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Stimulation by zinc of cultured vascular endothelial cell proliferation: Possible involvement of endogenous basic fibroblast growth factor
Life Sciences, Vol. 55, No. 23, pp. 1781-1787, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All fights reserved 0024-3205/94 $6.00 + .00
Pergamon 0024-3205(94)00350-5
STIMULATION BY ZINC OF CULTURED VASCULAR ENDOTHELIAL CELL PROLIFERATION : POSSIBLE INVOLVEMENT OF ENDOGENOUS BASIC FIBROBLAST GROWTH FACTOR
T o s h i y u k i Kaji ~, Y a s u y u k i F u j i w a r a ~, Chika Yamamoto% Michiko Sakamoto ~ and Hiroshi Kozuka b ~Department of E n v i r o n m e n t a l Science, F a c u l t y of Pharmaceutical Sciences, H o k u r i k u U n i v e r s i t y , Kanazawa and bDepartment of Toxicology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical U n i v e r s i t y , Toyama, JAPAN
(Received in final form September 26, 1994)
Summary Vascular endothelial cells from b o v i n e aorta were c u l t u r e d w i t h zinc s u l f a t e (20 MM or less) f o r 24 h and labeled w i t h [ a H ] t h y m i d i n e or [ ' 4 C ] l e u c i n e d u r i n g the last 3 h of t h e c u l t u r e . I t was f o u n d t h a t the cell n u m b e r was s i g n i f i c a n t l y increased by zinc alone ; t h e i n c o r p o r a t i o n of both [ 3 H ] t h y m i d i n e and [ ' ~ C ] l e u c i n e was also stimulated by t h e metal. The s t i m u l a t i o n by zinc o c c u r r e d in t h e presence or absence of exogenous basic f i b r o b l a s t g r o w t h f a c t o r (bFGF). However, o t h e r heavy metals i n c l u d i n g copper, manganese and nickel did not e x h i b i t such a s t i m u l a t o r y effect, s u g g e s t i n g t h a t zinc plays a p a r t i c u l a r role in e n d o t h e l i a l cell p r o l i f e r a t i o n . Stimulation of the [ 3 H ] t h y m i d i n e incorporation by zinc disappeared in the presence of e i t h e r c y c l o h e x i m i d e or a n t i - b F G F IgG ; in a d d i t i o n , a l i p o x y g e n a s e i n h i b i t o r n o r d i h y d r o g u a i a r e t i c acid d i m i n i s h e d the zinc s t i m u l a t i o n but a c y c l o o x y g e n a s e i n h i b i t o r indomethacin did not e x h i b i t such an i n h i b i t o r y effect. These r e s u l t s indicated t h a t zinc stimulated t h e p r o l i f e r a t i o n of endothelial cells t h r o u g h the l i p o x y g e n a s e pathway w h i c h mediates t h e s t i m u l a t i o n by endogenous bFGF. I n o t h e r words, t h e metal may a m p l i f y t h e endogenous b F G F - d e p e n d e n t p r o l i f e r a t i o n of t h e cells.
Key Words: basic fibroblast growth factor, endothelial cells, zinc, vascular endothelial cells The p r o l i f e r a t i o n of v a s c u l a r endothelial cells is postulated to c o n t r i b u t e to p r e v e n t i o n of v a s c u l a r lesions such as atherosclerosis. When t h e v a s c u l a r endothelium is damaged, e n d o t h e l i a l cells will migrate and p r o l i f e r a t e at t h e damaged area to r e - e n d o t h e l i a l i z e . However, if t h e r e p a i r of e n d o t h e l i u m is i n s u f f i c i e n t , t h e s u b e n d o t h e l i a l collagen may i n d u c e platelet a g g r e g a t i o n (1) ; p l a t e l e t - d e r i v e d g r o w t h f a c t o r (2) will cause a h y p e r p l a s i a of v a s c u l a r smooth-muscle cells w h i c h is a component of e a r l y stage of a t h e r o s c l e r o s i s (3). Correspondence a d d r e s s : T o s h i y u k i Kaji, Department of E n v i r o n m e n t a l Science, F a c u l t y of Pharmaceutical Sciences, H o k u r i k u U n i v e r s i t y , Ho-3 Kanagawa-machi, Kanazawa 920-11, Japan.
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We have s t u d i e d t h e damage of v a s c u l a r e n d o t h e l i a l cells caused by heavy metals using a cell c u l t u r e system. I t was revealed t h a t an essential t r a c e element zinc p r o t e c t s t h e cells from c a d m i u m - i n d u c e d d e s t r u c t i o n of v a s c u l a r e n d o t h e l i a l cell l a y e r (4-6). We h y p o t h e s i z e d t h a t zinc may have p h y s i o l o g i c a l a c t i v i t i e s f o r t h e maintenance of v a s c u l a r e n d o t h e l i a l cell monolayer. The p r e s e n t s t u d y was u n d e r t a k e n to c l a r i f y t h e e f f e c t of zinc on t h e p r o l i f e r a tion of c u l t u r e d v a s c u l a r e n d o t h e l i a l cells. We f o u n d t h a t t h e metal has a st im u l a t o r y e f f e c t on t h e p rol i f e r a t i o n .
Materials and Methods Materials. Vascular e n d o t h e l i a l cells d e r i v e d from b o v i n e a o r t a (7) were a g i f t from Drs. Katsuo Sueishi and Y u t a k a Nakashima ( F i r s t Department of Pathology, Faculty of Medicine, Kyushu U n i v e r s i t y , Fukuoka, Japan). Dulbecco's modified Eagle's medium and fetal b o v i n e serum were purchased from Nissui Pharmaceutical (Tokyo, Japan) and Cell C u l t u r e L a b o r a t o r i e s (Cleveland, OH, USA), r e s p e c t i v e l y . ASF 301 medium which c o n t a i n s o n l y n e g l i g i b l e amount of zinc was from Ajinomoto (Tokyo, Japan). Tissue c u l t u r e plates and dishes were o b t a i n e d from Costar (Cambridge, MA, USA). [/v/ethy/-'aH]Thymidine (740 GBq/mmol) and L - [ ' 4 C ( U ) ] l e u c i n e (13.6 GBq/mmol) were from New England Nuclear (Boston, MA, USA). Recombinant human bFGF (bFGF) and its a n t i b o d y (anti-bFGF IgG) were p u r chased from Genzyme (Cambridge, MA, USA) and C o l l a b o r a t i v e Biomedical Products (Bedford, MA, USA), r e s p e c t i v e l y . Cycloheximide, n o r d i h y d r o g u a i a r e t i c a c i d (NDGA) and indomethacin were from Sigma Chemical (St. Louis, MO, USA). The MTT cell g r o w t h assay kit was purchased from Chemicon I n t e r n a t i o n a l (Temecula, CA, USA). Zinc sulfate, c o p p e r c h l o r i d e , manganese c h l o r i d e and o t h e r r e a g e n t s were from Wako Purechemical I n d u s t r i e s (Osaka, Japan). Cell number. Vascular e n d o t h e l i a l cells (passages 36-43) were plated i n t o 2 4 - w e l l c u l t u r e plates at 1 x 104 c e l l s / w e l l and c u l t u r e d f o r 24 h in in Dulbecco's modified Eagle's medium supplemented with 10% fetal b o v i n e serum at 37 °C in a h u m i d atmos p h e r e of 5% CO~ in air. At t h i s time, t h e attached cell number was a p p r o x i m a t e l y 1 x 104 c e l l s / w e l l . A f t e r c u l t u r e , th e medium was d is c a r d e d and t h e cells were washed t w i c e with s e r u m - f r e e ASF 301 medium. The cells were then inc u b a t e d at 37 °C f o r 24 h in 0.25 ml of f r e s h s e r u m - f r e e ASF 301 medium in the presence of zinc s u l f a t e at 2.0, 5.0, 10 or 20;~N. The cell number was d e t e r m i n e d by t he MTT assay. DNA and p r o t e i n s y n t h e s i s . Endothelial cells were plated into 6-well c u l t u r e plates at 1 x 10 ~ c e l l s / w e l l and c u l t u r e d f o r 24 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal b o v i n e serum. A f t e r c u l t u r e , t h e medium was d i s c a r d e d and t h e cells were washed twice with s e r u m - f r e e ASF 301 medium. The cells were t h e n i n c u b a te d at 37 °C f o r 24 h in 1.0 ml of f r e s h s e r u m - f r e e ASF 301 medium in t h e presence of zinc sulfate, c o p p e r c h l o r i d e , manganese c h l o r i d e or nickel c h l o r i d e at 2.0, 5.0, 10 or 20 ~M combined with or w i t h o u t bFGF at 0.1, 1.0 or 10 ng/ml and labeled with [ 3 H ] t h y m i d i n e at 10 kBq/ml or [ ' ~ C ] l e u c i n e at 25 kBq/ml d u r i n g t h e last 3 h of t h e i n c u b a t i o n . A f t e r i n c u b a t i o n , t he medium was dis c ar ded and t h e cells were washed twice with Ca, M g - f r e e p h o s p h a t e - b u f f e r e d saline (CMF-PBS); t h e cells were scraped o f f with a r u b b e r policeman in the presence of 0.75 ml of CMF-PBS. The c u l t u r e well was washed with 0.75 ml of CNF-PBS and t h e wash was combined with t h e cell suspension. The cell h o m o g e n a t e w a s p r e p a r e d by sonication and th e i n c o r p o r a t i o n of [ ~ H ] t h y m i d i n e or [~4C]leucine into t h e 5% t r i c h l o r o a c e t i c a c i d - i n s o l u b l e f r a c t i o n of an a l i q u o t of t h e cell homogenate was d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n c o u n t i n g . A p o r t i o n of t h e cell homogenate was used f o r t h e d e t e r m i n a t i o n of DNA c o n t e n t by t h e f l u o r o m e t r i c method (8). In a n -
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o t h e r e x p e ri me n t , e n d o t h e l i a l ceils were t r e a t e d with zinc s u l f a t e at 10 ~M combined with or w i t h o u t c y c l o h e x i m i d e at 1.0 ~M, a n ti -bFG F IgG at 5.0 ~, NDGA at 0.01, 0.1 or 1.0 ~g/ml or indomethacin at 0.01, 0.1 or 1.0 pg/ml; t h e i n c o r p o r a t i o n of [ ~ H ] t h y m i d i n e into t h e a c i d - i n s o l u b l e f r a c t i o n was determined in a similar way. Statistical analysis. Results were analyzed f o r statistical s i g n i f i c a n c e by S t u d e n t ' s t t e s t . P values of less than 0.05 were considered to i n d i c a t e s t a t i s t i c a l l y significant differences.
Resu Its F i g u r e 1 shows t h e e f f e c t of zinc s u l f a t e on t h e number of g r o w i n g e n d o t h e l i a l cells, zinc at 2.0 ~M or more s i g n i f i c a n t l y increased t h e cell number a f t e r a 24-h incubation. Since a s t i m u l a t o r y e f f e c t of zinc s u l f a t e on t he p r o l i f e r a t i o n of e n d o t h e l i a l cells was indicated, t h e i n c o r p o r a t i o n of [ ~ H ] t h y m i d i n e and [' 4C]leucine was examined as a marker of DNA and p r o t e i n s y n t h e s i s , r e s p e c t i v e l y . As shown in Fig. 2, zinc s i g n i f i c a n t l y increased t h e i n c o r p o r a t i o n of both [ 3 H ] t h y m i d i n e and [ ' 4 C ] l e u c i n e into t h e a c i d - i n s o l u b l e f r a c t i o n of e n d o t h e l i a l cells. I t is possible t h a t t h e s u l f a t e from zinc s u l f a t e is r e s p o n s i b l e f o r t h e p r o l i f e r a t i v e e f f e c t of zinc sulfate. To exclude t h i s p o s s i b i l i t y , t h e e f f e c t of zinc sulfate, zinc c h l o r i d e and sodium s u l f a t e on t he [ a H ] t h y m i d i n e i n c o r p o r a t i o n was examined and compared. The r e s u l t was as follows : c o n t r o l , 8,785 +_934 dpm/pg DNA (n=4); zinc s u l f a t e at 10 ~M, 26,127 + 1,523 dpm/~g DNA (rf=4, P
Fig. 1
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Effect of zinc on t h e number of g r o w i n g v a s c u l a r e n d o t h e l i a l cells. Growing c u l t u r e s of e n d o t h e l i a l cells were in c ubat ed at 37 °C f o r 24 h in t h e presence of zinc s u l fate at 2.0, 5.0, 10 or 20 ~M. Values ar e means + SE of f o u r samples. S i g n i f i c a n t l y d i f f e r e n t from t h e c o r r e s p o n d in g c o n t r o l , * P
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Vol. 55, No. 23, 1994
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Fig. 2 Effect of zinc on t h e i n c o r p o r a t i o n of [ ~ H ] t h y m i d i n e (left panel) and [ ' 4 C ] l e u c i n e ( r i g h t panel) into t h e a c i d - i n s o l u b l e f r a c t i o n of g r o w i n g vasc u l a r e n d o t h e l i a l cells. Growing c u l t u r e s of e n d o t h e l i a l cells were i n c u bated at 37 °C f o r 24 h in t h e presence of zinc s u l f a t e at 2.0, 5.0, 10 or 20 pM and labeled with [ ~ H ] t h y m i d i n e or [ ' 4 C ] l e u c i n e d u r i n g t h e last 3 h of t h e i n c u b a t i o n . Values a r e means _+ SE of f o u r samples. S i g n i f i c a n t l y d i f f e r e n t from t h e c o r r e s p o n d i n g c o n t r o l , **P
TABLE
I
Effect of Zinc, Copper, Manganese and Nickel on t h e I n c o r p o r a t i o n of [ 3 H ] T h y m i d i n e into t h e A c i d - I n s o l u b l e Fraction of Growing Vascular Endothelial Cells
[ ~ H ] T h y m i d i n e I n c o r p o r a t i o n (dpm/j]g DNA)
Control Zinc Copper Manganese Nickel
Absence of bFGF
Presence of bFGF
4,440 + 130 11,822 + 1,387. 4,647+ 632 3,935+ 600 3,615+ 307
6,795 + 477 27,862 _+2,107.~ 7,275+ 102 7,187+ 365 6,910+ 422
Growing c u l t u r e s of e n d o t h e l i a l cells were in c ubat ed at 37 °C f o r 24 h in t h e pres ence of zinc sulfate, c o p p e r c h l o r i d e , manganese c h l o r i d e or nickel c h l o r i d e at 10 uM each combined with or w i t h o u t bFGF at 10 ng/ml and labeled with [ 3 H ] t h y m i d i n e d u r i n g t h e last 3 h of t h e i n c u b a t i o n . Values are m e a n s + SE of f o u r samples. Sign i f i c a n t l y d i f f e r e n t from t h e c o r r e s p o n d i n g c o n t r o l , *P
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I
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Fig. 3 Effect of c y c l o h e x i m i d e (left panel) and anti-bFGF IgG ( r i g h t panel) on z i n c - i n d u c e d increase in th e i n c o r p o r a t i o n of [ ~ H ] t h y m i d i n e into t h e a c i d - i n s o l u b l e f r a c t i o n of g r o w i n g v a s c u l a r e n d o t h e l i a l cells. Growing c u l t u r e s of e n d o t h e l i a l cells were incubated at 37 °C f o r 24 h in t he presence of zinc s u l f a t e at 10 ~N combined with or w i t h o u t c y c l o h e x i m i d e at 1.0 jim or a n t i - b F G F IgG at 5.0 ~g/ml and labeled with [ ~ H ] t h y m i d i n e d u r i n g t h e last 3 h of t h e i n c u b a t i o n. Values are means + SE of f o u r samples. S i g n i f i c a n t l y d i f f e r e n t from t h e c o r r e s p o n d i n g c o n t r o l , * * * P
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NI3(3A (ixg/ml) Fig. 4 Effect of NDGA (left panel) and indomethacin ( r i g h t panel) on z i n c - i n d u c e d increase in t h e i n c o r p o r a t i o n of [ ~ H ] t h y m i d i n e into t h e a c i d - i n s o l u b l e f r a c t i o n of g r o w i n g v a s c u l a r e n d o t h e l i a l cells. Growing c u l t u r e s of e n d o t h e l i a l cells were i n c u b a t e d at 37 °C f o r 24 h in t h e presence of zinc s u l f a t e at 10 pM combined with or w i t h o u t NDGA at 0.01, 0.1 or 1.0 ~g/ml or indomethacin at 0.01, 0.1 or 1.0 Mg/ml and labeled with [ ~ H ] t h y m i d i n e d u r i n g t h e last 3 h of t h e i n c u b a t i o n . Values are means + SE of f o u r samples. S i g n i f i c a n t l y d i f f e r e n t from t he c o r r e s p o n d i n g c o n t r o l , *P
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F i g u r e 3 shows the e f f e c t of c y c l o h e x i m i d e and a n t i - b F G F IgG on z i n c - i n d u c e d increase in the [ ~ H ] t h y m i d i n e i n c o r p o r a t i o n in e n d o t h e l i a l cells. Stimulation of the [ ~ H ] t h y m i d i n e i n c o r p o r a t i o n by zinc completely disappeared in the presence of e i t h e r c y c l o h e x i m i d e or a n t i - b F G F IgG. Since b o v i n e serum albumin at 5 . 0 , g / m l did not d i s t u r b t h e zinc s t i m u l a t i o n (not shown), p o s s i b i l i t y of n o n s p e c i f i c b i n d i n g of zinc to the a n t i b o d y could be excluded. I t was r e p o r t e d t h a t the s t i m u l a t i o n by bFGF of e n d o t h e l i a l cell p r o l i f e r a t i o n is mediated by l i p o x y g e n a s e p r o d u c t s of a r a c h i d o n i c acid (9), so t h a t the e f f e c t of zinc in the presence of l i p o x y g e n a s e i n h i b i t o r NDGA was examined and compared to t h a t in t h e presence of c y c l o o x y g e n a s e i n h i b i t o r indomethacin. As shown in Fig. 4, zinc s t i m u l a t i o n of t h e [ ~ H ] t h y m i d i n e i n c o r p o r a t i o n was s i g n i f i c a n t l y reduced by NDGA. In c o n t r a s t , indomethacin failed to change the [ ~ H ] t h y m i d i n e i n c o r p o r a t i o n in the presence or absence of zinc.
Discussion
Zinc is an essential t r a c e element w h i c h has a v a r i e t y of roles in the mammalian system (10). The metal p a r t i c i p a t e s in enzymatic systems r e q u i r e d f o r DNA and RNA s y n t h e s i s (1 1). In fact, a s t i m u l a t o r y e f f e c t of zinc on bone g r o w t h has been shown in experimental animals (12) and c u l t u r e d bone (13). In t h e p r e s e n t s t u d y , we showed t h a t zinc stimulates t h e p r o l i f e r a t i o n of c u l t u r e d v a s c u l a r endothelial cells. T h i s c l e a r l y indicated t h a t one of the p h y s i o l o g i c a l f u n c t i o n s of zinc is to promote t h e g r o w t h of v a s c u l a r e n d o t h e l i a l cells. Since o t h e r heavy metals failed to i n d u c e such a s t i m u l a t i o n , zinc is suggested to be a p a r t i c u l a r metal f o r e n d o t h e l i a l cell proliferation. Vascular e n d o t h e l i a l cells p r o d u c e bFGF to promote t h e i r own g r o w t h (14). Blockage by both a p r o t e i n s y n t h e s i s i n h i b i t o r c y c l o h e x i m i d e and a n t i - b F G F IgG i n dicates an i n v o l v e m e n t of endogenous bFGF s y n t h e s i z e d d u r i n g t h e e x p o s u r e to zinc in the s t i m u l a t i o n by t h e metal. The mitogenic e f f e c t of bFGF on e n d o t h e l i a l cells was r e p o r t e d to be mediated by l i p o x y g e n a s e p r o d u c t s but not by c y c l o o x y g e n a s e p r o d u c t s of a r a c h i d o n i c acid (9). I n h i b i t i o n of zinc s t i m u l a t i o n of t h e [ ~ H ] t h y m i d i n e i n c o r p o r a t i o n by NDGA but not by indomethacin s u g g e s t s t h a t the mitogenic e f f e c t of zinc is also mediated by the l i p o x y g e n a s e pathway and s u p p o r t s o u r h y p o t h e s i s t h a t z i n c - i n d u c e d p r o l i f e r a t i o n of e n d o t h e l i a l cells was mediated at least p a r t l y by e n d o g e n o u s bFGF. However, the p o s s i b i l i t y t h a t o t h e r e n d o t h e l i a l cell g r o w t h f a c t o r s can i n t e r a c t w i t h zinc is not excluded. T h e r e is no signal sequences in bFGF molecule w h i c h would d i r e c t its release by t h e normal s e c r e t a r y p a t h w a y and no defined mechanism f o r release of t h e g r o w t h f a c t o r has been shown o t h e r t h a n cell death (15). In o t h e r words, one of t h e p h y s i ological role of bFGF is to r e p a i r t h e damage of v a s c u l a r e n d o t h e l i u m via s t i m u l a t i o n of e n d o t h e l i a l cell p r o l i f e r a t i o n . In t h e p r e s e n t s t u d y , it was shown t h a t s t i m u l a t i o n of the [ ~ H ] t h y m i d i n e i n c o r p o r a t i o n by exogenous bFGF was potentiated by zinc, s u g g e s t i n g t h a t zinc would e x h i b i t its s t i m u l a t o r y a c t i v i t y on e n d o t h e l i a l cell p r o l i f e r a t i o n when bFGF is s u p p l i e d from damaged e n d o t h e l i a l cells. In o t h e r words, zinc may be an a u g m e n t a t i v e f a c t o r of bFGF on the r e p a i r of damaged endothelium. In c o n c l u s i o n , it was revealed t h a t zinc has a s t i m u l a t o r y effect on v a s c u l a r e n d o t h e l i a l cell p r o l i f e r a t i o n . This s t i m u l a t i o n was suggested to be mediated at least p a r t l y by e n d o g e n o u s bFGF. A l t h o u g h t h e p h y s i o l o g i c a l a c t i v i t i e s of bFGF on e n d o t h e l i a l cell f u n c t i o n s i n c l u d i n g the p r o d u c t i o n of plasminogen a c t i v a t o r s (16, 17) and t h e i r i n h i b i t o r (18) also may be modulated by zinc, t h i s remains to be
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e l u c i d a t e d . Zinc may be one of t h e f a c t o r s w h i c h is i n v o l v e d in t h e r e p a i r of v a s c u l a r endotheli urn.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
I. MATSUOKA, N. NAKAHATA and H. NAKANISHI, Biochem. Pharmacol. 38 1941-1844
(1989). R. ROSS, E. W. RAINES and D. F. BOWEN-POPE, Cell 46 155-169 (1986). R. ROSS and J. A. GLOMSET, N. Engl. J. Med. 295 420-425 (1976). T. KAJI, A. MISHIMA, C. YAMAMOTO, M. SAKAMOTO and F. KOIZUMI, Toxicology 71 267-276 (1992). T. KAJI, A. MISHIMA, E. KOYANAGI, C. YAMAMOTO, M. SAKAMOTO and H. KOZUKA, Toxicology 7-6 257-270 (1992). T. KAJI, A. MISHIMA, C. YAMAMOTO, M. SAKAMOTO, H. KOZUKA, Toxicol. Lett. 6-6 247-255 (1993). Y. NAKASHIMA, K. SUEISHI and K. TANAKA, F i b r i n o l y s i s 2 227-234 (1988). J . M . KISSANE and E. ROBINS, J. Biol. Chem. 233 184-188 (1958). V. FAFEUR, Z. P. JIANG and P. BOHLEN, J. Cell Physiol. 149 277-283 (1991). R. E. BURCH, H. K. HAHN and J. F. SULLIVAN, Clin. Chem. 21 501-520 (1975). A . F . P A R I S I a n d B . L . VALEE, Am.J. Clin. Nutr. 221222-1230 (1969). l . YAMAGUCHI, A. MOCHIZUKI and S. OKADA, J. Pharm. Dyn. 5_619-626 (1982). M. YAMAGUCHI, H. OISHI and Y. SUKETA, Biochem. Pharmacol. 36 4007-4012 (1987). L. SCWEIGERER, D. G. NEWFELD, J. FRIEDMAN, J. A. ABRAHAM, J. C. FIDDES and D. GOSPODAROWICZ, Nature 325 257-259 (1987). D. B. RIFKIN and D. MOSCATELLI, J. Cell Biol. 109 1-6 (1989). C. YAMAMOTO, T. KAJI, M. FURUYA, N1. SAKAMOTO, H. KOZUKA and KOIZUMI, Thromb. Res. 73 255-263 (1994). Q. SAKSELA, D. MOSCATELLIand D. B. RIFKIN, J. Cell Biol. 105 957-963 (1987). T. KAJI, C. YAMAMOTO, M. SAKAMOTO, H. KOZUKA and F. KOIZUMI, Life Sci. 64 1563-1569 (1994).
Pergamon 0024-3205(94)00350-5
STIMULATION BY ZINC OF CULTURED VASCULAR ENDOTHELIAL CELL PROLIFERATION : POSSIBLE INVOLVEMENT OF ENDOGENOUS BASIC FIBROBLAST GROWTH FACTOR
T o s h i y u k i Kaji ~, Y a s u y u k i F u j i w a r a ~, Chika Yamamoto% Michiko Sakamoto ~ and Hiroshi Kozuka b ~Department of E n v i r o n m e n t a l Science, F a c u l t y of Pharmaceutical Sciences, H o k u r i k u U n i v e r s i t y , Kanazawa and bDepartment of Toxicology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical U n i v e r s i t y , Toyama, JAPAN
(Received in final form September 26, 1994)
Summary Vascular endothelial cells from b o v i n e aorta were c u l t u r e d w i t h zinc s u l f a t e (20 MM or less) f o r 24 h and labeled w i t h [ a H ] t h y m i d i n e or [ ' 4 C ] l e u c i n e d u r i n g the last 3 h of t h e c u l t u r e . I t was f o u n d t h a t the cell n u m b e r was s i g n i f i c a n t l y increased by zinc alone ; t h e i n c o r p o r a t i o n of both [ 3 H ] t h y m i d i n e and [ ' ~ C ] l e u c i n e was also stimulated by t h e metal. The s t i m u l a t i o n by zinc o c c u r r e d in t h e presence or absence of exogenous basic f i b r o b l a s t g r o w t h f a c t o r (bFGF). However, o t h e r heavy metals i n c l u d i n g copper, manganese and nickel did not e x h i b i t such a s t i m u l a t o r y effect, s u g g e s t i n g t h a t zinc plays a p a r t i c u l a r role in e n d o t h e l i a l cell p r o l i f e r a t i o n . Stimulation of the [ 3 H ] t h y m i d i n e incorporation by zinc disappeared in the presence of e i t h e r c y c l o h e x i m i d e or a n t i - b F G F IgG ; in a d d i t i o n , a l i p o x y g e n a s e i n h i b i t o r n o r d i h y d r o g u a i a r e t i c acid d i m i n i s h e d the zinc s t i m u l a t i o n but a c y c l o o x y g e n a s e i n h i b i t o r indomethacin did not e x h i b i t such an i n h i b i t o r y effect. These r e s u l t s indicated t h a t zinc stimulated t h e p r o l i f e r a t i o n of endothelial cells t h r o u g h the l i p o x y g e n a s e pathway w h i c h mediates t h e s t i m u l a t i o n by endogenous bFGF. I n o t h e r words, t h e metal may a m p l i f y t h e endogenous b F G F - d e p e n d e n t p r o l i f e r a t i o n of t h e cells.
Key Words: basic fibroblast growth factor, endothelial cells, zinc, vascular endothelial cells The p r o l i f e r a t i o n of v a s c u l a r endothelial cells is postulated to c o n t r i b u t e to p r e v e n t i o n of v a s c u l a r lesions such as atherosclerosis. When t h e v a s c u l a r endothelium is damaged, e n d o t h e l i a l cells will migrate and p r o l i f e r a t e at t h e damaged area to r e - e n d o t h e l i a l i z e . However, if t h e r e p a i r of e n d o t h e l i u m is i n s u f f i c i e n t , t h e s u b e n d o t h e l i a l collagen may i n d u c e platelet a g g r e g a t i o n (1) ; p l a t e l e t - d e r i v e d g r o w t h f a c t o r (2) will cause a h y p e r p l a s i a of v a s c u l a r smooth-muscle cells w h i c h is a component of e a r l y stage of a t h e r o s c l e r o s i s (3). Correspondence a d d r e s s : T o s h i y u k i Kaji, Department of E n v i r o n m e n t a l Science, F a c u l t y of Pharmaceutical Sciences, H o k u r i k u U n i v e r s i t y , Ho-3 Kanagawa-machi, Kanazawa 920-11, Japan.
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We have s t u d i e d t h e damage of v a s c u l a r e n d o t h e l i a l cells caused by heavy metals using a cell c u l t u r e system. I t was revealed t h a t an essential t r a c e element zinc p r o t e c t s t h e cells from c a d m i u m - i n d u c e d d e s t r u c t i o n of v a s c u l a r e n d o t h e l i a l cell l a y e r (4-6). We h y p o t h e s i z e d t h a t zinc may have p h y s i o l o g i c a l a c t i v i t i e s f o r t h e maintenance of v a s c u l a r e n d o t h e l i a l cell monolayer. The p r e s e n t s t u d y was u n d e r t a k e n to c l a r i f y t h e e f f e c t of zinc on t h e p r o l i f e r a tion of c u l t u r e d v a s c u l a r e n d o t h e l i a l cells. We f o u n d t h a t t h e metal has a st im u l a t o r y e f f e c t on t h e p rol i f e r a t i o n .
Materials and Methods Materials. Vascular e n d o t h e l i a l cells d e r i v e d from b o v i n e a o r t a (7) were a g i f t from Drs. Katsuo Sueishi and Y u t a k a Nakashima ( F i r s t Department of Pathology, Faculty of Medicine, Kyushu U n i v e r s i t y , Fukuoka, Japan). Dulbecco's modified Eagle's medium and fetal b o v i n e serum were purchased from Nissui Pharmaceutical (Tokyo, Japan) and Cell C u l t u r e L a b o r a t o r i e s (Cleveland, OH, USA), r e s p e c t i v e l y . ASF 301 medium which c o n t a i n s o n l y n e g l i g i b l e amount of zinc was from Ajinomoto (Tokyo, Japan). Tissue c u l t u r e plates and dishes were o b t a i n e d from Costar (Cambridge, MA, USA). [/v/ethy/-'aH]Thymidine (740 GBq/mmol) and L - [ ' 4 C ( U ) ] l e u c i n e (13.6 GBq/mmol) were from New England Nuclear (Boston, MA, USA). Recombinant human bFGF (bFGF) and its a n t i b o d y (anti-bFGF IgG) were p u r chased from Genzyme (Cambridge, MA, USA) and C o l l a b o r a t i v e Biomedical Products (Bedford, MA, USA), r e s p e c t i v e l y . Cycloheximide, n o r d i h y d r o g u a i a r e t i c a c i d (NDGA) and indomethacin were from Sigma Chemical (St. Louis, MO, USA). The MTT cell g r o w t h assay kit was purchased from Chemicon I n t e r n a t i o n a l (Temecula, CA, USA). Zinc sulfate, c o p p e r c h l o r i d e , manganese c h l o r i d e and o t h e r r e a g e n t s were from Wako Purechemical I n d u s t r i e s (Osaka, Japan). Cell number. Vascular e n d o t h e l i a l cells (passages 36-43) were plated i n t o 2 4 - w e l l c u l t u r e plates at 1 x 104 c e l l s / w e l l and c u l t u r e d f o r 24 h in in Dulbecco's modified Eagle's medium supplemented with 10% fetal b o v i n e serum at 37 °C in a h u m i d atmos p h e r e of 5% CO~ in air. At t h i s time, t h e attached cell number was a p p r o x i m a t e l y 1 x 104 c e l l s / w e l l . A f t e r c u l t u r e , th e medium was d is c a r d e d and t h e cells were washed t w i c e with s e r u m - f r e e ASF 301 medium. The cells were then inc u b a t e d at 37 °C f o r 24 h in 0.25 ml of f r e s h s e r u m - f r e e ASF 301 medium in the presence of zinc s u l f a t e at 2.0, 5.0, 10 or 20;~N. The cell number was d e t e r m i n e d by t he MTT assay. DNA and p r o t e i n s y n t h e s i s . Endothelial cells were plated into 6-well c u l t u r e plates at 1 x 10 ~ c e l l s / w e l l and c u l t u r e d f o r 24 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal b o v i n e serum. A f t e r c u l t u r e , t h e medium was d i s c a r d e d and t h e cells were washed twice with s e r u m - f r e e ASF 301 medium. The cells were t h e n i n c u b a te d at 37 °C f o r 24 h in 1.0 ml of f r e s h s e r u m - f r e e ASF 301 medium in t h e presence of zinc sulfate, c o p p e r c h l o r i d e , manganese c h l o r i d e or nickel c h l o r i d e at 2.0, 5.0, 10 or 20 ~M combined with or w i t h o u t bFGF at 0.1, 1.0 or 10 ng/ml and labeled with [ 3 H ] t h y m i d i n e at 10 kBq/ml or [ ' ~ C ] l e u c i n e at 25 kBq/ml d u r i n g t h e last 3 h of t h e i n c u b a t i o n . A f t e r i n c u b a t i o n , t he medium was dis c ar ded and t h e cells were washed twice with Ca, M g - f r e e p h o s p h a t e - b u f f e r e d saline (CMF-PBS); t h e cells were scraped o f f with a r u b b e r policeman in the presence of 0.75 ml of CMF-PBS. The c u l t u r e well was washed with 0.75 ml of CNF-PBS and t h e wash was combined with t h e cell suspension. The cell h o m o g e n a t e w a s p r e p a r e d by sonication and th e i n c o r p o r a t i o n of [ ~ H ] t h y m i d i n e or [~4C]leucine into t h e 5% t r i c h l o r o a c e t i c a c i d - i n s o l u b l e f r a c t i o n of an a l i q u o t of t h e cell homogenate was d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n c o u n t i n g . A p o r t i o n of t h e cell homogenate was used f o r t h e d e t e r m i n a t i o n of DNA c o n t e n t by t h e f l u o r o m e t r i c method (8). In a n -
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o t h e r e x p e ri me n t , e n d o t h e l i a l ceils were t r e a t e d with zinc s u l f a t e at 10 ~M combined with or w i t h o u t c y c l o h e x i m i d e at 1.0 ~M, a n ti -bFG F IgG at 5.0 ~, NDGA at 0.01, 0.1 or 1.0 ~g/ml or indomethacin at 0.01, 0.1 or 1.0 pg/ml; t h e i n c o r p o r a t i o n of [ ~ H ] t h y m i d i n e into t h e a c i d - i n s o l u b l e f r a c t i o n was determined in a similar way. Statistical analysis. Results were analyzed f o r statistical s i g n i f i c a n c e by S t u d e n t ' s t t e s t . P values of less than 0.05 were considered to i n d i c a t e s t a t i s t i c a l l y significant differences.
Resu Its F i g u r e 1 shows t h e e f f e c t of zinc s u l f a t e on t h e number of g r o w i n g e n d o t h e l i a l cells, zinc at 2.0 ~M or more s i g n i f i c a n t l y increased t h e cell number a f t e r a 24-h incubation. Since a s t i m u l a t o r y e f f e c t of zinc s u l f a t e on t he p r o l i f e r a t i o n of e n d o t h e l i a l cells was indicated, t h e i n c o r p o r a t i o n of [ ~ H ] t h y m i d i n e and [' 4C]leucine was examined as a marker of DNA and p r o t e i n s y n t h e s i s , r e s p e c t i v e l y . As shown in Fig. 2, zinc s i g n i f i c a n t l y increased t h e i n c o r p o r a t i o n of both [ 3 H ] t h y m i d i n e and [ ' 4 C ] l e u c i n e into t h e a c i d - i n s o l u b l e f r a c t i o n of e n d o t h e l i a l cells. I t is possible t h a t t h e s u l f a t e from zinc s u l f a t e is r e s p o n s i b l e f o r t h e p r o l i f e r a t i v e e f f e c t of zinc sulfate. To exclude t h i s p o s s i b i l i t y , t h e e f f e c t of zinc sulfate, zinc c h l o r i d e and sodium s u l f a t e on t he [ a H ] t h y m i d i n e i n c o r p o r a t i o n was examined and compared. The r e s u l t was as follows : c o n t r o l , 8,785 +_934 dpm/pg DNA (n=4); zinc s u l f a t e at 10 ~M, 26,127 + 1,523 dpm/~g DNA (rf=4, P
Fig. 1
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Coneentration( ~M)
Effect of zinc on t h e number of g r o w i n g v a s c u l a r e n d o t h e l i a l cells. Growing c u l t u r e s of e n d o t h e l i a l cells were in c ubat ed at 37 °C f o r 24 h in t h e presence of zinc s u l fate at 2.0, 5.0, 10 or 20 ~M. Values ar e means + SE of f o u r samples. S i g n i f i c a n t l y d i f f e r e n t from t h e c o r r e s p o n d in g c o n t r o l , * P
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<
Vol. 55, No. 23, 1994
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Fig. 2 Effect of zinc on t h e i n c o r p o r a t i o n of [ ~ H ] t h y m i d i n e (left panel) and [ ' 4 C ] l e u c i n e ( r i g h t panel) into t h e a c i d - i n s o l u b l e f r a c t i o n of g r o w i n g vasc u l a r e n d o t h e l i a l cells. Growing c u l t u r e s of e n d o t h e l i a l cells were i n c u bated at 37 °C f o r 24 h in t h e presence of zinc s u l f a t e at 2.0, 5.0, 10 or 20 pM and labeled with [ ~ H ] t h y m i d i n e or [ ' 4 C ] l e u c i n e d u r i n g t h e last 3 h of t h e i n c u b a t i o n . Values a r e means _+ SE of f o u r samples. S i g n i f i c a n t l y d i f f e r e n t from t h e c o r r e s p o n d i n g c o n t r o l , **P
TABLE
I
Effect of Zinc, Copper, Manganese and Nickel on t h e I n c o r p o r a t i o n of [ 3 H ] T h y m i d i n e into t h e A c i d - I n s o l u b l e Fraction of Growing Vascular Endothelial Cells
[ ~ H ] T h y m i d i n e I n c o r p o r a t i o n (dpm/j]g DNA)
Control Zinc Copper Manganese Nickel
Absence of bFGF
Presence of bFGF
4,440 + 130 11,822 + 1,387. 4,647+ 632 3,935+ 600 3,615+ 307
6,795 + 477 27,862 _+2,107.~ 7,275+ 102 7,187+ 365 6,910+ 422
Growing c u l t u r e s of e n d o t h e l i a l cells were in c ubat ed at 37 °C f o r 24 h in t h e pres ence of zinc sulfate, c o p p e r c h l o r i d e , manganese c h l o r i d e or nickel c h l o r i d e at 10 uM each combined with or w i t h o u t bFGF at 10 ng/ml and labeled with [ 3 H ] t h y m i d i n e d u r i n g t h e last 3 h of t h e i n c u b a t i o n . Values are m e a n s + SE of f o u r samples. Sign i f i c a n t l y d i f f e r e n t from t h e c o r r e s p o n d i n g c o n t r o l , *P
Vol. 55, No. 23, 1994
Zinc and Endothelial Cell Proliferation
1785
I
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Fig. 3 Effect of c y c l o h e x i m i d e (left panel) and anti-bFGF IgG ( r i g h t panel) on z i n c - i n d u c e d increase in th e i n c o r p o r a t i o n of [ ~ H ] t h y m i d i n e into t h e a c i d - i n s o l u b l e f r a c t i o n of g r o w i n g v a s c u l a r e n d o t h e l i a l cells. Growing c u l t u r e s of e n d o t h e l i a l cells were incubated at 37 °C f o r 24 h in t he presence of zinc s u l f a t e at 10 ~N combined with or w i t h o u t c y c l o h e x i m i d e at 1.0 jim or a n t i - b F G F IgG at 5.0 ~g/ml and labeled with [ ~ H ] t h y m i d i n e d u r i n g t h e last 3 h of t h e i n c u b a t i o n. Values are means + SE of f o u r samples. S i g n i f i c a n t l y d i f f e r e n t from t h e c o r r e s p o n d i n g c o n t r o l , * * * P
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NI3(3A (ixg/ml) Fig. 4 Effect of NDGA (left panel) and indomethacin ( r i g h t panel) on z i n c - i n d u c e d increase in t h e i n c o r p o r a t i o n of [ ~ H ] t h y m i d i n e into t h e a c i d - i n s o l u b l e f r a c t i o n of g r o w i n g v a s c u l a r e n d o t h e l i a l cells. Growing c u l t u r e s of e n d o t h e l i a l cells were i n c u b a t e d at 37 °C f o r 24 h in t h e presence of zinc s u l f a t e at 10 pM combined with or w i t h o u t NDGA at 0.01, 0.1 or 1.0 ~g/ml or indomethacin at 0.01, 0.1 or 1.0 Mg/ml and labeled with [ ~ H ] t h y m i d i n e d u r i n g t h e last 3 h of t h e i n c u b a t i o n . Values are means + SE of f o u r samples. S i g n i f i c a n t l y d i f f e r e n t from t he c o r r e s p o n d i n g c o n t r o l , *P
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F i g u r e 3 shows the e f f e c t of c y c l o h e x i m i d e and a n t i - b F G F IgG on z i n c - i n d u c e d increase in the [ ~ H ] t h y m i d i n e i n c o r p o r a t i o n in e n d o t h e l i a l cells. Stimulation of the [ ~ H ] t h y m i d i n e i n c o r p o r a t i o n by zinc completely disappeared in the presence of e i t h e r c y c l o h e x i m i d e or a n t i - b F G F IgG. Since b o v i n e serum albumin at 5 . 0 , g / m l did not d i s t u r b t h e zinc s t i m u l a t i o n (not shown), p o s s i b i l i t y of n o n s p e c i f i c b i n d i n g of zinc to the a n t i b o d y could be excluded. I t was r e p o r t e d t h a t the s t i m u l a t i o n by bFGF of e n d o t h e l i a l cell p r o l i f e r a t i o n is mediated by l i p o x y g e n a s e p r o d u c t s of a r a c h i d o n i c acid (9), so t h a t the e f f e c t of zinc in the presence of l i p o x y g e n a s e i n h i b i t o r NDGA was examined and compared to t h a t in t h e presence of c y c l o o x y g e n a s e i n h i b i t o r indomethacin. As shown in Fig. 4, zinc s t i m u l a t i o n of t h e [ ~ H ] t h y m i d i n e i n c o r p o r a t i o n was s i g n i f i c a n t l y reduced by NDGA. In c o n t r a s t , indomethacin failed to change the [ ~ H ] t h y m i d i n e i n c o r p o r a t i o n in the presence or absence of zinc.
Discussion
Zinc is an essential t r a c e element w h i c h has a v a r i e t y of roles in the mammalian system (10). The metal p a r t i c i p a t e s in enzymatic systems r e q u i r e d f o r DNA and RNA s y n t h e s i s (1 1). In fact, a s t i m u l a t o r y e f f e c t of zinc on bone g r o w t h has been shown in experimental animals (12) and c u l t u r e d bone (13). In t h e p r e s e n t s t u d y , we showed t h a t zinc stimulates t h e p r o l i f e r a t i o n of c u l t u r e d v a s c u l a r endothelial cells. T h i s c l e a r l y indicated t h a t one of the p h y s i o l o g i c a l f u n c t i o n s of zinc is to promote t h e g r o w t h of v a s c u l a r e n d o t h e l i a l cells. Since o t h e r heavy metals failed to i n d u c e such a s t i m u l a t i o n , zinc is suggested to be a p a r t i c u l a r metal f o r e n d o t h e l i a l cell proliferation. Vascular e n d o t h e l i a l cells p r o d u c e bFGF to promote t h e i r own g r o w t h (14). Blockage by both a p r o t e i n s y n t h e s i s i n h i b i t o r c y c l o h e x i m i d e and a n t i - b F G F IgG i n dicates an i n v o l v e m e n t of endogenous bFGF s y n t h e s i z e d d u r i n g t h e e x p o s u r e to zinc in the s t i m u l a t i o n by t h e metal. The mitogenic e f f e c t of bFGF on e n d o t h e l i a l cells was r e p o r t e d to be mediated by l i p o x y g e n a s e p r o d u c t s but not by c y c l o o x y g e n a s e p r o d u c t s of a r a c h i d o n i c acid (9). I n h i b i t i o n of zinc s t i m u l a t i o n of t h e [ ~ H ] t h y m i d i n e i n c o r p o r a t i o n by NDGA but not by indomethacin s u g g e s t s t h a t the mitogenic e f f e c t of zinc is also mediated by the l i p o x y g e n a s e pathway and s u p p o r t s o u r h y p o t h e s i s t h a t z i n c - i n d u c e d p r o l i f e r a t i o n of e n d o t h e l i a l cells was mediated at least p a r t l y by e n d o g e n o u s bFGF. However, the p o s s i b i l i t y t h a t o t h e r e n d o t h e l i a l cell g r o w t h f a c t o r s can i n t e r a c t w i t h zinc is not excluded. T h e r e is no signal sequences in bFGF molecule w h i c h would d i r e c t its release by t h e normal s e c r e t a r y p a t h w a y and no defined mechanism f o r release of t h e g r o w t h f a c t o r has been shown o t h e r t h a n cell death (15). In o t h e r words, one of t h e p h y s i ological role of bFGF is to r e p a i r t h e damage of v a s c u l a r e n d o t h e l i u m via s t i m u l a t i o n of e n d o t h e l i a l cell p r o l i f e r a t i o n . In t h e p r e s e n t s t u d y , it was shown t h a t s t i m u l a t i o n of the [ ~ H ] t h y m i d i n e i n c o r p o r a t i o n by exogenous bFGF was potentiated by zinc, s u g g e s t i n g t h a t zinc would e x h i b i t its s t i m u l a t o r y a c t i v i t y on e n d o t h e l i a l cell p r o l i f e r a t i o n when bFGF is s u p p l i e d from damaged e n d o t h e l i a l cells. In o t h e r words, zinc may be an a u g m e n t a t i v e f a c t o r of bFGF on the r e p a i r of damaged endothelium. In c o n c l u s i o n , it was revealed t h a t zinc has a s t i m u l a t o r y effect on v a s c u l a r e n d o t h e l i a l cell p r o l i f e r a t i o n . This s t i m u l a t i o n was suggested to be mediated at least p a r t l y by e n d o g e n o u s bFGF. A l t h o u g h t h e p h y s i o l o g i c a l a c t i v i t i e s of bFGF on e n d o t h e l i a l cell f u n c t i o n s i n c l u d i n g the p r o d u c t i o n of plasminogen a c t i v a t o r s (16, 17) and t h e i r i n h i b i t o r (18) also may be modulated by zinc, t h i s remains to be
Vol. 55, No. 23, 1994
Zinc and Endothelial Cell Proliferation
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e l u c i d a t e d . Zinc may be one of t h e f a c t o r s w h i c h is i n v o l v e d in t h e r e p a i r of v a s c u l a r endotheli urn.
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