Cytotechnology 3: 21-29, 1990. 9 1990 Kluwer Academic Publishers. Printed in the Netherlands.

Characteristics of arachidonic acid metabolism of human endothelial cells in culture

Vincenta Martinez-Sales, Maria Jos6 Gomez-Lechrn and Juan Gilabertl Research Center and Department of Obstetrics and 1Gynecology, Hospital 'La Fe', Avda. Campanar 21, 46009 Valencia, Spain Received 18 October 1988; accepted in revised form 17 April 1989

Key words: endothelial cells, prostacyclin, thromboxane

A2

Abstract

Human umbilical endothelial cells in culture retain differentiated morphological and functional characterization in primary culture and even in the early subcultures, after which they begin to degenerate. We have studied the morphological and biochemical characterization (ability to produce prostacyclin, prostaglandin E2 and thromboxane A2 in culture) of endothelial cells in the first seven subcultures. In addition the influence of serum and endothelial cell growth factor added to the culture medium have been evaluated. With 20% normal human serum, cell proliferation is faster than with the same concentration of human fetal or bovine fetal serum. After the 3rd passage, morphological and growth alterations become observable in the endothelial cells. However, prostacyclin, prostaglandin E2 and thromboxane A2 production showed no variations during the study.

Introduction Endothelial cells (EC) are deeply involved in many important physiological and pathological processes (Czervionke et al., 1978; Gimbrone, 1986). These cells can be isolated from the vessels of man and experimental animals and maintained in vitro as a homogeneous population for prolonged periods. Cultures allow direct study of metabolic and physiological properties under experimentally controlled conditions. Cultured human umbilical vein EC are the experimental model most widely used in vascular research. However, if an 'in vitro' cellular model of this

kind is to be useful, cells must retain the expression of differentiated structural and biochemical functions in culture. In fact, EC keep their identity in primary culture and even in the early subcultures, but it has been reported that they lose this identity, after multiple passages (Gimbrone, 1986; Jaffe, 1984). In spite of the fact that useful information can be obtained from primary cultures, extended subcultures wilt facilitate larger quantities of cells for biochemical studies. It is, therefore, worthwhile to examine the maintenance of specific EC functions after several subcultures. We have examined the capacity of human umbilical endothe-

22 lial cells to produce prostaglandins, principally prostacyclin, an important biochemical specific function related to endothelial hemostatic/thrombotic balance that is not yet well understood, in culture after multiple passages. An understanding of endothelial cells' capacity to produce prostaglandin throughout several subcultures will provide an in vitro experimental model that will permit a close examination of the biochemical mechanisms that regulate arachidonic acid metabolism in the endothelial cell. Prostaglandins are involved in the regulation of vascular tone (Levin et aI., 1984). Prostacyclin, which is a major product of arachidonic metabolism in vascular EC, is a potent vasodilator and platelet aggregation inhibitor (Moncada et al., 1977). Endothelial cells also release, though in small quantities, other arachidonic acid metabolites, such as PGE 2, TXA z and P G F ~ (Schr6r, 1985). TXA2 is a very important vasoconstrictor and platelet aggregation stimulant (Needleman, et al., 1976; Moncada and Vane, 1979). An equilibrium between PGI 2 and TXAz is believed to play an important role in the maintenance of vascular tone and in platelet homeostasis. The aim of our work was to study the capacity of well identified human umbilical endothelial cells to synthesize and secrete prostacyclin, prostaglandin E2 and thromboxane A 2 during the first seven subcultures. We also present data on the influence of the type and concentration of serum (human or bovine), and the endothelial cell growth supplemen t (ECGS) on cell growth.

Material and methods

Endothelial cells were obtained from human umbilical cord veins by an adaptation of the method of Jaffe et al. (1973a). Umbilical cords were obtained immediately after normal vaginal delivery, placed in a sterile container without transport medium and kept at 4~ until processing (1-4 hr). The umbilical vein in undamaged segments of cord (20-30 cm in length) was cannulated with a 2 cm long plastic cannula attached to a one-way stop cock, and was rinsed with 100 ml of PBS to

remove residual blood. The other end of the umbilical vein was then clamped. The vein was filled with 0.08% collagenase solution (sp. act. 0.37 U/mg) in 0.8% NaC1, 0.03% KC1, 0.2% glucose and 0.24% HEPES at pH 7.4 and incubated at 37~ for 8-10 rain. After incubation the collagenase solution containing the endothelial cells was flushed from the cord and the vein was washed with 30 ml of serumfree culture medium. The effluent was centrifuged at 250 g for 10 min at room temperature and the cells were resuspendend in 20 ml of fresh culture medium and seeded in plastic Roux flasks. The first medium renewal was 2 hours after plating. Thereafter culture medium was replaced at 48 hr intervals. The yield from this procedure was in the range of 0.7 - 3 x 106 cells/cord. Cell viability was assessed by the trypan blue dye exclusion test (0.04 % final concn). Human fibronectin was used as a biomatrix. Flasks were coated with 500 gl of undiluted pooled human serum, left standing for 1 hour and then washed twice with ice-cold Phosphate Buffer Saline (PBS), pH 7.4, and the cells were seeded immediately. The culture medium was Medium 199 with 15 mM HEPES supplemented with 20% adult human serum (AHS, without heat inactivation), L-glutamine 2 mM, sodium pyruvate 1 mM, 50 U/ml penicillin, 50 gg/ml streptomicin sulfate, buffered with sodium bicarbonate pH 7.4. For subculture, confluent monolayers were harvested with 0.25% Trypsin - 0.01% EDTA in PBS without Ca++ or Mg++ at pH 7.4. The subculture ratio was usually 1:2 or 1:3.

Ultrastructure preparations

Monolayers were fixed in situ with 2.5% glutaraldehyde in 0.1 M cacodilate buffer, pH 7.4, postfixed with 2% osmium tetroxide, 1.8% potassium ferrocyanide in water and embedded in Epon. Sections were contrasted with uranyl acetate and lead citrate for examination by transmission electron microscopy.

23

Endothelial cell growth

Assay for PGI2, PGE2 and

All endothelial cell growth assays were performed with passage 1 cells plated on fibronectin 24-well coated plates at different densities. Triplicate cultures were fed every two days with the appropriate medium and the cells were harvested by trypsin-EDTA treatment, as described before, and counted in an haemocytometer. Data are reported as number of cells attached to wells as a function of time. Human endothelial cell growth in culture media supplemented with 20% adult human serum (AHS), 20% fetal human serum (FHS) and 20% fetal bovine serum (FBS) was compared. Cell growth was also evaluated as a function of the concentration of AHS. In this case the culture medium was supplemented with 2.5, 5, 10, 15 or 20% AHS. In both types of experiments, cells were plated at a density of 25 x 103 cell/ well. Stimulation of cell growth by supplementation of the culture medium with varying concentrations (0-300 ~tg/ml) of endothelial cell growth supplement (ECGS, Sigma; Maciag et al., 1982) and 15% AHS was also evaluated. Endothelial cells were plated in this case at a density of 4 x 103 cell/well. Cultures containing 15% AHS in the absence of ECGS served as control.

Cells were plated in 24-well culture plates at a density of approx. 25 x 103 cells/well. When the cells were confluent they were washed gently four times with Krebs-Ringer buffer (118 mM NaC1, 4.7 mM KC1, 1.2 mM KPO4H2, 1.2 mM MgaSO4, 25 m M NaCO3H, 10 m M glucose, pH 7.4) saturated with 95% 02 : 5% CO2 at 37~ The cells were then incubated for 5 minutes at 37~ with 0.4 ml/well of Krebs-Ringer buffer or 20 p.M arachidonate-enriched Krebs-Ringer buffer. The incubation was stopped by removing the buffer and adding indomethacine. Aliquots of incubation mixture were stored at -20~ until assayed. All experiments were performed in triplicate in primary culture cells and in the first seven subcultures. 6-Keto-PGFI~ (the stable metabolite of PGI2) and TxB 2 (the stable metabolite of TxA2) were measured by RIA, using our own specific antibodies (Martinez-Sales et al., 1987, 1989). A commercial (Amersham International) radioimmunoanalysis technique was used to measured PGE2. Cellular protein was determined using the Lowry method. All measurements were made at least in duplicate and results are expressed in terms of protein units.

TxA 2

Results

Immuno cyto che mis try Monolayers were fixed in situ with Kamowsky fixative, and the Von Willebrand factor was localized by indirect immunoperoxidase-labeled staining. Specific antibodies obtained in rabbit (Carmona et al., 1989) and goat anti-rabbit IgG-peroxidase were used. The peroxidase substrate was 3-amino-9-ethylcarbazol (Nakane, 1968). Preparations were lightly counterstained with diluted 0.003% toluidine blue in water and mounted in Karion F.

The fresh umbilical vein effluents contain small clumps of 5-15 rounded cells, which attach to the biomatrix and spread to form clusters within the first few hours in culture. These colonies increase in size and form monolayers by day 7-10.

Morphology Examination by optical and electron microscopy revealed no contamination with smooth muscle cells. Cells retain their typical morphology in the primary culture (Fig. 1 A,C). After the third passage morphological and growth alterations became observable, and the

24

Fig. 1. Endothelial ceils from human umbilical vein in monolayer culture at different stages of growth. A) Primary culture after seven days; mature monolayer of tightly-packed polygonal ceils. B) Monolayer after 8 days of the 5th subculture. C) Electron micrographs of peripherical cytoplasm of endothelial cells after the 1st subculture, Weibcl-Palade body (arrow). D) Demonstration of factor VIII by immunoperoxidase staining. Early monolayer after the 1st subculture and E) after 4th subculture.

doubling time increased. After the 5th passage, proliferation was slower (Fig. 1B). The von Willebrand factor remained steady

throughout the study, but after the 4th or 5th passage the intensity of the dye decreased greatly (Fig. 1. D,E).

25

A

•o

too,

....

IO ~ AHS

~ l S

~AH$ ]0 ~ AHS

- o

The results shown in Fig. 3 are reported as number of cells in the wells as a function of time. Optimal rates of endothelial cell growth in primary culture were reached by supplementing the culture media with adult homologous sera. Both fetal human serum pooled from the umbilical cord vein and fetal calf sera produced less growth.

.--~222

TxB2, PGE2 and 6-Keto-PGFI~production doys

of

culture.

B DO0 _.--------I

?O0

- -

% J~'S

?

sm

~0o io0 0

The TxB2, PGEz and 6-Keto-PGFla production by endothelial cell primary culture and by the seven subcultures studied, either in basal conditions or after stimulation of cells with 20 gM arachidonic acid, are shown in Table 1. Our results on TxB2, PGE2 and 6-Keto-PGFaa varied greatly depending on the different umbilical cords studied. None of the subcultures incubated with or without arachidonic acid showed statistically significant differences in the production of TxB2, PGE2 or 6-Keto PGFI~ when compared with the primary culture.

% AHS

Fig. 2. H u m a n endothelial cell, passage 1 (subcultttre ratio

1:2), growth as a function o f the concentration of adult h u m a n serum (AHS). The medium was supplemented with various concentrations o f AHS. Data are reported as number of cells/well x 10 3 (A) as a function o f time and (B) as a function of AHS concentration in the culture medium. Data are expressed as mean + SD of triplicate cultures.

20% AHS //

I/I

20"1. F H S

....

20q* F B S

/11II

x

//,t

Influence of concentration and origin of different sera on endothelial cell growth 30o

a) Human endothelial cell growth was evaluated as a function of the concentration of AHS. The cultures were fed every two days with varying concentrations (2.5, 5, 10, 15 or 20%) of AHS. The results, shown in Fig. 2, indicate that a concentration of AHS ranging from 15-20% produces the highest rates of endothelial cell growth. b) The influence of sera from different sources on cell growth was examined. The cultures were fed every two days with 20% AHS, FHS and FBS.

----

/.J i

i

2

4 r

of

cul~re.

Fig. 3. H u m a n endothelial cell, passage 1 (subculture ratio

1:2), growth in culture media supplemented with 20% of adult human serum (AHS), fetal h u m a n serum (FHS) and fetal bovine serum (FBS). Data are reported as number of cells/well x 103. Data are expressed as mean + SD of triplicate cultures.

26 Table 1. Prostacyclin, PGEz and TxA z production by human endothelial cells Subculture

6-Keto - PGFlc~ pg/~tg protein

PGEz pg/lxg protein

Control

20 I.tM AA

Control

20 gM AA

Control

20 IxM AA

0

51 + 9 (6)

493 + 240 (5)

NDV (3)

1.5 _+ 0.7 (3)

0.6 + 0.48 (4)

1.9 + 0.24 (4)

1

50 + 15 (11)

446 + 260 (11)

1.1 + 0.5 (3)

2.6 + 1.1 (7)

NDV (5)

1.9 + 0.92 (8)

36 + 14 (10)

478 + 118 (10)

NDV (6)

2.1 + 0.5 (6)

0.6 + 0.l (4)

2.9 + 0.9 (8)

3

30 + 19 (4)

487 + 250 (5)

NDV (3)

1.4 + 0.5 (3)

0.6 + 1 (3)

2.7 + 1.7 (4)

4

21 + 7 (6)

307 + 66 (5)

NDV (3)

1.2 _+ 0.6 (3)

0.4 + 0.2 (5)

3.2 + 1.7 (6)

5

34 + 5 (3)

260 + 50 (3)

NDV (3)

2.0 _+1.9 (3)

NDV (3)

2.0 + 1.3 O)

55 + 30 (3)

485 + 45 (3)

NDV (3)

2.5 + 0.1 (3)

NDV (3)

2.1 + 0.2 (3)

25 +_ 3 (3)

414 _+ 51 (3)

NDV (3)

1.4 _+ 0.2 (3)

NDV (3)

2.1 + 0.3 (3)

7

TxBz pg/lxg protein

6-Keto-PGFI~, PGF_,z and TxB 2 were measured with and without control stimulation with arachidonic acid. The results are expresed as mean + SD and the number in parenthesis represents the number of separate cell preparations of differont cords. NDV - non detectable value.

Influence of the concentration of endothelial cell growth supplement (ECGS) on endothelial growth Endothelial cells were plated at a density of 4 x 103 cell/well in 24-well cell culture plates. The cultures were fed every two days with varying concentrations (0-300 gg/ml) of ECGS and 15% AHS. The cultures were harvested after ten days. Cultures containing 15% AHS in the absence of the ECGS served as control. The results shown in Fig. 4A are reported as viable endothelial cell number per well as a function of time (days in culture). Cells retained their proliferation capacity longer when the ECGS was present. Fig. 4B shows the ECGS dose-response curve of the human umbilical endothelial cells after 10 days in culture. When the ECGS concentration was lower

than 10 gg/ml, no response was obtained. An ECGS concentrations of 300 gg/ml produced the maximal rates of endothelial cell growth.

Influence of the concentration of endothelial cell growth supplement on PGI2 production Endothelial cells were plated at a density of 25 x 103 cell/well in 24-well cell culture plate. The culture medium was supplemented with 15% AHS and 100 gg/ml ECGS. This concentration of ECGS was used to study how ECGS affects PGI2 synthesis because it is the growth factor concentration that produces half-maximal effect on growth of endothelial cells in culture. In addition, the initial inoculum and the cell concentration are

27 Discussion 70.

,7, o x

uo~,,~ a~:~I w ~ , ~ n:~ I 100~lOadR:~]

,~2 2s

do'f= of

culture.

70

~

~o ~ ~o j,~g

ECGF/ml

Fig. 4. Stimulation o f h u m a n endothelial cell (EC) growth by supplementation o f the culture medium with endothelial cell, growth supplement (ECGS). EC, passage 1, were plated at a density o f 4 x 10 ~ cells/well. A) Data are reported as a viable EC number per well as a function of time in culture; and B) the cultures were harvested after ten days in culture and the results are reported as viable EC number as a function of ECGS

(~tg/ml).

so large that they minimize the effect of ECGS on cell growth. In these conditions PGI2 production by the EC after stimulation of cells with 20 gM arachidonic acid is 486+63 pg/gg protein (n=3) in primary culture and 537+149 pg/gg protein (n=4) in the first subculture. When human umbilical cells were cultured without ECGS, the 6-ketoPGFla value was 493+240 pg/lxg protein (n=5) in primary culture and 446+260 pg/~tg protein ( n = l l ) in the first subculture. Comparison of these results with and without ECGS added to the culture medium shows that PGI2 production by the endothelial umbilical cells is not affected by the presence or absence of this factor.

One important factor affecting endothelial cell culture growth is the type and the concentration of the serum added to the culture medium, because serum supplementation of cells in vitro provides a complex series of mitogenic influences in the form of growth factors, hormones and attachment factors, which affect growth and maintenance of the cell culture (Hayashi and Sato, 1976). Several authors use fetal sera to culture endothelial cells, but our results show that better growth of human endothelial cells is obtained with human adult serum than with human fetal or bovine fetal serum. The presence of ECGS in the culture medium makes it possible for the cells to retain their proliferation capacity for a longer time (Maciag et al., 1984). According to previous reports and our results, the ECGS shows significant mitogenic activity for human umbilical vein endothelial cells at doses of 75-300 gg/ml in presence of human serum (Maciag et al., 1984). In contrast with previous reports (Maciag et al., 1984) we found the maximal rates of endothelial cell growth with ECGS concentrations of 300 ~tg/ml, whereas they obtained it with 100150 ~tg/ml. This discrepancy could be due to more prolific growth in the presence of AHS than in the presence of FBS, which probably requires a higher concentration of ECGS to stimulate growth. It has been reported (Maciag, et al., 1984) that ECGF supplementation is not necessary to achieve cell growth at cell densities greater than 1.25 x 104 cells/cm2; this is the case for subcultures at a ratio of 1:2 or 1:3, such as we use for routine studies (which represents about 8-10 x 103 cells/ cm~). These results suggest that it is possible to grow umbilical vein endothelial cells at high cell density in the absence of ECGS while supplementation with ECGS may be essential for growing at low cell density. The cells were identified as endothelial by: 1) their cobblestone morphology, cell growth pattern (forming a contact inhibited monolayer) (Jaffe, 1984) and the presence of Wei-

28 bel Palade bodies (Weibel and Palade, 1964); 2) over 90% positive immunoperoxidase staining for von Willebrand factor (Jaffe et al., 1973b; Seitz et al., 1987). 3) In addition it is characteristic of endothelial cells that prostacyclin production is greater than PGFa and TXB2 production, and changes in the balance between prostaglandin production were indicative of changes in the thromboresistant properties of the endothelial cells in culture (Lumdberg et al., 1986). Using these three criteria for identifying endothelial cells, we found, as have other authors (Jaffe, 1984), that human umbilical endothelial cells cultured in the absence of any special growth factors present morphological and growth alterations after the third or fourth subculture. However, human umbilical endothelial cells retain their function with respect to PGI z, PGE 2 and TxA2 in, the seven subcultures studied. These results do not coincide with those of Chesterman et al. (1983), who reported that the relative amount of PGF~ produced by porcine aortic endothelial cells increases with time, from 20% of the prostacyclin production shortly after isolation to 100% in subculture cells. The discrepancies between our results and those of Chesterman et al., (1983) are not unique; a comparison of studies on the endothelium in different animal species or anatomic sites shows many such differences. Chesterman et al. (1983), reported that generalizations about endothelial functions are not always justified, because if the endothelium subserves different functions in different vascular beds in vivo, it is not unreasonable to expect differences to appear in vitro. It is important to take into account the fact that when human umbilical endothelium cells are studied in culture, great variations from one cord to another are detected. In conclusion, the results of the present study indicate that although human umbilical endothelial cells retain their function with respect to prostagtandin synthesis throughout many subcultures, they undergo morphological alterations and growth retardation after the third passage in our experimental conditions.

Acknowledgements This work was supported in part by the research grant 88/1706 and 89/0686 from the Fondo de Investigaciones Sanitarias de la Seguridad Social. We greatly appreciate the technical assistance of M. Luisa Aragon6s, Guadalupe Manzano and Bel6n Rubio.

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29 14. Martinez-Salcs V, Llopis F and Aznar J (1989) Preparaci6n de un antisuero de alta especificidad para la determinaeidn de 6-keto-PGFla por radioinmunoensayo. Rev. Iberoamer. Tromb. Hemos. (in press). 15. Moncada S, Higgs EA and Vane JR (1977) Human arterial and venous tissues generate prostacyclin (Prostaglandin X), a potent inhibitor of platelet aggregation. Lancet i: 1822. 16. Moncada S and Vane JR (1979) Pharmacological and endogenous roles of prostaglandin endoperoxides thromboxane A 2 and prostacyclin. Pharmacol. Rev. 30: 293-331. 17. Nakane PK (1968) Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study in pituitary gland of the rat. J. Histochem. Cytochem. 16: 557-560.

18. Needleman P, Minkes M and Raz A (1976) Thromboxanes: selective biosynthesis and distinct biological properties. Science 193: 163-165. 19. Seitz ILl, Neven E, Henrich M, Schrader J and Wechsler W (1987) In: Cerv6s-Navarro J and Ferszt R (eds) Stroke and Microcirculation, pp. 111-115. Raven Press. 20. Schr6r K (1985) Prostaglandins, other eicosanoids and endothelial cells. Basic Res. Cardiol. 80: 502-514. 21. Weibel ER and Palade C (1964) New cytoplasmic components in arteria endothelia. J. Cell. Biol. 23: 101-112.

Address for offprints: V. Martinez-Sales, Centro de Investigaci6n, Hospital 'La Fe', Avd~ Campanar 21, 46009 Valencia, Spain

Characteristics of arachidonic acid metabolism of human endothelial cells in culture.

Human umbilical endothelial cells in culture retain differentiated morphological and functional characterization in primary culture and even in the ea...
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