0040.8166/9O,‘lx)22-0615/$10.(K)
TISSUE AND CELL, 1990 22 (5) 615-628 @ 1990 Longman Group UK Ltd.
S. FURUYA”,
C. EDWARD%
MORPHOLOGICAL BEHAVIOR BOVINE ADRENAL MEDULLA ENDOTHELIAL CELLS Keywords:
Bovine adrenal medulla, diaphragmed fcncstrae
and R. ORNBERGS
OF CULTURED CAPILLARY
cndothclial
cells.
cell
culture.
capillaq
fornwtwn.
ABSTRACT. Bovine adrenal medulla capillary cndothellal cells were isolated and cloned. and their morphological behaviors m uitro were examined. In the culture of primary or early passage, one type of colony formed intracellular lumina both on the dish and in the three dimensional collagen gel. Another type proliferated well and showed morphology ranging from slender-shape to cobblestone shape. and were easily cloned. Cloned cells which showed slcndcrshapes formed tubular network on plastic dish after addition of PMA. OAG or vanadate. and these cells also formed multicellular tubules in the three dimensional collagen gel. tlowcvcr. the formation of diaphragmed fenestrae by thcsc slender-shape clones war rare. One clone which showed cobblcstonc shape formed diaphragmcd fcncstrac, when cultured on collagen gel for more than one month. Isolated colonies or clones showed heterogeneity of cell \hapc. anglogenic behaviors and fcnestrac formatwn.
Introduction
1983; Montesano
and Orci, 1985: Olander and Danilov., 1986; Leibovich et al., 1987; Sato et al., 1987; Madri et al., 1988; Yen-Patton et al., 1988) and fenestrae formation (Milici et al., 1985; Lombardi et al., 1986,1988; Montesano and Orci. 1988). To study the formation of fenestrated capillaries in vitro, we isolated and cloned endothelial cells from bovine adrenal medulla. We observed the time course of disappearance of diaphragmed fenestrae in primary culture, and examined the induction of capillary formation and fenestrae formation in cloned cells. However. isolated clones showed heterogeneity of cell shape, angiogenic behaviors and fenestrae formation. Fenestrae formation in the clones which showed high angiogenic activity was rarely observed. In this report, we describe the differences of cell shape and morphological behavior of the clones, as they relate to angiogenesis and fenestrae formation (a preliminary note was reported previously).
Pt al., 1985; Allikmets
Endocrine organs have fenestrated capillaries with geometrically arranged diaphragmed pores in the highly attenuated endothelium (E&in, 1965). The correlation between these specialized structures and capillary permeability has been described (Simionescu, 1983). There have been many reports describing in vitro modulation of cell shape and angiogenesis (Folkman and Haudenschild, 1980; Maciag et al., 1982; Nicosia et al., 1982; Madri and Williams, 1983; Montesano et al., 1983, 1986; 1988; Schor et al., * National lnstitutc for Physiological Scicnccs. Myodaiji, Okazaki, 444, Japan. t Unwersity of South Florida Medical Center, College ol Medicine. Research and Graduate Affairs Box 40. 12901 North 30th Street, Tampa. FL 33612, USA. $ Baxter Healthcare Corporation, Baxter Technology Park WGZ-2s. Round Lake, Illinois 60073, USA. Address correspondence to: Sonoko Furuya, National Institute for Physiological Sciences. Myodaiji, Okazaki. 444. Japan. Received 8 January 1990. Revised 20 June 1990. 615
616
FURUYA
ET AL.
Materials and methods
al., 1985; Furuya et al., 1989). The cells in the
Materials used were collagen type 1 (Nitta Gelatin Co., Osaka); Percoll (Pharmacia), endothelial cell growth supplement [ECGS], basement membrane MatrigelTM and human fibronectin, (Collaborative Research); human transforming growth factor p (R & D Systems Inc.), DiI-acetylated LDL (Biomedical Technologies Inc.), sodium orthovanadate (Wako Pure Chemical Industries, LTD); Dibutyryl cyclic AMP (Yamasa Co.); collagenase type I, bovine serum albumin type V, phorbol myristate[PMA], l-oleoyl[cis]-9;C2:0) 2-acetyl-sn-glycerol(C18: 1, [OAG] and DMEM/F12 (Sigma); fetal calf serum (Flow Laboratories); glutaraldehyde (LADD); OsOl (Polysciences); ECA, RPA (E. Y Labo. Inc); LCA, GS-1, PHA-E4, PHA-L (Vector Labo. Inc.); RCA120, SBA, PNA (Honen Co.) and streptavidin-HRP (Amersham).
top band were inoculated on plastic dishes or dishes coated with 5 pg human fibronectin per cm* with DMEM/F12 containing 10% FCS, 100 U/ml penicillin and 100 pg/ml streptomycin. Several hours to 1 day later, chromaffin cells were removed by pipetting and cortical cells were removed by brief trypsinization (0.05% trypsin, l-5 min). Colonies of endoethelial cells were identified by the uptake of DiI-acetylated LDL (Voyta et al., 1984), and non-endothelial cells were removed as described by Folkman et al. (1979). In some experiments, ECGS (50 ,ug/ ml) + heparin (lU/ml) media, conditioned media of B16 melanoma, or conditioned media of endothelial cells were used until colonies of sufficient size were obtained. Cloned endothelial cells of the 3rd-8th subculture were used for experiments. In some experiments, the endothelial cell-rich primary cultures were dissociated with trypsin and immediately used for three-dimensional collagen culture without cloning.
Materials
Cell preparation
Cell culture in the three-dimensional collagen gel
The cells of bovine adrenal medulla were dissociated by a modification of the chromaffin cell preparation from adrenal glands, and were separated by Percoll centrifugation (Greenberg and Zinder, 1982; Banerjee et
Fig. 1. Fenestrated Figs 2-6. Primary
endothelial culture
Three dimensional collagen gel was prepared by the modification of Montesano et al. (1983). Seven volumes of collagen type I or
cell dissociated
of endothelial
Fig. 2. EndothLlial cells cultured thin processes. x250.
from bovine
cells inoculated
adrenal
medulla.
on collagen-coated
~7,500. plastic dishes.
for 1 day. Cell body is still round, and has begun to elongate
Fig. 3. Endothelial cell cultured for 1 day sectioned horizontally to the cell layer. Attenuated endothelium with diaphragmed fenestrae has been internalized into the round cell body and has begun to degrade. New processes have begun to elongate. Debris (arrows) has been released between processes. x 10,000. Fig. 4. Endothelial proliferate. X250.
cells cultured
for 4 days.
Ceils
have Aattened
and are beginning
to
Fig. 5. Endothelial cell cultured for 10 days and sectioned horizontally to the cell layer. Diaphragmed fenestrae have disappeared completely. Many ribosomes can be observed in the cytoplasm, and dense amorphous materials arc seen between the cell processes. ~5,700. Fig. 6. Colony of endothelial cells cultured the cells. Cells have become thick, reflective, from the dish. x250.
for 3 weeks (type l), showing large vacuoles in tubular in shape (arrow), and finally detached
Fig. 7. Cloned endothelial cells on collagen-coated dish at 4th subculture slender-shape and did not form large vacuoles as did in Fig. 6. x250.
(type 2). Cells were
.
05
618
FURUYA
rat tail collagen were mixed with 2 volumes of 5x DMEM/F12 followed by 1 volume of 0.05 N NaOH-0.2 M HEPES-0.26 M NaHCOs at 4°C. Dissociated endothelial cells were suspended in the cold collagen mixture at a density of 5-20 x 105/ml, and then laid on the top of base layer of previously gelled collagen by warming at 37°C in CO2 incubator. After 30 min-1 hr at 37°C 2 ml of the medium (DMEM/F12 + 10% FCS + antibiotics) was laid onto the collagen gel. In some cases, 1: 1 mixture of basement membrane matrigel and collagen gel was used for capillary formation. Photographs of the cells grown on the dishes or in the collagen gel were taken with a Nikon Diaphoto TMD inverted phasefluorescence microscope or a TMD equipped with Hoffman modulation contrast. Motility of cultured cells were observed by a timelapse video recorder (Panasonic, AG-6720) combined with TV camera with an image intensifier tube (Panasonic, WV-1900) and an image processor (Nippon Avionics, Image Z-II).
ET AL.
Binding of lectins
Endothelial cells grown on 15-mm coverslips or on collagen gel for 2-7 days were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer at 4°C for 1 hr-overnight. Coverslips of cultured cells and cryosections (10 pm thickness) of gels, were incubated with biotinized lectins diluted 1: 50 for 2 hr at 25°C or overnight at 4°C reincubated with 1: 200 diluted streptavidin HRP for 2 hr at 25°C and then processed for DAB-H202 reaction for 10 min at room temperature. Preparation for electron microscopy Cells cultured on dishes or in gel were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2 for 3 hr-overnight. After washing with 0.1 M cacodylate buffer containing 8% sucrose, cells were postfixed with 2% 0~0~ for 1 hr, washed with double distilled water (DDW) for 10 min x 3, blockstained with 1% uranyl acetate in DDW for 1 hr, dehydrated with a graded ethanol series and embedded in Epon 812 or Araldite. After polymerization, the plastic dish was
Figs Sll. Tubular structures formed on plastic dishes. Cells shown in Fig. 7 at 4th-8th subculture were incubated on the plastic dishes. After cells had grown to a multilayer, OAG (20 ng/ml), or vanadate (40 PM) was added to the culture. Fig. 8. Tubular network formed on the second layer of the cells. Lumen-like has formed. Cells treated with OAG for 15 days after confluence. X250.
space (arrow)
Fig. 9. Tubular structure sectioned horizontally to the cell layer. Dense materials, filamentous materials and debris were seen in the lumen-like space (*). Cells 15 days after confluence. x4,700. Fig. 10. Tubular structure sectioned perpendicularly to the cell layer. Dense materials like basal lamina were seen beneath the cells in the lumen-like space (*). Amorphous debris was also seen in the lumen. Pinocytotic vesicles can be observed. Various junctions (small arrows, arrow head) were seen between the adjacent cells. Cells treated with vanadate for 1 month after confluence. X 17,000. Fig. 11. Capillary-like structure formed between the coverslip and the plastic dish. Cell debris (arrow) was seen in the lumen. Cells treated with 1 ng/ml TGF-P for 3 weeks. X250. Fig. 12. Capillary-like structures formed in the three dimensional collagen subculture shown in Figure 7 were incubated for 4 days. Hoffman modulation
gel. Cells at 3rd contrast. X125.
Fig. 13. Capillary-like structure formed in the collagen gel. Cells of type 1 at second subcultures were cultured for 12 days. Large vacuoles formed in the cell, and then vacuoles were connected with those of adjacent cells forming larger lumina. Hoffman modulation contrast. X250. Fig. 14. Semi-thin section of capillary-like structure formed in the three-dimensional collagen gel. Uncloned cells at first subculture were incubated for 2 weeks. Toluidine blue staining. X500.
017
MORPHOLOGICAL
BEHAVIOR
OF ENDOTHELIAL
623
CELLS
022 Fig5 19, 20. 22. Lumina formed in a ccl1 of type I at second subculture in the collagen gel Cells which originally showed morphological character as shown in Figure 6 wcrc cultured in the collagen gel for 12 days. big. IY. Lumina formed in the cytoplasm of a cell. with much debris inside them. Mitochondria, rough ER. rihosomes and filaments were evident in the cytoplasm around the lumina. Pinocytotic vesicles arc abundant at the plasmalemma on the luminal hidl X 106/ml) into collagen gel, they migrated and branched in a longitudinal fashion. Slit-like luminal spaces appeared within l-2 weeks of culture (Fig. 14). Cell debris was often observed in the lumen (Figs 15, 16). The cells forming the tubular structure had many free ribosomes and large rough ER filled with dense materials. Many microvilli were located at the luminal side of the cells, and bundles of stress fibers were observed at the abluminal side (Fig. 16). Pinocytotic vesicles were present mostly at the abluminal plasmalemma, but also at the luminai plasmalemma (Fig. 17). A discontinuous basal lamina was observed at the abluminal side (Figs 17, 18). Junctions between cells consisted mostly of zonula adherence (Fig. 15). Gap junctions were occasionally observed (Fig. 18). The tubular structure in collagen gel was retained for 2 weeks, but generally disappeared at later times as cells died or migrated onto the collagen gel surface. Uncloned cells after first subculture routinely formed tubular structures. However, cloned cells of 3rd8th subculture occasionally formed tubular structures and these were not stable for more than 1 week. Most of the cells in the gel were dead, and remaining cells proliferated on the gel. Cells having intracellular lumina were occasionally seen, though the incidence was low. When type 1 cells of first or second subculture were used, more than half the cells in the gel were dead within 1 day. In most of the remaining cells, large vacuoles appeared within 2-3 days of culture, and the cells became thick and cylinder-shaped with intra-
MORPHOLOGICAL
BEHAVIOR
OF ENDOTHELIAL
cellular lumina (Figs 13, 19). Intracellular lumina in a cell fused to each other to form a single large lumen. Occasionally, lumina that had formed in a cell, fused with those from adjacent cells to form a large lumen surrounded by 2 or several cells (Fig. 20). Septa between the lumina in a cell were often seen, and many pinocytotic vesicles were present at the septal membrane and cytoplasmic membrane facing the intracellular lumina (Fig. 20). Plasmalemmal vesicles possessing a thin diaphragm and a central knob were rare. During the first week of culture, much debris was seen in the lumen, and the cytoplasmic envelope which circled the intracellular lumina contained mitochondria, ribosomes, rough ER and filaments. At this stage, basal lamina was rarely seen (Figs 19, 20). After l-2 weeks of culture, cell processes encircling the lumen separated, and the debris in the lumen passed through the open space to the outside (Fig. 21), leaving the lumen empty. The thinned envelope surrounding the empty intracellular lumen, had onlv pinocytotic vesicles and multivesicular bodies, and a continuous basal lamina were observed on the extracellular side (Fig. 22). The cells rarely survived more than two weeks in culture. Chunge of cell shape and formation of diaphragmed fenestrae
Attempts to induce fenestrae formation by modulator agents were net successful in the slender-shape clones which showed high angeogeneity as shown in Figure 7. But, profound morphological changes were observed. PMA caused the slender shape to thicken to spindle shape, and TGF-/3 induced slender shape cells to form a cobblestone appearance (Fig. 23). Dibutyryl cyclic AMP inhibited cell proliferation and caused the formation of numerous area of thin cytoplasm (Fig. 24). However, fenestrae observed in these cells were occasional and few. Significant number of fenestrae were observed in one clone which showed cobblestone shape rather than spindle shape at confluence, when cells were cultured on collagen gel with DMEM/F12 containing 5% horse serum and 1% FCS for more than 1 month. In the culture, cells had clusters of very thin areas surrounded by a thicker, ruffled cytoplasm (Fig. 25). Most of the cells free of other cells had the clusters of these thin areas.
h-5 i
CELLS
Observation with time-lapse video showed this thin area to be sticky to the substrate and to show very little movement. in contrast to ruffling at the cell edge and vigorous movement of thick area encircling the thin areas. Ultrathin sections cut horizontally to the cell monolayer showed that these thin areas were clusters of diaphragmed fenestrae in highly attenuated envelope (Fig. 26). Discussion During the isolation and cloning of capillary endothelial cells from bovine adrenal medulla, it was apparent that cell shapes and morphological behaviors varied among cultures and even in the same culture dish, although almost all endothelial cells were fenestrated at the beginning of primary culture. When examining the primary culture, cells exhibit two distinct forms of morphology. One is elongated cells which easily form intracellular lumina called type 1 cells, and the other does not form intracellular lumina, proliferates well and is called type 2 cells. Various cell shapes from a cobblestone-like shape to a slender shape. were observed in confluent type 2 colonies. Cells had preferential binding sites to GS1, RCA120, SBA and RPA lectins, which recognize D-galactosyl residues as reported in capillary endothelial cells of dog cerebral cortex (Gerhart et al., 1986) and newly formed capillaries of rat brain (Minamikawa et al., 1987). It is not clear that the character of type I cells and type 2 cells is inherently different or not. When cells dissociated from adrenal medulla were cultured, most of colonies showed type 1 character or type 2 character throughout, in individual case. In cloned type 2 cells subcultured more than several times, we occasionally observed that cells suddenly changed to a reflective, cylindrical shape and detached from the dish, despite intracellular lumina not being formed in a cell. Folkman et al. (1979) have reported that capillary endothelial cells of early passage became vacuolated and failed to proliferate, when cells were cultured on non-coated plastic dishes. It seems that expression of cell properties will be influenced by primary environments in vitro such as substrate. media, and other factors, and also change by cellular aging. Biochemical analysis will be
Fig. 23. Cells shown in Figure 7 were cultured for 6 days with 1 ngjml TGF-P after confluence. Cell changed to cobblestone shape from slender shape. x350. Fig. 24. Cells shown in Figure 7 were cultured for Y days with flat and very thin. x350. Figs 25, 26. Cells which showed cobble-stone like morphology on collagen gel with 5% horse serum and 1% FCS for 36 days.
1mM
Bt,cAMP.
Cells became
at confluence, were cultured
Fig. 25. Cells which formed the clusters of highly thinned areas (arrows) embedded in epoxy resin. Clusters of thin areas were seen in the part of cytoplasm, and corresponded to the clusters of diaphragmed fenestrae (*) in EM micrograph as shown in Figure 26 x525. Fig. 26. Clusters of diaphragmed fenestrae seen in a cell cut horizontally to the cell layer. The clusters of diaphragmed fenestrae (*) were so attenuated that only a small part of them is contained in this ultrathin section. Bundles of stress fiberswere always seen around the clusters. X9.600.
MOKPHOLOGICAL
BEHAVIOR
OF ENDOTHELIAL
necessary to elucidate the difference between type 1 or 2 cells. As for type 2 cells, we can observe heterogenous colonies of cell shapes in a culture dish of primary culture, such as a colony of cobblestone-like cells, a colony of slendershape and a colony consisted of cobblestonelike cells in the center and slender cells at the periphery. Slender-shaped clones seemed more angiogenic than cobblestone shape clones. Tsuboi et al. (1990) have reported that clones with the highest endogenous bFGF level exhibit an elongated morphology, while cells with low bFGF retained a cobblestone shape. Cell migration and invasion correlated directly with endogenous bFGF. Although we have not measured endogenous bFGF in our clones, our morphological observations seem to coincide well with their results. The formation of fenestrae in vitro has been described mostly in the clone isolated from bovine adrenal cortex by Furie er al. (1984). Their clone showed not only high angiogenic activity by the addition of PMA, vanadate and FGF, but also formed diaphragmed fenestrae significantly after the addition of PMA. retinoic acid and vanadate, (Lombardi et al., 1986; 1988; Montesano et
CELLS
027
al., 1988) or after inoculation on MDCK cell matrix (Milici et al., 1985). In contrast to these reports. our slender-shape clones which showed high angiogenic activity did not form diaphragmed fenestrae significantly with any additives, and a clone of cobblestone-like shape formed diaphragmed fenestrae without any treatment (process of fenestrae formation was described precisely; Furuva. 1990). It is likely that the angiogenic activity and the activity of fenestrae formation in the cells are not correlated directly, rather contradictory phenomenon in our clones. Comparison with isolated clones expressing distinct phenotypes will be required to explain what endogenous and exogenous factors are involved in cell differentiation.
Acknowledgement
We thank Dr. H. Pollard, National Institutes of Health, for his support, and Prof. Dr. G. Eguchi, National Institute for Basic Biology. for generously providing B16 melanoma. We are also grateful to Dr. T. Ozaki for the support to obtain bovine adrenal glands. and Dr. T. Nakayama and Dr. K. Furuya. for discussion.
References Allikmets. E. YU. and Danilov. S. M. 1986. Mitogen-induced disorganization of capillary-like structures formed h\ human large vcsscl endothclial cells in ok-o. Tissue & Cell. 18, 481-489. Bancrjcc, D. K.. Ornberg. R. L., Youdim, M. B. H., Heldman. E. and Pollard, H. B. 1985. Endothclial cells from bovine adrenal medulla develop capillary-like growth patterns in culture. Pro
TISSUE AND CELL, 1990 22 (5) 615-628 @ 1990 Longman Group UK Ltd.
S. FURUYA”,
C. EDWARD%
MORPHOLOGICAL BEHAVIOR BOVINE ADRENAL MEDULLA ENDOTHELIAL CELLS Keywords:
Bovine adrenal medulla, diaphragmed fcncstrae
and R. ORNBERGS
OF CULTURED CAPILLARY
cndothclial
cells.
cell
culture.
capillaq
fornwtwn.
ABSTRACT. Bovine adrenal medulla capillary cndothellal cells were isolated and cloned. and their morphological behaviors m uitro were examined. In the culture of primary or early passage, one type of colony formed intracellular lumina both on the dish and in the three dimensional collagen gel. Another type proliferated well and showed morphology ranging from slender-shape to cobblestone shape. and were easily cloned. Cloned cells which showed slcndcrshapes formed tubular network on plastic dish after addition of PMA. OAG or vanadate. and these cells also formed multicellular tubules in the three dimensional collagen gel. tlowcvcr. the formation of diaphragmed fenestrae by thcsc slender-shape clones war rare. One clone which showed cobblcstonc shape formed diaphragmcd fcncstrac, when cultured on collagen gel for more than one month. Isolated colonies or clones showed heterogeneity of cell \hapc. anglogenic behaviors and fcnestrac formatwn.
Introduction
1983; Montesano
and Orci, 1985: Olander and Danilov., 1986; Leibovich et al., 1987; Sato et al., 1987; Madri et al., 1988; Yen-Patton et al., 1988) and fenestrae formation (Milici et al., 1985; Lombardi et al., 1986,1988; Montesano and Orci. 1988). To study the formation of fenestrated capillaries in vitro, we isolated and cloned endothelial cells from bovine adrenal medulla. We observed the time course of disappearance of diaphragmed fenestrae in primary culture, and examined the induction of capillary formation and fenestrae formation in cloned cells. However. isolated clones showed heterogeneity of cell shape, angiogenic behaviors and fenestrae formation. Fenestrae formation in the clones which showed high angiogenic activity was rarely observed. In this report, we describe the differences of cell shape and morphological behavior of the clones, as they relate to angiogenesis and fenestrae formation (a preliminary note was reported previously).
Pt al., 1985; Allikmets
Endocrine organs have fenestrated capillaries with geometrically arranged diaphragmed pores in the highly attenuated endothelium (E&in, 1965). The correlation between these specialized structures and capillary permeability has been described (Simionescu, 1983). There have been many reports describing in vitro modulation of cell shape and angiogenesis (Folkman and Haudenschild, 1980; Maciag et al., 1982; Nicosia et al., 1982; Madri and Williams, 1983; Montesano et al., 1983, 1986; 1988; Schor et al., * National lnstitutc for Physiological Scicnccs. Myodaiji, Okazaki, 444, Japan. t Unwersity of South Florida Medical Center, College ol Medicine. Research and Graduate Affairs Box 40. 12901 North 30th Street, Tampa. FL 33612, USA. $ Baxter Healthcare Corporation, Baxter Technology Park WGZ-2s. Round Lake, Illinois 60073, USA. Address correspondence to: Sonoko Furuya, National Institute for Physiological Sciences. Myodaiji, Okazaki. 444. Japan. Received 8 January 1990. Revised 20 June 1990. 615
616
FURUYA
ET AL.
Materials and methods
al., 1985; Furuya et al., 1989). The cells in the
Materials used were collagen type 1 (Nitta Gelatin Co., Osaka); Percoll (Pharmacia), endothelial cell growth supplement [ECGS], basement membrane MatrigelTM and human fibronectin, (Collaborative Research); human transforming growth factor p (R & D Systems Inc.), DiI-acetylated LDL (Biomedical Technologies Inc.), sodium orthovanadate (Wako Pure Chemical Industries, LTD); Dibutyryl cyclic AMP (Yamasa Co.); collagenase type I, bovine serum albumin type V, phorbol myristate[PMA], l-oleoyl[cis]-9;C2:0) 2-acetyl-sn-glycerol(C18: 1, [OAG] and DMEM/F12 (Sigma); fetal calf serum (Flow Laboratories); glutaraldehyde (LADD); OsOl (Polysciences); ECA, RPA (E. Y Labo. Inc); LCA, GS-1, PHA-E4, PHA-L (Vector Labo. Inc.); RCA120, SBA, PNA (Honen Co.) and streptavidin-HRP (Amersham).
top band were inoculated on plastic dishes or dishes coated with 5 pg human fibronectin per cm* with DMEM/F12 containing 10% FCS, 100 U/ml penicillin and 100 pg/ml streptomycin. Several hours to 1 day later, chromaffin cells were removed by pipetting and cortical cells were removed by brief trypsinization (0.05% trypsin, l-5 min). Colonies of endoethelial cells were identified by the uptake of DiI-acetylated LDL (Voyta et al., 1984), and non-endothelial cells were removed as described by Folkman et al. (1979). In some experiments, ECGS (50 ,ug/ ml) + heparin (lU/ml) media, conditioned media of B16 melanoma, or conditioned media of endothelial cells were used until colonies of sufficient size were obtained. Cloned endothelial cells of the 3rd-8th subculture were used for experiments. In some experiments, the endothelial cell-rich primary cultures were dissociated with trypsin and immediately used for three-dimensional collagen culture without cloning.
Materials
Cell preparation
Cell culture in the three-dimensional collagen gel
The cells of bovine adrenal medulla were dissociated by a modification of the chromaffin cell preparation from adrenal glands, and were separated by Percoll centrifugation (Greenberg and Zinder, 1982; Banerjee et
Fig. 1. Fenestrated Figs 2-6. Primary
endothelial culture
Three dimensional collagen gel was prepared by the modification of Montesano et al. (1983). Seven volumes of collagen type I or
cell dissociated
of endothelial
Fig. 2. EndothLlial cells cultured thin processes. x250.
from bovine
cells inoculated
adrenal
medulla.
on collagen-coated
~7,500. plastic dishes.
for 1 day. Cell body is still round, and has begun to elongate
Fig. 3. Endothelial cell cultured for 1 day sectioned horizontally to the cell layer. Attenuated endothelium with diaphragmed fenestrae has been internalized into the round cell body and has begun to degrade. New processes have begun to elongate. Debris (arrows) has been released between processes. x 10,000. Fig. 4. Endothelial proliferate. X250.
cells cultured
for 4 days.
Ceils
have Aattened
and are beginning
to
Fig. 5. Endothelial cell cultured for 10 days and sectioned horizontally to the cell layer. Diaphragmed fenestrae have disappeared completely. Many ribosomes can be observed in the cytoplasm, and dense amorphous materials arc seen between the cell processes. ~5,700. Fig. 6. Colony of endothelial cells cultured the cells. Cells have become thick, reflective, from the dish. x250.
for 3 weeks (type l), showing large vacuoles in tubular in shape (arrow), and finally detached
Fig. 7. Cloned endothelial cells on collagen-coated dish at 4th subculture slender-shape and did not form large vacuoles as did in Fig. 6. x250.
(type 2). Cells were
.
05
618
FURUYA
rat tail collagen were mixed with 2 volumes of 5x DMEM/F12 followed by 1 volume of 0.05 N NaOH-0.2 M HEPES-0.26 M NaHCOs at 4°C. Dissociated endothelial cells were suspended in the cold collagen mixture at a density of 5-20 x 105/ml, and then laid on the top of base layer of previously gelled collagen by warming at 37°C in CO2 incubator. After 30 min-1 hr at 37°C 2 ml of the medium (DMEM/F12 + 10% FCS + antibiotics) was laid onto the collagen gel. In some cases, 1: 1 mixture of basement membrane matrigel and collagen gel was used for capillary formation. Photographs of the cells grown on the dishes or in the collagen gel were taken with a Nikon Diaphoto TMD inverted phasefluorescence microscope or a TMD equipped with Hoffman modulation contrast. Motility of cultured cells were observed by a timelapse video recorder (Panasonic, AG-6720) combined with TV camera with an image intensifier tube (Panasonic, WV-1900) and an image processor (Nippon Avionics, Image Z-II).
ET AL.
Binding of lectins
Endothelial cells grown on 15-mm coverslips or on collagen gel for 2-7 days were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer at 4°C for 1 hr-overnight. Coverslips of cultured cells and cryosections (10 pm thickness) of gels, were incubated with biotinized lectins diluted 1: 50 for 2 hr at 25°C or overnight at 4°C reincubated with 1: 200 diluted streptavidin HRP for 2 hr at 25°C and then processed for DAB-H202 reaction for 10 min at room temperature. Preparation for electron microscopy Cells cultured on dishes or in gel were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2 for 3 hr-overnight. After washing with 0.1 M cacodylate buffer containing 8% sucrose, cells were postfixed with 2% 0~0~ for 1 hr, washed with double distilled water (DDW) for 10 min x 3, blockstained with 1% uranyl acetate in DDW for 1 hr, dehydrated with a graded ethanol series and embedded in Epon 812 or Araldite. After polymerization, the plastic dish was
Figs Sll. Tubular structures formed on plastic dishes. Cells shown in Fig. 7 at 4th-8th subculture were incubated on the plastic dishes. After cells had grown to a multilayer, OAG (20 ng/ml), or vanadate (40 PM) was added to the culture. Fig. 8. Tubular network formed on the second layer of the cells. Lumen-like has formed. Cells treated with OAG for 15 days after confluence. X250.
space (arrow)
Fig. 9. Tubular structure sectioned horizontally to the cell layer. Dense materials, filamentous materials and debris were seen in the lumen-like space (*). Cells 15 days after confluence. x4,700. Fig. 10. Tubular structure sectioned perpendicularly to the cell layer. Dense materials like basal lamina were seen beneath the cells in the lumen-like space (*). Amorphous debris was also seen in the lumen. Pinocytotic vesicles can be observed. Various junctions (small arrows, arrow head) were seen between the adjacent cells. Cells treated with vanadate for 1 month after confluence. X 17,000. Fig. 11. Capillary-like structure formed between the coverslip and the plastic dish. Cell debris (arrow) was seen in the lumen. Cells treated with 1 ng/ml TGF-P for 3 weeks. X250. Fig. 12. Capillary-like structures formed in the three dimensional collagen subculture shown in Figure 7 were incubated for 4 days. Hoffman modulation
gel. Cells at 3rd contrast. X125.
Fig. 13. Capillary-like structure formed in the collagen gel. Cells of type 1 at second subcultures were cultured for 12 days. Large vacuoles formed in the cell, and then vacuoles were connected with those of adjacent cells forming larger lumina. Hoffman modulation contrast. X250. Fig. 14. Semi-thin section of capillary-like structure formed in the three-dimensional collagen gel. Uncloned cells at first subculture were incubated for 2 weeks. Toluidine blue staining. X500.
017
MORPHOLOGICAL
BEHAVIOR
OF ENDOTHELIAL
623
CELLS
022 Fig5 19, 20. 22. Lumina formed in a ccl1 of type I at second subculture in the collagen gel Cells which originally showed morphological character as shown in Figure 6 wcrc cultured in the collagen gel for 12 days. big. IY. Lumina formed in the cytoplasm of a cell. with much debris inside them. Mitochondria, rough ER. rihosomes and filaments were evident in the cytoplasm around the lumina. Pinocytotic vesicles arc abundant at the plasmalemma on the luminal hidl X 106/ml) into collagen gel, they migrated and branched in a longitudinal fashion. Slit-like luminal spaces appeared within l-2 weeks of culture (Fig. 14). Cell debris was often observed in the lumen (Figs 15, 16). The cells forming the tubular structure had many free ribosomes and large rough ER filled with dense materials. Many microvilli were located at the luminal side of the cells, and bundles of stress fibers were observed at the abluminal side (Fig. 16). Pinocytotic vesicles were present mostly at the abluminal plasmalemma, but also at the luminai plasmalemma (Fig. 17). A discontinuous basal lamina was observed at the abluminal side (Figs 17, 18). Junctions between cells consisted mostly of zonula adherence (Fig. 15). Gap junctions were occasionally observed (Fig. 18). The tubular structure in collagen gel was retained for 2 weeks, but generally disappeared at later times as cells died or migrated onto the collagen gel surface. Uncloned cells after first subculture routinely formed tubular structures. However, cloned cells of 3rd8th subculture occasionally formed tubular structures and these were not stable for more than 1 week. Most of the cells in the gel were dead, and remaining cells proliferated on the gel. Cells having intracellular lumina were occasionally seen, though the incidence was low. When type 1 cells of first or second subculture were used, more than half the cells in the gel were dead within 1 day. In most of the remaining cells, large vacuoles appeared within 2-3 days of culture, and the cells became thick and cylinder-shaped with intra-
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cellular lumina (Figs 13, 19). Intracellular lumina in a cell fused to each other to form a single large lumen. Occasionally, lumina that had formed in a cell, fused with those from adjacent cells to form a large lumen surrounded by 2 or several cells (Fig. 20). Septa between the lumina in a cell were often seen, and many pinocytotic vesicles were present at the septal membrane and cytoplasmic membrane facing the intracellular lumina (Fig. 20). Plasmalemmal vesicles possessing a thin diaphragm and a central knob were rare. During the first week of culture, much debris was seen in the lumen, and the cytoplasmic envelope which circled the intracellular lumina contained mitochondria, ribosomes, rough ER and filaments. At this stage, basal lamina was rarely seen (Figs 19, 20). After l-2 weeks of culture, cell processes encircling the lumen separated, and the debris in the lumen passed through the open space to the outside (Fig. 21), leaving the lumen empty. The thinned envelope surrounding the empty intracellular lumen, had onlv pinocytotic vesicles and multivesicular bodies, and a continuous basal lamina were observed on the extracellular side (Fig. 22). The cells rarely survived more than two weeks in culture. Chunge of cell shape and formation of diaphragmed fenestrae
Attempts to induce fenestrae formation by modulator agents were net successful in the slender-shape clones which showed high angeogeneity as shown in Figure 7. But, profound morphological changes were observed. PMA caused the slender shape to thicken to spindle shape, and TGF-/3 induced slender shape cells to form a cobblestone appearance (Fig. 23). Dibutyryl cyclic AMP inhibited cell proliferation and caused the formation of numerous area of thin cytoplasm (Fig. 24). However, fenestrae observed in these cells were occasional and few. Significant number of fenestrae were observed in one clone which showed cobblestone shape rather than spindle shape at confluence, when cells were cultured on collagen gel with DMEM/F12 containing 5% horse serum and 1% FCS for more than 1 month. In the culture, cells had clusters of very thin areas surrounded by a thicker, ruffled cytoplasm (Fig. 25). Most of the cells free of other cells had the clusters of these thin areas.
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Observation with time-lapse video showed this thin area to be sticky to the substrate and to show very little movement. in contrast to ruffling at the cell edge and vigorous movement of thick area encircling the thin areas. Ultrathin sections cut horizontally to the cell monolayer showed that these thin areas were clusters of diaphragmed fenestrae in highly attenuated envelope (Fig. 26). Discussion During the isolation and cloning of capillary endothelial cells from bovine adrenal medulla, it was apparent that cell shapes and morphological behaviors varied among cultures and even in the same culture dish, although almost all endothelial cells were fenestrated at the beginning of primary culture. When examining the primary culture, cells exhibit two distinct forms of morphology. One is elongated cells which easily form intracellular lumina called type 1 cells, and the other does not form intracellular lumina, proliferates well and is called type 2 cells. Various cell shapes from a cobblestone-like shape to a slender shape. were observed in confluent type 2 colonies. Cells had preferential binding sites to GS1, RCA120, SBA and RPA lectins, which recognize D-galactosyl residues as reported in capillary endothelial cells of dog cerebral cortex (Gerhart et al., 1986) and newly formed capillaries of rat brain (Minamikawa et al., 1987). It is not clear that the character of type I cells and type 2 cells is inherently different or not. When cells dissociated from adrenal medulla were cultured, most of colonies showed type 1 character or type 2 character throughout, in individual case. In cloned type 2 cells subcultured more than several times, we occasionally observed that cells suddenly changed to a reflective, cylindrical shape and detached from the dish, despite intracellular lumina not being formed in a cell. Folkman et al. (1979) have reported that capillary endothelial cells of early passage became vacuolated and failed to proliferate, when cells were cultured on non-coated plastic dishes. It seems that expression of cell properties will be influenced by primary environments in vitro such as substrate. media, and other factors, and also change by cellular aging. Biochemical analysis will be
Fig. 23. Cells shown in Figure 7 were cultured for 6 days with 1 ngjml TGF-P after confluence. Cell changed to cobblestone shape from slender shape. x350. Fig. 24. Cells shown in Figure 7 were cultured for Y days with flat and very thin. x350. Figs 25, 26. Cells which showed cobble-stone like morphology on collagen gel with 5% horse serum and 1% FCS for 36 days.
1mM
Bt,cAMP.
Cells became
at confluence, were cultured
Fig. 25. Cells which formed the clusters of highly thinned areas (arrows) embedded in epoxy resin. Clusters of thin areas were seen in the part of cytoplasm, and corresponded to the clusters of diaphragmed fenestrae (*) in EM micrograph as shown in Figure 26 x525. Fig. 26. Clusters of diaphragmed fenestrae seen in a cell cut horizontally to the cell layer. The clusters of diaphragmed fenestrae (*) were so attenuated that only a small part of them is contained in this ultrathin section. Bundles of stress fiberswere always seen around the clusters. X9.600.
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necessary to elucidate the difference between type 1 or 2 cells. As for type 2 cells, we can observe heterogenous colonies of cell shapes in a culture dish of primary culture, such as a colony of cobblestone-like cells, a colony of slendershape and a colony consisted of cobblestonelike cells in the center and slender cells at the periphery. Slender-shaped clones seemed more angiogenic than cobblestone shape clones. Tsuboi et al. (1990) have reported that clones with the highest endogenous bFGF level exhibit an elongated morphology, while cells with low bFGF retained a cobblestone shape. Cell migration and invasion correlated directly with endogenous bFGF. Although we have not measured endogenous bFGF in our clones, our morphological observations seem to coincide well with their results. The formation of fenestrae in vitro has been described mostly in the clone isolated from bovine adrenal cortex by Furie er al. (1984). Their clone showed not only high angiogenic activity by the addition of PMA, vanadate and FGF, but also formed diaphragmed fenestrae significantly after the addition of PMA. retinoic acid and vanadate, (Lombardi et al., 1986; 1988; Montesano et
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al., 1988) or after inoculation on MDCK cell matrix (Milici et al., 1985). In contrast to these reports. our slender-shape clones which showed high angiogenic activity did not form diaphragmed fenestrae significantly with any additives, and a clone of cobblestone-like shape formed diaphragmed fenestrae without any treatment (process of fenestrae formation was described precisely; Furuva. 1990). It is likely that the angiogenic activity and the activity of fenestrae formation in the cells are not correlated directly, rather contradictory phenomenon in our clones. Comparison with isolated clones expressing distinct phenotypes will be required to explain what endogenous and exogenous factors are involved in cell differentiation.
Acknowledgement
We thank Dr. H. Pollard, National Institutes of Health, for his support, and Prof. Dr. G. Eguchi, National Institute for Basic Biology. for generously providing B16 melanoma. We are also grateful to Dr. T. Ozaki for the support to obtain bovine adrenal glands. and Dr. T. Nakayama and Dr. K. Furuya. for discussion.
References Allikmets. E. YU. and Danilov. S. M. 1986. Mitogen-induced disorganization of capillary-like structures formed h\ human large vcsscl endothclial cells in ok-o. Tissue & Cell. 18, 481-489. Bancrjcc, D. K.. Ornberg. R. L., Youdim, M. B. H., Heldman. E. and Pollard, H. B. 1985. Endothclial cells from bovine adrenal medulla develop capillary-like growth patterns in culture. Pro
