Various culture conditions, such as the presence of ascorbic acid, initial plating density and the nature of the substratum (plastic, gelatin or native collagen gels), influenced the growth, morphology and migration of three cloned populations of adult bovine aorta endothelial cells.

Aorta endothelial cells showed two distinctive and reversible morphological phenotypes. Cells presenting a free apical surface were polygonal and formed sheets of overlapping or non-overlapping cells, depending on the culture conditions. When the cells were able to establish adhesive interactions over their entire cell surface they adopted an elongated shape and formed meshworks of interconnected ‘sprouting’ cells. The cells were capable of migrating into a collagen gel from both their basal and apical surfaces. Once in the gel, they formed characteristic, compact, threedimensional meshworks.

Many methods have been described for the isolation and culture of endothelial cells (Gimbrone, 1976; Davison, Bensch & Karasek, 1980; Eskin et al. 1978; Booyse, Sedlak & Rafelson, 1975; Folkman, Haudenschild & Zetter, 1979). These cells generally form a ‘cobblestone’ monolayer of polygonal cells at confluence. Several groups have reported the gradual appearance in postconfluent cultures of a secondary growth of cells with a distinctive, elongated morphology. These new cells form an interconnected network beneath the original monolayer, stain positively for factor VIII antigen and are therefore believed to be of endothelial origin. By analogy with the formation of capillary sprouts during neovascularization in vivo, these elongated cells have usually been referred to as ‘sprouting cells’ (Duthu & Smith, 1980; Schwartz, 1978; Cotta-Pereira et al. 1980; McAuslan, Hannan, Reilly & Stewart, 1980; Mueller, Rosen & Levine, 1980; Gospodarowicz, Mecher & Birdwell, 19786; Folkman & Haudenschild, 1980).

The extracellular matrix is now recognized as affecting a number of important aspects of cell behaviour (Hay, 1981). Endothelial cells in vivo rest upon a collagen-containing basement membrane and migrate through an extracellular matrix containing type I collagen during the course of neovascularization (Schoefl, 1963; Barnhart & Baechler, 1978; Mason et al. 1979; Ausprunk & Folkman, 1977). Cells may be grown on collagenous substrata in vitro and these have been shown to influence cell proliferation (S. L. Schor, 1980; Schor, Schor, Winn & Rushton, 1982; Kleinman et al. 1981), migration (Schor, 1980; Schor, Schor & Bazill, 1981a,b) and biosynthetic activity (Gallagher, Gasiunas & Schor, 1980; Meier & Hay, 1975).

Endothelial cells in culture also synthesize and deposit their own extracellular matrix (Jaffe et al. 1976; Howard, Macarak, Gunson & Kefalides, 1976), the nature of which may be influenced by soluble factors in the medium, such as ascorbic acid (Peterkofsky, 1972; De Clerck & Jones, 1980).

In this paper we present data concerning the effects of a collagenous substratum and ascorbic acid on the proliferation and morphology of bovine aortic endothelial cells, as well as the formation and behaviour of sprouting cells.

Culture media and substrata

Cells were cultured in Eagle’s MEM supplemented with 15 % donor calf serum, 50 μg/ml ascorbic acid, 2 mM-glutamine, 1 mM-sodium pyruvate, non-essential amino acids (Gibco-Biocult Ltd, Uxbridge) and 100 units/ml penicillin and streptomycin (this complete growth medium is referred to as 15% DCS-MEM). Occasionally this medium was used without ascorbic acid; this will be indicated as appropriate. Conditioned medium was obtained by incubating confluent cultures of either cloned or uncloned bovine aortic endothelial cells with 15% DCS-MEM for 3 days. The collected medium was Millipore-filtered and frozen at —20°C until used. ‘Cloning medium’ was prepared by mixing 3 parts 15 % DCS-MEM with 1 part conditioned medium.

Type I collagen was extracted from rat tail tendons with 0·5N-acetic acid, dialysed against distilled water and used to prepare an aqueous stock solution containing 2·2 mg collagen/ml. Threedimensional gels of native collagen fibres were prepared as previously described (Schor, 1980). Accordingly, 8·5 ml of the aqueous collagen stock solution were rapidly mixed with 1 ml of 10 × concentrated MEM and 0·5 ml of 7·5% sodium bicarbonate to form a gelling solution and 2 ml samples of this solution were rapidly pipetted into 35 mm plastic tissue culture dishes (Gibco Biocult, Uxbridge, Cat. no. 1–53066). Gels set within 5 min of incubation at 37°C in an humidified incubator gassed with 5 % CO2 in air. Gels were equilibrated with the appropriate medium for at least 3h before use. Heat-denatured collagen (i.e. gelatin) was made by heating samples of the aqueous stock collagen solution at 60 °C for 20 min. Gelatin-coated dishes were prepared by incubating 35 mm plastic tissue-culture dishes with 1 ml of the gelatin solution for 1 h at 37 °C, removing the gelatin solution and washing the dishes twice with Hanks’ balanced salt solution (HBSS). Solutions of 0·1 % gelatin (BDH, Poole, Dorset, Cat. no. 44045) in distilled water were also used to prepare the gelatin-coated dishes.

Clonal isolation and culture of adult bovine aorta endothelial cells (BAEC)

A segment of the descending thoracic aorta (approximately 25 cm) was obtained from adult cows and transported at ambient temperature from the local abattoir. The processing times were such that cells were put into culture within 2–3 h of the death of the animal. Working under sterile conditions, the aorta was trimmed of adventitial fat and then slit open along the intercostal arteries. With the aid of surgical clamps, the luminal surface was laid flat over the mouth of a beaker and washed with a stream of phosphate-buffered saline containing 200 units/ml of penicillin and streptomycin, until no traces of blood remained. A central area of the exposed luminal surface (approximately 17 cm × 3 cm) was then scraped with a scalpel blade and the dislodged cells were incubated with 4 ml of 0·2mg/ml collagenase (Millipore Co., Cat. no. CLS 41D264) at 37 °C for 20 min. The cell suspension was pipetted at regular intervals during this 20 min period in order to increase the yield of single cells. At the end of the collagenase incubation, 5 ml of 15 % DCS-MEM were added to the digestion mixture and the cells then centrifuged at 120 g for 5 min, resuspended in 15 % DCS-MEM and seeded into four 35 mm plastic tissue-culture dishes. After 2 h incubation at 37 °C, the medium with floating cells was discarded, the attached cells washed five times with HBSS containing 8 units/ml gentamycin and 100units/ml of penicillin and streptomycin and then incubated once again with 15 % DCS-MEM. The majority of attached cells at this stage (greater than 80%) were single and generally well-separated from their neighbours, with the remaining ones present in small groups consisting of two to four cells. Approximately 5 × 105 cells remained attached on day 1 from the cells originally scraped from the 51 cm2 aortic segment.

Cloned preparations of endothelial cells were obtained by a two-step procedure. The location of selected individual cells was marked on day 1 with a pen on the underside of the plastic dish. Neighbouring cells were then scraped off with the tip of a Pasteur pipette blown out to form a microneedle. Cultures were observed daily and neighbouring cells cleared away by this procedure until the selected colony contained between 150 and 200 cells (7–10 days later). At this stage all of the colonies in this initial culture consisted of a cobblestone monolayer of polygonal cells. The selected colony was then ring-cloned with 0·05 % crystalline trypsin (Sigma Chemical Co., London, Cat. no. T-0134) and 2mM-EGTA in Dulbecco’s phosphate-buffered saline (PBS) and the cells were plated onto a gelatin-coated 35 mm tissue-culture dish containing 1 ml of cloning medium. The selection of a colony and cloning procedure were repeated in this second dish, except that a larger colony (300–500 cells) was trypsinized this time. The small number of cells plated onto the second dish (i.e. 150–200) and the two-step nature of the cloning procedure optimize the likelihood of obtaining clonal cultures derived from a single parental cell. The use of conditioned medium (i.e. our ‘cloning medium’) was found to facilitate the growth of colonies.

Stock cultures of cloned cells were routinely grown on gelatin-coated 90 mm plastic tissue-culture dishes (Dibco Biocult, Uxbridge, Cat. no. 1–50350) in 15 % DCS-MEM and split in a 1: 3 ratio when confluent (usually once a week). Medium was changed three times per week. The results reported in this paper were obtained with three cloned cell populations, used between passages 5 and 13.

Uncloned primary cultures appeared to consist entirely of endothelial cells and some have been subcultured up to 10 times without loss of their typical endothelial cell morphology. In contrast, other uncloned cultures, which looked equally homogeneous in the first few passages, soon became heavily contaminated with what appeared to be smooth muscle cells.

Cell numbers in cultures growing on the various substrata were determined with a Coulter electronic particle counter as previously described (Schor, 1980). The endothelial nature of the cloned cells was confirmed in all cases by their positive staining for factor VIII antigen, as described by Schwartz (1978).

Histology

Cultures on collagen gels were fixed in 10 % formaldehyde at room temperature, embedded in Paraplast (Lancer) and, after routine processing, 5 μm sections were stained with haematoxylin/eosin.

Electron microscopy

Cultures were initially fixed by flooding the dish or gel with 3 % glutaraldehyde in M/15 Sorensen’s phosphate buffer (pH 7·2). After three washes with buffer, the cultures were post-fixed for 1 h in 1 % OsO4 in the same buffer. After three further washes, 15 mm diameter ‘plugs’ were cut from the gels with a cork-borer and processed as for tissue specimens. Monolayers for scanning electron microscopy (SEM) were bored out attached to the Petri dish with a warmed cork-borer. After dehydration, for transmission electron microscopy (TEM), collagen gel discs were infiltrated and embedded in Epon/Araldite via propylene oxide, and whole monolayers on plastic were embedded in situ, using HPMA and Lufts Epon. Sections were stained with uranyl acetate and lead citrate, and viewed at 80 kV in an AEI 801A TEM. For SEM, discs of collagen or bored-out plastic+cells were dehydrated through ethanol and critical-point dried from liquid CO2 using Freon 113 as transitional fluid. The specimens were sputter-coated with 10 nm gold and viewed in a Cambridge S410 SEM at an accelerating voltage of 20 kV.

Cell growth

The initial growth rates and final saturation cell densities were found to be influenced by the nature of the substratum. In the experiment shown in Fig. 1, 105 cells were plated onto plastic dishes, gelatin-coated dishes and three-dimensional collagen gels in 15% DCS-MEM, with or without 50 μg/ml ascorbic acid. The medium was changed three times per week during the course of the experiment.

Fig. 1.

Proliferation of bovine aorta endothelial cells in 15% DCS-MEM with (•) or without (○) ascorbic acid. 105 cells were plated on the different substrata. Freshly dissolved ascorbic acid (50 μg/ml) was added when the medium was changed (every 2–3 days). Each point represents the mean of duplicate cultures. The standard deviation is shown by a vertical bar, unless it is too small to be represented.

Fig. 1.

Proliferation of bovine aorta endothelial cells in 15% DCS-MEM with (•) or without (○) ascorbic acid. 105 cells were plated on the different substrata. Freshly dissolved ascorbic acid (50 μg/ml) was added when the medium was changed (every 2–3 days). Each point represents the mean of duplicate cultures. The standard deviation is shown by a vertical bar, unless it is too small to be represented.

Ascorbic acid produced a significant increase in the rate of cell proliferation during the logarithmic phase of growth but did not affect the final cell density achieved on any of the substrata. This stimulation of growth rate was more apparent on the collagen gels than on the other two substrata. Identical results were obtained in replicate experiments, in which the ascorbic acid was added daily instead of the usual three times per week when the medium was changed (data not shown). The magnitude of this increase in growth rate varied from experiment to experiment (from no apparent stimulation to values approximately double those shown in Fig. 1) and appeared to be dependent upon the particular batch of serum used (data not shown). Significant stimulation of growth rate by ascorbic acid (P < 0·01) was observed in six out of eight experiments, involving all three of the cloned cultures. The increase in cell number on plastic dishes that occurred at day 16 (Fig. 1) was due to the appearance of a secondary growth of cells (i.e. the sprouting cells) and will be discussed in the following section.

The effects of different initial plating densities on cell growth are presented in Fig. 2. Cells were plated onto plastic dishes and gelatin-coated dishes in 15 % DCS-MEM at initial densities between 2·5 × 104 and 2 × 105cells/dish. The plating efficiency of the endothelial cells is somewhat lower on the collagen gels compared to the other two substrata (unpublished observations); consequently, higher cell numbers were initially plated onto the gels (i.e. 4 × 104 and 6 × 105) in order to achieve comparable cell densities on all substrata 24 h after plating. Plating densities equal to or lower than 4 × 104 will be referred to as ‘low’ in the subsequent discussion, while densities greater than or equal to 105 will be referred to as ‘high’.

Fig. 2.

Proliferation of bovine aorta endothelial cells plated at different initial densities. The cells were grown on the different substrata in 15 % DCS-MEM containing ascorbic acid (50 μg/ml). Cells were plated at 2·5 × 104 (○), 5 × 104 (•), 105 (▵) and 2 × 105 (▴) cells/culture on plastic and gelatin and 4 × 104 (○), 105 (•), 2 × 105 (▵), 4 × 105 (▴) and 6 × 105 (▫) cells/culture on collagen. Every point represents the mean of duplicate cultures. The standard deviation was always less than 11 % of the mean.

Fig. 2.

Proliferation of bovine aorta endothelial cells plated at different initial densities. The cells were grown on the different substrata in 15 % DCS-MEM containing ascorbic acid (50 μg/ml). Cells were plated at 2·5 × 104 (○), 5 × 104 (•), 105 (▵) and 2 × 105 (▴) cells/culture on plastic and gelatin and 4 × 104 (○), 105 (•), 2 × 105 (▵), 4 × 105 (▴) and 6 × 105 (▫) cells/culture on collagen. Every point represents the mean of duplicate cultures. The standard deviation was always less than 11 % of the mean.

Doubling times during the logarithmic phase of growth decreased with increasing initial plating density. The results presented in Fig. 2 show that doubling times vary between 15 and 40 h on plastic and’gelatin-coated dishes and between 17 and 67 h on collagen gels. The saturation cell density increased with increasing plating density on all substrata. For a given plating density, cells on collagen gels reached a lower saturation density compared with the other two substrata. The maximum saturation cell density achieved was 8 × 10s cells/culture (105 cells/cm2) on collagen gels and 1·2 × 106cells/culture (1·5 × 105 cells/cm2) on plastic and gelatin. The cells reached confluency 2–3 days before the saturation cell density was achieved. The increase in cell number that occurred during this 2–3 day period resulted in the cells becoming more tightly packed and consequently occupying a smaller area of the substratum per cell.

Cell morphology in early confluent cultures

The morphology of cells at confluence was dependent upon the nature of the substratum, initial plating density and the presence of ascorbic acid. Allowing for slight variations depending on the individual clone examined (a total of three in this study) and passage number, distinctive cell morphologies can be obtained in a reproducible fashion by altering the culture conditions appropriately. These cell morphologies and the particular conditions under which they are obtained may be summarized as follows, (a) A cobblestone monolayer of non-overlapping, polygonal cells was produced when cells were plated onto gelatin-coated dishes at high cell density in the presence of ascorbic acid (Fig. 3A), or onto collagen gels at both high and low cell densities in the presence of ascorbic acid (Fig. 3B). (b) A highly disorganized layer of overlapping, elongated cells was produced when cells were plated onto plastic or gelatin-coated dishes at low initial cell density in the absence of ascorbic acid (Fig. 3c). (c) An intermediate monolayer of slightly elongated cells with some degree of overlapping was produced when cells were plated under any condition not listed above (Fig. 3D).

Fig. 3.

The morphology of bovine aorta endothelial cells at confluence varied according to culture conditions. A non-overlapping, cobblestone monolayer; is shown on gelatin (A) and collagen (B) substrata; (c) highly disorganized layer on gelatin; (D) intermediate monolayer on plastic. ×250.

Fig. 3.

The morphology of bovine aorta endothelial cells at confluence varied according to culture conditions. A non-overlapping, cobblestone monolayer; is shown on gelatin (A) and collagen (B) substrata; (c) highly disorganized layer on gelatin; (D) intermediate monolayer on plastic. ×250.

Cell morphology in late post-confluent cultures

The different morphologies in early confluent cultures (Fig. 3) are not stable. All cultures eventually reorganize themselves to produce a similar endpoint consisting of a confluent, cobblestone monolayer of polygonal cells occupying the entire surface of the substratum and an underlying meshwork of interconnected, elongated cells similar in appearance to the sprouting cells described by others. Data supporting our interpretation that the different cellular morphologies observed in culture result from the reversible, phenotypic modulation of a single cell type will be presented in the following section. The data in this section are concerned with the manner in which culture conditions influence the production and subsequent behaviour of sprouting cells in late, post-confluent cultures.

Cells plated onto plastic dishes under conditions that resulted in the formation of either the disorganized layer of elongated cells (Fig. 3c) or the intermediate monolayer (Fig. 3D) at early confluence were the first to reorganize themselves into a confluent cobblestone monolayer and a large number of sprouting cells distributed homogeneously over the dish. This reorganization usually began 5–6 days after confluency was reached and appeared to be completed within a short period of time (i.e. 24h). The number of sprouting cells increased rapidly during the next week in culture, a continuous network of such cells expanding throughout the entire culture under the cobblestone monolayer was ultimately produced (Fig. 4A). This increase in the number of sprouting cells may be measured and is apparent in Fig. 1 between days 16 and 20. The presence of ascorbic acid retarded the appearance of the sprouting cells by 3–4 days, although their subsequent development and increase in number was identical to that in cultures maintained in the absence of ascorbic acid.

Fig. 4.

Post-confluent cultures with sprouting cells on plastic (A) and gelatin (B–F) substrata; D, the same culture as in c 24 h later; E, F show detail of sprouting cell associations See text for details. ×250 (A–D); ×475 (E, F).

Fig. 4.

Post-confluent cultures with sprouting cells on plastic (A) and gelatin (B–F) substrata; D, the same culture as in c 24 h later; E, F show detail of sprouting cell associations See text for details. ×250 (A–D); ×475 (E, F).

Cells grown on gelatin-coated dishes behaved in a similar fashion. Again, cultures that produced a disorganized cell layer at confluence (Fig. 3c) reorganized themselves to produce a cobblestone monolayer plus sprouting cells during the next 5–6 days. On this substratum, however, very few sprouting cells were initially seen in the reorganized cultures; these sprouting cells were invariably present in only three to four isolated patches (Fig. 4B), which eventually expanded to cover the entire dish. The appearance of sprouting cells was greatly delayed (by 15–20 days) in cultures growing on gelatin-coated dishes under conditions that initially produced a cobblestone monolayer (i.e. plated at high cell density in the presence of ascorbic acid). When sprouting cells did eventually appear, they were also present in discrete patches and subsequently expanded to cover the dish (Fig. 4c).

When a given culture was examined sequentially for an extended period of time (i.e. 30–50 days post-confluence), it was common to observe the extensive network of sprouting cells (Fig. 4c) disappear within a period of 48 h to produce a culture containing very few, isolated sprouting cells and an apparent corresponding increase in the density of cells in the cobblestone monolayer (Fig. 4D); such cultures may, in turn, revert back to an extensive meshwork of sprouting cells indistinguishable from that shown in Fig. 4c within a 48 h period. These interconversions in cell morphology occur several times during extended periods in culture and are not accompanied by any measurable change in total cell number. Occasionally the disappearance of the sprouting cells resulted in the appearance of a second, nearly confluent, monolayer at the bottom of the dish, separated from the original monolayer by an extracellular matrix. In this situation, sprouting cells appeared between the two monolayers after a further 48 h of incubation.

The sprouting cells are often vacuolated and interconnected to form rings and loops in which the boundaries between individual cells are difficult to visualize (Fig. 4E, F). The appearance of a typical late, post-confluent culture in the SEM is shown in Fig. 5A. An extensive meshwork of sprouting cells may be seen in relief below the confluent monolayer of polygonal cells. The monolayer may be gently peeled back with a Pasteur pipette, thus revealing the network of sprouting cells growing on the substratum (Fig. 5B, C). Such results indicate that under these conditions the sprouting cells are more firmly attached to the substratum than to the overlying monolayer. Small patches of cell-produced extracellular matrix remained attached to the plastic substratum (Fig. 5c).

Fig. 5.

A. SEM of a typical monolayer of endothelial cells, under which the profile of sprouting cells is apparent, forming an irregular network of ridges, ×575. B. SEM of underlying network of sprouting cells after removal (pushed to right) of overlying cobblestone (nonolayer with the tip of a Pasteur pipette. The sprouting cells appear more firmly attached to the plastic substratum than the underside of the disturbed monolayer. ×360. c. Detail from exposed sprouting-cell network. The cells are extended in several directions and small patches of matrix (subendothelium) are apparent on the plastic substratum (arrowheads). ×750.

Fig. 5.

A. SEM of a typical monolayer of endothelial cells, under which the profile of sprouting cells is apparent, forming an irregular network of ridges, ×575. B. SEM of underlying network of sprouting cells after removal (pushed to right) of overlying cobblestone (nonolayer with the tip of a Pasteur pipette. The sprouting cells appear more firmly attached to the plastic substratum than the underside of the disturbed monolayer. ×360. c. Detail from exposed sprouting-cell network. The cells are extended in several directions and small patches of matrix (subendothelium) are apparent on the plastic substratum (arrowheads). ×750.

Sprouting cells also appeared in cultures growing on three-dimensional collagen gels. In the presence of ascorbic acid, they first appeared 12–25 days after confluence. The number of sprouting cells increased gradually during the next 2–4 weeks and they eventually formed compact, well-delineated patches under the cobblestone monolayer (Fig. 6A, arrows). In the absence of ascorbic acid sprouting cells first appeared 3—7 days earlier than when ascorbic acid was present and these were homogeneously distributed throughout the culture (Fig. 6B).

Fig. 6.

Post-confluent cultures, with sprouting cells, grown on collagen gels with (A, C) or without (B, D) ascorbic acid. On the gel surface, the sprouting cells may form compact patches (A, arrows) or disseminated networks, B. Sprouting cells migrated into the underlying gel where they formed three-dimensional meshworks (c, D; arrowheads in A, B). D. The same field as B, focussing deeper into the gel. ×250 (A, B); ×350 (C); ×150 (D).

Fig. 6.

Post-confluent cultures, with sprouting cells, grown on collagen gels with (A, C) or without (B, D) ascorbic acid. On the gel surface, the sprouting cells may form compact patches (A, arrows) or disseminated networks, B. Sprouting cells migrated into the underlying gel where they formed three-dimensional meshworks (c, D; arrowheads in A, B). D. The same field as B, focussing deeper into the gel. ×250 (A, B); ×350 (C); ×150 (D).

Sprouting cells remained confined to the gel surface for the first 5–10 days. After this initial period, a few cells started migrating down into the gel matrix. The number of cells within the gel matrix increased gradually and, irrespective of the presence or absence of ascorbic acid, they formed several three-dimensional meshworks of interconnected cells (usually with no direct continuity with cells at the gel surface) (Fig. 6c, D). Such three-dimensional cellular meshworks can be seen in Fig. 6A, B as well, although they are (necessarily) out of focus (arrowheads). Cells may be selectively removed from the gel surface (Schor, 1980), thus making it possible to count the number of cells present within the gel matrix; by using this approach we have estimated that in late, post-confluent cultures as many as 106 cells may be present within the gel matrix in the form of such compact, three-dimensional meshworks (i.e. more than the number of cells present at the gel surface at this time).

Histological sections of late, post-confluent cultures revealed that two to three layers of cells were present in certain regions of the gel surface (Fig. 7A, B). Occasionally, a cell that is apparently in the process of migrating into the gel matrix may be observed (Fig. 7B). Within the collagen matrix, cells are seen at various distances from the gel surface. Sections through the three-dimensional cellular meshworks within the gel matrix indicated that the constituent cells were in close contact with each other and often enclosed lumen-like spaces (Fig. 7A, C, D). The appearance of cells in thin sections of such gel cultures in the TEM is shown in Fig. 8. The monolayer of endothelial cells at the gel surface rests on a cell-produced extracellular matrix (i.e. subendothelium), which is clearly distinguishable from the subjacent collagen gel; details concerning the biosynthesis and ultrastructure of this subendothelium will be published elsewhere. A group of sprouting cells (three cellular profiles) is shown in Fig. 8 within the monolayer subendothelium, apparently pushing it down. The ultrastructure of the sprouting cells and the cells of the monolayer was indistinguishable.

Fig. 7.

Transverse histological sections of post-confluent cultures on collagen gels. Sprouting cells migrate from the two to three-cell layer at the surface (A, B) into the underlying gel (c, D) where they form various cellular connections. ×475.

Fig. 7.

Transverse histological sections of post-confluent cultures on collagen gels. Sprouting cells migrate from the two to three-cell layer at the surface (A, B) into the underlying gel (c, D) where they form various cellular connections. ×475.

Fig. 8.

Vertical section through cobblestone layer and underlying group of sprouting cells within the depth of the subendothelium. Junctional complexes are apparent between adjacent cells in both the monolayer and sprouting cells. ×7000.

Fig. 8.

Vertical section through cobblestone layer and underlying group of sprouting cells within the depth of the subendothelium. Junctional complexes are apparent between adjacent cells in both the monolayer and sprouting cells. ×7000.

Origin of the sprouting cells

Sprouting cells have been observed in late, post-confluent cultures of all the cloned bovine aortic endothelial cells examined, as well as in primary and uncloned cultures. In all cases, the culture conditions influenced the appearance and distribution of cells as described above. Primary cultures and late-passage cultures (up to passage 13 in this study) behaved in a similar fashion.

The two-step cloning method employed in this study makes it unlikely that sprouting cells and cobblestone cells in the monolayer are descendants of different parental cells. The following observations support the conclusion that the bovine aortic endothelial cells are capable of undergoing a reversible phenotypic interconversion between these two distinct morphologies, (i) The monolayer may be detached from cultures growing on plastic dishes or gelatin-coated dishes in a manner that leaves the network of sprouting cells firmly attached to the substratum (as in Fig. 5A). When dishes containing such an exposed network of sprouting cells were cultured for a further 24 h, all the cells adopted a cobblestone morphology and ultimately produced a typical monolayer, (ii) The three-dimensional cellular meshworks growing within the collagen matrix may be separated from the cells on the gel surface. This is accomplished simply by first detaching the surface cells by the sequential application of proteolytic enzymes and then recovering the cells within the gel by hydrolysing it with collagenase (Schor, 1980). Cells recovered from within the gel in this fashion were then plated at high density onto gelatin-coated dishes (in medium containing ascorbic acid) where they grew to produce a confluent, cobblestone monolayer, (iii) Cells may be plated as a single-cell suspension homogeneously distributed throughout the three-dimensional collagen matrix when the gel is initially cast (Schor, 1980). Cultures growing on gelatin-coated dishes consisting of a cobblestone monolayer with no visible sprouting cells were harvested with trypsin and so-plated within the gel matrix at a density of 2 × 105 cells per gel. Cells were spherical 10 min after plating and gradually assumed an elongated morphology within the next 3 h. After 3 days of incubation many of the cells were seen to be associated either in tandem arrays (Fig. 9A) or in anastomosing meshworks (Fig. 9B) similar in appearance to those observed in late, post-confluent gel cultures. These elongated cells were then harvested by hydrolysing the collagen gel and replated at high density onto gelatin-coated dishes, where they formed a typical cobblestone monolayer when confluent, (iv) An equally reversible morphological change was induced when the cells were overlaid with a collagen gel. To do this, the medium was removed from confluent or semi-confluent cultures (on plastic, gelatin or collagen substrata) and the attached cells were overlaid with 2ml of collagen gelling solution (Materials and Methods). Alternatively, a gel previously cast in a separate dish may be detached and placed directly on top of the cultures. Within 10–15 min, cells in the originally cobblestone monolayer retract from each other, becoming irregular in outline and exposing areas of the substratum (Fig. 9c). When overlaid, isolated cells (i.e. in semi-confluent cultures or at the edge of a colony) adopt an elongated shape reminiscent of the sprouting cells (Fig. 9D). The morphology of cells in these monolayers remained unchanged for extended periods in culture, as long as the overlaid gel was left in place. However, when the latter was removed, cells reformed a typical cobblestone monolayer within 3h (Fig. 9E).

Fig. 9.

Bovine aorta endothelial cells plated directly into the gel matrix (A, B) or overlaid with a collagen gel (c, D). E. Same culture as shown in c, 24 h after removing the overlaid gel. ×300 (A, D, E); ×275 (B); ×325 (C).

Fig. 9.

Bovine aorta endothelial cells plated directly into the gel matrix (A, B) or overlaid with a collagen gel (c, D). E. Same culture as shown in c, 24 h after removing the overlaid gel. ×300 (A, D, E); ×275 (B); ×325 (C).

Cells began to migrate up into the overlaid gel within 24h. After 10 days, approximately 6–10% of the total cells were within the overlaid gel matrix, where they formed tandem arrays and three-dimensional meshworks indistinguishable from those shown in Fig. 9A, B. These cells were recovered from the overlaid gel (by hydrolysis of the isolated gel with collagenase) and reformed a typical cobblestone monolayer when replated onto gelatin-coated dishes.

Overlaying cultures with a glass coverslip produced identical morphological changes, as shown in Fig. 9C, D.

Data are presented in this study concerning the effects of the substratum on the proliferation and morphology of bovine aortic endothelial cells. Cells were grown on plastic dishes, gelatin-coated dishes and three-dimensional gels of type I collagen fibres. Lower saturation cell densities and growth rates were consistently obtained on the three-dimensional gel cultures compared with the other two substrata. Similar results have previously been obtained with capillary endothelial cells (Schor, Schor & Kumar, 1979; Schor et al. 1980), human skin fibroblasts (Schor, 1980), chick chondrocytes (Gibson, Schor & Grant, 1981), and mouse 3T3 cells (Schor & Rushton, unpublished observations). In contrast, the proliferation of a number of transformed cell lines has been found to be identical on all three substrata (Schor, 1980; Schor et al. 1982).

The manner in which the substratum influences the proliferative behaviour of the bovine aortic endothelial cells is not understood. Previous studies have shown that a collagen gel substratum can determine the responsiveness of endothelial (Schor el al. 1979, 1980), and epithelial cells (Gospodarowicz, Greenburg & Birdwell, 1978a) to various exogenous growth factors. Bovine aortic endothelial cells synthesize a growth factor to which they are responsive (Astaldiet al. 1981 ; Gajdusek & Schwartz, 1982). The effects of the substratum on cell proliferation reported here may, therefore, result from alterations in the cell’s responsiveness to growth factors present in the medium and/or from changes in the synthesis and secretion of growth factors by the endothelial cells themselves.

Sprouting cells, which are identified on the basis of their distinctive morphology, have been observed in late, post-confluent cultures of bovine aortic endothelial cells (Schwartz, 1978; Gospodarowicz et al. These studies provided convincing evidence, in the form of positive staining for factor VIII antigen (an observation confirmed in this laboratory), regarding the endothelial nature of the sprouting cells. The cells used in our studies have been prepared by a two-step cloning procedure in order to optimize the likelihood of obtaining cultures derived from a single cell. The data we present indicate that such cloned bovine aortic endothelial cells (a total of three different lines examined) may adopt a number of distinctive morphologies and growth patterns in vitro in response to such experimental parameters as initial cell density, the nature of the substratum and the presence of ascorbic acid. These same experimental parameters have been shown to influence the nature of the extracellular matrix synthesized by cells in vitro (De Clerck & Jones, 1980; our unpublished observations), therefore suggesting that the extracellular matrix may contribute to the control of sprouting cell formation in some fashion.

Thin sections of post-confluent cultures growing on plastic, gelatin or collagen gels show that the endothelial cell monolayer rests on an extracellular matrix (i.e. subendothelium). When sprouting cells appear they invariably lie within or underneath the subendothelium of the monolayer and therefore no longer possess a free apical cell surface. Cells within the gel matrix are similarly surrounded by an isotropic environment of collagen fibres. Taken together, these observations lead us to suggest that the sprouting-cell phenotype is produced when endothelial cells are in an environment in which adhesive interactions may be established over the entire cell surface and the apical-basal polarity characteristic of cells growing on a two-dimensional surface can no longer be maintained. In support of this view are the observations that cells that resemble sprouting cells are produced when the endothelial cultures are overlaid with a collagen gel, glass coverslip (this paper) and fibrin clot (Kadish, Butterfield & Folkman, 1979).

Bovine aortic endothelial cells are capable of active migration both in vivo and in vitro (Harker & Ross, 1979; Glaser et al. 1980). The formation of capillary sprouts during angiogenesis has been shown to involve the migration of endothelial cells through their own basement membrane and into the surrounding extracellular matrix (Schoefl, 1963; Ausprunk & Folkman, 1977). Our data show that bovine aortic endothelial cells are capable of migration through their own subendothelium and, when cultured on a collagen gel, of continued migration into the three-dimensional collagen matrix. When overlaid with a collagen gel, these cells are also able to migrate up into gel matrix (i.e. from their apical surface).

Once within the three-dimensional gel matrix, the bovine aortic endothelial cells form characteristic meshworks of interconnected cells (Fig. 6). Mammary epithelial cells have been reported to form similar cellular networks within the gel matrix (Yang, Kube, Park & Furmanski, 1981). Other cell types, such as fibroblasts (both normal and transformed) and a variety of tumour-derived cell lines (e.g. melanoma), start migrating (as single cells) into the collagen matrix well before reaching confluency and do not form cell—cell associations within the gel (Schor, 1980; Schor, Allen & Harrison, 1980).

Folkman & Haudenschild (1980) have reported the formation of intercellular networks (similar in appearance to sprouting cells) and ‘capillary’ formation by capillary-derived endothelial cells growing on gelatin-coated dishes. The formation of such cellular networks required the presence of tumour-cell-conditioned medium and was not observed to occur with aortic endothelial cells. Our observations, as well as those of others (Schwartz, 1978), are at variance with these findings, in that highly vacuolated sprouting cells and intercellular networks (similar to the capillaries described by Folkman & Haudenschild) were obtained with aortic endothelial cells grown in the absence of tumour-cell-conditioned medium. These results raise the interesting question of whether aortic endothelial cells under appropriate environmental conditions are capable of expressing various phenotypic characteristics that are thought to be specifically associated with capillary endothelial cells in vivo.

The nature of the extracellular matrix is known to affect both the kinetics and pattern of formation of new blood vessels in vivo (Ryan, 1973). The three-dimensional collagen gels (as well as other more complex macromolecular matrices) provide an experimental model system with which to examine the effects of the extracellular matrix on particular aspects of endothelial cell behaviour that contribute to the process of angiogenesis in vivo (e.g. cell proliferation, migration and the formation of intercellular networks).

Previous studies have suggested that sprouting cells synthesize different genetic species of collagen molecules compared with cells in the cobblestone monolayer (Cotta-Pereira et al. 1980). The interpretation of these results is, however, complicated by the fact that sprouting cells were examined in ‘mixed’ cultures, which actually contained a greater number of cells organized within a cobblestone monolayer. The data we present here allow an improved degree of experimental control to be exercised over the expression of endothelial cell phenotype in vitro. For example, cells plated within a three-dimensional collagen gel uniformly adopt an elongated morphology, appear to be actively motile and become organized into characteristic cellular networks. Such homogeneous cultures will facilitate the identification of possible differences between the biosynthetic activity of such cells and those within a simple cobblestone monolayer.

Our results indicate that the kinetics and pattern of sprouting cell formation, the rate of cell proliferation and final saturation cell density on dishes coated with gelatin (i.e. denatured collagen) differ from those observed on gels of native collagen fibres. These findings indicate that the particular organization of collagen within the substratum plays an important role in the control of cell behaviour. Previous studies have indicated that the involvement of fibronectin in mediating cell adhesion (S. L. Schor & Court, 1979) and the response of endothelial cells to tumour angiogenesis factor (Schor et al. 1980) are also different on denatured and native collagen substrata.

We thank Mr G. Rushton and Mr B. Winn for excellent technical assistance and Ms E. Mercer for typing this manuscript.

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