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Factors controlling the in Vitro growth pattern of human microvascular endothelial cells
JOURNAL OF ULTRASTRUCTURE RESEARCH 82, 7 6 - 8 9
(1983)
Factors Controlling the in Vitro Growth Pattern of Human Microvascular Endothelial Cells K. G. BENSCH,* P. M. DAVISON, AND M. A. KARASEK Departments of *Pathology and Dermatology, Stanford University School of Medicine, Stanford, California 94305 Received April 15, 1982; and in revised form September 8, 1982 Monolayer cultures of endothelial cells of human dermal microvascular origin were exposed to a variety of culture conditions and in vitro differentiation of the cells assessed by light and electron microscopic examination. Restoration of a cytologic and fine structural appearance which resembled most closely that present in vivo was possible by raising the intracellular cAMP level. These cells formed junctional complexes seen in uncontracted microvessels and specialized attachment sites at their basal cell membrane, contained a complex network of bundled micro- and intermediate filaments and numerous Weibel-Palade bodies and accumulated electron-opaque deposits between the cells and the culture dish surface.
absence of these substances (Davison and Karasek, 1981). On fine-structural analysis, however, it is apparent that there are significant cytologic differences between the apparently similar ceils grown on the fibronectin substratum and those with elevated cytoplasmic cAMP levels. Analyses of these differences are presented in this report and comparisons are made with human microvascular endothelial cells in vivo.
Capillary endothelial cells act as a highly selective two-way filter, the function of which varies with the tissue they serve. Of equal importance for normal vascular function are specific metabolic properties of the endothelium, particularly those related to coagulative and inflammatory processes. In vivo study of transendothelial transport, diffusion, and metabolic processes is fraught with difficulties because of the large number of factors and variables that have to be taken into account in the interpretation of the findings. Thus models of microvascular endothelium lend themselves more readily to experimental analyses, although they may not be entirely representative of function under physiologic conditions. We have recently reported a method for growing human capillary endothelial cells for prolonged periods of time in vitro (Davison et al., 1980). Proliferation of these cells is dependent upon the presence of a high concentration of human serum in the culture medium and one of two factors, a fibronectin substratum or agents that elevate intracellular levels of cyclic AMP. Under either of these conditions the cells grow as expanding confluent colonies of cuboidal cells in marked contrast to the elongated, single, virtually nonproliferating cells seen in the
MATERIALS AND METHODS
Isolation of microvascular endothelial cells. Endothelial cells lining the microvessels of the newborn human foreskin dermis were isolated as described elsewhere (Davison et al., 1980). Briefly, the tissue was trimmed of underlying facia, flattened on a dissecting board and the epidermis removed using a Castroviejo keratotome set to cut at 0.1 mm. The epidermis was discarded and the dermis cut into 5-ram-square sections that were incubated for 40 rain at 37°C in 0~3% trypsin solution containing 1% EDTA. The sections were rinsed extensively with 0.9% saline, the endothelial cells expressed from the dermal tissue into Eagle's minimal essential medium (MEM) containing 10% pooled human serum using the back of a No. 10 scalpel blade, collected by centrifugation (800 g for 1 rain), and resuspended in MEM containing 10% pooled human serum and antibiotics. Cells were plated onto either plain plastic Petri dishes (Lux 35 × 10 ram) or dishes coated with fibroneetin (Davison et al., 1980). Cell culture. All ceils were maintained in Eagle's minimal essential medium supplemented with 10% 76
0022-5320/83/010076-14503.00/0 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Cells growing on plain dishes show a random pattern of attachment to the culture dish with little contact between cells, most of which are of irregular, elongate form. × 157. FIG. 2. In contrast to the culture shown in Fig. 1, cells exposed to a fibronectin covered surface form colonies of closely apposed cells, x 157. FIG. 3. A growth pattern, similar to that seen on exposure to llbronectin, is observed when cells are grown on a plain dish in a medium containing CT and IMX. x 157.
pooled human serum (Davison and Karasek, 1981). Cholera enterotoxin (CT) (1 x 10-9 M) together with a 3-isobutyl-l-methylaxanthine (IMX) (3.3 x 10-~ M) were added to half of the cultures that were plated onto plain plastic (Davison and Karasek, 1981). Cells were maintained at 37°C in an atmosphere of 94% air and 6% CO2. The culture medium was replaced twice weekly. Electron microscopy. Petri dishes containing the cells were fixed by overlaying the monolayer cultures with a 1:1 mixture of culture medium with 3% phosphatebuffered glutaraldehyde solution at room temperature (Sabatini et al., 1963; Gordon et al., 1963). This fixative was replaced 15 min later with a mixture of 2% phosphate-buffered glutaraldehyde solution. The cultures were rinsed with phosphate buffer prior to a 1-hr treatment with buffered 1% OsO 4 (Palade, 1952). After dehydration in ethanol, the cells were embedded in situ in epoxy plastic (Richardson et al., 1960). U1trathin sections cut perpendicular and parallel to the plastic culture dish were stained with uranyl and lead salts and examined in an Elmiskop 101 electron microscope (Venable and Coggeshall, 1965). Morphometric evaluations were carried out on 25 electron micrographs of cells grown under each of the three different culture conditions. Each random photograph (at 20 000 ×) of a cell included a section of the
cell membrane. A 285-point grid was used for the determination of the fractional volumes according to the method of Weibel (1963) and of Rohr et al. (1976).
RESULTS
Light Microscopy Human microvascular endothelial cells plated onto a fibronectin-coated substratum attach rapidly as small sheets of cuboidal cells. These circular colonies expand in size as the cells proliferate (Fig. 2). A similar pattern is observed when the cells are grown on a plain plastic surface but in a medium supplemented with CT and IMX (Fig. 3). These two agents have been found to promote a severalfold proliferation of human dermal microvascular endothelial cells and bring about a pronounced increase in intracellular cAMP levels; the extent of these changes is similar to that produced by addition of dibutyryl-cAMP to the medium (Davison and Karasek, 1981). The resulting
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organized growth pattern is in marked contrast with that seen when cells are plated onto a plain plastic culture dish and nourished by medium lacking CT and IMX. Although cells do attach initially as small cell sheets under this condition, the plating efficiency is less than half that obtained with the added growth factors. In addition, as the cells slowly proliferate they fail to remain as cohesive colonies and single cells are seen at the periphery of cell groups. These individual cells are fusiform to nearly triangular in outline, usually with one to three long narrow processes. After 5 to 7 days, such cultures are primarily composed of individual cells distributed in a random manner and with few lateral associations to other cells (Fig. 1).
Electron Microscopy Cell attachment. Sections cut through a cell layer perpendicular to the plane of the culture surface provide detailed information on the attachment of the cells to the substratum and the extent of cell spreading. Figure 4 shows the relatively poor adhesion of cells to the dish and lack of spreading of the cytoplasm of cells grown on plain plastic in the absence of medium supplements. The plump, solitary cells are intermediate in form between floating, round cells and those that have spread to form a typical, contiguous, thin cell layer as present with cells grown on a fibronectin-coated dish (Fig. 5). Endothelial cells plated onto plain plastic surfaces, but exposed to a medium containing CT and IMX, exhibit a spreading behavior nearly identical to that observed with cells exposed to fibronectin (Fig. 6). Under either of these conditions, cells are highly flattened, and the cytoplasm
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is extensively spread and closely adherent to the substratum. Junctions are formed between adjacent cells. In vivo endothelial cells have a polarity with a distinct vascular luminal and basal cell surface. The latter normally forms a basal lamina to which it is frmly attached. Formation of this lamina was not observed in our experiments. The cell membrane of cells cultured on plain dishes adheres closely to the plastic with little intervening space. In contrast, perpendicular sections through cells grown on the fibronectin coated dishes show a thin, finely, and usually uniformly granular layer between the plastic and the cell, even after prolonged culture. This material is most likely the fibronectin that was spread on the culture vessels prior to explanting the cells. At points of close association of microfilament bundles with the basal cell membrane, however, extracellular electron-opaque patches are observed (Fig. 9). These resemble structures usually referred to as focal adhesions and they possibly represent cell secretions. In the CT and IMX exposed cells there is a nonuniform, thin, and finely granular layer of electron-dense material between the cells and the plastic. It should be noted, that these cells were seeded on uncoated dishes.
Cytoskeletal Organization Microfilaments. At higher magnification, quantitative and organizational differences in the cytoplasmic organelles within cells maintained under each of the three growth conditions are apparent. Particularly striking are the differences in the distribution of the microfilaments (about 70 ~ diameter) and the intermediate (90-100/~) filaments. Cells grown on plain dishes contain a uni-
FIGS. 4-6. Electron micrographs of cells grown under conditions identical to those shown in Figs. 1-3. All of the sections were cut perpendicular to the culture dish surface; all magnifications are at × 4300. FIG. 4. Exposed to a plain culture dish, cells do not flatten and cell-to-cell contact does not occur. FIG. 5. In contrast to Fig. 4, a fibronectin substratum results in rapid, uniform spreading of cytoplasm with close lateral contact with neighboring cells. FIG. 6, Similar to the pattern shown in Fig. 5, cells exposed to a plain dish in a medium containing CT and IMX grow in a uniform, thin monolayer with formation of specialized cell junctions with neighboring cells.
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FIG. 7. Section parallel to the dish surface of ceils grown on an uncoated dish shows a uniform layer of subplasmalemmal microfilaments and an irregular array of intermediate filaments throughout the cytoplasm. x 5800.
form, relatively thick layer of microfilaments in the subplasmalemmal cytoplasm facing the culture medium; it is clearly visible as an electron-opaque band at the lateral cell margin in sections cut parallel to the plane of a dish (Fig. 7). Closer examination of the " l u m i n a l " cell wall shows these filaments to be in parallel arrays (Fig. 8). A similar microfilament layer is also present in cells grown on fibronectin. But in these cells, the filament layer consists of flat bundles and it is located subjacent to both the luminal and abluminal cell membrane. In the later location, the bundles of filaments often appear to make contact with the cell membrane. At such points, an illdefined, electron-opaque plaque can be seen on the outside of the cell membrane (Fig. 9). Cells treated with CT and IMX exhibit
a similar organization of their microfilaments in relation to the basal cell membrane. But, in addition, a number of these cells Show beneath the luminal surface a convergence of microfilaments resulting in dense bodylike structures (Fig. 10). Morphphometric analyses showed signifcant differences in the fractional volume of the filaments between cells grown under different conditions. In sections cut parallel to the substratum surface, the cytoplasmic fractional volume of filament bundles was 0.138 ___ 0.121, 0.031 _ 0.055, and 0.072 _+ 0.108 for cells grown on plain dishes, fibronectin-coated dishes, and cells treated with the drugs, respectively. It should be noted that these figures do not represent the absolute volumes of cytoplasm occupied by filaments because there is always nonfilamentous cytoplasmic substance between the
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FIG. 8. High magnification of the cell depicted in Fig. 7 shows the parallel array of the cell membraneassociated microfilaments. × 50 000.
individual threads of a bundle (see Fig. 9). There also was a difference in the size of the bundles; the fractional volume per bundle was 0.0035, 0.0019, and 0.0042 for cells grown on plain dishes, fibronectin c o v e r e d dishes, and cells e x p o s e d to the drugs. Intermediate filaments. Intermediate filaments (9-11 nm) are rare in cells grown on plain dishes. In contrast, in cells grown on fibronectin, they often form loose bundles of about a dozen filaments that crisscross the cytoplasm. T h e y are also present beneath the cell surface, although not as closely a p p o s e d to it as the microfilaments,
and frequently they run parallel to the micro filament bundles (Fig. 9). An a b u n d a n c e of intermediate filaments is one of the striking features of cells cultivated in the presence of CT and I M X . It is not unusual to find nuclei e n v e l o p e d in a thick layer of these filaments or to o b s e r v e bundles of them measuring as m u c h as 1.5 /xm in diameter coursing through the cytoplasm (Fig. 11). Cell Junctions. F o r m a t i o n of specialized cell junctions are an inherent p r o p e r t y of enthothelial cells as part of their function as a b l o o d - t i s s u e barrier. Intercellular junctions
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FIG. 10. Section similar to that shown in Fig. 8, of cell exposed to CT and IMX. Note the dense bodies formed by coverging bundles of microfilaments. Part of the cell surface is visible along the upper left margin of the illustration, x 22 400. are p r e s e n t in c u l t u r e d cells, their n u m b e r d e p e n d i n g g r e a t l y o n the c u l t u r e c o n d i t i o n s . E n d o t h e l i a l cells o n p l a i n p l a s t i c dishes h a v e v e r y few close c e l l - c e l l contacts a n d do n o t show a n y specialized forms of i n t e r c e l l u l a r c o n t a c t at t h e s e p o i n t s . I n c o n t r a s t , cells g r o w n o n f i b r o n e c t i n f o r m s p e c i a l i z e d cell j u n c t i o n s . A t p o i n t s of c o n tact t h e r e is a close a p p o s i t i o n o f the cell
m e m b r a n e s with a n i n c r e a s e in the e l e c t r o n d e n s i t y o f the a d j a c e n t c y t o p l a s m ; t h e s e j u n c t i o n s h a v e the a p p e a r a n c e of m a c u l a e or z o n u l a e o c c l u d e n t e s . I n a d d i t i o n to t h e s e s p e c i a l i z e d j u n c t i o n s , i n t e r d i g i t a t i o n s of the c y t o p l a s m of a d j a c e n t cells are f r e q u e n t l y o b s e r v e d in cells g r o w n in the p r e s e n c e of C T a n d I M X (Fig. 12). A t t h e s e p o i n t s o f j u x t a p o s i t i o n , b u n d l e s of microfilaments r u n
FIG. 9. Cells grown on a fibronectin-covered dish contain cell membrane-associated bundles of microfilaments (short arrows), two of which are particularly close to the basal cell membrane (double short arrows). Note the electron-opaque plaques at the cell outside at this point (hollow arrows). Intermediate filaments form loose bundles, several of which run parallel to the microfilament bundles (long arrows). Arrowheads point at a cross and a tangentially sectioned Weibel-Palade body. x 68 000.
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FIG. 12. Section perpendicular to dish surface of cell exposed to CT and IMX. Close apposition at point of contact between adjacent cells is marked by increased electron density. Arrowhead points at tangentially sectioned bundles of microfilaments running parallel to line of intercellular contact; arrow points at bundles of intermediate filaments. Note deposits between plastic and cells. × 30 000.
parallel to the line of contact between cells (Fig. 12). Cellular Organelles
There are other, albeit less obvious differences between the cytoplasmic organelles of cells grown under the different conditions. Cells of c u l t u r e s g r o w n on fibronectin and cells exposed to CT and IMX also have a large complement of Weibel-Palade bodies. In contrast, these bodies are very rare in endothelial cells grown on plain plastic dishes. Although there appear to be fewer pinocytotic vesicles in cells grown on plastic dishes, morphometric evaluation does not show a statistically significant difference between the three groups. DISCUSSION
There are at least half a dozen different types of vascular endothelia in mammals, with the main varieties being the continuous, the fenestrated, and the discontinous endothelial lining (Majno, 1965). Endothelial cells of human dermal microvessels were
used in our study. These vessels are lined by a continuous layer of low endothelium which readily detaches from its basal lamina by gentle treatment with trypsin in vitro. Recent progress in tissue culture techniques now permits long term cultivation of these cells (Davison et al., 1980). One of the major prerequisites for such growth is an in vitro environment which resembles the in vivo situation in several points: high serum concentrations of the culture medium and a substratum similar to that of the capillary wall. The latter, as shown by our studies, can be substituted for by exposure of the cells to pharmacological agents known to significantly raise the intracellular cAMP levels (Davison and Karasek, 1981). The aim of this study was a delineation of tissue culture conditions which result in the restoration of the functional ultrastructure of human microcapillary endothelial cells. In vitro cultivation in the presence of relatively high serum concentrations (1050%) allowed these cells to attach themselves to a Nain plasic culture surface and
FIG. 11. Cells exposed to CT and IMX contained large numbers of intermediate filaments, often surrounding the nucleus. Note the abundance of pinocytotic vesicles, x 24 000.
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to multiply slowly. Under these conditions, however, the cells are not capable of flattening out and establishing specialized cellcell junctions which are a prerequisite for the normal function of the dermal capillary endothelium and characteristic of these cells (Shepro, 1977). Furthermore, aside from a random pattern of distribution, there is a general paucity of micro- and intermediate cytoplasmic filaments and they are unable to form bundles as normally observed in vivo. In addition, cells in such a medium soon lose their specific markers, the Weibel-Palade bodies (Weibel and Palade, 1964). When plated on a substratum of fibronectin, however, the same cell population not only reorganizes into contiguous groups of flattened cells, but also forms intercellular junctions. In addition, a rearrangement of the cytoplasmic micro- and intermediate filaments also occurs. Thus, provision of a substratum results in a partial restoration of the normal appearance of these cells, a result that had been previously observed by others in in vitro studies of fibroblasts treated with fibronectin (Yamada and Olden, 1978). Two types of fibronectin are known, the fibronectin, presently in relatively high concentrations, and the cellular (cell surface) fibronectin which has been most extensively studied on fibroblasts (Yamada and Olden, 1978). Cellular fibronectin is also produced by several other cell types, including endothelial cells (Birdwell et al., 1978; Jaffe and Mosher, 1978; Macarak et al., 1978). Fibronectinl also called spreading factor, is a major, large cell surface and interstitial glycoprotein that plays an important role in cell morphology, mobility, cell-cell aggregation, cell-substratum adhesiveness, and other biological activities (Yamada and
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Olden, 1978; Grinnell, 1976). It is present, at least in fibroblasts, as an organized network on the cell surface, the maintenance of which appears to be interdependent with that of the cytoplasmic microfilament organization (Kurkinen et al., 1978; Chen et al., 1978). In addition, however, cell surface fibronectin can bind to collagen, thus mediating attachment of dissociated cells to collagen-containing substrata, including basement membranes (Pearlstein, 1976; Engvall and Ruoslahti, 1977). Fibronectin also attaches to plastic surfaces which would explain the observed spreading of the endothelial cells used in our experiments (Ali et al., 1977). Their aggregation into groups of cells is most likely not only caused by the known greater cell-cell adhesiveness after fibronectin treatment, but also the greater mobility of such cells due to more effective cell-substratum traction (Yamada et al., 1976). The closest ultrastructural resemblance of the cultured endothelial cells to those present in dermal microvessels was present in monolayer cultures treated with agents known to significantly raise the intracellular cAMP level. These cells formed a highly organized intracellular network of microand intermediate filaments, branches of which extended closely to intercellular cell junctions and to points of apparent cell attachment of the abluminal cell surface to extracellular, electron-opaque deposits; these were present between the originally plain culture dish surface and the cell. Thus, under such stimuli, endothelial cells are able to produce and presumedly also secrete fibronectin and possibly other substances in quantities sufficient for cell-cell aggregation, cell spreading, and formation of specialized sites of attachment to extracellular structures (Macarak and Kefalides, 1978). Our understanding of the morphology and
FIG. 13. Section o f foreskin microvessel. A n e t w o r k of microfilaments is p r e s e n t t h r o u g h o u t the c y t o p l a s m of the flat endothelial cells; note that it is particularly p r o m i n e n t near the abluminal cell m e m b r a n e (arrowhead). The short arrow points at a zonula a d h e r e n s , the hollow arrows at cross- and tangentially sectioned W e i b e l Palade bodies, x 37 000.
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the compounds responsible for focal sites of adherence between cells and structures in their surroundings is still incomplete. In vitro studies, particularly on fibroblasts, however, have shown that several types of such contact exist (Chen and Singer, 1980). Depending on the distance between a cell membrane and the substratum and the size of such attachment sites, at least two different types are now recognized, so-called focal adhesions and close contacts. In our experiments with agents that raise the intracellular cAMP level, we observed bundles of microfilaments in very close proximity to the basal cell membrane, sometimes even appearing to merge with it. At such points, there usually was a focal aggregation of extracellular, electron-opaque material very near the cell surface. The nature of these extracellular deposits is not known, but immunocytochemical studies carried out by others on sites of focal adhesions of fibroblasts indicate that it is most likely not fibronectin (Chen and Singer, 1980). The latter appears to localize as an extracellular matrix surrounding such focal adhesion sites and, possibly, also as extracellular strips paralleling the course of microfilarnent bundles where they lie close to the cell membrane. Intercellular junctions were well developed in our cultures treated with CT and IMX. These had the cross-sectional profile of junctional modifications of undilated vessels with imbrications of cytoplasmic ridges of neighboring cells (Tripathi and Tripathi, 1977). In addition, junctional complexes were prominent at these sites. Aside from tight junctions, there were adherent junctions with bundles of microfilaments in their immediate vicinity. Bundled intermediate filaments were not observed in these areas, but they were commonly encountered elsewhere and they are most likely composed of vimentin (Renner et al., 1981). The role of these and/or the microfilaments in the contraction of microvessels under physiological conditions and individual cells during the inflammatory response remains to be
established (Hammersen, 1976, 1977; Addicks et al., 1979; Goldman et al., 1979). In contrast to endothelial cells without elevated cAMP levels, Weibel-Palade bodies were prominent in the CT and IMX treated cells and they persisted on prolonged subculture. This increase in number may be related to the increased growth rate of endothelial cells observed in the presence of cyclic AMP in vitro (Davison and Karasek, 1981). During accelerated proliferation of the microvasculature in vivo, increase in number of Weibel-Palade bodies also occurs (Kumar et al., 1980; Lonchampte and Regnault, 1978; Takeshige and Fujimoto, 1977). An explanation for the interesting relationship between proliferation rate and Weibel-Palade bodies both in vivo and in vitro is not known. In conclusion, these experiments indicate that the use of monolayer cultures as an in vitro model for the study of endothelial cells requires fulfillment of several conditions for the establishment of the functional ultrastructure of this cell type. Serum supplements to the culture medium are insufficient for in vitro differentiation of endothelial cells. Addition of a fibronectin substratum leads to a partial restoration of the in vitro morphology. The latter, however, is only reached after elevation of the cytoplasmic cAMP level. The authors thank M. EUen Charlton and Irma Daehne for technical assistance. REFERENCES ADDICKS, K., WEIGELT, H., HANCK, G., LUEBBERS, D. W., AND KNOCHE, H. (1979) Bibl. Anat. 17, 21. ALI, I. V., MAUTNER, V. M., LANZA, R., AND HYNES, R . O . (1977). Cell 11, 115. BIRDWELL, C. R., GOSPODAROWICZ, D., AND NICKOLSON, G. L. (1978) Proc. Nat. Acad. Sci. USA 75, 3273. CHEN, L. B., MURRAY, A., SEGAL,R. A., BUSHNELL, A., AND WALSH, M. L. (1978) Cell 14, 377. CHEN, W. T., AND SINGER, S. J. (1980) Proc. Nat. Acad. Sci. USA 77, 7318. DAVISON, P. M., BENSCH, K., AND KARASEK, M. A. (1980) J. Invest. Dermatol. 75, 316. DAVISON, P. M., AND KARaSEK, M. A. (1981) J. Cell. Physiol. 106, 253.
MICROVASCULAR ENDOTHELIAL CELLS ENGVALL, E., AND RUOSLAHTI, E. (1977) Int. J. Cancer 20, 1. GOLDMAN, R. D., MILSTED, A., SCHLOSS, J. A., STARGER,J., AND YE~A, M.J. (1979) Annu. Rev. Physiol. 41,703. GORDON, G. B., MILLER, L. R., A N D BENSCH, K. G. (1963) Exp. Cell Res. 31, 440~43. GRINNELL, F. (1976) Exp. Cell Res. 97, 265. HAMMERSEN, F. (1976) Beitr. Pathol. 157, 327. HAMMERSEN, F. (1977) Bibl. Anat. 16, 370. JAFEE, E. A., AND MOSHER, D. F. (1978) J. Exp. Med. 147, 1779. KUMAR, P., KUMAR, S., MARSDEN, H. B., LYNCH, P. G., AND EARNSHAW, E. (1980) CancerRes, 40, 2010. KURKINEN, M., WARTIOVAARA,J., AND VAHERI, A. (1978) Exp. Cell. Res. 111, 127. LONCHAMPTE, M. O., AND REGNAULT, F. (1978) in REGNAULT, F., AND CUHAULT, J. (Eds.), Cellular and Biochemical Aspects in Diabetic Retinopathy. INSERM Symposium No. 7, Elsevier/North Holland, Amsterdam. LUFT, H. J. (1961) J. Biophys. Biochem. 9, 409. MACARAK, E. J., AND KEFALIDES, N . A . (1978) in KEFALIDES, N. A. (Ed.), Biology and Chemistry of Basement Membranes, p. 343, Academic Press, New York. MACARAK, E. J., KIRBY, E., KIRK, T., AND KEEALIDES, N. A. (1978) Proc. Nat. Acad. Sci. USA 75, 2621.
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MAJNO, G. (1965) in HAMILTON, W. F., AND DOW, P. (Eds.), Handbook of Physiology, Section 2, Vol. III, p. 2293, Amer. Physiol. Soc., Washington, D. C. PALADE, G. E. (1952) J. Exp. Med. 95, 285-298. PEARLSTEIN, E. (1976) Nature (London) 262, 497. RENNER, W., FRANKE, W. W., SCHMID, E., GEISLER, M., WEBER, K., AND MANDELKOW, E. (1981) J. Mol. Biol. 149, 285. RICHARDSON, K. C., JARRETT, L., AND EINKE, E. H. (1960) Stain Tech. 35, 313-323. ROHR, H., OBERHOLZER,M., BARTSCH,G., AND KELLER, M. (1976) Int. Rev. Exp. Pathol. 15,233-325. SABATINI, D. D., BENSCH, K., AND BARRNETT, R. J. (1963) J. CellBiol. 17, 19-58. SHEPRO, D. (1977) Bibl. Anat. 16, 384. TAKESHIGE,Y., AND FUJIMOTO,S. (1977) Bibl. Anat. 16, 364. TRIPATHI, R. C., AND TmPATHI, B. Y. (1977) Bibl. Anat. 16, 307. VENABLE, J. H., AND COGGESHALL, R. (1965) J. Cell Biol. 25, 407--408. WEIBEL, E. R., AND PALADE, G. E. (1964) J. Cell Biol. 23, 101. WEIBEL, E . R . (1963) Morphometry of Human Lung, Springer-Verlag, Heidelberg Academic Press, New York. YAMADA, K. M., AND OLDEN, K. (1978) Nature (London) 275, 179. YAMADA, K. M., YAMADA, S. S., AND PASEAN, I. (1976) Proc. Nat. Acad. Sci. USA 73, 1217.
(1983)
Factors Controlling the in Vitro Growth Pattern of Human Microvascular Endothelial Cells K. G. BENSCH,* P. M. DAVISON, AND M. A. KARASEK Departments of *Pathology and Dermatology, Stanford University School of Medicine, Stanford, California 94305 Received April 15, 1982; and in revised form September 8, 1982 Monolayer cultures of endothelial cells of human dermal microvascular origin were exposed to a variety of culture conditions and in vitro differentiation of the cells assessed by light and electron microscopic examination. Restoration of a cytologic and fine structural appearance which resembled most closely that present in vivo was possible by raising the intracellular cAMP level. These cells formed junctional complexes seen in uncontracted microvessels and specialized attachment sites at their basal cell membrane, contained a complex network of bundled micro- and intermediate filaments and numerous Weibel-Palade bodies and accumulated electron-opaque deposits between the cells and the culture dish surface.
absence of these substances (Davison and Karasek, 1981). On fine-structural analysis, however, it is apparent that there are significant cytologic differences between the apparently similar ceils grown on the fibronectin substratum and those with elevated cytoplasmic cAMP levels. Analyses of these differences are presented in this report and comparisons are made with human microvascular endothelial cells in vivo.
Capillary endothelial cells act as a highly selective two-way filter, the function of which varies with the tissue they serve. Of equal importance for normal vascular function are specific metabolic properties of the endothelium, particularly those related to coagulative and inflammatory processes. In vivo study of transendothelial transport, diffusion, and metabolic processes is fraught with difficulties because of the large number of factors and variables that have to be taken into account in the interpretation of the findings. Thus models of microvascular endothelium lend themselves more readily to experimental analyses, although they may not be entirely representative of function under physiologic conditions. We have recently reported a method for growing human capillary endothelial cells for prolonged periods of time in vitro (Davison et al., 1980). Proliferation of these cells is dependent upon the presence of a high concentration of human serum in the culture medium and one of two factors, a fibronectin substratum or agents that elevate intracellular levels of cyclic AMP. Under either of these conditions the cells grow as expanding confluent colonies of cuboidal cells in marked contrast to the elongated, single, virtually nonproliferating cells seen in the
MATERIALS AND METHODS
Isolation of microvascular endothelial cells. Endothelial cells lining the microvessels of the newborn human foreskin dermis were isolated as described elsewhere (Davison et al., 1980). Briefly, the tissue was trimmed of underlying facia, flattened on a dissecting board and the epidermis removed using a Castroviejo keratotome set to cut at 0.1 mm. The epidermis was discarded and the dermis cut into 5-ram-square sections that were incubated for 40 rain at 37°C in 0~3% trypsin solution containing 1% EDTA. The sections were rinsed extensively with 0.9% saline, the endothelial cells expressed from the dermal tissue into Eagle's minimal essential medium (MEM) containing 10% pooled human serum using the back of a No. 10 scalpel blade, collected by centrifugation (800 g for 1 rain), and resuspended in MEM containing 10% pooled human serum and antibiotics. Cells were plated onto either plain plastic Petri dishes (Lux 35 × 10 ram) or dishes coated with fibroneetin (Davison et al., 1980). Cell culture. All ceils were maintained in Eagle's minimal essential medium supplemented with 10% 76
0022-5320/83/010076-14503.00/0 Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Cells growing on plain dishes show a random pattern of attachment to the culture dish with little contact between cells, most of which are of irregular, elongate form. × 157. FIG. 2. In contrast to the culture shown in Fig. 1, cells exposed to a fibronectin covered surface form colonies of closely apposed cells, x 157. FIG. 3. A growth pattern, similar to that seen on exposure to llbronectin, is observed when cells are grown on a plain dish in a medium containing CT and IMX. x 157.
pooled human serum (Davison and Karasek, 1981). Cholera enterotoxin (CT) (1 x 10-9 M) together with a 3-isobutyl-l-methylaxanthine (IMX) (3.3 x 10-~ M) were added to half of the cultures that were plated onto plain plastic (Davison and Karasek, 1981). Cells were maintained at 37°C in an atmosphere of 94% air and 6% CO2. The culture medium was replaced twice weekly. Electron microscopy. Petri dishes containing the cells were fixed by overlaying the monolayer cultures with a 1:1 mixture of culture medium with 3% phosphatebuffered glutaraldehyde solution at room temperature (Sabatini et al., 1963; Gordon et al., 1963). This fixative was replaced 15 min later with a mixture of 2% phosphate-buffered glutaraldehyde solution. The cultures were rinsed with phosphate buffer prior to a 1-hr treatment with buffered 1% OsO 4 (Palade, 1952). After dehydration in ethanol, the cells were embedded in situ in epoxy plastic (Richardson et al., 1960). U1trathin sections cut perpendicular and parallel to the plastic culture dish were stained with uranyl and lead salts and examined in an Elmiskop 101 electron microscope (Venable and Coggeshall, 1965). Morphometric evaluations were carried out on 25 electron micrographs of cells grown under each of the three different culture conditions. Each random photograph (at 20 000 ×) of a cell included a section of the
cell membrane. A 285-point grid was used for the determination of the fractional volumes according to the method of Weibel (1963) and of Rohr et al. (1976).
RESULTS
Light Microscopy Human microvascular endothelial cells plated onto a fibronectin-coated substratum attach rapidly as small sheets of cuboidal cells. These circular colonies expand in size as the cells proliferate (Fig. 2). A similar pattern is observed when the cells are grown on a plain plastic surface but in a medium supplemented with CT and IMX (Fig. 3). These two agents have been found to promote a severalfold proliferation of human dermal microvascular endothelial cells and bring about a pronounced increase in intracellular cAMP levels; the extent of these changes is similar to that produced by addition of dibutyryl-cAMP to the medium (Davison and Karasek, 1981). The resulting
78
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organized growth pattern is in marked contrast with that seen when cells are plated onto a plain plastic culture dish and nourished by medium lacking CT and IMX. Although cells do attach initially as small cell sheets under this condition, the plating efficiency is less than half that obtained with the added growth factors. In addition, as the cells slowly proliferate they fail to remain as cohesive colonies and single cells are seen at the periphery of cell groups. These individual cells are fusiform to nearly triangular in outline, usually with one to three long narrow processes. After 5 to 7 days, such cultures are primarily composed of individual cells distributed in a random manner and with few lateral associations to other cells (Fig. 1).
Electron Microscopy Cell attachment. Sections cut through a cell layer perpendicular to the plane of the culture surface provide detailed information on the attachment of the cells to the substratum and the extent of cell spreading. Figure 4 shows the relatively poor adhesion of cells to the dish and lack of spreading of the cytoplasm of cells grown on plain plastic in the absence of medium supplements. The plump, solitary cells are intermediate in form between floating, round cells and those that have spread to form a typical, contiguous, thin cell layer as present with cells grown on a fibronectin-coated dish (Fig. 5). Endothelial cells plated onto plain plastic surfaces, but exposed to a medium containing CT and IMX, exhibit a spreading behavior nearly identical to that observed with cells exposed to fibronectin (Fig. 6). Under either of these conditions, cells are highly flattened, and the cytoplasm
79
is extensively spread and closely adherent to the substratum. Junctions are formed between adjacent cells. In vivo endothelial cells have a polarity with a distinct vascular luminal and basal cell surface. The latter normally forms a basal lamina to which it is frmly attached. Formation of this lamina was not observed in our experiments. The cell membrane of cells cultured on plain dishes adheres closely to the plastic with little intervening space. In contrast, perpendicular sections through cells grown on the fibronectin coated dishes show a thin, finely, and usually uniformly granular layer between the plastic and the cell, even after prolonged culture. This material is most likely the fibronectin that was spread on the culture vessels prior to explanting the cells. At points of close association of microfilament bundles with the basal cell membrane, however, extracellular electron-opaque patches are observed (Fig. 9). These resemble structures usually referred to as focal adhesions and they possibly represent cell secretions. In the CT and IMX exposed cells there is a nonuniform, thin, and finely granular layer of electron-dense material between the cells and the plastic. It should be noted, that these cells were seeded on uncoated dishes.
Cytoskeletal Organization Microfilaments. At higher magnification, quantitative and organizational differences in the cytoplasmic organelles within cells maintained under each of the three growth conditions are apparent. Particularly striking are the differences in the distribution of the microfilaments (about 70 ~ diameter) and the intermediate (90-100/~) filaments. Cells grown on plain dishes contain a uni-
FIGS. 4-6. Electron micrographs of cells grown under conditions identical to those shown in Figs. 1-3. All of the sections were cut perpendicular to the culture dish surface; all magnifications are at × 4300. FIG. 4. Exposed to a plain culture dish, cells do not flatten and cell-to-cell contact does not occur. FIG. 5. In contrast to Fig. 4, a fibronectin substratum results in rapid, uniform spreading of cytoplasm with close lateral contact with neighboring cells. FIG. 6, Similar to the pattern shown in Fig. 5, cells exposed to a plain dish in a medium containing CT and IMX grow in a uniform, thin monolayer with formation of specialized cell junctions with neighboring cells.
80
BENSCH, DAVISON, AND KARASEK
FIG. 7. Section parallel to the dish surface of ceils grown on an uncoated dish shows a uniform layer of subplasmalemmal microfilaments and an irregular array of intermediate filaments throughout the cytoplasm. x 5800.
form, relatively thick layer of microfilaments in the subplasmalemmal cytoplasm facing the culture medium; it is clearly visible as an electron-opaque band at the lateral cell margin in sections cut parallel to the plane of a dish (Fig. 7). Closer examination of the " l u m i n a l " cell wall shows these filaments to be in parallel arrays (Fig. 8). A similar microfilament layer is also present in cells grown on fibronectin. But in these cells, the filament layer consists of flat bundles and it is located subjacent to both the luminal and abluminal cell membrane. In the later location, the bundles of filaments often appear to make contact with the cell membrane. At such points, an illdefined, electron-opaque plaque can be seen on the outside of the cell membrane (Fig. 9). Cells treated with CT and IMX exhibit
a similar organization of their microfilaments in relation to the basal cell membrane. But, in addition, a number of these cells Show beneath the luminal surface a convergence of microfilaments resulting in dense bodylike structures (Fig. 10). Morphphometric analyses showed signifcant differences in the fractional volume of the filaments between cells grown under different conditions. In sections cut parallel to the substratum surface, the cytoplasmic fractional volume of filament bundles was 0.138 ___ 0.121, 0.031 _ 0.055, and 0.072 _+ 0.108 for cells grown on plain dishes, fibronectin-coated dishes, and cells treated with the drugs, respectively. It should be noted that these figures do not represent the absolute volumes of cytoplasm occupied by filaments because there is always nonfilamentous cytoplasmic substance between the
MICROVASCULAR ENDOTHELIAL CELLS
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FIG. 8. High magnification of the cell depicted in Fig. 7 shows the parallel array of the cell membraneassociated microfilaments. × 50 000.
individual threads of a bundle (see Fig. 9). There also was a difference in the size of the bundles; the fractional volume per bundle was 0.0035, 0.0019, and 0.0042 for cells grown on plain dishes, fibronectin c o v e r e d dishes, and cells e x p o s e d to the drugs. Intermediate filaments. Intermediate filaments (9-11 nm) are rare in cells grown on plain dishes. In contrast, in cells grown on fibronectin, they often form loose bundles of about a dozen filaments that crisscross the cytoplasm. T h e y are also present beneath the cell surface, although not as closely a p p o s e d to it as the microfilaments,
and frequently they run parallel to the micro filament bundles (Fig. 9). An a b u n d a n c e of intermediate filaments is one of the striking features of cells cultivated in the presence of CT and I M X . It is not unusual to find nuclei e n v e l o p e d in a thick layer of these filaments or to o b s e r v e bundles of them measuring as m u c h as 1.5 /xm in diameter coursing through the cytoplasm (Fig. 11). Cell Junctions. F o r m a t i o n of specialized cell junctions are an inherent p r o p e r t y of enthothelial cells as part of their function as a b l o o d - t i s s u e barrier. Intercellular junctions
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FIG. 10. Section similar to that shown in Fig. 8, of cell exposed to CT and IMX. Note the dense bodies formed by coverging bundles of microfilaments. Part of the cell surface is visible along the upper left margin of the illustration, x 22 400. are p r e s e n t in c u l t u r e d cells, their n u m b e r d e p e n d i n g g r e a t l y o n the c u l t u r e c o n d i t i o n s . E n d o t h e l i a l cells o n p l a i n p l a s t i c dishes h a v e v e r y few close c e l l - c e l l contacts a n d do n o t show a n y specialized forms of i n t e r c e l l u l a r c o n t a c t at t h e s e p o i n t s . I n c o n t r a s t , cells g r o w n o n f i b r o n e c t i n f o r m s p e c i a l i z e d cell j u n c t i o n s . A t p o i n t s of c o n tact t h e r e is a close a p p o s i t i o n o f the cell
m e m b r a n e s with a n i n c r e a s e in the e l e c t r o n d e n s i t y o f the a d j a c e n t c y t o p l a s m ; t h e s e j u n c t i o n s h a v e the a p p e a r a n c e of m a c u l a e or z o n u l a e o c c l u d e n t e s . I n a d d i t i o n to t h e s e s p e c i a l i z e d j u n c t i o n s , i n t e r d i g i t a t i o n s of the c y t o p l a s m of a d j a c e n t cells are f r e q u e n t l y o b s e r v e d in cells g r o w n in the p r e s e n c e of C T a n d I M X (Fig. 12). A t t h e s e p o i n t s o f j u x t a p o s i t i o n , b u n d l e s of microfilaments r u n
FIG. 9. Cells grown on a fibronectin-covered dish contain cell membrane-associated bundles of microfilaments (short arrows), two of which are particularly close to the basal cell membrane (double short arrows). Note the electron-opaque plaques at the cell outside at this point (hollow arrows). Intermediate filaments form loose bundles, several of which run parallel to the microfilament bundles (long arrows). Arrowheads point at a cross and a tangentially sectioned Weibel-Palade body. x 68 000.
84
MICROVASCULAR ENDOTHELIAL CELLS
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FIG. 12. Section perpendicular to dish surface of cell exposed to CT and IMX. Close apposition at point of contact between adjacent cells is marked by increased electron density. Arrowhead points at tangentially sectioned bundles of microfilaments running parallel to line of intercellular contact; arrow points at bundles of intermediate filaments. Note deposits between plastic and cells. × 30 000.
parallel to the line of contact between cells (Fig. 12). Cellular Organelles
There are other, albeit less obvious differences between the cytoplasmic organelles of cells grown under the different conditions. Cells of c u l t u r e s g r o w n on fibronectin and cells exposed to CT and IMX also have a large complement of Weibel-Palade bodies. In contrast, these bodies are very rare in endothelial cells grown on plain plastic dishes. Although there appear to be fewer pinocytotic vesicles in cells grown on plastic dishes, morphometric evaluation does not show a statistically significant difference between the three groups. DISCUSSION
There are at least half a dozen different types of vascular endothelia in mammals, with the main varieties being the continuous, the fenestrated, and the discontinous endothelial lining (Majno, 1965). Endothelial cells of human dermal microvessels were
used in our study. These vessels are lined by a continuous layer of low endothelium which readily detaches from its basal lamina by gentle treatment with trypsin in vitro. Recent progress in tissue culture techniques now permits long term cultivation of these cells (Davison et al., 1980). One of the major prerequisites for such growth is an in vitro environment which resembles the in vivo situation in several points: high serum concentrations of the culture medium and a substratum similar to that of the capillary wall. The latter, as shown by our studies, can be substituted for by exposure of the cells to pharmacological agents known to significantly raise the intracellular cAMP levels (Davison and Karasek, 1981). The aim of this study was a delineation of tissue culture conditions which result in the restoration of the functional ultrastructure of human microcapillary endothelial cells. In vitro cultivation in the presence of relatively high serum concentrations (1050%) allowed these cells to attach themselves to a Nain plasic culture surface and
FIG. 11. Cells exposed to CT and IMX contained large numbers of intermediate filaments, often surrounding the nucleus. Note the abundance of pinocytotic vesicles, x 24 000.
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MICROVASCULAR ENDOTHELIAL CELLS
to multiply slowly. Under these conditions, however, the cells are not capable of flattening out and establishing specialized cellcell junctions which are a prerequisite for the normal function of the dermal capillary endothelium and characteristic of these cells (Shepro, 1977). Furthermore, aside from a random pattern of distribution, there is a general paucity of micro- and intermediate cytoplasmic filaments and they are unable to form bundles as normally observed in vivo. In addition, cells in such a medium soon lose their specific markers, the Weibel-Palade bodies (Weibel and Palade, 1964). When plated on a substratum of fibronectin, however, the same cell population not only reorganizes into contiguous groups of flattened cells, but also forms intercellular junctions. In addition, a rearrangement of the cytoplasmic micro- and intermediate filaments also occurs. Thus, provision of a substratum results in a partial restoration of the normal appearance of these cells, a result that had been previously observed by others in in vitro studies of fibroblasts treated with fibronectin (Yamada and Olden, 1978). Two types of fibronectin are known, the fibronectin, presently in relatively high concentrations, and the cellular (cell surface) fibronectin which has been most extensively studied on fibroblasts (Yamada and Olden, 1978). Cellular fibronectin is also produced by several other cell types, including endothelial cells (Birdwell et al., 1978; Jaffe and Mosher, 1978; Macarak et al., 1978). Fibronectinl also called spreading factor, is a major, large cell surface and interstitial glycoprotein that plays an important role in cell morphology, mobility, cell-cell aggregation, cell-substratum adhesiveness, and other biological activities (Yamada and
87
Olden, 1978; Grinnell, 1976). It is present, at least in fibroblasts, as an organized network on the cell surface, the maintenance of which appears to be interdependent with that of the cytoplasmic microfilament organization (Kurkinen et al., 1978; Chen et al., 1978). In addition, however, cell surface fibronectin can bind to collagen, thus mediating attachment of dissociated cells to collagen-containing substrata, including basement membranes (Pearlstein, 1976; Engvall and Ruoslahti, 1977). Fibronectin also attaches to plastic surfaces which would explain the observed spreading of the endothelial cells used in our experiments (Ali et al., 1977). Their aggregation into groups of cells is most likely not only caused by the known greater cell-cell adhesiveness after fibronectin treatment, but also the greater mobility of such cells due to more effective cell-substratum traction (Yamada et al., 1976). The closest ultrastructural resemblance of the cultured endothelial cells to those present in dermal microvessels was present in monolayer cultures treated with agents known to significantly raise the intracellular cAMP level. These cells formed a highly organized intracellular network of microand intermediate filaments, branches of which extended closely to intercellular cell junctions and to points of apparent cell attachment of the abluminal cell surface to extracellular, electron-opaque deposits; these were present between the originally plain culture dish surface and the cell. Thus, under such stimuli, endothelial cells are able to produce and presumedly also secrete fibronectin and possibly other substances in quantities sufficient for cell-cell aggregation, cell spreading, and formation of specialized sites of attachment to extracellular structures (Macarak and Kefalides, 1978). Our understanding of the morphology and
FIG. 13. Section o f foreskin microvessel. A n e t w o r k of microfilaments is p r e s e n t t h r o u g h o u t the c y t o p l a s m of the flat endothelial cells; note that it is particularly p r o m i n e n t near the abluminal cell m e m b r a n e (arrowhead). The short arrow points at a zonula a d h e r e n s , the hollow arrows at cross- and tangentially sectioned W e i b e l Palade bodies, x 37 000.
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BENSCH, DAVISON, AND KARASEK
the compounds responsible for focal sites of adherence between cells and structures in their surroundings is still incomplete. In vitro studies, particularly on fibroblasts, however, have shown that several types of such contact exist (Chen and Singer, 1980). Depending on the distance between a cell membrane and the substratum and the size of such attachment sites, at least two different types are now recognized, so-called focal adhesions and close contacts. In our experiments with agents that raise the intracellular cAMP level, we observed bundles of microfilaments in very close proximity to the basal cell membrane, sometimes even appearing to merge with it. At such points, there usually was a focal aggregation of extracellular, electron-opaque material very near the cell surface. The nature of these extracellular deposits is not known, but immunocytochemical studies carried out by others on sites of focal adhesions of fibroblasts indicate that it is most likely not fibronectin (Chen and Singer, 1980). The latter appears to localize as an extracellular matrix surrounding such focal adhesion sites and, possibly, also as extracellular strips paralleling the course of microfilarnent bundles where they lie close to the cell membrane. Intercellular junctions were well developed in our cultures treated with CT and IMX. These had the cross-sectional profile of junctional modifications of undilated vessels with imbrications of cytoplasmic ridges of neighboring cells (Tripathi and Tripathi, 1977). In addition, junctional complexes were prominent at these sites. Aside from tight junctions, there were adherent junctions with bundles of microfilaments in their immediate vicinity. Bundled intermediate filaments were not observed in these areas, but they were commonly encountered elsewhere and they are most likely composed of vimentin (Renner et al., 1981). The role of these and/or the microfilaments in the contraction of microvessels under physiological conditions and individual cells during the inflammatory response remains to be
established (Hammersen, 1976, 1977; Addicks et al., 1979; Goldman et al., 1979). In contrast to endothelial cells without elevated cAMP levels, Weibel-Palade bodies were prominent in the CT and IMX treated cells and they persisted on prolonged subculture. This increase in number may be related to the increased growth rate of endothelial cells observed in the presence of cyclic AMP in vitro (Davison and Karasek, 1981). During accelerated proliferation of the microvasculature in vivo, increase in number of Weibel-Palade bodies also occurs (Kumar et al., 1980; Lonchampte and Regnault, 1978; Takeshige and Fujimoto, 1977). An explanation for the interesting relationship between proliferation rate and Weibel-Palade bodies both in vivo and in vitro is not known. In conclusion, these experiments indicate that the use of monolayer cultures as an in vitro model for the study of endothelial cells requires fulfillment of several conditions for the establishment of the functional ultrastructure of this cell type. Serum supplements to the culture medium are insufficient for in vitro differentiation of endothelial cells. Addition of a fibronectin substratum leads to a partial restoration of the in vitro morphology. The latter, however, is only reached after elevation of the cytoplasmic cAMP level. The authors thank M. EUen Charlton and Irma Daehne for technical assistance. REFERENCES ADDICKS, K., WEIGELT, H., HANCK, G., LUEBBERS, D. W., AND KNOCHE, H. (1979) Bibl. Anat. 17, 21. ALI, I. V., MAUTNER, V. M., LANZA, R., AND HYNES, R . O . (1977). Cell 11, 115. BIRDWELL, C. R., GOSPODAROWICZ, D., AND NICKOLSON, G. L. (1978) Proc. Nat. Acad. Sci. USA 75, 3273. CHEN, L. B., MURRAY, A., SEGAL,R. A., BUSHNELL, A., AND WALSH, M. L. (1978) Cell 14, 377. CHEN, W. T., AND SINGER, S. J. (1980) Proc. Nat. Acad. Sci. USA 77, 7318. DAVISON, P. M., BENSCH, K., AND KARASEK, M. A. (1980) J. Invest. Dermatol. 75, 316. DAVISON, P. M., AND KARaSEK, M. A. (1981) J. Cell. Physiol. 106, 253.
MICROVASCULAR ENDOTHELIAL CELLS ENGVALL, E., AND RUOSLAHTI, E. (1977) Int. J. Cancer 20, 1. GOLDMAN, R. D., MILSTED, A., SCHLOSS, J. A., STARGER,J., AND YE~A, M.J. (1979) Annu. Rev. Physiol. 41,703. GORDON, G. B., MILLER, L. R., A N D BENSCH, K. G. (1963) Exp. Cell Res. 31, 440~43. GRINNELL, F. (1976) Exp. Cell Res. 97, 265. HAMMERSEN, F. (1976) Beitr. Pathol. 157, 327. HAMMERSEN, F. (1977) Bibl. Anat. 16, 370. JAFEE, E. A., AND MOSHER, D. F. (1978) J. Exp. Med. 147, 1779. KUMAR, P., KUMAR, S., MARSDEN, H. B., LYNCH, P. G., AND EARNSHAW, E. (1980) CancerRes, 40, 2010. KURKINEN, M., WARTIOVAARA,J., AND VAHERI, A. (1978) Exp. Cell. Res. 111, 127. LONCHAMPTE, M. O., AND REGNAULT, F. (1978) in REGNAULT, F., AND CUHAULT, J. (Eds.), Cellular and Biochemical Aspects in Diabetic Retinopathy. INSERM Symposium No. 7, Elsevier/North Holland, Amsterdam. LUFT, H. J. (1961) J. Biophys. Biochem. 9, 409. MACARAK, E. J., AND KEFALIDES, N . A . (1978) in KEFALIDES, N. A. (Ed.), Biology and Chemistry of Basement Membranes, p. 343, Academic Press, New York. MACARAK, E. J., KIRBY, E., KIRK, T., AND KEEALIDES, N. A. (1978) Proc. Nat. Acad. Sci. USA 75, 2621.
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MAJNO, G. (1965) in HAMILTON, W. F., AND DOW, P. (Eds.), Handbook of Physiology, Section 2, Vol. III, p. 2293, Amer. Physiol. Soc., Washington, D. C. PALADE, G. E. (1952) J. Exp. Med. 95, 285-298. PEARLSTEIN, E. (1976) Nature (London) 262, 497. RENNER, W., FRANKE, W. W., SCHMID, E., GEISLER, M., WEBER, K., AND MANDELKOW, E. (1981) J. Mol. Biol. 149, 285. RICHARDSON, K. C., JARRETT, L., AND EINKE, E. H. (1960) Stain Tech. 35, 313-323. ROHR, H., OBERHOLZER,M., BARTSCH,G., AND KELLER, M. (1976) Int. Rev. Exp. Pathol. 15,233-325. SABATINI, D. D., BENSCH, K., AND BARRNETT, R. J. (1963) J. CellBiol. 17, 19-58. SHEPRO, D. (1977) Bibl. Anat. 16, 384. TAKESHIGE,Y., AND FUJIMOTO,S. (1977) Bibl. Anat. 16, 364. TRIPATHI, R. C., AND TmPATHI, B. Y. (1977) Bibl. Anat. 16, 307. VENABLE, J. H., AND COGGESHALL, R. (1965) J. Cell Biol. 25, 407--408. WEIBEL, E. R., AND PALADE, G. E. (1964) J. Cell Biol. 23, 101. WEIBEL, E . R . (1963) Morphometry of Human Lung, Springer-Verlag, Heidelberg Academic Press, New York. YAMADA, K. M., AND OLDEN, K. (1978) Nature (London) 275, 179. YAMADA, K. M., YAMADA, S. S., AND PASEAN, I. (1976) Proc. Nat. Acad. Sci. USA 73, 1217.