JOURNAL OF ULTRASTRUCTURERESEARCH 50, 22 32

(1975)

Fine Structure of Vascular Endothelium in Culture I CHRISTIAN C. HAUDENSCHILD, RAMZI S. COTRAN,2 MICHAEL A. GIMBRONE, JR., a n d JUDAH FOLKMAN

Departments of Surgery and Pathology, Harvard Medical School, the Children's Hospital Medical Center, and the Peter Bent Brigham Hospital, Boston, Massachusetts 02115 Received January 25, 1974, and in revised form July 3, 1974 Human umbilical cord vein endothelial cells were harvested by collagenase perfusion, cultured in Medium 199 with 20% fetal calf serum, and subcultured for 19 passages over one year. By phase microscopy, cultured endothelial cells were small and polygonal and in successful cultures, formed a monolayer of closely packed cells. By electron microscopy, cultured endothelium retained fine structural characteristics of in vivo umbilical vein endothelium. Specific endothelial granules first described by Weibel and Palade (30), were found regularly in all passages. Microtubules (250 A), two types of filaments with diameters of 100 A and 60 70 A, respectively, pinocytotic vesicles, multivesicular bodies, and cellular junctions were distributed in a way that cultured endothelium could be distinguished by morphologic criteria from the most probable contaminants, smooth muscle cells, and fibroblasts. INTRODUCTION Vascular endothelium constitutes the m a i n and u l t i m a t e barrier between blood a n d t i s s u e s a n d as s u c h p l a y s a n i m p o r t a n t role i n a n u m b e r of physiologic a n d p a t h o logic processes (2, 17). U n t i l r e c e n t l y it has b e e n d i f f i c u l t to s t u d y e n d o t h e l i u m in vitro b e c a u s e of t h e u n a v a i l a b i l i t y of m e t h o d s to i s o l a t e a n d c u l t u r e e n d o t h e l i u m in p u r e form. S c a t t e r e d e a r l i e r a t t e m p t s (6, I9, 2I) were n o t p u r s u e d . I n t h e p a s t two years, however, s e v e r a l r e p o r t s d e s c r i b i n g e n d o t h e l i a l c u l t u r e s h a v e a p p e a r e d (8, 9, 13, 14, 16, 29), a n d m o r p h o l o g i c as well as i m m u nologic c r i t e r i a were u s e d to i d e n t i f y cult u r e d cells as e n d o t h e l i u m (10, 14, 18). I n p r e v i o u s c o m m u n i c a t i o n s (8, 10) we rep o r t e d a t e c h n i q u e for p r i m a r y c u l t u r e a n d s u b c u l t u r e of h u m a n u m b i l i c a l v e i n e n d o thelium, and characterized the population b e h a v i o r of s u c h c u l t u r e s , u s i n g p h a s e contrast microscopy and autoradiography

after e x p o s u r e to t r i t i u m - l a b e l e d t h y m i d i n e . I n t h i s p a p e r we d e m o n s t r a t e t h e p r e s e n c e of t h e u n i q u e u l t r a s t r u c t u r a l m a r k e r of e n d o t h e l i u m , t h e specific e n d o t h e l i a l g r a n u l e (30, 31) or W e i b e l - P a l a d e b o d y ( W P B ) 3 t h r o u g h 19 p a s s a g e s in vitro a n d d e s c r i b e t h e fine s t r u c t u r e of h u m a n e n d o t h e l i u m in p r i m a r y c u l t u r e s a n d in successive subcultures. MATERIALS AND METHODS Endothelial cells were harvested from umbilical cord veins by collagenase treatment as previously described (8, 10). The cultures were maintained on plastic surfaces (Falcon) at a density of approximately 100,000 cells/cm2: Medium 199 containing 20% fetal calf serum (Grand Island Biological Co., Grand Island, New York), 15 mmole per liter of HEPES buffer (N-2-hydroethylpiperazine-N'-ethanesulfonic acid), pH 7.4, 100 mg of penicillin and 100 mg of streptomycin per liter was used. The medium of the primary cultures was changed every 2 days while that of subsequent passages was changed twice a week. For subculturing, the cells were detached from the plate by exposure to 0.25% trypsin-0.05% EDTA in modified Puck's saline A for 2-3 min and passed into fresh medium at a density of approximately 5 × 105 cells/ml. After 10 passages, some of the cultures were

1Supported by grants from the National Heart and Lung Institute (HL 08251), and the National Cancer Institute (CA08185). 2Please send reprint requests to Dr. Ramzi S. Cotran, Department of Pathology, Peter Bent Brigham Hospital, Boston, Massachusetts 02115. Copyright © 1975by Academic Press. Inc. All rights of reproduction in any form reserved.

Sin the text, figures, and legends, the terms specific endothelial granule and Weibel-Palade body (WPB) are used interchangeably. 22

ENDOTHELIUM IN CULTURE frozen in presence of 7.5% DMSO (dimethyl sulfoxide) in medium 199 containing 20% fetal calf serum. These cells maintained their viability at 70°K for several months. For electron microscopic studies the cultured cells were fixed in situ at room temperature with 2.5% glutaraldehyde-0.1 M cacodylate buffer (pH 7.4) for 1 hr, washed in the same buffer, postfixed in 2% OsO4in distilled water for 1 hr at room temperature, stained with 0.1% aqueous uranyl acetate, dehydrated with ethanol containing 0.1% toluidine blue stain, and infiltrated with 1:1 mixture of Epon and ethanol for 1 hr, Epon alone for 1 hr at room temperature, followed by Epon at 55°C. Temperatures over 55°C melted the plastic dishes and glued them irreversibly to the Epon. After 12-14 hr of curing the 2-3 mm thick Epon layer containing the cells was only partially polymerized and still elastic. The brittle plastic dish was then cracked and the warm Epon layer was peeled off. The Epon layer was turned upside down and cured again at 60°C for at least 24 hr. A flat glossy Epon casting containing every cell of the dish was routinely obrained by this procedure. Areas selected with an inverted phase contrast microscope were cut, mounted on Epon blocks, and sectioned parallel or perpendicular to the plane of the cell monolayer.Thin sections were stained with uranyl acetate and lead citrate and examined on Philips EM 200 or EM 300 electron microscopes. OBSERVATIONS

Primary Cultures T h e following account describes the ult r a s t r u c t u r e of endothelial cells after 6 hrs, 24 hr, and 6-7 days of p r i m a r y culture. Six hours after isolation from h u m a n umbilical veins, small clusters of 5-12 spindle-shaped cells began to spread on the s u b s t r a t u m . The light-microscopic appearance of these clusters has been reported in detail in previous publications (8, 10). T h e fine structure of individual cells in the clusters (Figs. 1-3) appeared identical to t h a t of native umbilical vein e n d o t h e l i u m (20). Cells were characterized by oval or flattened nuclei, pinocytosis vesicles, a perinuclear region containing Golgi complexes, small arrays of rough endoplasmic reticulum and mitochondria, and m a n y rod-shaped m e m b r a n e b o u n d granules known as specific endothelial granules or Weibel-Palade bodies (30, 31) (Fig. 2). T h e

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electron density of these granules varied even in single sections of the same cell (Fig. 3) ; their i n t e r n u m consisted of from 6 to 20 tubules a p p r o x i m a t e l y 150 A in width; the t u b u l a r nature was especially evident in cross sections (see Fig. 7). Specific endothelial granules were always b o u n d by a unit m e m b r a n e . They were often clustered toward the periphery of the cytoplasm but occasionally were present in the region of the Golgi a p p a r a t u s . The cytoplasm also c o n t a i n e d variable n u m b e r s of filaments, a p p r o x i m a t e l y 100 A in width. These filaments occasionally occurred in bundles adjacent to the nucleus and sometimes exhibited a criss-cross p a t t e r n (Figs. 1 and 2). Some cells showed increased n u m b e r s of multivesicular bodies and vacuoles (Fig. 3). At 24 hr, endothelial cells had spread to more t h a n twice their original surface area (Fig. 4). T h y m i d i n e incorporation studies reported earlier (8, 10) showed t h a t only little D N A synthesis h a d occurred in the cell clusters at this time. T h e nuclei were now more rounded or oval, exhibited prominent nucleoli, and less peripherally placed dense chromatin. The flat extensions of the cytoplasm consisted of free ribosomes, microtubules (approximately 250 A in width) as well as m i c r o p i n o c y t o s i s vesicles 500-1000 A in diameter. Specific endothelial granules, identical to those seen at 6 hr were present (Fig. 4). An additional feature at this stage was the presence of small bundles of thin filaments 60-70 A in width, which exhibited occasional fusiform dense bodies (Fig. 4). These filaments were present almost exclusively toward the periphery of the cells. Vacuoles and multivesicular bodies as seen at 6 hr were still present, and in areas the vacuoles became confluent. By 3 days an increase of D N A synthesis was noted (10) and cells at the periphery of the individual clusters underwent mitosis. By 6-7 days most of the clusters were confluent. T h e y consisted of a monolayer

FIas. 1-3. Electron micrographs of primary cultures of endothelium fixed 6 hr after plating. The sections are parallel to the plane of the monolayer. FIG. 1. Periphery of a cluster of endothelium. The cells are not completely spread out. The nuclei show infoldings and dense chromatin borders. 100 A filaments are located close to the nuclei. Note Weibel-Palade bodies in all ceils (arrows, see also Fig. 2). x 3 600. Fro. 2. Edge of a spreading endothelial cell shows Weibel-Palade bodies (W), pinocytotic vesicles (arrows), mitochondria (MI) and a crisscross pattern of 100 A filaments (F) close to the nucleus, x 25 100. 24

FIG. 3. Spreading edges of two endothelial cells. Weibel-Palade bodies (W) show different electron densities. Note areas of increased density along the cell junctions (J). M, multivesicular body; T, microtubules; V, vacuole. × 25 200. FIG. 4. Section close to the bottom of an endothelial cell after 24 hours in culture. 60-70 A filaments are at the periphery (arrow), 100 A filaments (F) are close to the nucleus. The cytoplasm contains abundant clusters of free ribosomes. W, Weibel-Palade bodies. × 5 760. 25

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(Fig. 5) of polygonal, closely packed cells showing an epithelioid pattern. These cells had many morphologic characteristics of endothelium in vivo. Typical WeibelPalade bodies occurred in 30-70% of cells in single thin sections. The 60-70 A fila-

ments were most prominent at the cell periphery, often at sites of cellular interdigitations (Fig. 6). Their number had increased considerably, and in sections cut perpendicular to the surface of the culture dishes, they could be localized to both free

Fro. 5. Monolayer after 6 days in culture. Part of an endothelial cell sectioned perpendicular to the plane (P) of the culture dish. W, Weibel-Palade body; G, Golgi complex; A, amorphous extracellular material. × 20 700. FIG. 6. Confluent primary culture (6 days), sectioned in a plane slightly oblique to the plane of the culture dish. 60-70/~ filaments with dense zones (arrows) are accumulated at the periphery of the lower cell. Some bundles of similar filaments in the upper cell are located at dense junctional complexes (J) and seem to be oriented in the same direction. The reason for the difference in filament contents of both cells is t h a t the lower cell is sectioned in a plane closer to the culture dish t h a n the upper cell. x 21 600.

ENDOTHELIUM IN'CULTURE and attached surfaces. The 100 A filaments seen in earlier cultures were also present. A distinct basal lamina was not seen. Occasionally a small amount of amorphous material was present between the intact plasma membrane of the endothelium and the surface of the plastic dish (Fig. 5). Adjacent cells were separated by a space about 200 A in width in most areas (Fig. 6). Condensations of cytoplasm were occasionally located adjacent to the junctions. The exact morphology of the junctional complexes was not studied.

Subcultures

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multinucleate cells (Fig. 10), 5-10 times larger than the typical epithelioid cells, appeared and increased in number. The fine structure of these large multinucleate cells was heterogeneous (Fig. 11). The cytoplasm contained a variety of the usual organelles, including "lipid" droplets, many yacuoles and multivesicular bodies, and prominent Golgi complexes. Bundles of 60-70 • filaments similar to those seen in the periphery of endothelial cells or abundantly in cultured smooth muscle (9, 24) were rare. Specific endothelial granules were not observed in these large cells. Cultures of two independent primary isolates have now been passed 10 and 19 times, respectively. Each shows approximately 50% of the dish forming monolayers of small epithelioid cells with the typical fine structure of endothelium.

Phase contrast observations of first and second passage subcultures showed progression from individual clusters to confluent monolayers of closely packed polygonal cells within 6-7 days by spreading and cell division, as described for primary cultures. DISCUSSION With subsequent passages, many areas of The ultrastructure of cells cultured after the cultures still contained monolayers of small epithelioid cells similar in all re- collagenase treatment of human umbilical spects to those seen in primary cultures. In cord veins was investigated to identify all subcultures electron microscopic stud- these cells as vascular endothelium and to ies of areas selected from the epithelioid determine their subsequent behavior in monolayers revealed cells that were very subcultures. As observed by phase contrast microssimilar to those in confluent primary subcultures and to umbilical vein endothelium copy, primary cultures as well as the first in situ, respectively. In particular, they two subcultures exhibited a growth pattern contained specific endothelial granules of a uniform epithelioid monolayer of po(Fig. 7), peripheral pinocytosis vesicles lygonal cells. At confluence these were (Fig. 8), vacuoles, multivesicular bodies densely packed; previous autoradiographic (Fig. 8), and both thick (100 A) and thin studies using tritium labeled thymidine (60-70 A) filaments (Fig. 8). Figures 7 and have established that such cultures exhibit 9 are portions of endothelial cells from post-confluence inhibition of cell division different cell strains in the second and (10, 18). This study demonstrates that the nineteenth subcultures, documenting the individual endothelial cells in culture presence of specific endothelial granules. showed ultrastructural features that were After the second passage, however, the virtually identical with those of native following morphologic changes were ob- umbilical vein endothelium (20), and that served in some areas of the cultures: (1) the endothelial cells were strikingly unicells became more elongated, did not cover form in the various cultures examined. A unique marker for endothelium, the the available surface completely, and began to pile up; (2) some cells exhibited Weibel-Palade body was present in apreticulation and increased numbers of proximately 30-70% of cells. These specific droplets in the cytoplasm; and (3) irregular endothelial granules have been described

Fro. 7. W e i b e l - P a l a d e bodies in c u l t u r e d e n d o t h e l i u m (second passage) contain t u b u l e s with a d i a m e t e r of a p p r o x i m a t e l y 150 ~ ; s o m e of t h e t u b u l e s show a dense core in cross section (arrow). × 56 250. Fla. 8. Periphery of an endothelial cell in culture after 5 passages. 60-70 A f i l a m e n t s with dense zones (arrows), 100 A f i l a m e n t s (F), a b u n d a n t pinocytotic vesicles, s o m e cisternae of s m o o t h e n d o p l a s m a t i c r e t i c u l u m a n d a m u l t i v e s i c u l a r body are shown. × 31 050. 28

FIG. 9. Cytoplasm of human endothelium in culture after 19 passages. Weibel-Palade bodies (W), 250 A tubules (arrows) and 100 A filaments (F) look similar to primary cultures. In this part of the cytoplasm there is a slight increase of vacuoles and multivesicular bodies, x 27 000. 29

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in vascular endothelium of human, rat, rabbit, monkey, pig, chicken, dog, shark, and frog (7, 15, 27, 30, 31). They are particularly numerous in human umbilical

vein and artery endothelium. Since every passage represents approximately a doubling of the number of cells it can be assumed that cultured endothelium pro-

FIG. 10. Phase contrast light micrograph of a multinucleated large cell, 17th passage. × 360. FIG. 11. Portion of the cytoplasm of a large multinucleated cell, 19th passage, showing fat droplets (D), Golgi complexes (G), and a b u n d a n t vacuoles (V). × 6 570.

ENDOTHEL1UM IN CULTURE duced new specific endothelial granules. The granules were identified by their rodshape, their single limiting membrane, and their content of from 6 to 20 tubules each approximately 150 A in width. There are few other structures which may have a similar appearance under the electron microscope. The "spindle-shaped body" described in fibroblasts (28) is smaller and shows a fibrillar rather than a tubular internum. Fingertiplike pseudopodia invaginating into adjacent cells may sometimes be mistaken for specific endothelial granules in oblique sections; however, such pseudopodia contain 60-70 A filaments and exhibit two limiting membranes since they represent an intrusion of part of a cell into another. Using these as criteria, we have not observed specific endothelial granules in cultured human fibroblasts, 3T3 cells, or umbilical vein smooth muscle cells. The specific endothelial granules were sometimes seen close to the Golgi areas, but fusion of these areas as reported by others (25) was not observed. The function of the specific endothelial granules is unknown. Their lysosomal nature has been denied because of their negative reaction for acid phosphatase (•5); they have been thought to play a role in the coagulation process (1). It is hoped that endothelial cultures may enable us to collect and isolate enough of these granules to characterize their function and content more precisely. Other ultrastructural features of cultured endothelium also resemble those seen in native umbilical vein endothelium, but are also found in other cultured mesenchymal cells. In particular, the presence of peripheral pinocytosis vesicles, microtubules, 100 A filaments as well as 60-70 A filaments is characteristic of different cell types in culture, including fibroblasts. Franks and Cooper (5) suggested that many tissue culture cell lines established from normal embryonic tissue derived either from endothelium or from pericytes.

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Because of the close similarity between the scanning electron microscopic appearance of cultured 3T3 fibroblasts and endothelial cells, Porter et al. have recently suggested that the former may be endothelial in origin (23). To our knowledge, none of these lines contain specific endothelial granules. The presence, in cultured endothelium, of bundles of 60-70 • filaments, with fusiform densities deserves consideration, since these bundles are characteristic of smooth muscle cells. Our previous study (10) indicates that some of the cultures may be contaminated by smooth muscle cells from the umbilical vein wall. However, in contrast to cultured vascular smooth muscle in which the entire cytoplasm contains this type of filament (9, 24), bundles in endothelial cells are localized almost exclusively to the periphery of the cell. In addition, cultured vascular smooth muscle cells have a characteristic light microscopic appearance, pile up into several layers in older cultures, and are associated with abundant extracellular material (9, 24). The distribution of 60-70 A filaments on the top and on the bottom of cultured cells (close to adherent points to the plastic), is similar to that observed in endothelium in vivo. A distinct basal lamina similar to that seen in small-vessel endothelium was uniformly absent in our cultures. However, occasionally amorphous basal laminalike material was seen in sections perpendicular to the plane of the monolayer. Whether this is similar to the amorphous material seen in the subendothelium of large vessels (12) in vivo has not been established. The identity of the large pleomorphic multinucleate cells which appeared in later passages is unknown. They were significantly different in phase morphology and in ultrastructure from cultured vascular smooth muscle cells, which also are large and occasionally multinucleate. They lacked specific endothelial granules but contained all other organelles found in the

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s m a l l e r e n d o t h e l i a l cells. M u l t i n u c l e a t e e n d o t h e l i a l cells i n v i v o a r e o f t e n o b s e r v e d a t t h e e d g e of i n t i m a l l e s i o n s (4, 22, 26) a n d in a o r t a s of a g i n g h u m a n s (3). I t h a s b e e n s h o w n t h a t c o l c h i c i n e t r e a t m e n t of B H K 2 1 cells p r o d u c e s m u l t i n u c l e a t e g i a n t cells ( 1 1 ) . I t is p o s s i b l e t h e n t h a t s u c h m u l t i n u c l e a t e c e l l s w e r e i n d u c e d b y a g i n g or b y s o m e t o x i c effect. H o w e v e r , e v e n in t h e nineteenth passage the small polygonal cells w h i c h f o r m e d e p i t h e l i o i d s h e e t s cont i n u e d to e x h i b i t t h e t y p i c a l W e i b e l P a l a d e b o d i e s c h a r a c t e r i s t i c of e n d o t h e lium. We are grateful to the staff of the Boston Hospital for Women (Lying-In Division) for their kind cooperation in obtaining umbilical cords, and to Ms. Claire Baldwin and Ms. Christine Keller for their expert technical assistance. REFERENCES 1. BURRI, P. H. AND WEIBEL, E. R., Z. Zellforsch. Mikrosk. Anat. 88, 426 (1968). 2. COTRAN,R. S., in REEVE,E. B., ANDGUYTON,A. C. (Eds.), Physical Basis of Circulatory Transport, Regulation and Exchange, p. 249. Saunders, Philadelphia, Pennsylvania, 1967. 3. COTTON, R. AND WARTMAN,W. B., Arch Pathol. 71, 3 (1961). 4. FALLON,J. T. ANDSTEHBENS,W. E., Circ. Res. 31, 546 (1972). 5. FRANKS,L. M. AND COOPER,T. W., Int. J. Cancer 9, 19 (1972). 6. FRYER,D, G., BIRNBAUM,G. ANDLUTTRELL,C. N., J. Atheroscler. Res. 6, 151 (1966). 7. FUCHS, A. AND WEIREL, E. R., Z. Zellforsch. Mikrosh. Anat. 108, 105 (1970). 8. GIMBRONE,M. A., COTRAN,R. S. ANDFOLKMAN,J., Ser. Haematol. IV, 435 (1973).

9. GIMBRONE,M. A., COTRAN,R. S., HAUDENSCHILD, C. C. AND FOLKMAN,J. J. Cell Biol. 59, 1O9a,

(1973). 10. GIMBRONE,M. A., COTRAN,R. S. ANDFOLKMAN,J., J. Cell Biol. 60, 673 (1974). 11. GOLDMAN,R. D., J. Cell Biol. 51,752 (1971). 12. HAUDENSCHILD,C. C. ANDSTUDER,A., Eur. J. Clin. Invest. 2, 1 (1971). 13. JAFFE, E. A., NACHMAN,R. L., BECKER,C. G. AND MINICK, R. C., Circulation 46, II-252 (1972). 14. JAFFE, E. A., NACHMAN,R. L., BECKER, C. G., AND MINICK,R. C., J. Clin. Invest. 52, 2745 (1973). 15. LEMEUNIER,A., BURRI, P. H. AND WEIBEL, E. R., Histochemie 20, 143 (1969). 16. LEWIS,L. J., HOAK,J. C., MACA,R. D. ANDFRY,G. L., Science 181,454 (1973).

17. MAJNO, G., Handbook of Physiology, Sec. II, Circulation, Vol. III, p. 2293 (1965). 18. MARTZ,E. ANDSTEINBERG,M. S., J. Cell Physiol. 79, 189 (1972). 19. MARUYAMA,Y., Z. ZeUforsch. Mikrosk. Anat. 60, 69 (1963). 20. PARRY, E. W. AND ABRAMOVICH,D. R., J. Anat. 111, 29 (1972). 21. POMERAT,C. M. AND SLICK, W. C., Nature (London) 198, 859 (1963). 22. POOLE, J. C. F., SANDERS,A. G. AND FLOREY, H. W., J. Pathol. Bacteriol. 75, 133 (1958). 23. PORTER, K. R., TODARO, G. J. AND FONTE, V., J. Cell Biol. 59, 633 (1973). 24. Ross, R., J. Cell Biol. 50, 172 (I971). 25. SENGEL,A. ANDSTOEBNER,P., J. Cell Biol. 44, 223

(1970). 26. SINAPIUS,D., Arch. Anat. Pathol. 322,622 (1952). 27. STEINSIEPE,K. F. ANDWEIBEL,E. R., Z. Zellforsch. Mikrosh. Anat. 108, 105 (1970). 28. VOELZ,H., J. Cell Biol. 20, 333 (1964). 29. WECHEZAK,A. R. ANDMANSFIELD,P. B., In Vitro 9,

39 (1973). 30. WEIBEL,E. R. ANDPALADE,G. E., J. Cell Biol. 23,

101 (1964). 31. WEmEL, E. R., Acta Anat. 59,390 (1964).

Fine structure of vascular endothelium in culture.

JOURNAL OF ULTRASTRUCTURERESEARCH 50, 22 32 (1975) Fine Structure of Vascular Endothelium in Culture I CHRISTIAN C. HAUDENSCHILD, RAMZI S. COTRAN,2...
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