177

J Physiology (1992) 86, 177-184 © Elsevier, Paris

C h a r a c t e r i z a t i o n o f c u l t u r e d rat aortic e n d o t h e l i a l cells P AndrE, M Michel, C Schott, JC Stoclet Laboratoire de Pharmacologie Cellulaire et Mol~culaire, CNRS URA 600, Universit~ Louis Pasteur de Strasbourg, BP 24, 67401 lllkirch, France (Received 25 March 1992; accepted 5 August 1992)

S u m m a r y - We describe a new method to obtain rat aortic endothelial cells without contamination by vascular smooth muscle cells. The endothelial cells were characterized up to the 20th passage by low density lipoprotein incorporation, the absence of c~-smooth muscle actin, the production of endothelium derived relaxing factor, and an elevation in intracellular free calcium concentration in response to bradykinin and ATP but not to AMP and angiotensin II. c~-smooth muscle actin / angiotensin II / A T P / bradykinin / endothelium derived relaxing factor / intracellular calcium / rat aorta / endothelial cells

Introduction Endothelial cells play a key role in maintaining normal arterial structural and functional integrity. Endothelial abnormalities have been associated with thrombosis and arteriosclerosis (Van Mourick, 1988). It is therefore interesting to possess and maintain identifiable endothelial cells in vitro to study the functional and metabolic response of these cells. Endothelial cell cultures are commonly obtained from elastic arteries of large mammals, such as bovine aorta (Booyse et al, 1975; Hecker et al, 1990; Schwartz, 1978), bovine pulmonary artery (Ryan et al, 1980), porcine aorta (Booyse et al, 1979) and the human umbilical vein (Jaffe et al, 1973; Thornton et al, 1983). Because of the size of these vessels, the techniques most frequently used to obtain endothelial cells are either physical removal of the intima by scraping, generally followed by a collagenase treatment, or direct enzymatic treatment of the vessels (Glassberg et al, 1982). It was possible to obtain capillary endothelial cells only by collagenase treatment (Folkman et al, 1979; Dropulic et al, 1987). In contrast, the isolation of endothelial cells from rat aorta, a smaller mammalian species, has proved to be more difficult. Because of the small size of the rat aorta, a sufficient amount of endothelial cells cannot be obtained by intimal

scraping. Furthermore, only one group has successfully used a collagenase treatment to obtain rat aortic endothelial cells (Smith, 1989), whereas others reported that these cells resist removal from the vascular wall by collagenase treatment (Merrilees and Scott, 1981; McGuire and Orkin, 1987). An explant technique to obtain rat aorta endothelial cells (REC) in culture has been developed by Merrilees and Scott (1981). In that case the rapid growth of endothelial cells ensured their predominance over vascular smooth muscle cells (VSMC), which first appeared at the margin of the explant after about 14 days. This technique has also been used by McGuire and Orkin (1987) to obtain REC. The explant technique, although productive, selects a migratory sub-population of endothelial cells and there is also co-development of different cells types: VSMC and endothelial cells grow together even if endothelial cells are predominant on the 17th day. We report here an efficient and reproducible technique for establishing in vitro cultures of endothelial cells from rat aorta, free of VSMC, characterized by their morphological aspect, the uptake of acetylated low density lipoproteins (DiI-Ac-LDL), the absence of c~-smooth muscle actin, the calcium response to several agonists and their ability to produce endothelium derived relaxing factor (EDRF).

178

Materials and methods

Isolation of REC and VSMC VSMC and REC were prepared according to a modification of the method described by Travo et al (1980) for VSMC cultures. Briefly, 9-10-week-old Wistar rats were killed by cervical dislocation and the aorta was dissected out under sterile conditions from the left coronary artery down to the diaphragm. Aortae (2-6) were then treated with collagenase 175 U/ml (Eurobio, France) in Hanks' balanced salt solution (Eurobio, France) for 30 min at 37°C. The adventitia was stripped off mechanically. The aorta was longitudinally opened but the endothelium was not scraped off. The aorta with its endothelium was transferred to an enzyme solution containing collagenase 87.5 U/ml, elastase 3 U/ml (Biosys, France) in Hanks' balanced salt solution and incubated for 75 min at 37°C with a slow shaking movement. After mechanical disruption by repetitive pipetting, single cell suspension containing VSMC and REC was obtained. This suspension was centrifuged for 10 rain at 100 g. The pellet was resuspended in Ham's F I 2 (Eurobio, France) and MEM (Eurobio, France) in the ratio of 1:1 with 2% ultroser G (IBF, France), 0.05 mM ascorbic acid (Sigma, France), 0.01 mM proline (Sigma, France), and 2 mM glutamine (Merck, Germany). To obtain REC, the cell suspension was pre-plated for 2 h. The supernatant was used to obtain VSMC in culture. It was then transferred into a new flask and the cultures were kept in a 37"C humidified incubator with an atmosphere of 95% air-5% CO2. The medium was changed every three days and confluent VSMC were obtained 15 days later. In the pre-plating flask, REC and some VSMC remained attached to the plastic dish. New specific medium for endothelial cells was then added containing: Dulbecco's modified Eagle's medium (Eurobio, F r a n c e ) H a m F12 in ratio 1:1, 20% foetal calf serum (Jacques Boy, France), 0.05 mM ascorbic acid, 0.01 mM proline, 2 m M glutamine, 100 U/ml specilline G (Specia, France), and 30 U/ml heparin (Biosys, France) to inhibit the growth of VSMC. Three or four days later, the REC were growing in clones while VSMC grew separately in single cells. To enrich the primary cultures for REC and to remove contaminating non-endothelial cells, the flask was placed under a microscope (objective x 10) and only pure clones of REC, based on their morphological aspect were mechanically separated, using a rubber policeman. REC, floating in the medium, were then transferred into a new flask and this operation was repeated once more three or four days later. Confluent endothelial cells were obtained seven days later. The medium was changed every three days and REC were replated by trypsinization every five days with a split-ratio of 1:4. REC were maintained and characterized until the 20th passage. Primary cultures were used for VSMC characterization.

Uptake of LDL REC cultured on glass coverslips in culture flasks were characterized using the selective uptake of acetylatedlow density lipoprotein. Confluent cells were incubated with 10 I.tg/ml lipoprotein labeled with the fluorescent probe 1,1' dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI-Ac-LDL), (Biomedical Technologies, USA) in culture medium for 4 h at 37°C. Cells were then washed twice with physiological salt solution (PSS, mM: NaC1 112, KCI 5, NaHCO325, KH2 PO4 1, MgSO4 1.2, CaC12 1.25, glucose 11.5) for 10 min, fixed in 4% paraformaldehyde in 0.12 M Na/Kphosphate buffer (pH 7.0), for 20rain and finally washed in phosphate-buffered saline (PBS, raM: NaCI 145, K phosphate 25, pH 7.4). For viewing, coverslips were mounted in glycerol-PBS (ratio 1:1). The samples were examined with a Zeiss Photoscope II using 25 x and 63 x Neofluor objectives. DiI-Ac-LDL was visualised using rhodamine excitation/emission filters. Images were recorded on llford HP 5 Plus film developed for a 400 ASA setting.

s-Smooth muscle actin immunofluorescence assays REC were cultured on glass coverslips in culture flasks. After washing with PBS-0.5% bovine serum albumin (BSA), confluent cells were fixed in methanol-acetic acid 5% and washed again in PBS-0.5% BSA. The cells were then incubated for 30 min in PBS-3% BSA at 37°C. After washing three times with PBS-0.5% BSA, the mouse monoclonal antibody anti c~-smooth muscle actin (Skalli et al, 1986) (a gift from G Gabbiani, Geneva) was added at a concentration of 1:50. Sixty min later the glass coverslip was washed three times at room temperature with PBS-0.5% BSA. Conjugated antimouse antibody FITC (Institut Pasteur Production, France), the second antibody, was added at a concentration of 1:50 for 20 min at room temperature. After washing, coverslips were mounted in glycerol-PBS (ratio 1:1) then examined with a Zeiss Photoscope II using 25 × and 63 × Neofluor objectives. Fluorosceine excitation/emission filters were used to visualise c~smooth muscle actin. Images were recorded on Ilford HP 5 Plus film developed for a 400 ASA setting.

Production of EDRF REC were cultured in 225-cm 2 plastic flasks (Nunc, Denmark). Confluent cells were detached mechanically with a rubber policeman and washed twice with modified Krebs solution (Krebs, mM: NaCI 118; NaHCO~ 25; glucose 10; KC1 4.7; CaC12 1.25; MgSO4 1.19; KH2 PO4 1.14). After 10 min centrifugation at 100 g, the cells were resuspended in 1 ml Krebs and counted with a Thomas naget. Two mm-long aortic rings from 9-11-week-old male Wistar rats were rubbed with a fine forceps to remove the endothelium and were mounted under an initial ten-

179 sion of 2 g. The bath containing l0 ml of Krebs was maintained at 37°C and bubbled with a mixture of 95% 02-5% CO2. After 60 rain equilibration, tension was readjusted to 2g. The absence of functional endothelium was assessed by the absence of any relaxing effect of acetylcholine 10-6M (Sigma, France) added when the maximal tension had been reached with l0 6M noradrenaline (Sigma, France). After a further washing period of 60 rain, the tissues were incubated for 15 rain with superoxide dismutase 100 U/ml (SOD) (Sigma, France), prior to readdition of noradrenaline 10-6 M. SOD was present during subsequent experiments in order to protect nitric oxide (NO) from degradation by superoxide anions possibly produced by cultured cells (Gryglewski et al, 1986; Rubanyi and Vanhoutte, 1986). Cumulative addition of cells to the bath showed that 3 x l0 s REC/ml caused 50% relaxation. This concentration was used for further experiments. After stable relaxation was reached, either an inhibitor of NO production from L-arginine, L-N~-nitroL-arginine methyl ester (k-NAME) 3 × 10-5 M (Sigma, France) or an inhibitor of guanylate cyclase, methylene blue 10 5 M (Sigma, France) was added to characterize the involvement of EDRF production and subsequent activation of guanylate cyc[ase, respectively. lntracellular calcium m e a s u r e m e n t The effects of bradykinin (Sigma, France), ATP (Sigma. France) and angiotensin II (AngII), (Sigma, France) on intracellular free calcium concentration Ca2+~ were measured according to the method used by Cornwell and Lincoln (1989). Briefly, confluent REC grown on coverslips, were washed three times with 3 ml of balanced salt solution (BSS, mM: Hepes 20, pH 7.5, NaCI 135, KCI 5, MgCI2 1, CaC12 1.25, glucose 10, BSA 0.025%). The glass coverslip was inserted into a quartz spectrophotometric cuvette. Three ml of Fura-2/acetoxymethyl ester (Fura-2/AM), (Molecular Probe, USA), 5 ~tM in calcium free BSS, were added to the cells and incubated 20 rain at 37°C. The cells were then washed three times with BSS and incubated for an additional 10 rain to ensure complete hydrolysis of Fura-2/AM. Ca2+i measurements were made in a SPEX 1681 spectrometer. Fluorescence was measured at two different excitation wavelengths, 340 and 380 nm. The emission was measured at 510 nm. Calibration was carried out in the presence of ionomycin (Calbiochem, France) and EGTA. Calculation of Ca2*i was made as described previously by Grynkiewicz et al (1985).

pearance that distinguished them from other cell types: they were in close contact with each other and always appeared to be circled by a carpet of b a s e m e n t m e m b r a n e . In addition, REC grew in clones and had a small cytoplasm, while VSMC appeared with distinct cell borders, a very large cytoplasm and with one or two nuclei per cell. At this stage, based on their morphological aspect, endothelial clones were scraped off and transferred into a new flask in which c o n f l u e n c e was reached approximately seven days later. At c o n f l u e n c e , REC (fig 1) presented a typical cobblestone appearance, f o r m i n g a m o n o l a y e r of p o l y g o n a l tightly packed cells. REC were seeded (4.5 x 105 cells/25 cm 2 ) and the subcultures reached c o n f l u e n c e after five days (1.8 × 106 cells/25 cm 2 ). All the characteristics described above and below were verified at the 5, 7, 9, 13 and 18th passages and were conserved until the 18th. LDL incorporation After i n c u b a t i o n with D i I - A c - L D L , c o n f l u e n t REC (fig 2A) were stained and the fluorescence was p r e d o m i n a n t l y associated with the perinuclear region as described by Voyta et al (1985) for bovine endothelial cells. In contrast, VSMC presented a low b a c k g r o u n d of fluorescence p r i n c i p a l l y localized on the cytoplasmic border and slightly associated with the c y t o p l a s m (fig 2B). lmmunostaining I m m u n o s t a i n i n g of slides c o n t a i n i n g c o n f l u e n t REC (fig 3A) which had been incubated with the m o n o c l o n a l anti o~-smooth muscle actin revealed that no cells were marked. In comparison, V S M C (fig 3B) treated with the same m o n o c l o n a l antibody showed an intense staining. I m m u n o f l u o r e s cence was associated with the cytoplasm and localizated on the o~-smooth muscle actin fibers. In no case was specific o~-actin fluorescence observed in REC cultures, showing no evidence of c o n t a m i n a t i o n by VSMC.

Results

Production

Cell morphology

Figure 4 illustrates a representative relaxing effect of 3 × 105 REC/ml on n o r a d r e n a l i n e - c o n t r a c t e d e n d o t h e l i u m denuded rat aortic rings, in the presence of SOD. 3 x 105 R E C / m l i n d u c e d about 50% relaxation of the aortic ring in 2 min (n = 5).

Initial cellular characterization was made by phase contrast microscopy. After two days in the pre-plating flask, REC showed a characteristic ap-

of

EDRF

180

Fig 1. Confluent REC in 7-day-old subculture. This relaxation was rapidly reversed by 3 x l0 5 M t - N A M E , a blocker of NO-synthetase, and by methylene blue 10-5 M, an inhibitor of cyclic GMP production by soluble guanylate cyclase. In the absence of SOD, no relaxing effect was observed (data not shown). In the presence of SOD, VSMC had no relaxing effect on rat aortic rings.

M e a s u r e m e n t of Ca2+i Representative traces of the effects of bradykinin (in the presence of captopril 10-7 M added 10 min before) and of ATP on Ca2+i in REC are illustrated in figure 5. The two agonists induced a rapid rise in Ca2+i, from 1.08 + 0.3 x 10-7M to 11.4 + 3 x 10 7 M for ATP (fig 5A) (n = 6) and from 1.26 + 0.2 x 10-7 M to 4.95 _+ 0.9 x 10 7 M for bradykinin (fig 5B) (n = 4). In both cases, the peak elevation in Ca2+i was followed by a decrease to a plateau phase which remained higher than the basal level. AMP (fig 5A) and AngII 10-8 M (data not shown) had no effect on these cells.

Discussion The aim of the experiments presented here was to obtain cultured REC from rat aorta.

REC were maintained as a homogeneous population of polygonal and mononucleated cells with a typical cobblestone appearance for 4 months up to the 20th passage. The kinetics of REC culture growth were similar to those described by Merrilees and Scott (1981) for REC obtained by the explant technique, except that the cell number which adhered at confluence was much higher using the explant technique. Several authors did not obtain viable REC following collagenase, trypsin, dispase II or elastase treatment (Merrilees and Scott, 1981; Cole et al, 1986). Several explanations may account for this difference from our results: a) we used an enzymatic mixture containing collagenase and elastase, instead of the use of one enzyme to obtain REC and VSMC simultaneously; b) VSMC were present in the experimental conditions used here as in the explant technique, at the beginning of the growth phase of REC, perhaps allowing the starting of the REC clone development in culture. In order to characterize them, the REC cultures were treated with the metabolic probe DiI-AcLDL which accumulates more rapidly in endothelial cells than in other cells (Voyta et al. 1985). Until the 20th passage, all the REC incubated with DiI-Ac-LDL were brightly stained. To observe the lack of contamination of REC by VSMC, c~-smooth muscle actin immunostaining of REC and VSMC was carried out. The use of a

181

Fig 2. Cellular labelling by DiI-Ac-LDLstaining. A. Confluent REC (x 25). B. Confluent VSMC (x 25).

monoclonal antibody against s-smooth muscle actin revealed the presence of c~-smooth muscle actin fibers in VSMC but not in REC. No cells present in REC cultures possessed c~-smooth muscle actin fibers showing no evidence of contamination of REC cultures by VSMC.

The goal of the pharmacological study reported here was to look for cytosolic calcium responses induced by reference agonists known to stimulate either REC or VSMC through specific receptors. In REC, both bradykinin and ATP induced a rapid rise in Ca2+i while AMP and AnglI had no effect.

182

Fig 3. Cellular labelling by immunostaining with a monoclonal anti or-smooth muscle actin antibody. A. Confluent RE(7 (×25) B. Confluent VSMC (x 63).

These results are in good agreement with previous findings showing that bradykinin also induced a "~+ rise in C a " i in bovine pulmonary artery endothelial cells (Myers et al, 1989) and that ATP produced the same effect in endothelial cells from different species and various vascular beds (Hal-

lam and Pearson 1986; Luckhoff and Busse, 1986; Carter et al, 1988). The presence of B2 bradykinin binding sites coupled with a G protein (Keravis et al, 1991) and of P2 purinoreceptors (Piroton et al, 1987) has been reported in endothelial cells.

183 NA

RI~? 3 1(15/inl

I0 tim

rig? 3 ]05/nil

ff

.

I. N A M E / . l [ I 5M

A

A

Illcl jl~ ~clIC blnc 141 5M

NaATI'

6.10-7"

j~

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~3

AMP

VSM(" 105/ml

2.10-7-

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2 illill

Fig 4. Representative contraction experiments showing the t-claxing effect of REC (A, B) and its reversion by L-NAME (A) or methylene blue (B) on endothelium-denuded rat aortic rings• In C, lack of effect of VSMC in the same conditions (n = 4). Rings were contracted with noradrenaline (NA).

i

0

I00

200

300 time, s.

B Bradykinin 4.8.10-7

It is well k n o w n that e n d o t h e l i a l cells release a diffusible r e l a x i n g substance called E D R F w h i c h stimulates g u a n y l a t e c y c l a s e and has been i d e n t i f i e d as N O ( P a l m e r e t al, 1988) or as a nitrogen o x i d e - r e l a t e d c o m p o u n d (Myers e t al, 1989). We show here that R E C , but not V S M C , were able to p ro d u ce relaxation o f n o r a d r e n a l i n e pre-contracted e n d o t h e l i u m d e n u d e d rat aortic rings in the p r e s e n c e o f SOD. This e f f e c t was a b o l ish ed by the N O synthetase inhibitor, L - N A M E and by m e t h y l e n e blue, an in h i b i to r o f the soluble guanylate cyclase, consistent with the p r o d u c t i o n o f a N O - l i k e r e l a x i n g factor by R E C .

t_)

J

2.4.10-7

0

100 •

.

200 .

[ilne,

s.

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Fig 5. Representative increases in Ca- i produced in REC by AMP 10~M and ATP 10-6M (n=6) (A), and bradykinin 10-8 M (17 = 4) (B).

Acknowledgment Financial support from RIOM-Laboratoires CERM and from Inserm-CRE 892018, is gratefully acknowledged.

References

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