EXPERIMENTAL
CELL
RESEARCH
192,
87-92
(1991)
Selective Conversion and Esterification of Monohydroxyeicosatetraenoic Acids by Human Vascular Smooth Muscle Cells: Relevance to Smooth Muscle Cell Proliferation’ H. J. M. BRINKMAN, Central
Laboratory
M. F.
of the Netherlands
VAN
BUUL-WORTELBOER,
Red Cross Blood
Transfusion
AND Seruice,
J. A. VAN MOURIK’ Amsterdam,
The Netherlands
heparin-like substances and prostaglandins, suppress the growth of smooth muscle cells under physiological conditions [Z-7]. Vascular smooth muscle cells start to proliferate and migrate upon exposure to a variety of stimuli, including platelet-derived growth factor [8] and interleukin 1 [9]. From several studies it appears that HETEs are also able to modulate the proliferation and migration of vascular smooth muscle cells. Both 12HETE and 15-HETE, major lipoxygenase metabolites of blood platelets [lo] and eosinophilic leukocytes [ll], respectively, are reported to be chemotactic for rat aortic smooth muscle cells [12]. The major lipoxygenase metabolite of neutrophilic leukocytes, 5-HETE [ 11,131, had no influence on rat aortic smooth muscle cell migration [12]. 12-HETE has also been shown to inhibit rabbit aorta smooth muscle cell proliferation [6]. Furthermore, HETEs have been shown to modulate the proliferation and migration of other cell types, including endothelial cells [14-171. These compounds also affect other cellular processes, such as the release of inflammatory mediators from mast cells [lS] and the endotoxininduced thromboplastin synthesis by monocytes [191. A mechanism by which HETEs modulate cellular processes might be esterification of these HETEs into cellular lipids. Incorporation of HETEs into cellular lipids has been reported for a variety of cell types, including bovine aortic endothelial cells, bovine aortic smooth muscle cells, and human umbilical vein endothelial cells [20-271. Here we describe a study of both the metabolism of exogenous HETEs by human umbilical artery smooth muscle cells and the influence of these HETEs on the serum-induced proliferation of growth-arrested human umbilical artery smooth muscle cells.
12-, and 15-hydroxyeicosatetraenoic acid 5-y (HETE), lipoxygenase metabolites of arachidonic acid that may modulate cell proliferation, were examined for their ability to affect the [3H]thymidine incorporation of human umbilical artery smooth muscle cells. We found that these hydroxy fatty acids inhibited the serum-induced [3H]thymidine incorporation of growtharrested vascular smooth muscle cells in a similar dosedependent manner. The inhibitory effect was dependent on the serum concentration used to stimulate cell growth. The higher the serum concentration, the lower the inhibitory effect of the HETE. In parallel experiments, the incorporation of HETEs into lipids of the smooth muscle cells was examined. After 20 h of incubation, we found that in the presence of 0.4% serum 70% of 3H-labeled 5-HETE was esterified into human vascular smooth muscle cell lipids. Twelve and eight percent, respectively, of 12- and 15-HETE were incorporated into smooth muscle cell lipids. Furthermore, we found that during the 20-h incubation of human umbilical artery smooth muscle cells with 12- and 15-HETE, these compounds were converted into metabolites with a chromatographic behavior on HPLC similar to that of diHETEs. 5-HETE was not converted into these polar metabolites. Increasing the serum concentration resulted in a decreased metabolism of all HETEs tested. Thus, the distinct differences between the metabolism of different HETEs by vascular smooth muscle cells does not reflect the proliferation inhibitory effect of these HETEs. L 1991 Academic Press, Inc.
INTRODUCTION In the normal noninjured blood vessel, smooth muscle cells have a low mitotic rate [l]. It seems likely that compounds secreted by the vascular lining, including
MATERIALS
AND METHODS
Cell culture. Human umbilical artery smoot,h muscle cells were isolated and cultured as previously described [7]. For experiments, cells from passages 3 to 10 were used. Experiments were performed with quiescent cells arrested in the GO/G1 phase of the cell cycle. Quiescent smooth muscle cells were obtained by maintaining the cells for 3 days in culture medium supplemented with 0.220.4% serum [7]. Measurement of cell proliferation. [3H]Thymidine-incorporation measurements were performed in 0.32~cm* wells as described [7].
’ This study was supported by the Netherlands Heart Foundation (Grant 28.004). * To whom reprint requests should be addressed c/o Publication Secretariat, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O. Box 9406,1006 AK Amsterdam, The Netherlands. 87
0014.4827191
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
88
BRINKMAN,
VAN
BUULWORTELBOER,
AND
VAN r
Thymidine (2 Ci/mmol, 0.2 &i/well; Amersham, Bucks, UK), mitogen (serum), and HETEs were added simultaneously to quiescent smooth muscle cells and incubated for 28 h. Before use, HETEs (Calhiochem, San Diego, CA, U.S.A.) were dried under a constant stream of nitrogen and redissolved and diluted in culture medium supplemented with human serum. Before the smooth muscle cells were harvested for [“Hlthymidine-incorporation measurements, the cultures were always microscopically examined. No morphological differences were observed between HETE-incubated cultures and controls. Metabolism of eicosanoids by smooth muscle cell cultures. “H-labeled 5-HETE (37 kBq; 6.36 TBq/mmol), 12.HETE (37 kBq; 5.92 TBq/mmol), 15.HETE (37 kBq; 8.62 TBq/mmol), and PGE, (37 kBq; 5.92 TBq/mmol) (Amersham, UK) were dried under a constant stream of nitrogen, dissolved in 5 ml of culture medium supplemented with human serum, and added to 25cm’cultures of quiescent smooth muscle cells. After incubation at 37°C for t,he indicated time, the medium was collected in tubes containing 7.5 ml of methanol. Cells were washed with 2.5 ml of PBS containing 0.5% bovine serum albumin (essentially fatty acid-free, Sigma Chemical Co., St. Louis, MO, U.S.A.). The wash fluid was added to the conditioned medium. After adding 2.5 ml of ice-cold EDTA/NaCl solution (0.9% NaCl, 77 mM EDTA, pH 7.0), the cells were scraped from the flask with a cell scraper (Costar Europe, Ltd., Badhoevedorp, The Netherlands) and transferred into tubes containine 5 ml of methanol. The flasks were washed with another 2.5 ml of EDTA/NaCl solution. The wash fluid was added to the harvested cells. Samples were stored at -20°C until further processing. Determination of cellular lipids by thin-layer chromatography. Samples were acidified with acetic acid to pH 4 and extracted once with 2 vol of chloroform. The lower phase was dried under a constant stream of nitrogen. The residue was dissolved in chloroform/methanol (2/l, v/v) and spotted on an TLC plate (silica gel G60, Merck, Darmstadt, FRG). Separation was achieved with chloroform/H,O/methanol (741 4/25, v/v/v). Standards were run simultaneously on parallel lanes. The separated compounds were visualized by I, staining. Radioactive spots were either detected by autoradiography or detected and quantitated using a two-dimensional radioscanner (system 200 imagine scanner, Packard, Downers Grove, IL, USA). ‘H-labeled HETEs and PGE, (Amersham) andunlabeled lipids (Sigma) and HETEs (Calhiothem) were used as standards. Because of the low pH, 5-HETE might he converted into the &lactone during the extraction procedure. To examine this possibility, 3H-laheled 5-HETE was dissolved in culture medium followed by lipid extraction and TLC as described. We found that only 1% of 5-HETE was converted into the lactone form. In this study, the formation of the &lactone of 5-HETE was therefore neglected. Determination of eicosanoids by HPLC’. After extraction with chloroform and evaporation, the residue was dissolved in the mobile phase for HPLC: methanol/H,O/acetic acid, 75/25/0.1 (v/v/v), for hydroxy-acid separation and H,O/acetonitrile/phosphatic acid, 701 30/0.1 (v/v/v), for prostaglandin separation. After filtration (dual 20 +M frits, J. T. Baker Co., Philipsburg, NJ, USA), reversed-phase HPLC was performed on an PBondapak Cl8 column (Waters, Milford, MA, USA). Prostaglandins were separated isocratically at a flow rate of 1.6 ml/min; hydroxy acids were separated isocratically at a flow rate of 1.0 ml/min. Fractions were collected and counted for radioactivity. Unlabeled prostaglandins (Sigma), 5, 12., and 15. HETE (Calhiochem), 5(S),12(S)-diHETE (Paesel, Frankfurt, FRG), and 12.HHT (a gift from G. Kivits and Dr. D. H. Nugteren, Unilever Research Center, Vlaardingen, The Netherlands) were used as standards. The procedure used for analyzing hydroxy acids is not effective for detecting phospholipids. We found that only 0.7% of [1-i*C]phosphatidyl serine was recovered after HPLC. Miscellaneous. Hydrolysis of lipid extracts were performed with 0.2 N NaOH in 80% methanol according to Bonser et al. [22]. Lactate dehydrogenase (LDH) was measured according to Bergmeyer and Brent [28]. Analysis of significance was done by Student’s t test.
MOURIK 1
1
I
I
5-HETE
0 :
lo
-
12.HETE
b I E P
a:
0
-
10
15.HETE
F %
I
5
-
0
-
I 1
R
I
I
I
I
10
20
30
40
J
FRACTION
FIG. 1. Conversion of HETEs by smooth muscle cells. Cultures of quiescent human umbilical artery smooth muscle cells were incubated with “H-labeled HETEs in the presence of 0.4% serum during 20 h (0). Control incubation without smooth muscle cells (0). Media were extracted and subjected to HPLC. Fractions of 0.8 ml were collected and counted for radioactivity.
RESULTS
Metabolism of Eicosanoids When 3H-labeled 5-HETE was incubated with cultures of quiescent human umbilical artery smooth muscle cells in the presence of 0.4% human serum, the fatty acid was efficiently cleared from the culture medium. Of the total radioactivity recovered in extracts of cells and media, 24% was found in the cell extract after 1 h, 47% after 4 h, and 70% after 20 h of incubation. HPLC analysis of extracts of the culture medium after 20 h of incubation revealed that hardly any 5-HETE was left (Fig. 1; Table 1). Increasing the serum concentration resulted in a decreased uptake of 5-HETE from the culture medium (Table 2). Thin-layer chromatography of the cell extracts revealed that virtually all radiolabel was associated with the neutrallipid and phospholipid fraction.
HETE
METABOLISM
AND
SMOOTH
MUSCLE
TABLE Conversion
and
76 of total radioactivity recovered (n = 2, mean -t range) Medium 5HETE 12.HETE 15.HETE PGE,
30 88 92 99
* k A 2
1
of Eicosanoids
Eicosanoid added
70 f 2 12 k 4 822 1 ?O
by Smooth
Unknown
6k6 59f3 62 f 1 100 f 0
94 -+ 6 41 *3 38 * 1 ND
Note. Cultures of quiescent human umbilical artery smooth muscle cells were medium with 0.4% serum. Cell extracts were analvzed bv” thin-laver chromatography ” under Materials and Methods. ND, not detectable.
No free 5HETE was detected in the cell extracts (Fig. 2, inset, lane 1). After 20 h of incubation, 12% of the total radioactivity recovered in the phospholipid fraction was associated with phosphatidylinositol and phosphatidyl-
TABLE Influence
of Serum Concentration and HETE-Inhibited llptake (1)
5-HETE 0.5 1 2.5 5 12.HETE 0.5 1 2.n 5 15.HETE 0.5 1 2.5 5
2 on HETE Proliferation
Metabolism
Conversion
Proliferation
(2)
(3)
(7; serum) 67.2 42.8
ND ND
12.7
ND
3.3 2.0
3.6 1.9
1.2
0.4
3.6 2.2
6.8 4.3
1.0
1.4
12 * 35 rt_ 86 f 97f
7* 5’ 9*’ 9
(‘96 serum) 9t 4’ 18 k 2’ 86 f 14 102f 1
(% serum) 15 * 2* 57 rt_ 6* 92k 8 107 f 11
Note. Cultures of quiescent human umbilical artery smooth muscle cells were incubated with “H-labeled HETEs for 20 h in the presence of different serum concentrations. The serum batch used was the same as that in the experiment shown in Fig. 3. Cell extracts were counted for radioactivity. Media extracts were analyzed by HPLC as described under Materials and Methods. In parallel incubations, the influence of 25 FM HETEs on smooth muscle cell proliferation was determined by [3H]thymidine-incorporation measurements as described under Materials and Methods. Results are expressed as (1) percentage of total radioactivity recovered in cell and supernatant detected in the cellular fraction; (2) percentage of total radioactivity recovered in cell and supernatant detected as polar metabolites in the supernatant; and (3) percentage of [3H]thymidine incorporated during the control incubation without HETE with the same serum concentration (mean + SD; controls: n = 6; + HETE: n = 3). Statistical difference between HETE-treated group and control group: *P < 0.0005, **P < 0.01. ND, not detectable.
89
PROLIFERATION
Muscle
% of total compounds detected in medium extracts (n = 2, mean * range)
Cells 2 4 2 0
Uptake
CELL
Cells % of total compounds detected in cell extracts (n = 3, mean *SD)
Free eicosanoid ND ND ND ND
Neutral liquid 43 i 17 25 * 21 26k 16 ND
incubated for 20 h with “H-labeled and media extracts were analyzed
Phospholipid 57 f 17 75 * 21 74 k 16 ND eicosanoids in culture by HPLC as described
serine, 18% with phosphatidylethanolamine, and 70% with phosphatidylcholine. Evidence for esterification of 5HETE into cellular lipids was further obtained by alkaline hydrolysis of lipid extracts of cells incubated for 20 h with 3H-labeled 5-HETE. Thin-layer chromatography after alkaline hydrolysis revealed that all of the phospholipid-associated radioactivity and most of the neutrallipid-associated radioactivity was released. Most of the radioactivity present in the hydrolysate eluted on TLC in the 5-HETE region (Fig. 2, inset). HPLC confirmed that indeed 5HETE was released (Fig. 2). The degree of incorporation of 12- and 15-HETE into smooth muscle cell lipids was much lower than that observed for 5-HETE. After 20 h of incubation in the presence of 0.4% serum, less than 12% ofthe total radioactivity recovered was found in the cell extract (Table 1). Thin-layer chromatography of the cell extracts revealed that, like 5-HETE, all radiolabel was associated with the neutrallipid and phospholipid fraction (Table 1). The distribution of 12- and 15-HETE among the different phospholipids was similar to that of 5-HETE, as judged by autoradiography (not shown). In addition to differences in the degree of incorporation into cellular lipids, there was another difference between the metabolism of 5-HETE and 12- and 15-HETE by smooth muscle cells. As shown in Fig. 1, 12- and 15-HETE were converted into metabolites with retention times on HPLC similar to that of 12(S),5(S)-diHETE. 5-HETE was not converted into metabolites with such a polar behavior on HPLC. Both the esterification and the conversion of 12- and 15-HETE were dependent on the serum concentration. The higher the serum concentration, the lower the metabolism of the HETEs added (Table 2). In contrast to the HETEs tested, we found that exogenous PGE, was not metabolized by smooth muscle cells to any extent. After 20 h of incubation of smooth muscle cell cultures with 3H-labeled PGE, in the presence of 0.4% serum, only 1% of the total radioactivity recovered
90
BRINKMAN,
VAN
BUUL-WORTELBOER,
AND
VAN
MOURIK
0 TAG+DAG
50
l
-
MAG
0 5-HETE F; ‘0 ,-40
-
; P 230 > I> ;
c
-
t
l
PE
0 PC 20
2 0 ;:
-
0 PIiPS 0 ORIGIN
10
-
I
I
I
I
I
1
10
20
30
40
FRACTION
FIG. 2. Esterification of 5.HETE into incubated with 3H-labeled 5-HETE in the Extracts were analyzed both by TLC (inset) with NaOH; (inset lane 3) standards. TAG, phosphatidylcholine; PI, phosphatidylinositol;
smooth muscle cell lipids. Cultures of quiescent human umbilical artery smooth muscle cells were presence of 0.4% serum during 20 h. Cells were extracted, followed by hydrolysis with NaOH. and HPLC. (0 and inset lane 1) Extract not treated with NaOH; (* and inset lane 2) extract treated triacylglycerol; DAG, diacylglycerol; MAG, monoacylglycerol; PE, phosphatidylethanolamine; PC, PS, phosphatidylserine.
was found in the cell extracts (Table 1). The radioactivity recovered from the culture medium chromatographed as PGE, (not shown). Influence
of HETEs
on Cell Proliferation
As shown in Fig. 3,5-, 12-, and 15-HETE inhibited the serum-induced [3H]thymidine incorporation of growtharrested vascular smooth muscle cells with a similar dose response. The inhibitory effect of the HETEs appeared to be serum-batch-dependent. 5-HETE was examined at a concentration of 25 pM in the presence of 5% human serum. Using five different serum batches, inhibition ranging from 3 to 55% (28 & 20%; mean t SD, n = 5) was observed. In addition, the inhibitory effect of the HETEs was dependent on the serum concentration. The higher the serum concentration, the lower the inhibitory effect of the HETE (Table 2). Significant inhibition of serum-induced smooth muscle cell proliferation was observed only at serum concentrations suboptimal for smooth muscle cell growth (serum concentrations at which half-maximal or less [3H]thymidine incorporation of growth-arrested cells was observed; Fig. 3, inset). DISCUSSION
Our results clearly demonstrate that there is a distinct difference between the metabolism of different ei-
cosanoids by human umbilical artery smooth muscle cells. PGE, was not converted by and incorporated into lipids of human umbilical artery smooth muscle cells to any extent (Table 1). On the other hand, at low serum concentrations 5-HETE was efficiently cleared from the culture medium (Fig. 1) and esterified into cellular lipids (Table 1, Fig. 2). 12- and 15-HETE were much less incorporated into smooth muscle cell lipids (Table 1). However, in contrast to 5-HETE, both 12- and 15HETE were converted into metabolites with retention times on HPLC in the diHETE region (Fig. 1). Both the esterification of 5-, 12-, and 15-HETE and the conversion of 12- and 15-HETE into polar metabolites decreased with increasing serum concentrations (Table 2). It has been reported that cultures of bovine thoracic smooth muscle cells take up more 5-HETE than 12HETE [25]. These data are in agreement with our observation showing that human umbilical artery smooth muscle cells preferably incorporate 5-HETE into their lipids (Table 1). Furthermore, we observed that phosphatidylcholine was the major phospholipid in which HETEs were esterified (Fig. 2). Similar results were reported for human umbilical vein endothelial cells and bovine coronary artery endothelial cells [26]. It has been reported that in human umbilical vein endothelial cells esterification of 5-HETE is inhibited by polyunsaturated fatty acids like arachidonic acid [27]. These data may explain our observation that the
HETE
n^
30
METABOLISM
SMOOTH
-
b I E Q 25 20’ ra
AND
SERVM
($1
-I
2o
-
E a : v z
15
-
0
0.025
I
I
I
0.25 HETE
2.5 t//M)
25
FIG. 3. Influence of HETEs on smooth muscle cell proliferation. Cultures of quiescent human umbilical artery smooth muscle cells were incubated with [3H]thymidine and 5-HETE (O), 12.HETE (m), and 15-HETE (0) in the presence of 1% serum. After 28 h, [3H]thymidine incorporation was determined. Results are expressed as means i SD of six determinations. Differences vs control: *P < 0.0005. Cell viability was judged by LDH measurements. Twenty five micromolar 5-, 12., and 15.HETE did not enhance the release of LDH during a 28-h incubation in the presence of 1% serum, as compared to the control incubations without HETE. (Inset) Effect of serum concentration on smooth muscle cell proliferation.
uptake of HETEs by human umbilical artery smooth muscle cells decreased with increasing serum concentrations (Table 2). Another explanation might be binding of HETEs to serum proteins. This might also explain our observation that the conversion of 12- and 15HETE into polar metabolites (Fig. 1) was decreased when the serum concentrations were increased (Table 2). The chromatographic behavior of the metabolites of 12- and 15HETE, synthesized by human umbilical artery smooth muscle cells (Fig. l), suggests that these metabolites are diHETEs. However, previously we have shown that when these cells were incubated with [l14C]arachidonate, no lipoxygenase-derived monoHETEs were formed [29]. Therefore, it seems most likely that the observed conversion of 12- and 15HETE (Fig. 1) is not due to lipoxygenase activity. One of the polar metabolites of 12-HETE, synthesized by human umbilical artery smooth muscle cells, eluted between 5(S),12(S)-diHETE and 12-HHT (Fig. 1). The chro-
MUSCLE
CELL
91
PROLIFERATION
matographic behavior of this 12-HETE metabolite is similar to that of one of the 12-HETE metabolites synthesized by human umbilical vein smooth muscle cells [30, 311. This metabolite was identified as an 8-hydroxyhexadecatrienoic acid and is probably formed by P-oxidation [30, 311. Conversion of HETEs into polar metabolites has also been reported for neutrophils [20, 321 and macrophages [23]. In neutrophils, the enzyme responsible for the conversion of 12-HETE into 12,20diHETE has been identified as a cytochrome P450 monooxygenase [33]. Further experiments need to be carried out to identify the enzyme(s) in human vascular smooth muscle cells responsible for the conversion of 12- and 15-HETE and to unravel the structure of the polar metabolites formed. The specific metabolism of exogenous HETEs by human vascular smooth muscle cells may implicate a relevant role of both the conversion and esterification of HETEs in smooth muscle cell function. For instance, it has been reported that esterification of 5-HETE into lipids of human umbilical vein endothelial cells inhibits prostaglandin synthesis in these cells [26]. HETEs have also been shown to modulate a variety of cellular processes [31], including the proliferation of rabbit aortic smooth muscle cells [6]. Here we show that 5-, 12-, and 15-HETE inhibited the serum-induced [3H]thymidine incorporation in a similar manner, when added at micromolar concentrations to quiescent human umbilical artery smooth muscle cells (Fig. 2). Similar concentrations of these HETEs are reported to affect the proliferation of a variety of other cell types [6, 15-171. Inhibition of smooth muscle cell proliferation was related to the serum concentration used (Table 2). Inhibition was observed only at serum concentrations suboptimal for smooth muscle cell growth. An excess of growth-stimulat,ing compounds, counteracting the inhibitory action of the HETE at higher serum concentrations, may explain these observations. On the other hand, a relationship between metabolism of 5-, 12-, and 15-HETE and HETE-inhibited proliferation was observed (Table 2); both processes decreased with increasing serum concentrations. With similar effects on smooth muscle cell proliferation, the uptake of 5-HETE was much higher than those of 12- and 15-HETE. Thus, if the growth inhibition is the result of accumulation of esterified HETE, then 12- and 15-HETE are more potent inhibitors than 5HETE. REFERENCES 1. 2.
3.
Spraragen, S. C., Bond, V. F’., and Dahl, 11,329. van Buul-Wortelboer, M. F., Brinkman, K. I’., de Groot, Ph. G., van Aken, W. C., (1986) Exp. Cell Res. 162, 151. Castellot, J. J., Favreau, L. V., Karnovsky, R. D. (1982) J. Bid. Chem. 257, 11,256.
L. K. (1962)
Circ. Res.
H. J. M., Dingemans, and van Mourik, J. A. M. J., and Rosenberg,
92
BRINKMAN,
VAN
BUUL-WORTELBOER.
AND
4.
de Groot, Ph. G., Brinkman, H. J. M., Gonsalves, M. D., and van Mourik, J. A. (1985) Biochim. Biophys. Acta 846, 342.
19.
5
CELL
RESEARCH
192,
87-92
(1991)
Selective Conversion and Esterification of Monohydroxyeicosatetraenoic Acids by Human Vascular Smooth Muscle Cells: Relevance to Smooth Muscle Cell Proliferation’ H. J. M. BRINKMAN, Central
Laboratory
M. F.
of the Netherlands
VAN
BUUL-WORTELBOER,
Red Cross Blood
Transfusion
AND Seruice,
J. A. VAN MOURIK’ Amsterdam,
The Netherlands
heparin-like substances and prostaglandins, suppress the growth of smooth muscle cells under physiological conditions [Z-7]. Vascular smooth muscle cells start to proliferate and migrate upon exposure to a variety of stimuli, including platelet-derived growth factor [8] and interleukin 1 [9]. From several studies it appears that HETEs are also able to modulate the proliferation and migration of vascular smooth muscle cells. Both 12HETE and 15-HETE, major lipoxygenase metabolites of blood platelets [lo] and eosinophilic leukocytes [ll], respectively, are reported to be chemotactic for rat aortic smooth muscle cells [12]. The major lipoxygenase metabolite of neutrophilic leukocytes, 5-HETE [ 11,131, had no influence on rat aortic smooth muscle cell migration [12]. 12-HETE has also been shown to inhibit rabbit aorta smooth muscle cell proliferation [6]. Furthermore, HETEs have been shown to modulate the proliferation and migration of other cell types, including endothelial cells [14-171. These compounds also affect other cellular processes, such as the release of inflammatory mediators from mast cells [lS] and the endotoxininduced thromboplastin synthesis by monocytes [191. A mechanism by which HETEs modulate cellular processes might be esterification of these HETEs into cellular lipids. Incorporation of HETEs into cellular lipids has been reported for a variety of cell types, including bovine aortic endothelial cells, bovine aortic smooth muscle cells, and human umbilical vein endothelial cells [20-271. Here we describe a study of both the metabolism of exogenous HETEs by human umbilical artery smooth muscle cells and the influence of these HETEs on the serum-induced proliferation of growth-arrested human umbilical artery smooth muscle cells.
12-, and 15-hydroxyeicosatetraenoic acid 5-y (HETE), lipoxygenase metabolites of arachidonic acid that may modulate cell proliferation, were examined for their ability to affect the [3H]thymidine incorporation of human umbilical artery smooth muscle cells. We found that these hydroxy fatty acids inhibited the serum-induced [3H]thymidine incorporation of growtharrested vascular smooth muscle cells in a similar dosedependent manner. The inhibitory effect was dependent on the serum concentration used to stimulate cell growth. The higher the serum concentration, the lower the inhibitory effect of the HETE. In parallel experiments, the incorporation of HETEs into lipids of the smooth muscle cells was examined. After 20 h of incubation, we found that in the presence of 0.4% serum 70% of 3H-labeled 5-HETE was esterified into human vascular smooth muscle cell lipids. Twelve and eight percent, respectively, of 12- and 15-HETE were incorporated into smooth muscle cell lipids. Furthermore, we found that during the 20-h incubation of human umbilical artery smooth muscle cells with 12- and 15-HETE, these compounds were converted into metabolites with a chromatographic behavior on HPLC similar to that of diHETEs. 5-HETE was not converted into these polar metabolites. Increasing the serum concentration resulted in a decreased metabolism of all HETEs tested. Thus, the distinct differences between the metabolism of different HETEs by vascular smooth muscle cells does not reflect the proliferation inhibitory effect of these HETEs. L 1991 Academic Press, Inc.
INTRODUCTION In the normal noninjured blood vessel, smooth muscle cells have a low mitotic rate [l]. It seems likely that compounds secreted by the vascular lining, including
MATERIALS
AND METHODS
Cell culture. Human umbilical artery smoot,h muscle cells were isolated and cultured as previously described [7]. For experiments, cells from passages 3 to 10 were used. Experiments were performed with quiescent cells arrested in the GO/G1 phase of the cell cycle. Quiescent smooth muscle cells were obtained by maintaining the cells for 3 days in culture medium supplemented with 0.220.4% serum [7]. Measurement of cell proliferation. [3H]Thymidine-incorporation measurements were performed in 0.32~cm* wells as described [7].
’ This study was supported by the Netherlands Heart Foundation (Grant 28.004). * To whom reprint requests should be addressed c/o Publication Secretariat, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P.O. Box 9406,1006 AK Amsterdam, The Netherlands. 87
0014.4827191
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
88
BRINKMAN,
VAN
BUULWORTELBOER,
AND
VAN r
Thymidine (2 Ci/mmol, 0.2 &i/well; Amersham, Bucks, UK), mitogen (serum), and HETEs were added simultaneously to quiescent smooth muscle cells and incubated for 28 h. Before use, HETEs (Calhiochem, San Diego, CA, U.S.A.) were dried under a constant stream of nitrogen and redissolved and diluted in culture medium supplemented with human serum. Before the smooth muscle cells were harvested for [“Hlthymidine-incorporation measurements, the cultures were always microscopically examined. No morphological differences were observed between HETE-incubated cultures and controls. Metabolism of eicosanoids by smooth muscle cell cultures. “H-labeled 5-HETE (37 kBq; 6.36 TBq/mmol), 12.HETE (37 kBq; 5.92 TBq/mmol), 15.HETE (37 kBq; 8.62 TBq/mmol), and PGE, (37 kBq; 5.92 TBq/mmol) (Amersham, UK) were dried under a constant stream of nitrogen, dissolved in 5 ml of culture medium supplemented with human serum, and added to 25cm’cultures of quiescent smooth muscle cells. After incubation at 37°C for t,he indicated time, the medium was collected in tubes containing 7.5 ml of methanol. Cells were washed with 2.5 ml of PBS containing 0.5% bovine serum albumin (essentially fatty acid-free, Sigma Chemical Co., St. Louis, MO, U.S.A.). The wash fluid was added to the conditioned medium. After adding 2.5 ml of ice-cold EDTA/NaCl solution (0.9% NaCl, 77 mM EDTA, pH 7.0), the cells were scraped from the flask with a cell scraper (Costar Europe, Ltd., Badhoevedorp, The Netherlands) and transferred into tubes containine 5 ml of methanol. The flasks were washed with another 2.5 ml of EDTA/NaCl solution. The wash fluid was added to the harvested cells. Samples were stored at -20°C until further processing. Determination of cellular lipids by thin-layer chromatography. Samples were acidified with acetic acid to pH 4 and extracted once with 2 vol of chloroform. The lower phase was dried under a constant stream of nitrogen. The residue was dissolved in chloroform/methanol (2/l, v/v) and spotted on an TLC plate (silica gel G60, Merck, Darmstadt, FRG). Separation was achieved with chloroform/H,O/methanol (741 4/25, v/v/v). Standards were run simultaneously on parallel lanes. The separated compounds were visualized by I, staining. Radioactive spots were either detected by autoradiography or detected and quantitated using a two-dimensional radioscanner (system 200 imagine scanner, Packard, Downers Grove, IL, USA). ‘H-labeled HETEs and PGE, (Amersham) andunlabeled lipids (Sigma) and HETEs (Calhiothem) were used as standards. Because of the low pH, 5-HETE might he converted into the &lactone during the extraction procedure. To examine this possibility, 3H-laheled 5-HETE was dissolved in culture medium followed by lipid extraction and TLC as described. We found that only 1% of 5-HETE was converted into the lactone form. In this study, the formation of the &lactone of 5-HETE was therefore neglected. Determination of eicosanoids by HPLC’. After extraction with chloroform and evaporation, the residue was dissolved in the mobile phase for HPLC: methanol/H,O/acetic acid, 75/25/0.1 (v/v/v), for hydroxy-acid separation and H,O/acetonitrile/phosphatic acid, 701 30/0.1 (v/v/v), for prostaglandin separation. After filtration (dual 20 +M frits, J. T. Baker Co., Philipsburg, NJ, USA), reversed-phase HPLC was performed on an PBondapak Cl8 column (Waters, Milford, MA, USA). Prostaglandins were separated isocratically at a flow rate of 1.6 ml/min; hydroxy acids were separated isocratically at a flow rate of 1.0 ml/min. Fractions were collected and counted for radioactivity. Unlabeled prostaglandins (Sigma), 5, 12., and 15. HETE (Calhiochem), 5(S),12(S)-diHETE (Paesel, Frankfurt, FRG), and 12.HHT (a gift from G. Kivits and Dr. D. H. Nugteren, Unilever Research Center, Vlaardingen, The Netherlands) were used as standards. The procedure used for analyzing hydroxy acids is not effective for detecting phospholipids. We found that only 0.7% of [1-i*C]phosphatidyl serine was recovered after HPLC. Miscellaneous. Hydrolysis of lipid extracts were performed with 0.2 N NaOH in 80% methanol according to Bonser et al. [22]. Lactate dehydrogenase (LDH) was measured according to Bergmeyer and Brent [28]. Analysis of significance was done by Student’s t test.
MOURIK 1
1
I
I
5-HETE
0 :
lo
-
12.HETE
b I E P
a:
0
-
10
15.HETE
F %
I
5
-
0
-
I 1
R
I
I
I
I
10
20
30
40
J
FRACTION
FIG. 1. Conversion of HETEs by smooth muscle cells. Cultures of quiescent human umbilical artery smooth muscle cells were incubated with “H-labeled HETEs in the presence of 0.4% serum during 20 h (0). Control incubation without smooth muscle cells (0). Media were extracted and subjected to HPLC. Fractions of 0.8 ml were collected and counted for radioactivity.
RESULTS
Metabolism of Eicosanoids When 3H-labeled 5-HETE was incubated with cultures of quiescent human umbilical artery smooth muscle cells in the presence of 0.4% human serum, the fatty acid was efficiently cleared from the culture medium. Of the total radioactivity recovered in extracts of cells and media, 24% was found in the cell extract after 1 h, 47% after 4 h, and 70% after 20 h of incubation. HPLC analysis of extracts of the culture medium after 20 h of incubation revealed that hardly any 5-HETE was left (Fig. 1; Table 1). Increasing the serum concentration resulted in a decreased uptake of 5-HETE from the culture medium (Table 2). Thin-layer chromatography of the cell extracts revealed that virtually all radiolabel was associated with the neutrallipid and phospholipid fraction.
HETE
METABOLISM
AND
SMOOTH
MUSCLE
TABLE Conversion
and
76 of total radioactivity recovered (n = 2, mean -t range) Medium 5HETE 12.HETE 15.HETE PGE,
30 88 92 99
* k A 2
1
of Eicosanoids
Eicosanoid added
70 f 2 12 k 4 822 1 ?O
by Smooth
Unknown
6k6 59f3 62 f 1 100 f 0
94 -+ 6 41 *3 38 * 1 ND
Note. Cultures of quiescent human umbilical artery smooth muscle cells were medium with 0.4% serum. Cell extracts were analvzed bv” thin-laver chromatography ” under Materials and Methods. ND, not detectable.
No free 5HETE was detected in the cell extracts (Fig. 2, inset, lane 1). After 20 h of incubation, 12% of the total radioactivity recovered in the phospholipid fraction was associated with phosphatidylinositol and phosphatidyl-
TABLE Influence
of Serum Concentration and HETE-Inhibited llptake (1)
5-HETE 0.5 1 2.5 5 12.HETE 0.5 1 2.n 5 15.HETE 0.5 1 2.5 5
2 on HETE Proliferation
Metabolism
Conversion
Proliferation
(2)
(3)
(7; serum) 67.2 42.8
ND ND
12.7
ND
3.3 2.0
3.6 1.9
1.2
0.4
3.6 2.2
6.8 4.3
1.0
1.4
12 * 35 rt_ 86 f 97f
7* 5’ 9*’ 9
(‘96 serum) 9t 4’ 18 k 2’ 86 f 14 102f 1
(% serum) 15 * 2* 57 rt_ 6* 92k 8 107 f 11
Note. Cultures of quiescent human umbilical artery smooth muscle cells were incubated with “H-labeled HETEs for 20 h in the presence of different serum concentrations. The serum batch used was the same as that in the experiment shown in Fig. 3. Cell extracts were counted for radioactivity. Media extracts were analyzed by HPLC as described under Materials and Methods. In parallel incubations, the influence of 25 FM HETEs on smooth muscle cell proliferation was determined by [3H]thymidine-incorporation measurements as described under Materials and Methods. Results are expressed as (1) percentage of total radioactivity recovered in cell and supernatant detected in the cellular fraction; (2) percentage of total radioactivity recovered in cell and supernatant detected as polar metabolites in the supernatant; and (3) percentage of [3H]thymidine incorporated during the control incubation without HETE with the same serum concentration (mean + SD; controls: n = 6; + HETE: n = 3). Statistical difference between HETE-treated group and control group: *P < 0.0005, **P < 0.01. ND, not detectable.
89
PROLIFERATION
Muscle
% of total compounds detected in medium extracts (n = 2, mean * range)
Cells 2 4 2 0
Uptake
CELL
Cells % of total compounds detected in cell extracts (n = 3, mean *SD)
Free eicosanoid ND ND ND ND
Neutral liquid 43 i 17 25 * 21 26k 16 ND
incubated for 20 h with “H-labeled and media extracts were analyzed
Phospholipid 57 f 17 75 * 21 74 k 16 ND eicosanoids in culture by HPLC as described
serine, 18% with phosphatidylethanolamine, and 70% with phosphatidylcholine. Evidence for esterification of 5HETE into cellular lipids was further obtained by alkaline hydrolysis of lipid extracts of cells incubated for 20 h with 3H-labeled 5-HETE. Thin-layer chromatography after alkaline hydrolysis revealed that all of the phospholipid-associated radioactivity and most of the neutrallipid-associated radioactivity was released. Most of the radioactivity present in the hydrolysate eluted on TLC in the 5-HETE region (Fig. 2, inset). HPLC confirmed that indeed 5HETE was released (Fig. 2). The degree of incorporation of 12- and 15-HETE into smooth muscle cell lipids was much lower than that observed for 5-HETE. After 20 h of incubation in the presence of 0.4% serum, less than 12% ofthe total radioactivity recovered was found in the cell extract (Table 1). Thin-layer chromatography of the cell extracts revealed that, like 5-HETE, all radiolabel was associated with the neutrallipid and phospholipid fraction (Table 1). The distribution of 12- and 15-HETE among the different phospholipids was similar to that of 5-HETE, as judged by autoradiography (not shown). In addition to differences in the degree of incorporation into cellular lipids, there was another difference between the metabolism of 5-HETE and 12- and 15-HETE by smooth muscle cells. As shown in Fig. 1, 12- and 15-HETE were converted into metabolites with retention times on HPLC similar to that of 12(S),5(S)-diHETE. 5-HETE was not converted into metabolites with such a polar behavior on HPLC. Both the esterification and the conversion of 12- and 15-HETE were dependent on the serum concentration. The higher the serum concentration, the lower the metabolism of the HETEs added (Table 2). In contrast to the HETEs tested, we found that exogenous PGE, was not metabolized by smooth muscle cells to any extent. After 20 h of incubation of smooth muscle cell cultures with 3H-labeled PGE, in the presence of 0.4% serum, only 1% of the total radioactivity recovered
90
BRINKMAN,
VAN
BUUL-WORTELBOER,
AND
VAN
MOURIK
0 TAG+DAG
50
l
-
MAG
0 5-HETE F; ‘0 ,-40
-
; P 230 > I> ;
c
-
t
l
PE
0 PC 20
2 0 ;:
-
0 PIiPS 0 ORIGIN
10
-
I
I
I
I
I
1
10
20
30
40
FRACTION
FIG. 2. Esterification of 5.HETE into incubated with 3H-labeled 5-HETE in the Extracts were analyzed both by TLC (inset) with NaOH; (inset lane 3) standards. TAG, phosphatidylcholine; PI, phosphatidylinositol;
smooth muscle cell lipids. Cultures of quiescent human umbilical artery smooth muscle cells were presence of 0.4% serum during 20 h. Cells were extracted, followed by hydrolysis with NaOH. and HPLC. (0 and inset lane 1) Extract not treated with NaOH; (* and inset lane 2) extract treated triacylglycerol; DAG, diacylglycerol; MAG, monoacylglycerol; PE, phosphatidylethanolamine; PC, PS, phosphatidylserine.
was found in the cell extracts (Table 1). The radioactivity recovered from the culture medium chromatographed as PGE, (not shown). Influence
of HETEs
on Cell Proliferation
As shown in Fig. 3,5-, 12-, and 15-HETE inhibited the serum-induced [3H]thymidine incorporation of growtharrested vascular smooth muscle cells with a similar dose response. The inhibitory effect of the HETEs appeared to be serum-batch-dependent. 5-HETE was examined at a concentration of 25 pM in the presence of 5% human serum. Using five different serum batches, inhibition ranging from 3 to 55% (28 & 20%; mean t SD, n = 5) was observed. In addition, the inhibitory effect of the HETEs was dependent on the serum concentration. The higher the serum concentration, the lower the inhibitory effect of the HETE (Table 2). Significant inhibition of serum-induced smooth muscle cell proliferation was observed only at serum concentrations suboptimal for smooth muscle cell growth (serum concentrations at which half-maximal or less [3H]thymidine incorporation of growth-arrested cells was observed; Fig. 3, inset). DISCUSSION
Our results clearly demonstrate that there is a distinct difference between the metabolism of different ei-
cosanoids by human umbilical artery smooth muscle cells. PGE, was not converted by and incorporated into lipids of human umbilical artery smooth muscle cells to any extent (Table 1). On the other hand, at low serum concentrations 5-HETE was efficiently cleared from the culture medium (Fig. 1) and esterified into cellular lipids (Table 1, Fig. 2). 12- and 15-HETE were much less incorporated into smooth muscle cell lipids (Table 1). However, in contrast to 5-HETE, both 12- and 15HETE were converted into metabolites with retention times on HPLC in the diHETE region (Fig. 1). Both the esterification of 5-, 12-, and 15-HETE and the conversion of 12- and 15-HETE into polar metabolites decreased with increasing serum concentrations (Table 2). It has been reported that cultures of bovine thoracic smooth muscle cells take up more 5-HETE than 12HETE [25]. These data are in agreement with our observation showing that human umbilical artery smooth muscle cells preferably incorporate 5-HETE into their lipids (Table 1). Furthermore, we observed that phosphatidylcholine was the major phospholipid in which HETEs were esterified (Fig. 2). Similar results were reported for human umbilical vein endothelial cells and bovine coronary artery endothelial cells [26]. It has been reported that in human umbilical vein endothelial cells esterification of 5-HETE is inhibited by polyunsaturated fatty acids like arachidonic acid [27]. These data may explain our observation that the
HETE
n^
30
METABOLISM
SMOOTH
-
b I E Q 25 20’ ra
AND
SERVM
($1
-I
2o
-
E a : v z
15
-
0
0.025
I
I
I
0.25 HETE
2.5 t//M)
25
FIG. 3. Influence of HETEs on smooth muscle cell proliferation. Cultures of quiescent human umbilical artery smooth muscle cells were incubated with [3H]thymidine and 5-HETE (O), 12.HETE (m), and 15-HETE (0) in the presence of 1% serum. After 28 h, [3H]thymidine incorporation was determined. Results are expressed as means i SD of six determinations. Differences vs control: *P < 0.0005. Cell viability was judged by LDH measurements. Twenty five micromolar 5-, 12., and 15.HETE did not enhance the release of LDH during a 28-h incubation in the presence of 1% serum, as compared to the control incubations without HETE. (Inset) Effect of serum concentration on smooth muscle cell proliferation.
uptake of HETEs by human umbilical artery smooth muscle cells decreased with increasing serum concentrations (Table 2). Another explanation might be binding of HETEs to serum proteins. This might also explain our observation that the conversion of 12- and 15HETE into polar metabolites (Fig. 1) was decreased when the serum concentrations were increased (Table 2). The chromatographic behavior of the metabolites of 12- and 15HETE, synthesized by human umbilical artery smooth muscle cells (Fig. l), suggests that these metabolites are diHETEs. However, previously we have shown that when these cells were incubated with [l14C]arachidonate, no lipoxygenase-derived monoHETEs were formed [29]. Therefore, it seems most likely that the observed conversion of 12- and 15HETE (Fig. 1) is not due to lipoxygenase activity. One of the polar metabolites of 12-HETE, synthesized by human umbilical artery smooth muscle cells, eluted between 5(S),12(S)-diHETE and 12-HHT (Fig. 1). The chro-
MUSCLE
CELL
91
PROLIFERATION
matographic behavior of this 12-HETE metabolite is similar to that of one of the 12-HETE metabolites synthesized by human umbilical vein smooth muscle cells [30, 311. This metabolite was identified as an 8-hydroxyhexadecatrienoic acid and is probably formed by P-oxidation [30, 311. Conversion of HETEs into polar metabolites has also been reported for neutrophils [20, 321 and macrophages [23]. In neutrophils, the enzyme responsible for the conversion of 12-HETE into 12,20diHETE has been identified as a cytochrome P450 monooxygenase [33]. Further experiments need to be carried out to identify the enzyme(s) in human vascular smooth muscle cells responsible for the conversion of 12- and 15-HETE and to unravel the structure of the polar metabolites formed. The specific metabolism of exogenous HETEs by human vascular smooth muscle cells may implicate a relevant role of both the conversion and esterification of HETEs in smooth muscle cell function. For instance, it has been reported that esterification of 5-HETE into lipids of human umbilical vein endothelial cells inhibits prostaglandin synthesis in these cells [26]. HETEs have also been shown to modulate a variety of cellular processes [31], including the proliferation of rabbit aortic smooth muscle cells [6]. Here we show that 5-, 12-, and 15-HETE inhibited the serum-induced [3H]thymidine incorporation in a similar manner, when added at micromolar concentrations to quiescent human umbilical artery smooth muscle cells (Fig. 2). Similar concentrations of these HETEs are reported to affect the proliferation of a variety of other cell types [6, 15-171. Inhibition of smooth muscle cell proliferation was related to the serum concentration used (Table 2). Inhibition was observed only at serum concentrations suboptimal for smooth muscle cell growth. An excess of growth-stimulat,ing compounds, counteracting the inhibitory action of the HETE at higher serum concentrations, may explain these observations. On the other hand, a relationship between metabolism of 5-, 12-, and 15-HETE and HETE-inhibited proliferation was observed (Table 2); both processes decreased with increasing serum concentrations. With similar effects on smooth muscle cell proliferation, the uptake of 5-HETE was much higher than those of 12- and 15-HETE. Thus, if the growth inhibition is the result of accumulation of esterified HETE, then 12- and 15-HETE are more potent inhibitors than 5HETE. REFERENCES 1. 2.
3.
Spraragen, S. C., Bond, V. F’., and Dahl, 11,329. van Buul-Wortelboer, M. F., Brinkman, K. I’., de Groot, Ph. G., van Aken, W. C., (1986) Exp. Cell Res. 162, 151. Castellot, J. J., Favreau, L. V., Karnovsky, R. D. (1982) J. Bid. Chem. 257, 11,256.
L. K. (1962)
Circ. Res.
H. J. M., Dingemans, and van Mourik, J. A. M. J., and Rosenberg,
92
BRINKMAN,
VAN
BUUL-WORTELBOER.
AND
4.
de Groot, Ph. G., Brinkman, H. J. M., Gonsalves, M. D., and van Mourik, J. A. (1985) Biochim. Biophys. Acta 846, 342.
19.
5
