1). b: 1 minute: CF binding is decreased in interpatch areas, as compared to that observed at 15 sec. (Note: The patch of ferritin particles at the far left probably represents a cross-section through a surface imagination, rather than an endocytotic vesicle.) C: 5 minkes: CF patches persist, but scattered binding is apparent in some interpatch areas. X60.000.

disaggregation of previously CF-bound anionic sites from patches. Lateral rnigration of anionic sites, patch formation and repopulation of interpatch areas were similar in live-transformed and primary endothelial cell cultures. However, because of the multilayered arrangement of the transformed cell cultures, CF was more frequently observed in intercellular clefts, as illustrated (Fig. 4).

3a

3b

Fig. 3. Live endothelial cells incubated with 0.16 mg CF/ml PBS for 22 min (X55.000). There is a marked increase in interpatch binding, with CF patches still discernible in some cells (a). while in other Cells. an CF is also visible in some intracelhda essentially uniform linear pattern of binding has reappeared (b). vesicles (ano~s). The patterns of CF binding and movement illustrated in this and the preceding figure were observed

in both primary

and transformed

Cultures.

4

Fig. 4. Transformed endothelial There is a marked accumulation to the free cell surface.

cells incubated of CF particles

0.16 mg CF/ml PBS for 22 min (X60.000). live with in the intercellular cleft and noticeably less CF binding

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Distribution of CF on free and attached surfaces of cultured endothelial cells In order to compare the CF-binding sites on the free (upper) and attached (substratum-bound) surfaces of cultured endothelial cells, the following experiments were done. Primary and transformed cultures were fixed in 2.5% glutaraldehyde for 30 min and then stained with CF (0.16 mg/ml for 3 min). Cells were then removed from the culture dish with a rubber policeman, centrifuged

5C Fig. 5. Primary endothelial cells were fixed and stained in situ (0.16 mg CF/ml PBS, 3 min), then detached from the culture dish, and resuspended in 0.16 mg CF/ml PBS for an additional 3 min. Cross-section through nucleus of pelleted cell (a) shows CF binding to both upper and lower cell surfaces (X20,000). At higher magnification (X55.000). the presumptive free cell surface (b) is identified by its dense CF staining, roughly twice that seen on the opposite suxface which previously was attached to the culture dish (c).

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(10,000 X g for 3 min), resuspended, and further incubated either in phosphatebuffered saline (control) or in phosphate-buffered saline containing 0.16 mg/ml of CF for 3 min at 20°C. The suspensions were again pelleted, fixed for an additional 30 min, and prepared for electron microscopy. In control cells, resuspended in phosphate-buffered saline without CF, CF binding was observed on only one side of the cell. In cells which were resuspended in PBS containing CF, binding occurred on both cell surfaces (Fig. 5a). Furthermore, one surface usually appeared to be more densely coated with CF than the opposite surface of the same cell (Figs. 5b and 5~). Since the free surface of cells suspended in CF was exposed to CF twice (once while attached to the substratum and once while in suspension) and the attached surface only once, it was assumed that the free surface was the site of the cell with the heaviest CF coating. Discussion Using cell electrophoresis various vertebrate cells have been shown to have a net negative charge at physiological pH [25]. In erythrocytes, leukocytes and Ehrlich ascites tumor cells, the majority of anionic surface charges are carboxyl groups of N-acetyl-neuraminic (sialic) acid residues in membrane glycoproteins. Ribonuclease-sensitive phosphate groups are also present in lesser amounts. The ratios and absolute amounts of anionic groups vary in different cell types [ 261. The contribution of these membrane components to surface-negative charge, as well as their intrinsic functional properties, may be relevant to the biology of a given cell. In previous experiments, Skutelsky et al. [14] utilized cationized ferritin to label anionic sites on the luminal front of aldehyde-fixed guinea pig and rabbit vessel explants. Neuraminidase treatment removed approximately 50% of CF binding on endothelial surfaces, but did not reduce the more intensive staining observed on exposed subendothelial connective tissue. In our experiments with human endothelial cultures, we have examined the anionic sites on both free and attached cell surfaces, using CF as an ultrastructural marker. That binding of CF to the cell surface is electrostatic was confirmed by the absence of binding of native (anionic) ferritin. However, because of such factors as variation in CF particle charge density [15] and possible differences in the mobility of, and charge distribution within, anionic membrane components, the actual number of surface anionic sites labeled by each CF molecule is unknown and probably variable. In living cells, polyvalent CF can induce the aggregation of anionic membrane components [20]. Therefore, to study the native distribution of anionic charges, we fixed the cells with glutaraldehyde prior to exposure to CF. This produced a linear pattern of CF binding, in both primary and transformed endothelial cells, which was qualitatively proportional to the concentration of CF in the medium and the time of incubation (Fig. 1). No special topographical organization in this binding was apparent in adequately fixed cells. We interpret this to mean that anionic molecules normally are evenly distributed over the unattached surfaces of cultured endothelial cells. In unfixed endothelial cultures incubated with CF, surface-bound CF aggre-

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gated into discrete patches in less than one minute, leaving broad expanses of cell membrane apparently devoid of negative charge (Fig. 2). However, within minutes, CF binding capacity reappeared in these interpatch areas (Fig. 2), and endocytosis (Fig. 3) and movement of CF into intercellular clefts (Fig. 4) were observed. Skutelsky and Danon [ 201, in their study of unfixed guinea pig aorta and vena cava explants, observed a similar pattern of redistribution of endothelial surface anionic sites. They also noted extensive shedding, interiorization and subendothelial deposition of CF complexes, features which were not especially prominent in unfixed endothelial cultures. In addition, in guinea pig vessels, no CF was seen between cells beyond tight junctions, whereas in cultured cells, CF tended to accumulate in intercellular clefts without apparent restriction. The differences in response to CF binding observed in vessel explants and endothelial cultures may reflect differences in species, vessel type, incubation conditions, and tissue architecture. In the live cell, a uniform distribution of anionic surface sites may result from mutual electrostatic repulsion of membrane components which are free to diffuse in the phospholipid bilayer [27], or it may reflect the influence of transmembrane cytoskeletal control [28]. Thus, polyvalent CF may cause patch formation in endothelial cells by cross-linking mobile anionic surface molecules, as suggested by Skutelsky and Danon [ 201. In addition, CF binding [ 19, may stimulate cytoskeletal rearrangement of transmembrane proteins 201, thus triggering a complex cellular reaction, including cell membrane alterations and endocytosis [ 291. Further studies with drugs and compounds which influence cytoskeletal elements and contractile proteins may help clarify the mechanisms of CF-induced endothelial cell surface alterations. In a comprehensive review [30] Nicholson discusses cell surface changes associated with malignancy and transformation. Various studies have documented changes in cell surface composition, receptors and antigens, and the mobility of lectin and antibody binding sites. These differences vary with cell type and the phase of cell cycle, and none appear to be consistently characteristic of the transformed state. In our experiments, the distribution and movement of surface anionic sites was qualitatively similar in primary and SV40transformed endothelial cultures. Although CF accumulation was especially prominent in the clefts between transformed cells, this may simply reflect the broader areas of intercellular apposition which occur in these non-contactinhibited cultures. However, quantitative differences in the density or mobility of CF binding sites in normal and transformed cells cannot be ruled out by the methods used in the present study. Little is known about differences in the luminal and basal surfaces of endothelial cells. Our observations indicate that anionic sites are present on both free and attached cell surfaces in endothelial cultures. Since these experiments were performed on prefixed cells, differences in the mobility of these sites could not be evaluated. It has been suggested that negative surface charge may account for endothelial non-thrombogenicity [ 161, but the presence of anionic sites in thrombogenic subendothelial connective tissues [ 141, and more recent data on endothelial metabolism [22,31,32], make this explanation unlikely. However, anionic sites on the basal surface of the endothelial cell may be involved in its attachment to the vessel wall, a process which has important im-

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plications for thrombosis and atherogenesis [ 32,33,34]. Other potential roles of charged surface components in endothelial physiology deserve consideration. Recent morphological and physiological studies [17] indicate that anionic sites in the renal glomerular-capillary unit may be important determinants of its permselectivity. Since most plasma macromolecules are anionic, electrostatic repulsion by surface charge in endothelial intercellular junctions and pinocytotic vesicles conceivably may also play a role in systemic vascular permeability. Low density lipoproteins (LDL), which have been implicated in the pathogenesis of atherosclerosis [34], normally are anionic at physiological pH [ 351. Cells cultured from patients with homozygous familial hypercholesterolemia, which lack LDL receptors, internalize cationized LDL but not normal LDL [36]. Similarly, enhanced cellular uptake has been observed with poly-L-lysine (cationized) conjugates of albumin and horseradish peroxidase [37]. Whether naturally occurring alterations in the charge density of macromolecules, or, conversely, cell membrane components, can influence endothelial endocytosis remains to be investigated. Acknowledgements The authors wish to thank helpful advice and discussions, excellent technical assistance.

Drs. M.A. Venkatachalam and H. Rennke and Ms. Gilda Vretea and Ethel J. Shefton

for for

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