Biochimica et Biophysica Acta. 1082 (1991) 33-36 ,c~ 1991 Elsevier Science Publishers B.V. 0005-2760/91/$03.50 ADONIS 0005276091001001
33
BBALIP 53596
Effect of TNF on triacylglycerol in cultured vascular smooth muscle cells O. Stein 1, E. Shiloni 2 and Y. Stein 3 I Department of Experimental Medicine and Cancer Research. Hebrew Unwersity-Hadassah Medical School. Jerusalem (Israel), 2 Department of Surgery. B. Hadassah Universtty Hospital, Jeru-~alem (Israel) and ~ Lipid Research Laboratory. Department of Medicine B, Hadassah University Hospital. Jerusalem (Israel)
(Received 14 August 19aO)
Key words: Cholesteryl ester; Phospholipid; Arteriosclerosis; Macrophage
Smooth muscle cells (SMC) isolated from bovine aorta or human saphenous vein were cultured and used to study the putative effect of recombinant human tumor necrosis factor (TNF) on lipid metabolism in vascular cells. Addition of T N F to the culture medium for 24-48 h resulted in an increase of 13Hloleie acid uptake and esterification into lipids. The effect could be seen already with 0.3 n g / m l and was maximal with 30 n g / m l . The effect of T N F was mainly on the incorporation of 13H]oleic acid into triacylglycerol which increased by 140% in the bovine cells. There was also a significant increase in [3Hlehelesteryl ester, in the human S M C there was a 40% increase in 13Hloleic acid into total liplds, while the rise in [3Hltriacylglycerol ranged between 60-90%. T N F did not modulate cellular triacylglycerol synthesis in cultured mouse peritoneal macrophages. Since T N F was shown to be synthesized and secreted not only by macrophages but also by smooth muscle cells, it could play an autocrine role in lipid metabolism during development of atherosclerotic lesions. The cellular population of the lesions, i.e., predominance of macrophages or smooth muscle cells, could determine the relative proportion of triaeylglycerol accumulation.
Introduction Lipid accumulation in the arterial wall is an important facet of atherosclerosis. The predominant lipid in the aortic atheroma is cholesteryl ester; however, in severe coronary atherosclerosis there is also marked accumulation of triacylglycerol [1-3]. The foam cells in the atheroma are derived from both macrophages and smooth muscle cells. While more progress was made with respect to the accumulation of lipids in macrophages, the mechanism of transformation of smooth muscle cells into foam cells is not well understood. In our previous study [4], we have investigated a possible interaction between macrophages and smeoth muscle cells which could result in an increased cholesteryl ester synthesis by the latter. In that study, we have shown that free cholesterol released from cholesteryl ester filled macrophages enriches plasma lipoproteins which stimulate cholesteryl ester synthesis in smooth muscle cells [4].
Correspondence: Y. Stein. Lipid Research Laboratory. Department of Medicine B, Hadassah University Hospital. P.O. Box 12000, Jerusalem 91120. Israel.
Macrophages secrete a host of various agents, among them also tumor necrosis factor (TNF) [5]. During the past few years, several studies have shown that injection of T N F into rats results in enhanced lipogenesis in the liver [6,7] and subcutaneous adipose tissue [8]. We have shown also an increased VLDL triacylglycerol secretion by rat liver after injection of r H u T N F in vivo [9]. Accumulation of cellular triacylglycerol in bovine aortic smooth muscle cells can be achieved by exposure to media enriched in free fatty acids [10] and therefore we have used these cells to study a putative effect of T N F on lipid metabolism in vascular cells. Materials and Methods Cell culture Bovine aortic smooth muscle cells were grown from explants of aortae and were subcuitured in DulbeccoVogt medium supplemented with 10% fetal bovine serum [10]. H u m a n vascular smooth muscle cells were grown from explants of saphenous vein and subcuitured in the same medium, supplemented with 107o human serum. The cells (7.104) were seeded in 35-mta pctri dishes and cultured for 5 days with a medium change on day 3. ThioglycoUate elicited mouse peritoneal macrophages were prepared as described [11].
34
Experimental design T N F was a d d e d to s e r u m - c o n t a i n i n g m e d i u m (where a p p r o p r i a t e ) a n d the cells were i n c u b a t e d for 24 h. Thereafter, the m e d i u m was removed, the ceils w a s h e d with p h o s p h a t e - b u f f e r e d saline (PBS) a n d i n c u b a t e d with serum-free m e d i u m c o n t a i n i n g 5 m g p r o t e i n / m l of lipoprotein deficient s e r u m (LPDS), 50 ,aM [3H]oleic acid c o m p l e x e d to d e l i p i d a t e d s e r u m a l b u m i n , T N F a n d L D L , 150 ,ttg p r o t e i n / m l , where, a p p r o p r i a t e . I n c u b a tion was carried o u t for 24 h, the m e d i u m w a s removed, the cell layer w a s h e d three times with 0.2% a l b u m i n in PBS a n d then three times with PBS. T h e cell layer w a s s c r a p e d with 1 ml 50% m e t h a n o l a n d 2 ml of 100% m e t h a n o l using a Teflon p o l i c e m a n . A f t e r a d d i t i o n of an equal volume of c h l o r o f o r m , the lipids were ext r a c t e d a n d the d e l i p i d a t e d residue used for d e t e r m i n a tion o f protein. Preparation o f fatty acid albumin complex, low.density lipoprotein and lipoprotein-deficient serum Oleic acid a n d [3H]oleic acid were c o m p l e x e d to d e l i p i d a t e d s e r u m a l b u m i n [12] at a m o l a r r a t i o of 4 : 1. L o w - d e n s i t y l i p o p r o t e i n s ( L D L ) were isolated f r o m hum a n p l a s m a at d = 1 . 0 1 9 - 1 . 0 6 3 g / m l [13]. L i p o p r o t e i n deficient s e r u m w a s p r e p a r e d b y r e m o v a l o f all l i p o p r o teins at d = 1.25 g / m l [10]. Chemical and chromatographic procedures Lipids were e x t r a c t e d a n d purified a c c o r d i n g to F o l c h et al. [14]. S e p a r a t i o n of labeled lipids w a s c a r r i e d o u t b y t h i n - l a y e r c h r o m a t o g r a p h y o n silica gel plates u s i n g a solvent system of p e t r o l e u m e t h e r / e t h y l e t h e r / a c e t i c acid (80 : 20 : 1). T h e c o m p o u n d s were identified a c c o r d ing to s t a n d a r d s a n d visualized b y e x p o s u r e to iodine vapor. R a d i o a c t i v i t y w a s d e t e r m i n e d with a T r i c a r b fl-scintillation s p e c t r o m e t e r ( M i n o x y , P a c k a r d ) with a b solute activity analyser. Protein w a s d e t e r m i n e d a c c o r d ing to L o w r y et al. [15]. Materials R e c o m b i n a n t h u m a n t u m o r necrosis f a c t o r ( r H u T N F ) , specific activity 107 U / m g , w a s f r o m C e t u s C o r p . (Emeryville, C A , U.S.A.). [9,10(n)-3H]Oleic acid (4.2 C i / m m o l ) a n d [~4C]choline (50 m C i / m m o l ) were f r o m A m e r s h a m I n t e r n a t i o n a l (U.K.). All c u l t u r e m e d i a a n d bovine fetal s e r u m were f r o m G i b c o ( G r a n d Island, N.Y., U.S.it.). Results E x p o s u r e o f c u l t u r e d bovine aortic s m o o t h muscle cells to r H u T N F for 48 h resulted in a n increase in the i n c o r p o r a t i o n of [3H]oleic acid into total lipids (Table 1). W h e n the d i s t r i b u t i o n of label in the cells w a s f u r t h e r analyzed, it c o u l d be seen t h a t this increase w a s a c c o u n t e d for m a i n l y b y a higher i n c o r p o r a t i o n of the
TABLE l Effect of TNF on [ 3H]oleic acid uptake and metabolism b~' bovine aortic smooth muscle cells Conditions: Bovine aortic smooth muscle cells were cultured in 35-ram petri dishes for 5 days with a medium change on day 3. On day 5, the medium was replaced by medium containing 5% LPDS and 60 ng/ml of TNF. On day 6, the medium was changed to the same medium but supplemented, in addition to TNF, with 50 /LM [3H]oleic acid complexed to albumin and LDL (150/tg protein/ml) where appropriate. Incubation was carried out for 24 h. At the end of the experiment, there was 220±0.3 ug of cellular protein/dish. PL, phospholipids: TG, triacylglycerol; CE. cholesteryl ester. Values are means±S.E, of triplicates or quadruplicates, a vs. b, i vs. k. j vs. I, P < 0.05; c vs. d, e vs. f. P < 0.0l; g vs. h, P < 0.001. Additions
None LDL TNF LDL+TNF
['~H]oleicacid uptake (nmol/dish) Total PL TG lipids 20.3±0.9 a 13.5-t-0.5 6.2+0.4 e 19.5±0.7 ¢ 12.5±0.1 5.7±0.5 s 31.0±3.9 b 12.0±1.9 17.0+1.4 t 29.5±1.0 'j 11.4±0.4 15.0±0.7 s
CE 0.25+0.05 i 0.5 ±0.09 J 0.4 ±0.01 k 1.0 ±0.051
labeled f a t t y a c i d i n t o triacyiglycerols. A t the s a m e time, there w a s also a slight increase in labeled cholesteryl ester, b o t h in the p r e s e n c e a n d a b s e n c e o f e x o g e n o u s L D L (Table l). U s i n g the s a m e c o n d i t i o n s , we then varied the concentration o f the T N F in the i n c u b a t i o n m e d i u m . T h e data presented in T a b l e II s h o w t h a t the lowest effective d o s e was 0.3 n g / m l and t h a t m a x i m a l effect was seen between 3 and 30 n g / m l . W h e n the e x p o s u r e t i m e to T N F was shortened t o 2 4 h, there w a s a 2.5-fold i n c r e a s e in the i n c o r p o r a t i o n o f [3H]oleic acid i n t o triacylglyceroi a n d c h o l e s t e r y l ester ( f r o m 4.0 + 0.1 to 10.6 + 0.2 a n d f r o m 0.4 + 0.02 to 1.0 4- 0.03 n m o l , respectively). T h e increase in i n c o r p o r a t i o n o f [3H]oleic a c i d i n t o triacylglycerol c o u l d b e d e m o n s t r a t e d also in cells p r e t r e a t e d for 24 h w i t h T N F
TABLE II
Effect of TNF concentration on incorporation of [3H]oleic acid into triacylglycerolby bovine aortic smooth muscle cells Conditions: as in Table L except for the amounts of TNF added and the cellular protein/dish which was 190 ± 5 pg. Values are means ± S.E. of quadruplicates, a vs. b, P < 0.05; a vs. c, d vs. e, g vs. h, P < 0.0l; d vs. f, P < 0.001. TNF, ng/ml 0 0.03 0.3 3.0 15.0 30.0 60.0
[3Hloleic acid uptake (nmol/dish) Total PL TG lipids 12.9±0.5 a 6.5±0.2 5.4±0.3 d 12.7±0.2 6.7±0.2 4.9±0.3 15.2±0.5 b 6.5±0.1 7.4±0.3" 18.7±1.3 6.1±0.3 11.2±0.7 199±0.3 5.3±0.2 12.8±0.2 20.5±1.4 ~ 5.9±0.2 13.0±0.4 f 20.5±1.1 4.7±0.15 13.5±0.6
CE 0,4±0.01 g 0.3±0.05 0.5±0.1 0.6±0.1 0.8±0.~ 1.0±0.1 s l.O±0.~
35 TABLE Ill Incorporation of [ t4C]choline into bpids by bovine aortic smooth muvcle cells
Conditions: Cells were cultured as in Table 1 and exposed to TNF for 24 h and to [14Clcholine 0 7 nmol/ml) for 4 h or to TNF for 48 h and to labeled choline for 24 h. Values are means± S.E. of triplicates. PC, phosphatidylcholine: PL, phospholipids. Exp.
I
TNF,
[14C]choline uptake
ng/ml
Total PL (% of medium label)
IlaCIPC (% of total l14CIPL)
4h
24h
4h
24h
5.0±6.2 4.6±0.6
93.2±0.3 95.0±1.0 93.8±1.0 92.0±2.0
88.3±1.5 87.9±2.0
0 30 0 30
II
2.4±0.1 2.3±0.3 1.1±0.1 1.0±0.2
a n d e x p o s e d to [3H]oleic acid for 2 or 4 h ( ! . 2 4 _+ 0.2 vs. 3.0 + 0.08 a n d 2.55 + 0.05 vs. 5.3 + 0.7 n g / d i s h for c o n t r o l vs. T N F treated, respectively). W e d e t e r m i n e d also i n c o r p o r a t i o n o f [t4Clcholine into p h o s p h o l i p i d s a n d results o f t w o e x p e r i m e n t s are s h o w n in T a b l e Ill. A f t e r 4 h i n c u b a t i o n , there w a s n o d i f f e r e n c e in the i n c o r p o r a t i o n o f ~4C i n t o total phosp h o l i p i d s b e t w e e n S M C e x p o s e d to 30 n g / m l o f T N F for 24 h a n d c o n t r o l cells. A f t e r 48 h e x p o s u r e to T N F a n d 24 h i n c u b a t i o n with [~4C]choline, a slight b u t statistically n o t significant r e d u c t i o n in ~aCdabeled p h o s p h o l i p i d w a s seen. O n thin-layer c h r o m a t o g r a p h y , m o r e t h a n 9070 o f [ t a C l c h o l i n e were recovered in phosp h a t i d y l c h o l i n e a f t e r 4 h a n d 8370 a f t e r 24 h in b o t h the T N F treated a n d c o n t r o l cells (Table Ill). T h e effect o f T N F w a s s t u d i e d also with S M C derived f r o m h u m a n s a p h e n o u s vein. As seen in T a b l e IV, there w a s a b o u t 4070 increase in i n c o r p o r a t i o n o f [3H]oleic a c i d i n t o total lipids; in b o t h e x p e r i m e n t s the increase w a s relatively m o r e p r o m i n e n t in triacylglycerol. T h e slightly h i g h e r i n c o r p o r a t i o n of [3H]oleic a c i d i n t o cholester)'l ester in T N F - t r e a t e d cells in exp. 11
did not reach statistical significance. W h e n T N F w a s a d d e d to m o u s e p e r i t o n e a l m a c r o p h a g e s , there w a s a slight increase in i n c o r p o r a t i o n of oleic acid i n t o phoso p h o l i p i d s w h i c h c o m p r i s e d a b o u t 90% o f total labeled lipids. T h e r e w a s n o c h a n g e in the i n c o r p o r a t i o n o f [3H]oleic acid i n t o triacylglycerol with 30 n g o f T N F / m l a n d p r a c t i c a l l y n o cholesteryl ester w a s f o u n d u n d e r these e x p e r i m e n t a l c o n d i t i o n s (Table V). Discussion T h e results p r e s e n t e d a b o v e indicate t h a t T N F c a n affect lipid m e t a b o l i s m in a o r t i c b o v i n e s m o o t h muscle cells. T h e m a i n f i n d i n g is a n increased i n c o r p o r a t i o n of fatty a c i d i n t o triacylglycerol a n d to a lesser extent i n t o cholesteryl ester. T h e r e w a s n o significant effect o f T N F o n p h o s p h o l i p i d m e t a b o l i s m as s t u d i e d with [3H]oleic acid o r [14C]choline. In s m o o t h muscle cells derived f r o m h u m a n s a p h e n o u s vein, the higher c o n c e n t r a t i o n of T N F (125 n g / m l ) c a u s e d a c o n c o m i t a n t increase of i n c o r p o r a t i o n o f [3H]-oleic acid into triacylglycerol a n d p h o s p h o l i p i d s . W h e n p a i r e d a n a l y s i s o f the effect of T N F o n the i n c o r p o r a t i o n o f [3H]oleic acid i n t o C E w a s
TABLE IV Effect of TNF on [JH]oleic acid uptake by human venous smooth muscle cells
Conditions: Human saphenous vein SMC were cultured as in Table I and exposed to TNF for 48 h and to [:~H]olcicacid for 24 h. In exp. I, no LDL was added and in exp. IL 150 lag protcin/ml of LDL was present during the incubation with [3H]oleic acid. There was 104±0.4 lag and 71.0±2.6 lag cellular protein/dish in exp. I and exp. IL respectively. Values are mear,s + S.E. of triplicates, a vs. b, P < 0.05: c vs. d, • vs. f, P < 0.01. Additions TNF, Exp. ns/ml None 125 None 3 30
I I II I! li
[3Hloleic acid uptake (nmol/dish) Total PL TG lipids 10.5±0.8 6.5±0.5 a 3.1±0.2 c 14.3±0.2 8.5±0.2 b 5.1±0.2 d 1.2±0.~ ~ 7.0±0.4 5.0±0.5 2.0±0.1 8.8±0.8 5.7±0.2 2.3±0.2 t 9.6±0.2 6.2±0.3
TABLE V Effect of TNF on [)H]olt, ie acid intwrporation into lipids I~)" mouse peritoneal macrophuges
Conditions: Thioglycollate elicited peritoneal macrophages were cultured for 18-20 h and then exposed to TNF for either 24 or 48 h. Incubation with [3Hloleic acid was carried out for 24 h. Cellular protein/dish was 400 + 29/tg. Values are means + S,E. of quadruplicates. [ 3H]olcic acid uptake (nmol/dish)
TNF ng/ml
h
0 3 30 3 30 60
24 ~ 48 48 ~
CE 6.35±0.05 0.35±0.05 ~50±0.08 0.60±0.02 0.70±0.~
Total lipids 20.7±1.0 23.4±~6 25.4±0.8 ~.0±l.l 23.2±0.5 23.1±1.6
FL
TG
17.4±0.8 ~.5±0.5 21.4±0.6 21.0±1.2 18.9±0.8 19.0±l.1
2.6±0.5 2.3±0.4 2.7±0.2 2.3±0.15 2.5±0.15 3.5±0.5
36 p e r f o r m e d , c o m b i n i n g d a t a f r o m b o t h bovine a n d hum a n cells, the difference ( A ) between the treated a n d c o n t r o l values was 0.41 + 0 . 0 8 n m o l (mean_+ S.E.), which was significantly different f r o m 0 ( P < 0 . 0 1 ) . W h e n aortic slices derived f r o m various species were i n c u b a t e d with labeled fatty acids, a b o u t a third of the labeled c o m p o u n d w a s i n c o r p o r a t e d i n t o triacylglycerol [16]. With [3H]glyeerol as precursor, 4 0 - 5 0 % o f label were i n c o r p o r a t e d into triacylglycerol b y c u l t u r e d b o v i n e o r r a b b i t aortic s m o o t h muscle ceils exposed to f a t t y acid enriched s e r u m [10]. In h u m a n arterial s m o o t h muscle a m a r k e d increase in triacylglycerol c o n t e n t w a s r e p o r t e d w h e n the cells were c u l t u r e d in the presence of sera c o n t a i n i n g elevated nonesterified fatty acids [17]. T N F a p p e a r s to be a p l u r i p o t e n t c y t o k i n e w h i c h affects v a s c u l a r a n d n o n v a s c u l a r cells [5]. Hc,wever, the effect of T N F o n lipid m e t a b o l i s m w a s seen m a i n l y in the liver [6-9]. In the arterial a t h e r o s c l e r o t i c wall, T N F c o u l d be synthesized a n d secreted b y the m a c r o p h a g e [5] a n d the s m o o t h muscle cell itself [18]. A recent s t u d y has s h o w n t h a t h u m a n s a p h e n o u s vein s m o o t h muscle cells express the TNF-t~ gene a n d secrete biologically active T N F - a [18]. Secretion of T N F w a s also i n d u c e d b y l i p o p o l y s a c c h a r i d e in c u l t u r e d m e s e n c h y m a l cells derived f r o m n e w b o r n rat h e a r t [11]. T h e effect o f T N F o n triacylglycerol d e s c r i b e d in this s t u d y c o u l d b e relev a n t to the f i n d i n g of a n increased triacylglycerol c o n tent of h u m a n a o r t a with age [19]. In h u m a n a t h e r o m a t o u s aorta, relatively m o r e triacyiglycerol w a s f o u n d in fibrous p l a q u e s t h a n in fatty streaks [20]. T h i s c o u l d be related to the different cellular p o p u l a t i o n , i.e., in the fibrous p l a q u e there are m a i n l y s m o o t h muscle cell derived f o a m cells [21], while in the f a t t y s t r e a k they o r i g i n a t e f r o m m a c r o p h a g e s [22], w h i c h h a v e a m u c h l o w e r triacylglycerol c o n t e n t . Acknowledgement T h i s s t u d y w a s s u p p o r t e d in p a r t b y a g r a n t f r o m the M a r i o S h a p i r o F u n d , T h e H e b r e w University o f Jerusalem.
References 1 Boncher, C.J.F., Woodford, F.P., Ter Haar Romeny, C.Ch., Boelsma, E. and Van Gent, C.M. (1959) Nature, 183, 47-48. 2 Dayton, S., Hashimoto, S. and Pearce, M.L. (1962) Exp. Mol. Pathol. 1. 481-496. 3 Meyer, B,J., Meyer, A.C.. Pepler, W.J. and Theron, J.J. (1966) Am. Heart J. 71, 68-78. 4 Stein, O., Halperin. G. and Stein, Y. (~.981) Biochim. Biophys. Acta 665, 477-490. 5 Sherry, B. and Cerami, A. (1988) J. Cell Biol. 107, 1269-1277. 6 Feingold, K.R. and Granfeld, C. (1987) J. Clin. Invest. 80,184-190. 7 Feingold, K.R,, Soued, M., Serio. M.K., Moser, A.H., Dinarello, C. and Grunfeld, C. (1989) Endocrinology 125, 267-274. 8 Grunfeld, C., Wilking, H., Neese, R., Gavin, L.A., Moser, A.H.. Gulli, R., Serio, M.K. and Feingold, K.R. (1989) Cane. Res. 49, 2554-2560. 9 Chajek-Shaul, T.. Friedman, G., Stein, O., Shiloni, E., Etienne, J. and Stein, Y. (1989) Biochim. Biophys. Aeta 1001, 316-324. 10 Stein, O., Coetzee, G.A. and Stein, Y. (1981) Canad. J. Biochem. 59, 662-667. 11 Friedman, G., Gallily, R., Chajek-Shaal, T., Stein, O., Shiloni, E., Etienne, J. and Stein, Y. (1988) Biochim. Biophys. Acta 960, 220-228. 12 Hasin, Y., Sapoznikov, D.. Stein, O. and Stein. Y. (1982) J. Mol. Cell. CardioL 14, 163-172. 13 Havel, R.J., Eder, H.A. and Bragdon, J.H. (1955) J. Clin. Invest. 34, 1345-1353. 14 Foleh, J., Lees, M. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509. 15 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, RJ. (1951) J. Biol. Chem. 193, 265-275. 16 Stein, Y. and Stein, O. (1962) J. Alheroscler. Res. 2, 400-412. 17 Laughton, C.W., Ruddl¢, D.L,, Bedford, C..L and Alderman, E.L. (1988) Atherosclerosis 70, 233-246. 18 Warner, S.J.C. and Libby, P. 0989) J. Immunol. 142, 100-109. 19 Smith, E.B., Evans, P.H. and Downham, M.D. (1967) J. Atheroscler. Res. 7, 171-186. 20 Katz, S.S., Shipley, G.G. and Small, D.M. (1976) J. C~in. Invest. 58, 200-211. 21 Rosenfeld, M.E., Tsukada, T., Chait, A. et al. (1987) Arteriosclerosis 7, 24-34. 22 Stary, H.C. (1987) Atherosclerosis 64, 91-108.
33
BBALIP 53596
Effect of TNF on triacylglycerol in cultured vascular smooth muscle cells O. Stein 1, E. Shiloni 2 and Y. Stein 3 I Department of Experimental Medicine and Cancer Research. Hebrew Unwersity-Hadassah Medical School. Jerusalem (Israel), 2 Department of Surgery. B. Hadassah Universtty Hospital, Jeru-~alem (Israel) and ~ Lipid Research Laboratory. Department of Medicine B, Hadassah University Hospital. Jerusalem (Israel)
(Received 14 August 19aO)
Key words: Cholesteryl ester; Phospholipid; Arteriosclerosis; Macrophage
Smooth muscle cells (SMC) isolated from bovine aorta or human saphenous vein were cultured and used to study the putative effect of recombinant human tumor necrosis factor (TNF) on lipid metabolism in vascular cells. Addition of T N F to the culture medium for 24-48 h resulted in an increase of 13Hloleie acid uptake and esterification into lipids. The effect could be seen already with 0.3 n g / m l and was maximal with 30 n g / m l . The effect of T N F was mainly on the incorporation of 13H]oleic acid into triacylglycerol which increased by 140% in the bovine cells. There was also a significant increase in [3Hlehelesteryl ester, in the human S M C there was a 40% increase in 13Hloleic acid into total liplds, while the rise in [3Hltriacylglycerol ranged between 60-90%. T N F did not modulate cellular triacylglycerol synthesis in cultured mouse peritoneal macrophages. Since T N F was shown to be synthesized and secreted not only by macrophages but also by smooth muscle cells, it could play an autocrine role in lipid metabolism during development of atherosclerotic lesions. The cellular population of the lesions, i.e., predominance of macrophages or smooth muscle cells, could determine the relative proportion of triaeylglycerol accumulation.
Introduction Lipid accumulation in the arterial wall is an important facet of atherosclerosis. The predominant lipid in the aortic atheroma is cholesteryl ester; however, in severe coronary atherosclerosis there is also marked accumulation of triacylglycerol [1-3]. The foam cells in the atheroma are derived from both macrophages and smooth muscle cells. While more progress was made with respect to the accumulation of lipids in macrophages, the mechanism of transformation of smooth muscle cells into foam cells is not well understood. In our previous study [4], we have investigated a possible interaction between macrophages and smeoth muscle cells which could result in an increased cholesteryl ester synthesis by the latter. In that study, we have shown that free cholesterol released from cholesteryl ester filled macrophages enriches plasma lipoproteins which stimulate cholesteryl ester synthesis in smooth muscle cells [4].
Correspondence: Y. Stein. Lipid Research Laboratory. Department of Medicine B, Hadassah University Hospital. P.O. Box 12000, Jerusalem 91120. Israel.
Macrophages secrete a host of various agents, among them also tumor necrosis factor (TNF) [5]. During the past few years, several studies have shown that injection of T N F into rats results in enhanced lipogenesis in the liver [6,7] and subcutaneous adipose tissue [8]. We have shown also an increased VLDL triacylglycerol secretion by rat liver after injection of r H u T N F in vivo [9]. Accumulation of cellular triacylglycerol in bovine aortic smooth muscle cells can be achieved by exposure to media enriched in free fatty acids [10] and therefore we have used these cells to study a putative effect of T N F on lipid metabolism in vascular cells. Materials and Methods Cell culture Bovine aortic smooth muscle cells were grown from explants of aortae and were subcuitured in DulbeccoVogt medium supplemented with 10% fetal bovine serum [10]. H u m a n vascular smooth muscle cells were grown from explants of saphenous vein and subcuitured in the same medium, supplemented with 107o human serum. The cells (7.104) were seeded in 35-mta pctri dishes and cultured for 5 days with a medium change on day 3. ThioglycoUate elicited mouse peritoneal macrophages were prepared as described [11].
34
Experimental design T N F was a d d e d to s e r u m - c o n t a i n i n g m e d i u m (where a p p r o p r i a t e ) a n d the cells were i n c u b a t e d for 24 h. Thereafter, the m e d i u m was removed, the ceils w a s h e d with p h o s p h a t e - b u f f e r e d saline (PBS) a n d i n c u b a t e d with serum-free m e d i u m c o n t a i n i n g 5 m g p r o t e i n / m l of lipoprotein deficient s e r u m (LPDS), 50 ,aM [3H]oleic acid c o m p l e x e d to d e l i p i d a t e d s e r u m a l b u m i n , T N F a n d L D L , 150 ,ttg p r o t e i n / m l , where, a p p r o p r i a t e . I n c u b a tion was carried o u t for 24 h, the m e d i u m w a s removed, the cell layer w a s h e d three times with 0.2% a l b u m i n in PBS a n d then three times with PBS. T h e cell layer w a s s c r a p e d with 1 ml 50% m e t h a n o l a n d 2 ml of 100% m e t h a n o l using a Teflon p o l i c e m a n . A f t e r a d d i t i o n of an equal volume of c h l o r o f o r m , the lipids were ext r a c t e d a n d the d e l i p i d a t e d residue used for d e t e r m i n a tion o f protein. Preparation o f fatty acid albumin complex, low.density lipoprotein and lipoprotein-deficient serum Oleic acid a n d [3H]oleic acid were c o m p l e x e d to d e l i p i d a t e d s e r u m a l b u m i n [12] at a m o l a r r a t i o of 4 : 1. L o w - d e n s i t y l i p o p r o t e i n s ( L D L ) were isolated f r o m hum a n p l a s m a at d = 1 . 0 1 9 - 1 . 0 6 3 g / m l [13]. L i p o p r o t e i n deficient s e r u m w a s p r e p a r e d b y r e m o v a l o f all l i p o p r o teins at d = 1.25 g / m l [10]. Chemical and chromatographic procedures Lipids were e x t r a c t e d a n d purified a c c o r d i n g to F o l c h et al. [14]. S e p a r a t i o n of labeled lipids w a s c a r r i e d o u t b y t h i n - l a y e r c h r o m a t o g r a p h y o n silica gel plates u s i n g a solvent system of p e t r o l e u m e t h e r / e t h y l e t h e r / a c e t i c acid (80 : 20 : 1). T h e c o m p o u n d s were identified a c c o r d ing to s t a n d a r d s a n d visualized b y e x p o s u r e to iodine vapor. R a d i o a c t i v i t y w a s d e t e r m i n e d with a T r i c a r b fl-scintillation s p e c t r o m e t e r ( M i n o x y , P a c k a r d ) with a b solute activity analyser. Protein w a s d e t e r m i n e d a c c o r d ing to L o w r y et al. [15]. Materials R e c o m b i n a n t h u m a n t u m o r necrosis f a c t o r ( r H u T N F ) , specific activity 107 U / m g , w a s f r o m C e t u s C o r p . (Emeryville, C A , U.S.A.). [9,10(n)-3H]Oleic acid (4.2 C i / m m o l ) a n d [~4C]choline (50 m C i / m m o l ) were f r o m A m e r s h a m I n t e r n a t i o n a l (U.K.). All c u l t u r e m e d i a a n d bovine fetal s e r u m were f r o m G i b c o ( G r a n d Island, N.Y., U.S.it.). Results E x p o s u r e o f c u l t u r e d bovine aortic s m o o t h muscle cells to r H u T N F for 48 h resulted in a n increase in the i n c o r p o r a t i o n of [3H]oleic acid into total lipids (Table 1). W h e n the d i s t r i b u t i o n of label in the cells w a s f u r t h e r analyzed, it c o u l d be seen t h a t this increase w a s a c c o u n t e d for m a i n l y b y a higher i n c o r p o r a t i o n of the
TABLE l Effect of TNF on [ 3H]oleic acid uptake and metabolism b~' bovine aortic smooth muscle cells Conditions: Bovine aortic smooth muscle cells were cultured in 35-ram petri dishes for 5 days with a medium change on day 3. On day 5, the medium was replaced by medium containing 5% LPDS and 60 ng/ml of TNF. On day 6, the medium was changed to the same medium but supplemented, in addition to TNF, with 50 /LM [3H]oleic acid complexed to albumin and LDL (150/tg protein/ml) where appropriate. Incubation was carried out for 24 h. At the end of the experiment, there was 220±0.3 ug of cellular protein/dish. PL, phospholipids: TG, triacylglycerol; CE. cholesteryl ester. Values are means±S.E, of triplicates or quadruplicates, a vs. b, i vs. k. j vs. I, P < 0.05; c vs. d, e vs. f. P < 0.0l; g vs. h, P < 0.001. Additions
None LDL TNF LDL+TNF
['~H]oleicacid uptake (nmol/dish) Total PL TG lipids 20.3±0.9 a 13.5-t-0.5 6.2+0.4 e 19.5±0.7 ¢ 12.5±0.1 5.7±0.5 s 31.0±3.9 b 12.0±1.9 17.0+1.4 t 29.5±1.0 'j 11.4±0.4 15.0±0.7 s
CE 0.25+0.05 i 0.5 ±0.09 J 0.4 ±0.01 k 1.0 ±0.051
labeled f a t t y a c i d i n t o triacyiglycerols. A t the s a m e time, there w a s also a slight increase in labeled cholesteryl ester, b o t h in the p r e s e n c e a n d a b s e n c e o f e x o g e n o u s L D L (Table l). U s i n g the s a m e c o n d i t i o n s , we then varied the concentration o f the T N F in the i n c u b a t i o n m e d i u m . T h e data presented in T a b l e II s h o w t h a t the lowest effective d o s e was 0.3 n g / m l and t h a t m a x i m a l effect was seen between 3 and 30 n g / m l . W h e n the e x p o s u r e t i m e to T N F was shortened t o 2 4 h, there w a s a 2.5-fold i n c r e a s e in the i n c o r p o r a t i o n o f [3H]oleic acid i n t o triacylglyceroi a n d c h o l e s t e r y l ester ( f r o m 4.0 + 0.1 to 10.6 + 0.2 a n d f r o m 0.4 + 0.02 to 1.0 4- 0.03 n m o l , respectively). T h e increase in i n c o r p o r a t i o n o f [3H]oleic a c i d i n t o triacylglycerol c o u l d b e d e m o n s t r a t e d also in cells p r e t r e a t e d for 24 h w i t h T N F
TABLE II
Effect of TNF concentration on incorporation of [3H]oleic acid into triacylglycerolby bovine aortic smooth muscle cells Conditions: as in Table L except for the amounts of TNF added and the cellular protein/dish which was 190 ± 5 pg. Values are means ± S.E. of quadruplicates, a vs. b, P < 0.05; a vs. c, d vs. e, g vs. h, P < 0.0l; d vs. f, P < 0.001. TNF, ng/ml 0 0.03 0.3 3.0 15.0 30.0 60.0
[3Hloleic acid uptake (nmol/dish) Total PL TG lipids 12.9±0.5 a 6.5±0.2 5.4±0.3 d 12.7±0.2 6.7±0.2 4.9±0.3 15.2±0.5 b 6.5±0.1 7.4±0.3" 18.7±1.3 6.1±0.3 11.2±0.7 199±0.3 5.3±0.2 12.8±0.2 20.5±1.4 ~ 5.9±0.2 13.0±0.4 f 20.5±1.1 4.7±0.15 13.5±0.6
CE 0,4±0.01 g 0.3±0.05 0.5±0.1 0.6±0.1 0.8±0.~ 1.0±0.1 s l.O±0.~
35 TABLE Ill Incorporation of [ t4C]choline into bpids by bovine aortic smooth muvcle cells
Conditions: Cells were cultured as in Table 1 and exposed to TNF for 24 h and to [14Clcholine 0 7 nmol/ml) for 4 h or to TNF for 48 h and to labeled choline for 24 h. Values are means± S.E. of triplicates. PC, phosphatidylcholine: PL, phospholipids. Exp.
I
TNF,
[14C]choline uptake
ng/ml
Total PL (% of medium label)
IlaCIPC (% of total l14CIPL)
4h
24h
4h
24h
5.0±6.2 4.6±0.6
93.2±0.3 95.0±1.0 93.8±1.0 92.0±2.0
88.3±1.5 87.9±2.0
0 30 0 30
II
2.4±0.1 2.3±0.3 1.1±0.1 1.0±0.2
a n d e x p o s e d to [3H]oleic acid for 2 or 4 h ( ! . 2 4 _+ 0.2 vs. 3.0 + 0.08 a n d 2.55 + 0.05 vs. 5.3 + 0.7 n g / d i s h for c o n t r o l vs. T N F treated, respectively). W e d e t e r m i n e d also i n c o r p o r a t i o n o f [t4Clcholine into p h o s p h o l i p i d s a n d results o f t w o e x p e r i m e n t s are s h o w n in T a b l e Ill. A f t e r 4 h i n c u b a t i o n , there w a s n o d i f f e r e n c e in the i n c o r p o r a t i o n o f ~4C i n t o total phosp h o l i p i d s b e t w e e n S M C e x p o s e d to 30 n g / m l o f T N F for 24 h a n d c o n t r o l cells. A f t e r 48 h e x p o s u r e to T N F a n d 24 h i n c u b a t i o n with [~4C]choline, a slight b u t statistically n o t significant r e d u c t i o n in ~aCdabeled p h o s p h o l i p i d w a s seen. O n thin-layer c h r o m a t o g r a p h y , m o r e t h a n 9070 o f [ t a C l c h o l i n e were recovered in phosp h a t i d y l c h o l i n e a f t e r 4 h a n d 8370 a f t e r 24 h in b o t h the T N F treated a n d c o n t r o l cells (Table Ill). T h e effect o f T N F w a s s t u d i e d also with S M C derived f r o m h u m a n s a p h e n o u s vein. As seen in T a b l e IV, there w a s a b o u t 4070 increase in i n c o r p o r a t i o n o f [3H]oleic a c i d i n t o total lipids; in b o t h e x p e r i m e n t s the increase w a s relatively m o r e p r o m i n e n t in triacylglycerol. T h e slightly h i g h e r i n c o r p o r a t i o n of [3H]oleic a c i d i n t o cholester)'l ester in T N F - t r e a t e d cells in exp. 11
did not reach statistical significance. W h e n T N F w a s a d d e d to m o u s e p e r i t o n e a l m a c r o p h a g e s , there w a s a slight increase in i n c o r p o r a t i o n of oleic acid i n t o phoso p h o l i p i d s w h i c h c o m p r i s e d a b o u t 90% o f total labeled lipids. T h e r e w a s n o c h a n g e in the i n c o r p o r a t i o n o f [3H]oleic acid i n t o triacylglycerol with 30 n g o f T N F / m l a n d p r a c t i c a l l y n o cholesteryl ester w a s f o u n d u n d e r these e x p e r i m e n t a l c o n d i t i o n s (Table V). Discussion T h e results p r e s e n t e d a b o v e indicate t h a t T N F c a n affect lipid m e t a b o l i s m in a o r t i c b o v i n e s m o o t h muscle cells. T h e m a i n f i n d i n g is a n increased i n c o r p o r a t i o n of fatty a c i d i n t o triacylglycerol a n d to a lesser extent i n t o cholesteryl ester. T h e r e w a s n o significant effect o f T N F o n p h o s p h o l i p i d m e t a b o l i s m as s t u d i e d with [3H]oleic acid o r [14C]choline. In s m o o t h muscle cells derived f r o m h u m a n s a p h e n o u s vein, the higher c o n c e n t r a t i o n of T N F (125 n g / m l ) c a u s e d a c o n c o m i t a n t increase of i n c o r p o r a t i o n o f [3H]-oleic acid into triacylglycerol a n d p h o s p h o l i p i d s . W h e n p a i r e d a n a l y s i s o f the effect of T N F o n the i n c o r p o r a t i o n o f [3H]oleic acid i n t o C E w a s
TABLE IV Effect of TNF on [JH]oleic acid uptake by human venous smooth muscle cells
Conditions: Human saphenous vein SMC were cultured as in Table I and exposed to TNF for 48 h and to [:~H]olcicacid for 24 h. In exp. I, no LDL was added and in exp. IL 150 lag protcin/ml of LDL was present during the incubation with [3H]oleic acid. There was 104±0.4 lag and 71.0±2.6 lag cellular protein/dish in exp. I and exp. IL respectively. Values are mear,s + S.E. of triplicates, a vs. b, P < 0.05: c vs. d, • vs. f, P < 0.01. Additions TNF, Exp. ns/ml None 125 None 3 30
I I II I! li
[3Hloleic acid uptake (nmol/dish) Total PL TG lipids 10.5±0.8 6.5±0.5 a 3.1±0.2 c 14.3±0.2 8.5±0.2 b 5.1±0.2 d 1.2±0.~ ~ 7.0±0.4 5.0±0.5 2.0±0.1 8.8±0.8 5.7±0.2 2.3±0.2 t 9.6±0.2 6.2±0.3
TABLE V Effect of TNF on [)H]olt, ie acid intwrporation into lipids I~)" mouse peritoneal macrophuges
Conditions: Thioglycollate elicited peritoneal macrophages were cultured for 18-20 h and then exposed to TNF for either 24 or 48 h. Incubation with [3Hloleic acid was carried out for 24 h. Cellular protein/dish was 400 + 29/tg. Values are means + S,E. of quadruplicates. [ 3H]olcic acid uptake (nmol/dish)
TNF ng/ml
h
0 3 30 3 30 60
24 ~ 48 48 ~
CE 6.35±0.05 0.35±0.05 ~50±0.08 0.60±0.02 0.70±0.~
Total lipids 20.7±1.0 23.4±~6 25.4±0.8 ~.0±l.l 23.2±0.5 23.1±1.6
FL
TG
17.4±0.8 ~.5±0.5 21.4±0.6 21.0±1.2 18.9±0.8 19.0±l.1
2.6±0.5 2.3±0.4 2.7±0.2 2.3±0.15 2.5±0.15 3.5±0.5
36 p e r f o r m e d , c o m b i n i n g d a t a f r o m b o t h bovine a n d hum a n cells, the difference ( A ) between the treated a n d c o n t r o l values was 0.41 + 0 . 0 8 n m o l (mean_+ S.E.), which was significantly different f r o m 0 ( P < 0 . 0 1 ) . W h e n aortic slices derived f r o m various species were i n c u b a t e d with labeled fatty acids, a b o u t a third of the labeled c o m p o u n d w a s i n c o r p o r a t e d i n t o triacylglycerol [16]. With [3H]glyeerol as precursor, 4 0 - 5 0 % o f label were i n c o r p o r a t e d into triacylglycerol b y c u l t u r e d b o v i n e o r r a b b i t aortic s m o o t h muscle ceils exposed to f a t t y acid enriched s e r u m [10]. In h u m a n arterial s m o o t h muscle a m a r k e d increase in triacylglycerol c o n t e n t w a s r e p o r t e d w h e n the cells were c u l t u r e d in the presence of sera c o n t a i n i n g elevated nonesterified fatty acids [17]. T N F a p p e a r s to be a p l u r i p o t e n t c y t o k i n e w h i c h affects v a s c u l a r a n d n o n v a s c u l a r cells [5]. Hc,wever, the effect of T N F o n lipid m e t a b o l i s m w a s seen m a i n l y in the liver [6-9]. In the arterial a t h e r o s c l e r o t i c wall, T N F c o u l d be synthesized a n d secreted b y the m a c r o p h a g e [5] a n d the s m o o t h muscle cell itself [18]. A recent s t u d y has s h o w n t h a t h u m a n s a p h e n o u s vein s m o o t h muscle cells express the TNF-t~ gene a n d secrete biologically active T N F - a [18]. Secretion of T N F w a s also i n d u c e d b y l i p o p o l y s a c c h a r i d e in c u l t u r e d m e s e n c h y m a l cells derived f r o m n e w b o r n rat h e a r t [11]. T h e effect o f T N F o n triacylglycerol d e s c r i b e d in this s t u d y c o u l d b e relev a n t to the f i n d i n g of a n increased triacylglycerol c o n tent of h u m a n a o r t a with age [19]. In h u m a n a t h e r o m a t o u s aorta, relatively m o r e triacyiglycerol w a s f o u n d in fibrous p l a q u e s t h a n in fatty streaks [20]. T h i s c o u l d be related to the different cellular p o p u l a t i o n , i.e., in the fibrous p l a q u e there are m a i n l y s m o o t h muscle cell derived f o a m cells [21], while in the f a t t y s t r e a k they o r i g i n a t e f r o m m a c r o p h a g e s [22], w h i c h h a v e a m u c h l o w e r triacylglycerol c o n t e n t . Acknowledgement T h i s s t u d y w a s s u p p o r t e d in p a r t b y a g r a n t f r o m the M a r i o S h a p i r o F u n d , T h e H e b r e w University o f Jerusalem.
References 1 Boncher, C.J.F., Woodford, F.P., Ter Haar Romeny, C.Ch., Boelsma, E. and Van Gent, C.M. (1959) Nature, 183, 47-48. 2 Dayton, S., Hashimoto, S. and Pearce, M.L. (1962) Exp. Mol. Pathol. 1. 481-496. 3 Meyer, B,J., Meyer, A.C.. Pepler, W.J. and Theron, J.J. (1966) Am. Heart J. 71, 68-78. 4 Stein, O., Halperin. G. and Stein, Y. (~.981) Biochim. Biophys. Acta 665, 477-490. 5 Sherry, B. and Cerami, A. (1988) J. Cell Biol. 107, 1269-1277. 6 Feingold, K.R. and Granfeld, C. (1987) J. Clin. Invest. 80,184-190. 7 Feingold, K.R,, Soued, M., Serio. M.K., Moser, A.H., Dinarello, C. and Grunfeld, C. (1989) Endocrinology 125, 267-274. 8 Grunfeld, C., Wilking, H., Neese, R., Gavin, L.A., Moser, A.H.. Gulli, R., Serio, M.K. and Feingold, K.R. (1989) Cane. Res. 49, 2554-2560. 9 Chajek-Shaul, T.. Friedman, G., Stein, O., Shiloni, E., Etienne, J. and Stein, Y. (1989) Biochim. Biophys. Aeta 1001, 316-324. 10 Stein, O., Coetzee, G.A. and Stein, Y. (1981) Canad. J. Biochem. 59, 662-667. 11 Friedman, G., Gallily, R., Chajek-Shaal, T., Stein, O., Shiloni, E., Etienne, J. and Stein, Y. (1988) Biochim. Biophys. Acta 960, 220-228. 12 Hasin, Y., Sapoznikov, D.. Stein, O. and Stein. Y. (1982) J. Mol. Cell. CardioL 14, 163-172. 13 Havel, R.J., Eder, H.A. and Bragdon, J.H. (1955) J. Clin. Invest. 34, 1345-1353. 14 Foleh, J., Lees, M. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509. 15 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, RJ. (1951) J. Biol. Chem. 193, 265-275. 16 Stein, Y. and Stein, O. (1962) J. Alheroscler. Res. 2, 400-412. 17 Laughton, C.W., Ruddl¢, D.L,, Bedford, C..L and Alderman, E.L. (1988) Atherosclerosis 70, 233-246. 18 Warner, S.J.C. and Libby, P. 0989) J. Immunol. 142, 100-109. 19 Smith, E.B., Evans, P.H. and Downham, M.D. (1967) J. Atheroscler. Res. 7, 171-186. 20 Katz, S.S., Shipley, G.G. and Small, D.M. (1976) J. C~in. Invest. 58, 200-211. 21 Rosenfeld, M.E., Tsukada, T., Chait, A. et al. (1987) Arteriosclerosis 7, 24-34. 22 Stary, H.C. (1987) Atherosclerosis 64, 91-108.
