Heterogeneity in endothelial cells from large vessels and microvessels

Differentiation

Differentiation (1987) 36: 57-70

0 Springer-Verlag 1987

Heterogeneity in endothelial cells from large vessels and microvessels Shant Kumar', David C. West', and Ann Ager2 Christie Hospital and Holt Radium Institute, Manchester, M20 9 BX, England Manchester University, Manchestcr, Immunology Department, MI 3 9 PL, England

Abstract. The successful isolation and culture of vascular endothelial cells has led to an upsurge of interest in their role in such diverse processes as thrombogenesis, atherosclerosis and tumour growth. In this article we have outlined methods for the culture and characterization of endothelial cells from large vessels, capillary and post-capillary venules of lymph nodes. Comparison of their immunological and metabolic properties illustrates the heterogeneity within the vasculature. The effect of growth and angiogenic factors on these cells and the efficacy of their use in culture medium is considered.

Introduction

The vascular system arises from blood islands which differentiate from the splanchopleuric mesoderm of the area vasculosa. They consist of blood cell precursors in the centre and immature endothelial cells at the periphery which grow to form the primary capillary plexus. After the initial capil-

lary plexus has been formed, the vascular system is extended by sprouting, growth and migration of the endothelial cells [6, 9, 15, 581. The term vascular endothelium has been assigned to the layer of cells which forms the inner lining of the blood and lymphatic vessels at all levels of the circulatory system. The endothelium is heterogeneous, i.e. the endothelial cells which comprise it differ in structure, function, antigenic composition, metabolic properties and in their response to growth factors. Such heterogeniety is apparent between different organs, as well as in different parts of the same organ and even in different segments of a single microcirculatory loop [35, 46, 68, 721. In situ, endothelial cells have been shown to be orientated longitudinally in the direction of blood flow [7, 161. Endothelial cells lining microvessels are flattened and elongated, while those lining large vessels are polygonal. However, these two cell types have been noted to co-exist. Generally, aortic endothelial cells are thicker than those of capillaries and veins [66]. Differences occur in the relative numbers of a unique type of endothelial cell specific inclusion the Weibel-Palade body (Fig. 3 ; [30, 75]),these being

Fig. I. Transmission electron micrograph of Weibel-Palade (W-P) bodies in endothelial cells: longtudinal and cross section (inset)

58

highest in number close to the heart and lowest in the microvessels. The relative number of plasmalemmal vesicles is much lower in arteriolar endothelium than in capillaries [66].Continuous endothelium is characteristic of brain, retina, and muscular capillaries, while fenestrated endothelium is found in endocrine glands and kidney. Venules have the most and arterioles the least elaborate junctional system, whereas capillaries have no communicating junctions 167, 691. Furthermore, interspecies differences in endothelial morphology have been reported [45, 571. The protective function of the immune system depends on the continual recirculation of lymphocytes between the vascular and tissue compartments 1191. During this circuit lymphocytes adhere to the luminal surface of post-capillary venule endothelium and subsequently migrate between endothelial cells to enter the extravascular space. In lymph nodes lymphocytes migrate from post-capillary venules, which are lined with a specialised type of high endothelial cell (HEC) [28]. The histological appearance of this endothelium is cuboidal and these post-capillary venules are commonly termed high endothelial venules (HEV). Apart from their characteristic morphology, HEV are readily distinguished from other post-capillary venules by biochemical studies. HEV have substantially higher levels of cytoplasmic non-specific esterase than other vascular endothelium [3]. The metabolism of sodium sulphate by lymphoid tissues has identified a unique biosynthetic pathway leading to the continuous secretion of a sulphated glycolipid. Autoradiographic studies localise this pathway solely to HEV in lymphoid tissue [4, 51. The rapid, preferential uptake of 35Ssulphate by HEV in rat lymph node may also be used to distinguish HEV from all other vascular and stromal cells of the lymph node [l]. Studies of recirculating lymphocytes have identified surface receptors which mediate lymphocyte adhesion to HEV [4, 14, 231 and the adhesion of lymphocytes to HEC is the basic functional assay for these cells in culture. HEV do not stain with VWF: Ag (factor VIII) antiserum. However, in lymph nodes the luminal surfaces of the hilar artery and vein are positive for VWF: Ag, as are arterioles, venules and capillaries lined with flat endothelium. Historically the endothelial cell was one of the earliest cell types to be tissue cultured (for review, see [48, 501). Lewis [41, 421 grew endothelial cells from explant cultures and observed that the cells appeared to retain the capacity to form capillary-like channels. It was Robertson [59] who applied cloning techniques to separate populations of cells from arterial explants. Maruyama [47] obtained endothelial cells by tryptic digestion of the internal lining of human umbilical cord veins and Fryer et al. [21] from umbilical artery intima. In the early 1970s, Jaffe et al. [34] and Gimbrone et al. 1241 substituted collagenase digestion for tryptic digestion in procedures for the isolation of endothelial cells and were able to grow homogeneous populations of endothelial cells identifiable by their cobblestone appearance, and the presence of factor VIII antigen and Weibel-Palade bodies. In 1974, we began to culture endothelial cells from microvessels of bovine brain white matter. In the mammalian brain, large blood vessels are almost exclusively limited to the grey matter and the blood supply to the white matter is largely via capillaries. Furthermore, normal nerve and glial cells from adult brain are very fastidious and will rarely grow in short-term cultures. Thus, white matter proved to

be a unique source of endothelial cells. Using these cells we have studied the effect of angiogenesis factors from various sources [ l l , 35, 37, 53 -55, 62, 64, 65, 76, 771. Numerous methods have been published to obtain cultures of endothelial cells of both large vessels and microvessels from a variety of organs. In this paper we have given a general scheme which is applicable for the preparation of endothelial cells from microvessels of solid tissues. In addition, separate sections on the isolation of endothelial cells from large vessels (human umbilical vein and bovine aorta), and post-capillary venules of lymph nodes, have been included. Materials and methods

Handling of tissues from the operating theatre or abbatoir was under aseptic, if not sterile, conditions. For general details of tissue culture procedure, see Paul [52]. Isolation of endothelial cells f r o m large vessels

Two common sources of large vessel endothelial cells are human umbilical vein and bovine aorta. Human umbilical vein ( H U V ) . The original method of Jaffe et al. [34] has proved perfectly adequate for the isolation of HUV cells. One end of a fresh umbilical vein, obtained within a few hours of delivery, was cannulated with a blunt 14-gauge needle, secured by artery forceps. The vein was gently washed with 100 ml phosphate-buffered saline (PBS), the open end closed with artery forceps, and 10 ml of 0.25% collagenase solution in basal medium 199 infused. After incubation at 37" C, for 30 min, the collagenase solution was collected and the released cells recovered by centrifugation at 600 g for 5 min. The pellet was then washed with complete medium 199 (medium 199 containing 2mM glutamine, 15% fetal calf serum, penicillin 100 IU/ml and streptomycin 50 pg/ml), resuspended in 10 ml fresh medium and transferred to a tissue culture flask (75 cm'). This was incubated overnight at 37" C in a 5% CO,/air mixture. The following day the medium was replaced and thereafter every 72 h. Usually, the cultures were ready to be used within 5-7 days. The cells were subcultured by rinsing twice with prewarmed PBS and incubation at 37" C, for 20 min with 8 ml PBS containing 2 m M EGTA. Then 2 m l PBS-trypsin (0.25%) was introduced, giving a final concentration of 0.05% trypsin. Cell detachment began within a few seconds. Detachment was aided by gentle shaking. If the release of cells appeared slow, the flasks were returned to the incubator and cell detachment was monitored every minute or so, by visual inspection under the inverted phase contrast microscope. It was absolutely vital that the cells were in contact with trypsin for as short a time as possible. The cell suspension was then mixed with 10 ml complete medium 199, in order to stop the action of trypsin, and washed by centrifugation. Cell numbers were determined using a haemocytometer or a Coulter counter. Viability was assessed by mixing a drop of trypan blue with a drop of cell suspension; live cells excluded the dye. In our experiments we have generally used primary or early passage cultures. In practice, umbilical cells cannot be easily passaged more than 2 or 3 times, without the presence of additional growth factors. In agreement with other workers we have found that the addition of endothelial cell growth factor

59

(ECGF) (Collaboratieve Research : 50 pg/ml) and porcine heparin (Sigma: sodium salt, grade 1 : 100 pg/ml) may allow better growth of these cells. Bovine aortic endotheliul cells (BAEC). Using a pair of large scissors, a cow aorta was freed of fat and other adherent tissue. The aorta was opened longitudinally, washed with 500ml PBS, and the intimal lining gently scraped with a scalpel to remove the uppermost cell layer. Care was taken not to penetrate the sub-endothelial layer. The intimal scrapings were transferred to 10 ml serum-free DMEM containing 0.25% collagenase, and digested for 10 min at 37" C. The resulting cell suspension was centrifuged at 600 g for 5 min and the pellet washed with complete medium (DMEM containing 10% newborn calf serum and antibiotics). Finally, the pellet was resuspended in 10 ml complete medium and transferred to a tissue culture flask (75 cm'). Within 7-10 days, cells had grown to sufficient density to be used for experimental studies. Endothelial cells f r o m microvessels of bovine brain white mutter

The following simple procedure has been successfully employed by us to obtain microvascular endothelial cells of acceptable purity [54, 551. Cow brains from freshly slaughtered animals were transported to the laboratory in Betadine (providone iodine solution). The brain was dissected under sterile conditions to collect 10-40 g white matter. This was washed with copious amounts of PBS. The tissue was finely minced with scissors, incubated with 30 ml 0.25% trypsinPBS for 5 min, and pipetted vigorously for several minutes. The action of trypsin was halted by the addition of an equal volume of complete medium 199 and the cells collected by centrifugation (600 g for 5 rnin). A myelin plug at the top of the supernatant fluid was discarded. The cell pellet was then resuspended in complete medium 199 and distributed between 30 tissue culture flasks (75 cm'). It was critical that the flasks were left undisturbed for 1 week. After the initial medium change, it took 2-3 weeks for the cells to become confluent. General procedure f o r obtaining endothelial cells of microvusculature

The following procedure can be adopted to obtain pure cultures of microvascular endothelial cells. The method was based on several papers describing the isolation of endothelial cells, for instance, from brain [13, 261, bovine adrenal [I81 and bovine retina [25, 631. 1. The tissue was chopped with a scalpel into small (3-5 mm3) pieces, a few drops of basal medium 199 added and a very fine mince made with curved scissors. Five volumes of medium 199 was added and the suspension syringed vigorously through a large bore needle. 2. Centrifugation was performed at 600 g for 10 min and the pellet resuspended in 3 vols medium 199. 3. The supernatant was homogenised with a Dounce homogeniser using 15-50 strokes brain tissue required fewer strokes than more solid organs such as kidney. 4. The mixture was passed through a 150 pm nylon cloth sieve and the filtrate collected. 5. The filtrate was centrifuged and the resulting pellet resuspended in 5 vols collagenase (Sigma type I, 0.5 mg/ml in basal medium 199). -

6. The mixture was incubated at 37" C for 6-16 h. 7. The contents were syringed through a wide-bore needle, filtered through 50 pm mesh nylon cloth and the filtrate collected. 8. The filtrate was centrifuged and the pellet resuspended in complete medium 199 for inoculation into flasks (10 m1/75 cm' flask). 9. Two days later, the supernatant was poured off and replaced with fresh complete medium 199 containing 1,l'dioctadecyl-3,3,3',3'-tetra-methylindocarbocyanine percholorate-labelled acetylated-low density lipoprotein (Dil-AcLDL) (10 pg/ml) (see below). 10. The following day, the cells were trypsinised into a single cell suspension (see above), resuspended in complete medium 199 at 4" C and sorted under sterile conditions using a fluorescence activated cell sorter (excitation wavelength 514 nm; emission wavelength above 550 nm) [22, 741. The Dil-Ac-LDL positive cell fraction was washed twice with complete medium 199 and plated into 75 cm' flasks. For greater purity, sorted cells were allowed to grow for 1-2 weeks, again incubated with Dil-Ac-LDL and resorted [74]. Should the facility for cell sorting not be available, it is possible to use a cloning technique [IS]. The cell pellet obtained at stage 8 was plated in 10 cm2 dishes. When colonies of " cobblestone-like'' cells had reached a reasonable size (5-10 cells) they were either removed using a cloning ring or all cells surrounding the desired colony were scraped away with the aid of a rubber policeman or wire [18, 791. Usually within 2-4 weeks, the cells had multiplied sufficiently to cover most areas of the dish. Identification of endothelial cells Factor VIII. Factor-VIII-related antigen is regarded as a universal marker of endothelial cells. As far as human endothelial cells are concerned, several commercial polyclonal antisera (Dakopatts, Behringwerke, Calbiochem or Atlantic Antibodies) have been found satisfactory for the localisation of VWF: Ag. More recently monoclonal antibodies against factor VIII have been raised by H.J. Reinders [56] (Central Laboratory of the Netherlands Red Cross Transfusion Service, PO Box 9190 1006 AD, Amsterdam, The Netherlands) and A.H. Goodall (Royal Free Hospital, London, England). Human factor VIII polyclonal antiserum has been successfully used to demonstrate factor VIII in bovine, rat and mouse endothelial cells. However, rabbit antibovine factor VIII (provided by Dr. E. Kirby, Temple University, School of Medicine, Philadelphia, USA and Dr. J.E. Brown, University of California, San Diego, USA) has been used by a number of workers, to identify bovine endothelial cells. To demonstrate the presence of factor VIII antigen, coverslip cultures were rinsed in PBS, and fixed in pre-cooled (-20" C) acetone for 5 min, air dried, and treated with a few drops of diluted factor VIII antiserum (e.g. Dr.E. Kirby's antiserum diluted 1 in 20). The coverslips were incubated at room temperature for 30min and washed with gentle shaking. They were incubated with fluorescein isothiocyanate (F1TC)-conjugated goat anti-rabbit immunoglobulins (Miles-Yeda Ltd., Israel: diluted 1 in 40) for 30 min. Those cultures treated with mouse monoclonal factor VIII antiserum were treated with FITC-conjugated rabbit anti-mouse immunoglobulins (Dakopatts, diluted 1 in

60

40). The cells were washed for 40 min, mounted in Citifluor (Agar Aids, Cambridge, U.K.) on a glass microscope slide, sealed with nail varnish and examined under a fluorescence microscope. Labelling of cells with Dil-Ac-LDL. Coverslip cultures were fed with fresh complete medium 199 containing a Dil-AcLDL probe (10 pg/ml; [22, 74]), now available from Biomedical Technologies, Cambridge, Mass., USA). The cells were incubated for 16 h, washed free of probe, mounted and examined under the fluorescence microscope using rhodamine filters. Angiogenesis assay and angiogenesis factors

Chicken chorioallantoic membrane assay was used to assess angiogenesis activity. A sample was considered angiogenic if it produced a "spoke-wheel'' pattern of vessels radiating from the site of its application [77]. Low-molecular-weight ( 400) angiogenesis factor was purified from rat Walker 256 carcinoma, as described previously [38]. Partial degradation products of sodium hyaluronate produced by the action of testicular hyaluronidase were obtained following the method of West et al. [76, 771. The effect of angiogenesis factors on tissue cultured endothelial cell proliferation was assessed either by directly counting cell numbers or by measuring DNA content [35, 62, 76, 781.

-

High endothelial cells Isolation and culture of HEC. Popliteal lymph node (LN) or cervical LN were dissected out from 250 g F, hybrid rats, 96 h after footpad injections of lo7 parental lymphocytes (four rats; 15-200 mg tissue). The tissue was washed twice with Dulbecco's A + B (Oxoid Ltd, Basingstoke, Hants, UK) plus antibiotics (50 IU/ml penicillin + 50 pg/ml streptomycin: DAB), incubated in 10% Betadine in DAB for 10 rnin and washed three times with 20 ml DAB. The tissue was minced finely and the fragments allowed to settle out of the DAB for 1-2 min. The supernatant was discarded and the tissue washed three times with 20 ml DAB, resuspended to a concentration of 50 mg tissue/ml in RPMI 1640 containing 10 mM NaHCO, , 20 mM HEPES, 2 mM glutamine and antibiotics (RPMI,,,), with 0.5% collagenase (type 11; Sigma Chemical Co, Poole, Dorset, UK). After shaking for 60 rnin at 37" C the tissue was dispersed with a Pasteur pipette, resuspended in 10 ml RPMIincand filtered through 100 pm nylon mesh (Simon Special Products Ltd, Stockport, Cheshire, UK) supported in a 23-mm-diameter Millipore Swinnex filter holder. Isolated cells were collected by centrifugation at 250 g for 5 rnin and plated in RPMI 1640 containing 10 mM NaHCO, , 2 mM glutamine, antibiotics (RPMIgro) and 20% heat-inactivated (56" C; 30min) fetal calf serum (FCS) (Sera-Lab Ltd, Crawley Down, Sussex, UK) in a 25 cm2 tissue culture flask (Nunc, Denmark). Cells were grown at 37" C in a humidified atmosphere of 5% CO, in air. After 60 min, non-adherent cells were removed and fresh growth medium added. Primary HEC cultures were routinely rinsed twice with DAB and RPMI plus 20% FCS was added every 48-72 h. Cells were sub-cultured using 0.1% trypsin (Difco 1 :250; Detroit, Mich., USA), 0.025% EDTA in PBS and plated at 50% of their confluent density.

[35S]-Sulphatelabelling of high endothelial cells in lymphatic tissue. In order to identify HEC isolated from LN, these cells were labelled prior to enzyme digestion. Slices (1-2 mm) of popliteal LN (100 mg total) were incubated with 0.8 ml 50 pCi/ml sodium [35S]-sulphate (> 5 mCi/pg S; Amersham International, UK) in RPMIincat 37" C for 30 min. Type I1 collagenase was added to a final concentration of 0.5% and HEC isolation continued as above. Isolated cells were plated on 13 mm Thermanox cover slips (Miles Laboratories Inc, Naperville, Ill., USA) in 24-well tissue culture plates (Linbro Space Saver, Flow Laboratories, UK). After 60 min adherent cells were washed three times with DAB, fixed with neutral buffered formalin for 30 min, processed for autoradiography and counterstained with methyl green and pyronin. Non-lymphoid cells were scored for size and 3sS-label. Lymphocyte-endothelial cell adhesion assay. HEC were plated at confluent density in 9-mm-diameter wells of 8-well multichamber glass slides (Lab-Tek, Miles Scientific Ltd) and used within 5 days. HEC were rinsed once with DAB and preincubated in RPMI,,, plus 1% FCS for 60 rnin at 37" C. Thoracic duct lymphocytes (TDL) were collected over a period of 2 h at room temperature via thoracic duct cannulae into 20 ml DAB plus 5 units/ml heparin [20], centrifuged at 250 g for 10 rnin and resuspended in RPMI,,, plus 1% FCS. TDL were suspended at a density of 3 x lo6 above HEC and incubated at 37" C in static culture. Non-adherent TDL were removed after agitation and HEC washed once with RPMI,,,. This wash procedure was repeated 5 times and cultures were fixed with 3.0% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 for 30 min at 37" C. Cells were stained with toluidine blue. TDL and HEC nuclei were scored using high-power light microscopy. One hundred fields of view at x 1000 magnification were studied and the ratio of TDL: HEC was calculated.

Results Vascular endothelial cells

The major problem with microvascular cultures is contamination of endothelial cells with pericytes. In cultures from large vessels smooth muscle cells could be a source of contamination. Readers are referred to Zetter [79] and Jaffe [33] who have addressed this problem at length. Cultured endothelial cells from human umbilical veins and bovine aortas grew to form a regular, contact-inhibited monolayer of closely adherent, polygonal cells with very little extracellular space (cobblestone morphology) as shown in Fig. 2 b and c. However, it was normal for cultures of bovine aortic endothelial cells, maintained at confluence for 5-14 days, to contain endothelial cells with fibroblastlike morphology which grow and migrate above or below the original monolayer. This phenomenon, termed " sprouting", has been reported several times in the literature for bovine aortic endothelial cells and more recently for bovine retinal capillary endothelial cells [63]. In our experience heparin (100 pg/ml) promoted this effect, culturing the cells on a gelatin substratum in the presence of ascorbate (50 pg/ ml) retarded the sprouting. In cultures of bovine brain white matter, isolated flattened cell clumps were evident 3-4 days after the initial

Fig. 2 a-c. Scanning electron photomicrographs of tissue cultured endothelial cells. a Bovine brain white matter capillary endothelial cells growing on plastic. b Confluent culture of bovine aortic endothelial cells growing on collagen substratum. c Same as b, at higher magnification

62

Fig. 3. Demonstration of uptake of l,l,dioctadecyL3,3,3’,3’-tetramelthylindocarbocyanine perchloratelabellcd acetylated-low density lipoprotein (Dil-Ac-LDL) by tissue cultured bovine aortic endothelial cells. Bovine brain white matter capillary endothelial cells did not label with DilAc-LDL (not shown)

Fig. 4. Staining of tissue cultured bovine aortic endothelial cells for V W F : Ag. Bovine brain capillary endothelial cells were also decorated by factor VIII antiserum (not shown)

plating. Subsequent growth was rapid, with cells migrating out as a loose sheet, and cultures normally becoming confluent in 2-3 weeks. Confluent cultures, although contact inhibited, were less polygonal than either aortic or umbilical vein endothelial cells and appeared more elongated or fusiform (Fig. 2 a). This morphology was maintained through several passages (1 :3 split). Light microscopy showed the presence of a few optically dense granules in the perinuclear region. Our studies on angiogenesis factors has involved culturing both bovine aortic and brain capillary endothelial cells on renatured type I collagen gels [35, 38, 651. However, these cell-types show different growth characteristics on col-

lagen gels in comparison to plastic. Both grow essentially on the surface of the gel with doubling times of approximately 72 h, as compared with growth on plastic: 24 and 48 h for aortic and brain capillary cells, respectively. On plastic, proliferation was evident within 24 h, but on collagen an initial lag phase is apparent. The length of this lag phase is dependent on the serum concentration in the growth medium. Concentrations below 15% proved inadequate to support growth. It appears that the collagen matrix removes an essential component from the serum, as preincubation of the gel with complete medium shortens the lag phase. Cell attachment differs between plastic and collagen gel substrata, being >SO% for plastic and 40%-45%

63 Table 1. Some characteristics of endothelial cells" Endothelial cell marker Source endothelial cell

Factor VIII

Uptake Prostacyclins Angiotensin y-Glutamyl Alkaline Weibel-Palade Morphology of Dilconverting transpeptidase phosphatase bodies Ac-LDL enzyme

+

+

+

+

+ b

+ b

+

+ b

3. Bovine adrenal capillary

+

+

+

+

?

Typical (Cobblestone)

4. Bovine retinal capillary

+

+

+

+

?

Typical (Cobblestone)

+

+

+

+

?

Typical (Cobblestone)

+

-

Negligible

Negligible

Rare

Atypical

-

-

?

?

?

Atypical

+

+

?

?

Absent

?

1. Human umbilical vein

2. Bovine aorta

5 . Bovine whole brain or

grey matter capillary 6. Bovine brain white matter capillary 7. Lymph node post capillary venule 8. Rat bone marrow

More common Typical (Cobblestone) More common Typical (Cobblestone)

Relevant references: [l, 8, 9, 15, 17, 21, 24-26, 32, 33, 37, 46, 47, 51, 52, 55, 60, 61, 63, 71, 791 Rone and Goodman [60] obscrvcd that tissue cultured rabbit aortic endothelial cells contained two populations of cells : elongated tightly packed ones, which were factor VIII positive but negative for ACE and failed to take up Dil-Ac-LDL; the second cell type with cobblestone morphology was factor VIIT negative but ACE and Dil-Ac-LDL positive a

+ : Positive;

& : weakly positive;

- : negative;

for collagen, a fact that must be borne in mind when comparing growth on these substrata. It was stated above that endothelial cells grow on the surface of the collagen matrix. Recently, we have confirmed the work of Montesano and Orci [49] in that, addition of phorbol myristyl acetate (20 pg/ml) to confluent cultures of aortic cells caused them to penetrate the collagen gel as vessel-like tubes. No effect was apparent in sparse cultures. Immunological and histochemical staining of cultured umbilical, aortic and brain capillary cells revealed differences between large vessel and capillary-derived cells. However, comparison with published results on capillary endothelial cells from other sources suggests that white matter capillary cells were atypical in the fact that they are factor VIII positive, do not endocytose Dil-Ac-LDL but do stain for y-glutamyl transpeptidase, which is a brain capillary marker (Figs. 3, 4; Table 1). Both purified TAF [38] and hyaluronic acid fragments [77] induced angiogenesis in the in vivo chicken chorioallantoic membrane assay (Fig. 5). TAF treatment enhanced the growth of capillary but not aortic endothelial cells, and only when the cells were growing on a native collagen gel (Fig. 6). HA fragments increased the growth of both capillary and aortic endothelial cells. Unlike TAF, for the stimulation to occur, it was not necessary to culture cells on collagen substratum (Fig. 7; [36, 38, 781). High endothelial cells

Autoradiographs revealed three distinct populations of cells in a collagenase digest of 35S-labelledLN: (1) 10- to 30-pmdiameter 35S-labelled, lightly staining cells; (2) 10- to

? : not known or controversial

15-pm-diameter non-lymphoid cells, which were unlabelled and lightly stained; and (3) lymphocytes, which were unlabelled and darkly stained (Fig. 8 a). Of the two populations of non-lymphoid cells (1 and 2), 71.7*4.9% (kSD, n=4) were large and labelled and were therefore HEC. The majority (>95%) of the remaining unlabelled cells were of medium size. HEC in fixed and stained preparations were round cells and were found singly, occasionally with 1 or 2 lymphocytes attached. Phase contrast microscopy of cells showed three distinct populations of cells, which could be directly identified with those in autoradiographs (Fig. 8 b). HEC were large, round, single cells with vesiculated cytoplasm and occasionally with 1-3 lymphocytes attached to their surface. During the first 2 days of culture the lymphocytes were washed away and HEC started to migrate. After 2 days most HEC were no longer vesiculated and the cells started to proliferate (Fig. 9a, b). At confluent densities HEC underwent a dramatic morphological change, becoming bipolar and tending to align in parallel arrays with substantial cytoplasmic, but no nuclear overlap (Fig. 9c). To date, 54 primary HEC cultures have been established and maintained to first confluence, taking an average of 10 days to double 7-8 times. HEC proliferation was FCS-dependent and the rate of proliferation was 10-fold less in newborn calf serum. Rat serum did not support HEC proliferation. HEC proliferated readily in RPMI 1640 and MEM growth media. The results of adhesion of lymphocytes to cultured HEC showed that the ratio of lymphocytes: HEC in fixed and stained preparations was 0.6 ( k 0 . 2 SE):I (n=12). In contrast, aortic endothelial cells showed a 40-fold lower affinity for lymphocytes than HEC. The ratios of lympho-

64

Fig. 5. In chicken chorioallantoic membrane (CAM) assay: a showing “spoke-wheel’’ pattern of capillaries following treatment with tumour angiogenesis factor (TAF) (b) Control untreated CAM

Plastic

x

u

20 -

20 -

15-

15-

Collagen

m

20-

0

HSF

15-

Fig. 6. In vitro stimulation of various types of cells with purified tumour angiogenesis factor. TAF enhanced the proliferation of bovine brain white matter capillary endothelial cells (BCEC) only when the cells were cultured on native collagen gel. N o concentration of TAF produced an increase in the number of capillary cells on plastic dishes. Bovine aortic endothelial cells (BAEC) or human skin fibroblasts ( H S q were not stimulated by TAF either on plastic or native collagen gel

5

$

-

BAEC

BCEC

20.

15

10

0

25

50

75

100

0

25

50

TAF (pgldish)

75

100

0

25

50

75

100

65 Table 2. Differences in the volumetric density of Weibel-Palade bodies (WPB) in capillary endothelial cells"

b C 0

w

g

100

Source of capillary endothelial cell

WPB Median (range) Statistical analysis

1. Human joint tissue (a) Normal joint

0 (0.000-0.017)

(b) Rheumatoid joint

0.006 (O.OOCr0.081)

(c) Osteoarthritic joint

0.005 (0.000-0.079)

..-I

LI

m

-

L a ..4 +. 0

&

.l

.5

i

5

10

50

100

C

.l

.5

1

5

10

50

100

C

ug u r o n a t e / well

C05200lM/N

Fig. 7a, b. Effect of hyaluronic acid fragments on endothelial cell number. Bovine aortic (a) and bovine brain capillary cells (b) were incubated for 72 h with HA fragments at 0- 100 pg uronate/well. At the end of this period, the DNA content of each well was assessed fluorimetrically. The levels of DNA are expressed relative to that of control wells. In this representative experiment each har corresponds to the average of triplicate wells kSD. C represents control wells with no additions

cytes: HEC were not significantly different when primary cultures or HEC subcultured (up to 8 passages) were used in the adhesion assay. Discussion The purpose of this communication was to describe practical methods for obtaining pure cultures of endothelial cells from large and microvessels. We also wanted to highlight the fact that endothelial cells are heterogeneous. Undoubtedly, cell morphology (i.e. cobblestone appearance) and cell markers such as the presence of factor VIII antigen, angiotensin converting enzyme, Weibel-Palade (W-P) bodies, capacity to take up Ac-Dil-LDL and the production of prostacylins are most valuable in establishing the identity of endothelial cells. However, in certain exceptional circum-

2. Chicken chorioallantoic membrane (CAM) 0.002 (0.000-0.010) (a) Normal (CAM) (untreated) (b) CAM treated with TAF

0.005 (0.000-0.066)

Normal vs rheumatoid P < 0.005 Normal vs osteoarthritic P< 0.005

Normal CAM vs TAF treated P<0.008 Normal CAM vs RAF treated P<0.008

(c) CAM treated with RAF" 0.008 (0.000-0.033) For details, see Kumar et al. 1391 . .and Sattar 1621 Purified tumour Both these factors stimulate angiogenesis factor (TAF) neovascularisation Purified rheumatoid in the CAM assay angiogenesis factor (RAF)

a

~~

stances these markers may fail to identify endothelial cells. It is also worth remembering that endothelial cells from normal and pathological tissues may differ markedly from each other. We have shown that W-P body volumetric density was significantly increased in capillary endothelial cells of both human brain tumours and rheumatoid joints com-

Fig. 8. a Autoradiograph of cells isolated from lymph node (LN) by collagenase digestion after 1 h in culture showing labelled high endothelial cell (HEC) : unlabelled substrate-dependent non-lymphoid cell ( M ) and unlabelled lymphocytes ( L ) .Methyl green and pyronin. b Phase contrast micrograph of isolated cells after 24 h in culture showing large, vesiculated HEC, medium-sized macrophages ( M ) and lymphocytes ( L )

66

Fig. 9a-c. Phase contrast micrographs of HEC in culture. a Primary HEC culture after 2 days, showing a flat, vesiculated cell (curved arrow), amoeboid cells (open arrowv) and a bipolar cell with fcw vesicules (solid arrow). b Primary HEC culture after 5 days, showing few vesiculated cells (curved arrow). c Primary HEC culture after 10 days, showing confluent bipolar cells aligned in a parallel array

pared with their normal counterparts ([29, 621 and Table 2). It is also apparent from the Results section that the source of endothelial cells and their requirement for a particular substatum may be crucial in determining their responsiveness to angiogenesis factors. HEC line post-capillary venules and are therefore a type of microvascular endothelium. The commonly used criteria

for identification of vascular endothelial cells were not applicable to this cell type. However, the use of a specific biochemical marker for HEC has allowed positive identification of isolated HEC after enzymic digestion of lymph nodes. Further, the use of a lymphoctye adhesion assay has permitted the demonstration of functional HEC in long-term culture. The ratio of lymphocytes bound to HEC b

Fig. IOa, b. Confluent and sparse cultures of bovine aortic endothelial cells were transferred to methionine free DMEM containing 5% new born calf serum, with or without hyaluronate fragments (10 pg/ml, 8-10 disaccharides in length). The cells were incubated for 24 h and then labelled with [3’S]-methionine (100 pCi/T75 flask) for a further 24 h. Integral membrane proteins were extracted using Triton X-114 in the presence of protease inhibitors, and 2.5 x 10’ cpm were analysed by two-dimensional electrophoresis (pH 4-8, PI gradient; 5%-15% acrylamide gradient). PI was determined by equilibrating I-cm slices of two gels in 0.5 ml boiled deionised double-distilled water and measuring the pH. Gels were fixed, impregnated with PPO/acetic acid and dricd for fluorography. Comparison of the fluorograms of confluent (a) and sparse cultures stimulated with hyaluronate fragments (b) showed six new proteins present in the latter. Four of these proteins were present in other sparse cultures containing no HA fragments. However, the hyaluronate fragments specifically induced two 42 kDa proteins of PI 5.0 and 4.0 (arrow) (b)

68

was remarkably similar to ratios measured in histological preparations of normal rat lymph nodes [2, 291. The strong tendency for cultured HEC to adopt a bipolar “fibroblastic” morphology and to align in parallel arrays at confluent density was maintained in long-term culture. This morphology is distinct from the polygonal shape or large vessel endothelial cells in culture. It is worth noting that cultured HEC from rat lymph nodes display a very similar morphology to that of HUV endothelial cells which have been subjected to prolonged treatment with immune interferon [70]. The demonstrated lack of vWF: Ag localisation in HEV in vivo in comparison with its presence in other microvascular endothelial cells of rat LN suggests that the precise relationship between these two types of microvascular endothelium is not straightforward with respect to their roles in haemostasis. Realising the need for an adequate endothelial marker, Lincoln et al. [43] employed two-dimensional electrophoresis to compare the polypeptide profiles of bovine aortic endothelial cells, smooth muscle cells and fibroblasts. They found proteins that were unique to each cell-type and identified a train of nine related proteins, with a molecular mass (M,) between 43 and 47 kDa and isoelectric points (PI) of 4.8-6.0, which appeared to be specific for endothelial cells. We have examined the proteins secreted by cultured bovine retinal capillary endothelial cells. A major component secreted into the medium was a non-collageneous glycoprotein of M, 47000, which was composed of nine related polypeptides of PI 4.65.5 [12], a pattern similar to that of Lincoln et al. and also to that of an endothelial cell inhibitor of plasminogen activators [44]. From these results, it appears that the presence of such a charge train is a useful indication that cultured cells are endothelial, at least for those of bovine origin. Media supplements, such as ECGF, endothelial cell conditioned medium, and tumour conditioned medium, are commonly employed to facilitate the growth and selection of endothelial cells from several sources. However, scant attention has been given to the effect of these on any subsequent metabolic studies. Gospodarowicz et al. [27] have compared the polypeptide profiles of endothelial cells cultured in the presence of epithelial growth factor and fibroblast growth factor. Both growth factors induced significant, but different changes in the protein patterns. Recently, we have examined the composition of integral membrane proteins in confluent and sparse cultures of bovine aortic endothelial cells (West and Kumar, unpublished results). Sparse cultures showed several changes in their polypeptide profile, when compared with confluent cells. Three proteins were induced : two apparently related polypeptides of M, 70 kDa, PI 6.2 and 6.3, and a protein of 21 kDa and PI 5.5. Also, a protein of 21 kDa, PI 5.7 was greatly reduced in concentration. Sparse cultures grown in the presence of either ECGF (50 &ml) or hyaluronate oligosaccharides (3-10 disaccharide units; 10 pgiml) exhibited these same alterations in the protein pattern, together with further changes unique to each growth factor. ECGF induced one additional polypeptide of 41 kDa, PI 5.4, and hyaluronate two 41 kDa proteins with PI values of 5.2 and 4.0 (Fig. 10). These studies highlight the problem of comparing results of studies on endothelial cells grown in the presence of different medium supplements. Recently, several workers have reported that both large vascular and capillary endothelial cells synthesise P-fibroblast growth factor [lo, 731 and this known autocrine factor, or endothelial cell condi-

tioned medium, would seem to be more “natural” media supplements than either ECGF or tumour conditioned medium. In conclusion, the presence of WVF: Ag, uptake of AcDil-LDL, W-P bodies, angiotensin converting enzyme, yglutamyl transpeptidase, alkaline phophatase, prostacyclin, and characteristic cell behaviour on various substrata have been used to aid identification of endothelial cells. Many workers have found that the use of special media and substrata is essential for the isolation and maintenance of endothelial cells in vitro. For instance, the mixing of fresh and spent medium from tissue cultured tumour or endothelial cells has been used to obtain cultures from bovine adrenal and bovine retina [18, 31, 631. Similarly, the addition of exogenous heparin, ECGF, ascorbic acid etc. has been used to enrich tissue culture media. Coating of plastic dishes with fibronectin, gelatin and other extracellular matrix components has been noted to help endothelial cell growth. We have found serum batches differ profoundly in their ability to sustain endothelial cell growth. That the endothelial cell is not a single entity is now beyond controversy. There is no standard endothelial cell and no universal marker for endothelial cell. Nonetheless, we would emphasise that in vast majority of cases it is feasible to use any one type of the tissue cultured endothelial cell as an in vitro model for a variety of different in vivo situations [40, 791. Acknowledgement. We are grateful to Dr. A. Sattar for his help with transmission and scanning electron microscopy. D.C.W. is a CRC fellow, and S.K. is in receipt of a grant from the British Heart Foundation.

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Received October 1987 / Accepted October 28, 1987