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Effect of vitamin E on the development of atherosclerosis
Toxicology 148 (2000) 179 – 185 www.elsevier.com/locate/toxicol
Effect of vitamin E on the development of atherosclerosis Nesrin Kartal O8 zer a,*, Angelo Azzi b a
Department of Biochemistry, Faculty of Medicine, Marmara Uni6ersity, 81326 Haydarpasa, Istanbul, Turkey b Institute of Biochemistry and Molecular Biology, Uni6ersity of Bern, 3012 Bern, Switzerland Accepted 14 September 1999
Abstract The development of atherosclerosis is a multifactorial process in which both elevated plasma cholesterol levels and proliferation of smooth muscle cells play a central role. Numerous studies have suggested the involvement of oxidative processes in the pathogenesis of atherosclerosis and especially of oxidised low density lipoproteins. Some epidemiological studies have shown an association between high dietary intake or high serum concentrations of vitamin E and lower rates of ischemic heart disease. Recently, the Cambridge Heart Antioxidant Study (CHAOS) reported strong protection by high vitamin E doses against the risk of fatal and non fatal myocardial infarction. Here we have shown that incubation of vascular smooth muscle cells in the presence of a-tocopherol resulted in inhibition of cell proliferation and protein kinase C activity. Since b-tocopherol and probucol are not inhibitory, the effect of a-tocopherol is considered due to a non-oxidant mechanism. In order to understand the protective role of a-tocopherol against atherosclerosis in vivo the following rabbit studies were carried out. Atherosclerosis was induced by a vitamin E poor diet containing 2% cholesterol in a group of rabbit. The other groups had 2% cholesterol in the diet plus 50 mg/kg vitamin E i.m. or 1% probucol or 50 mg/kg vitamin E plus 1% probucol. After 4 weeks, aortas were removed and analysed by microscopy for atherosclerotic lesions. Samples of the media were analysed for protein kinase C activity. The aortas of cholesterol-fed rabbits showed typical atherosclerotic lesions, detected by microscopic examination, their media smooth muscle cells exhibited an increase in protein kinase C activity. Vitamin E fully prevented cholesterol induced atherosclerotic lesions and the induction of protein kinase C activity while probucol was not effective. These results show that the protective effect of vitamin E against hypercholesterolemic atherosclerosis is not produced by an other antioxidant such as probucol and, therefore, may not be linked to the antioxidant properties of this vitamin. The effects observed at the level of smooth muscle cells in vitro and ex-vivo suggests an involvement of signal transduction events in the protective effect of vitamin E against atherosclerosis. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vitamin E; Probucol; Protein kinase C; Vascular smooth muscle cell; Atherosclerosis
1. Introduction * Corresponding author. Tel.: + 90-216-4144733; fax: +90216-4181047. E-mail address: [email protected] (N.K. O8 zer).
The development of atherosclerosis is a multifactorial process in which both elevated plasma
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cholesterol levels and proliferation of smooth muscle cells play a central role. Numerous studies have suggested the involvement of oxidative processes in the pathogenesis of atherosclerosis and especially of oxidised low-density lipoprotein (Raines and Ross, 1993; Ross, 1993; Steinberg, 1995). Some epidemiological studies have shown an association between high dietary intake and high serum concentrations of vitamin E and lower rates of ischemic heart disease (Gey, 1990; Gey et al., 1993; Rimm et al., 1993; Stampfer et al., 1993). The Cambridge Heart Antioxidant Study (CHAOS) reported strong protection by high vitamin E doses against the risk of fatal and non fatal myocardial infarction (Stephens et al., 1996). One of the mechanisms of a-tocopherol action is to protect low-density lipoproteins from oxidation by peroxy-radicals (Carew et al., 1987; Esterbauer et al., 1992). Smooth muscle cell proliferation is controlled by growth factor receptors, which, via a cascade of kinases and phosphatases, produce activation and expression of proteins necessary for the progression and completion of the cell cycle (Muller et al., 1993). As a part of the main signal transduction path initiated by growth factors or hormones (Azzi et al., 1992), protein kinase C has a complex activation mechanism superimposed to a permissive phosphorylation (Bornfeldt et al., 1994; Dutil et al., 1994; Newton, 1995; Bornancin and Parker, 1996). Cell culture studies have shown that a-tocopherol brings about inhibition of smooth muscle cell proliferation. The latter event takes place via inhibition of protein kinase C activity. a-Tocopherol also inhibits low density lipoprotein stimulated smooth muscle cell proliferation and protein kinase C activity (O8 zer et al., 1993). Moreover, increased protein kinase C activity and expression in atherosclerotic rabbits was also shown (Sirikci et al., 1996). The following cell culture and animal studies were carried out to investigate the mechanism of a-tocopherol action on smooth muscle cell proliferation and on the protection against atherosclerosis; the effect of a-tocopherol was also compared with that of b-tocopherol and probucol.
2. Materials and methods Tissue culture materials were purchased from Falcon Labware (Becton Dickinson) and growth media and serum for cell culture obtained from Gibco Laboratories. [g-32P]ATP was from Amersham International. D-a-Tocopherol and D-b-tocopherol were generous gifts from Hofmann La Roche (Basel). The peptide FKKSFKL (custom synthesised by Dr Rolli, Institut fu¨r Klinische Immunologie, Inselspital Bern) was used as a substrate for the PKC assay. Vitamin E-poor rabbit diet and vitamin E (Ephynal) was kindly donated from Hoffman La Roche (Basel, Switzerland and Istanbul, Turkey), and probucol was a gift from Marion Merrell Dow Pharmaceuticals, Switzerland. The peptide PLSRTLSVAAKK, used as a substrate for the protein kinase C assay, was synthesised by Dr C. Servis, Epalinges (Switzerland). The kit for measurement of protein kinase C activity was obtained from Upstate Biotechnology, Lake Placid, NY, USA. All other chemicals used were of the purest grade commercially available.
2.1. Cell culture studies The cell line used in this work, A7r5 rat aortic smooth muscle cells was obtained from the American Type Culture Collection. The cells were grown in Dulbecco’s modified minimum Eagle medium. Following 48 h serum deprivation the cells were restimulated to growth by addition of 10% fetal calf serum (FCS) in the presence or absence of D-a, D-b-tocopherols and probucol. The cells were detached by trypsinization and counted in a hemocytometer in triplicate. The viability of the cells was assessed by the trypan blue dye exclusion method. PKC activity was measured in permeabilized smooth muscle cells according to the procedure of Alexander et al. (1990). Briefly the cells after 48 h serum deprivation were incubated for 7 h in the presence or absence of 50 mM D-a-tocopherol, D-b-tocopherol and probucol and for the last 2 h in the presence (when specified) of 100 nM phorbol myristate acetate (PMA). Afterwards they were washed and assayed for PKC activity by
N.K. O8 zer, A. Azzi / Toxicology 148 (2000) 179–185
adding [g-32P]ATP (250 mM), peptide substrate (100 mM) and streptolysin-O (0.6 I.U.). After 10 min incubation at 37°C the reaction was stopped by 100 ml trichloroacetic acid and the samples centrifuged for 5 min and spotted on P81 ion exchange chromotography paper and the radioactivity counted. The background phosphorylation, measured in the absence of substrate, was subtracted.
Fig. 1. Differential effects of a-tocopherol and b-tocopherol on smooth muscle cell proliferation and protein kinase C activity. (A) Quiescent cells are restimulated to grow with FCS (10%) in the presence of a-tocopherol and/or b-tocopherol (50 mM). After 48 h restimulation cells were counted with a hemocytometer. Viability was greater than 95%. (B) Quiescent cells are restimulated to grow with FCS (10%) in the presence of a-tocopherol and/or b-tocopherol (50 mM). After 7 h restimulation the cells are permeabilized and protein kinase C is measured as described in Section 2. The basal kinase activity is subtracted in all samples and only the PMA-stimulated activity is shown.
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2.2. Animal experiments Thirty male New Zealand albino rabbits of 2–4 months of age were used in this study. They were fed 100 g per day of a diet poor in vitamin E. Probucol and cholesterol were added to the diet as diethyl ether solution. The control diet was treated with the same amount of pure solvent. All diets were dried of the solvent before use. The concentrations of cholesterol, vitamin E and probucol used were based on previous literature reports (Stein et al., 1989; Ferns et al., 1992; Bocan et al., 1993; Mantha et al., 1993; Konneh et al., 1995). The rabbits were randomly assigned to one of the following six groups. One group of rabbits was only fed with the diet, without additions or treatments. The second group received injections of 50 mg/kg vitamin E intramuscularly on alternate rear legs, once daily. The diet of the third group contained 2% cholesterol. The diet of the fourth group 2% cholesterol and the rabbits received injections of 50 mg/kg vitamin E per day. The fifth group of rabbits received 1% probucol in addition to 2% cholesterol in the daily diet. The sixth group had 2% cholesterol and 1% probucol in the diet and was injected with 50 mg/kg vitamin E per day. After 4 weeks plasma samples were taken and thoracic aortas were removed. Samples were taken from the media of thoracic aortas to measure smooth muscle cell protein kinase C activity by using a non-radioactive kit (Upstate Biotechnology). The plasma cholesterol level were determined using an automated enzymatic technique. Vitamin E levels were measured by reverse phase high-pressure liquid chromatography (Nierenberg and Nann, 1992).
3. Results The effect of a-tocopherol on smooth muscle cell proliferation and protein kinase C is shown in Fig. 1. a-Tocopherol, at concentrations of 50 mM, inhibits rat A7r5 smooth muscle cell proliferation, while b-tocopherol is ineffective. When a-tocopherol and b-tocopherol are added together, no inhibition of cell growth is seen (Fig. 1A). Both
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Fig. 2. Effect of probucol on smooth muscle cell proliferation and protein kinase C activity. Quiescent cells are restimulated to grow with FCS (10%) in the presence of the indicated amounts of probucol. Cell proliferation and protein kinase C activity were measured as indicated in Section 2.
Table 1 Effect of cholesterol, probucol and vitamin E treatment on their plasma levels in rabbitsa Group
Cholesterol (mM)
Vitamin E (mM)
Control Vitamin E Cholesterol Cholesterol +vitamin E Cholesterol +probucol Cholesterol +probucol +vitamin E
1.0 9 0.5 0.9 90.1 21.1 910.7* 12.4 96.1*
5.2 9 2.4 50.7 9 10.2* 14.69 6.5** 216.29 70.2*
13.7 9 6.6*
12.49 4.5**
11.69 3.5*
254.79 64.0*
a The plasma level of cholesterol and vitamin E have been measured in all the five rabbits of the six diet groups. The numbers (mean9 S.D.) represent the plasma values measured after 1 month of diet. * PB0.01. ** Not statistically significant.
compounds are transported equally in cells and do not compete with each other for the uptake. The prevention by b-tocopherol of the growth inhibition by a-tocopherol suggests that a site-directed event is the basis of a-tocopherol inhibition rather than a general radical scavenging reaction. In smooth muscle cells permeabilized with streptolysin-O, to permit the entry of a peptide substrate (Fig. 1B), a-tocopherol inhibits protein kinase C activity, whereas b-tocopherol is ineffective. When both are present b-tocopherol prevents the inhibitory effect of a-tocopherol. Fig. 2 addresses the question whether or not probucol, a potent lipophilic antioxidant, exerts an effect on smooth muscle cell proliferation and protein kinase C activity. Probucol (10–50 mM) does not inhibit smooth muscle cell proliferation and protein kinase C activity. The following animal experiments addresses the question if vitamin E can prevent development of atherosclerosis in vivo and whether probucol can mimic the effect of vitamin E. Cholesterol and vitamin E plasma concentrations of the six animal groups are shown in Table 1. The 2% cholesterol diet supplementation for 4 weeks resulted in an approximately 20-fold increase of plasma cholesterol. After additional supplementation with vitamin E or probucol, plasma cholesterol increased 12- and 13-fold, respectively, relative to control. Plasma vitamin E concentrations were higher in the cholesterol fed rabbits. In this study (Table 2) in the total absence of vitamin E in the diet, the activity of smooth muscle cell protein kinase C was 8.4 D-absorbance units/min per mg protein. After vitamin E treatment an approximately 50% reduction in protein kinase C activity was seen. With cholesterol supplementation the activity of protein kinase C increased to 10.2 D-absorbance units/min per mg protein. Vitamin E treatment was able in this case to reduce the values for protein kinase C activity to those measured in the absence of cholesterol (4.5 D-absorbance units/ min per mg protein). Smooth muscle cells from probucol plus cholesterol treated rabbits showed a protein kinase C activity lower than that in the presence of cholesterol alone, although the data were not statistically significant. Instead, when the rabbits were also treated with vitamin E the
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protein kinase C value decreased to 5.2 D-absorbance units/min per mg protein and reached significance relative to the cholesterol fed rabbit group.
4. Discussion An early event in the development of atherosclerosis is the intimal deposition of lipids, including cholesterol. This deposition arises from a combination of extra- and intra-cellular processes and has been shown, in some animal models, to be inhibited by antioxidants (Esterbauer et al., 1992). We have observed a specific inhibition of proliferation in vitro of smooth muscle cells by a-tocopherol at physiological concentrations. Of particular relevance was the finding of an antiproliferative effect of a-tocopherol on rat and human
Table 2 Protein kinase C activity from smooth muscle cell homogenates obtained from differently treated rabbitsa Group
Total protein kinase C activity (D-absorbance/min per mg protein)
Control Vitamin E Cholesterol Cholesterol +vitamin E Cholesterol +probucol Cholesterol +probucol +vitamin E
8.49 1.1 4.5 92.5 10.2 9 2.4 4.5 91.0
B0.02c
6.89 1.8
NSc
5.2 91.4
B0.04c
P
B0.01b
a Aortic medias were minced, homogenised and nuclei sedimented by centrifugation. Supernatants were centrifuged again at 100 000×g to obtain cytosolic fractions. Pellets were used for preparation of membrane fractions. Protein kinase C activity was measured in both fractions. Since they did not show significant membrane/cytosol distribution changes, only the values of total protein kinase C activity are reported. Results are expressed as mean 9S.D. (n= 5). NS, not statistically significant. b Referres to the control group. c Referres to the cholesterol group.
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smooth muscle cells. Proliferation of these cells is fundamental in the onset of accelerated atherosclerosis, after cardiac transplantation and restenosis. Multiple pathways are involved in the proliferative response of smooth muscle cell to an extracellular stimulus, which has rendered it difficult to identify precise targets for antiproliferative agents. One of the important elements in signal transduction cascades is protein kinase C and its inhibition appears to be sufficient to cause inhibition of smooth muscle cell proliferation in vitro. The inhibition of smooth muscle cell proliferation in vitro by a-tocopherol at physiological concentrations may explain the finding that in vivo smooth muscle cell only proliferate under stress situations (Clowes and Schwartz, 1985; Raines and Ross, 1993). A local or generalised diminution of a-tocopherol concentration, caused by dietary or oxidative factors, can lead to cell growth stimulation and atherosclerosis progress. b-Tocopherol, an antioxidant almost as potent as a-tocopherol, not only does not show any effect at the level of cell proliferation or protein kinase C activity, but rather it prevents the effects of a-tocopherol. The oxidised product of a-tocopherol, a-tocopherylquinone, is not inhibitory of smooth muscle cell proliferation (Azzi et al., 1995).Thus, it is legitimate to conclude that the mechanism of action of a-tocopherol, as a regulator of smooth muscle cell proliferation, is not due to its antioxidant properties. The data discussed above speak for the existence of an a-tocopherolbinding protein, reacting with a-tocopherol as an agonist and b-tocopherol as an antagonist. b-Tocopherol exhibits only a minor effect on cell growth or protein kinase C activity, despite the fact that it is an antioxidant almost as potent as a-tocopherol. Moreover, since b-tocopherol prevents the effects of a-tocopherol it seems logical to conclude that the mechanism of action of a-tocopherol as a regulator of smooth muscle cell proliferation cannot be associated with its antioxidant properties. The inhibition by a-tocopherol and the lack of inhibition by b-tocopherol of cell proliferation and protein kinase C activity shows that the mechanism involved is not related to the
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radical scavenging properties of these two molecules, which are essentially equal (Pryor et al., 1993). Moreover, the above results can be interpreted in terms of the existence of a common intermediate, a putative a-tocopherol-binding protein, able to bind a- and b-tocopherol with similar affinity, a-tocopherol acting as an agonist and b-tocopherol as an antagonist. By comparing a-tocopherol with analogous compounds exhibiting similar antioxidant properties such as b-tocopherol or probucol it was concluded that a-tocopherol exerts its action independently of its free-radical scavenger capacity and most probably by interacting with a yet not characterised receptor molecule in smooth muscle cells (Boscoboinik et al., 1991, 1994, 1995). Several groups have reported that antioxidant vitamins and especially vitamin E have an important anti-atherogenic role (Stahelin et al., 1992; Gey et al., 1993; Rimm et al., 1993; Stampfer et al., 1993; Steinberg 1995). The question posed in this study has been if cholesterol and vitamin E can regulate molecular events at the level of signal transduction. Since the importance of protein kinase C in smooth muscle cell proliferation was reported in many studies (Castellot et al., 1989; Matsumoto and Sasaki, 1989; Chatelain et al., 1993; Newby et al., 1995) and its proliferation is inhibited by a-tocopherol (Boscoboinik et al., 1991, 1995), the activity in the rabbit aorta smooth muscle cells was measured. The present in vivo data for cholesterol stimulation and vitamin E inhibition of protein kinase C activity agree with previous results both obtained in vitro (Boscoboinik et al., 1991; O8 zer et al., 1993; Boscoboinik et al., 1995; O8 zer et al., 1995;) and in vivo (Sirikci et al., 1996). It appears that probucol does not act as a protective agent against atherosclerotic plaque formation in the absence of vitamin E. It also appears that among the molecular events that may be responsible for the protection by vitamin E against atherosclerotic plaque formation, inhibition of smooth muscle protein kinase C may play a crucial role. In summary, these studies provide a molecular interpretation to the epidemiological information linking a decrease of plasma a-tocopherol with an increased risk of ischemic heart disease. a-Tocopherol effects are
indicative of a site-directed recognition mechanism such as the binding of a-tocopherol to a ‘receptor protein’. This would be followed by a series of events including activation (or expression) of a protein phosphatase, dephosphorylation of protein kinase C, inhibition of its activity and finally by affecting gene transcription inhibition of cell proliferation.
References Alexander, D.R., et al., 1990. A method for measuring protein kinase C activity in permeabilized T lymphocytes by using peptide substrates. Evidence for multiple pathways of kinase activation. Biochem. J. 268, 303 – 308. Azzi, A, et al., 1992. The protein kinase C family. Eur. J. Biochem. 208, 547 – 557. Azzi, A., et al., 1995. Vitamin E: a sensor and an information transducer of the cell oxidation state. Am. J. Clin. Nutr. 62 (Suppl.), 1337S – 1346S. Bocan, T.M., et al., 1993. The relationship between the degree of dietary-induced hypercholesterolemia in the rabbit and atherosclerotic lesion formation. Atherosclerosis 102, 9 – 22. Bornancin, F., Parker, P.J., 1996. Phosphorylation of threonine 638 critically controls the dephosphorylation and inactivation of protein kinase Ca. Curr. Biol. 6, 1114 – 1123. Bornfeldt, K.E., et al., 1994. Insulin-like growth factor-I and platelet-derived growth factor-BB induce directed migration of human arterial smooth muscle cells via signaling pathways that are distinct from those of proliferation. J. Clin. Invest. 93, 1266 – 1274. Boscoboinik, D., et al., 1991. Inhibition of cell proliferation by alpha-tocopherol. Role of protein kinase C. J. Biol. Chem. 266, 6188 – 6194. Boscoboinik, D., et al., 1994. Inhibition of protein kinase C activity and vascular smooth muscle cell growth by d-alpha-tocopherol. Biochim. Biophys. Acta 1224, 418 – 426. Boscoboinik, D., et al., 1995. Tocopherols and 6-hydroxychroman-2-carbonitrile derivatives inhibit vascular smooth muscle cell proliferation by a nonantioxidant mechanism. Arch. Biochem. Biophys. 318, 241 – 246. Carew, T.E., et al., 1987. Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc. Natl. Acad. Sci. USA 84, 7725 – 7729. Castellot, J.J., et al., 1989. Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells. J. Cell Biol. 109, 3147 – 3155.
N.K. O8 zer, A. Azzi / Toxicology 148 (2000) 179–185 Chatelain, E., et al., 1993. Inhibition of smooth muscle cell proliferation and protein kinase C activity by tocopherols and tocotrienols. Biochim. Biophys. Acta 1176, 83–89. Clowes, A.W., Schwartz, S.M., 1985. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ. Res. 56, 139 – 145. Dutil, E.M., et al., 1994. In vivo regulation of protein kinase C by trans-phosphorylation followed by autophosphorylation. J. Biol. Chem. 269, 29359–29362. Esterbauer, H., et al., 1992. Inhibition of LDL oxidation by antioxidants. EXS 62, 145–157. Ferns, G.A., et al., 1992. Probucol inhibits neointimal thickening and macrophage accumulation after balloon injury in the cholesterol-fed rabbit. Proc. Natl. Acad. Sci. USA 89, 11312 – 11316. Gey, K.F., 1990. The antioxidant hypothesis of cardiovascular disease: epidemiology and mechanisms. Biochem. Soc. Trans. 18, 1041 – 1045. Gey, K.F., et al., 1993. Increased risk of cardiovascular disease at suboptimal plasma concentrations of essential antioxidants: an epidemiological update with special attention to carotene and vitamin C. Am. J. Clin. Nutr. 57, 787S–797S. Konneh, M.K., et al., 1995. Vitamin E inhibits the intimal response to balloon catheter injury in the carotid artery of the cholesterol-fed rat. Atherosclerosis 113, 29–39. Mantha, S.V., et al., 1993. Antioxidant enzymes in hypercholesterolemia and effects of vitamin E in rabbits. Atherosclerosis 101, 135–144. Matsumoto, H., Sasaki, Y., 1989. Staurosporine, a protein kinase C inhibitor interferes with proliferation of arterial smooth muscle cells. Biochem. Biophys. Res. Commun. 158, 105 – 109. Muller, R., et al., 1993. Signals and genes in the control of cell-cycle progression. Biochim. Biophys. Acta 1155, 151– 179. Newby, A.C., et al., 1995. Inhibition of rabbit aortic smooth muscle cell proliferation by selective inhibitors of protein kinase C. Br. J. Pharmacol. 114, 1652–1656. Newton, A.C., 1995. Protein kinase C: structure, function, and regulation. J. Biol. Chem. 270, 28495–28498.
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Nierenberg, D.W., Nann, S.L., 1992. A method for determining concentrations of retinol, tocopherol, and five carotenoids in human plasma and tissue samples. Am. J. Clin. Nutr. 56, 417 – 426. O8 zer, N.K., et al., 1993. d-alpha-Tocopherol inhibits low density lipoprotein induced proliferation and protein kinase C activity in vascular smooth muscle cells. FEBS Lett. 322, 307 – 310. O8 zer, N.K., et al., 1995. New roles of low density lipoproteins and vitamin E in the pathogenesis of atherosclerosis. Biochem. Mol. Biol. Int. 35, 117 – 124. Pryor, A.W., et al., 1993. A rapid screening test to determine the antioxidant potencies of natural and synthetic antioxidants. J. Org. Chem. 58, 3521 – 3532. Raines, E.W., Ross, R., 1993. Smooth muscle cells and the pathogenesis of the lesions of atherosclerosis. Br. Heart J. 69, S30 – S37. Rimm, E.B., et al., 1993. Vitamin E consumption and the risk of coronary heart disease in men (see comments). New Engl. J. Med. 328, 1450 – 1456. Ross, R., 1993. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362, 801 – 809. Sirikci, O8 ., et al., 1996. Dietary cholesterol-induced changes of protein kinase C and the effect of vitamin E in rabbit aortic smooth muscle cells. Atherosclerosis 126, 253 – 263. Stahelin, H.B., et al., 1992. Nutritional factors correlating with cardiovascular disease: results of the Basel Study. Bibl. Nutr. Dieta. 49, 24 – 35. Stampfer, M.J., et al., 1993. Vitamin E consumption and the risk of coronary disease in women (see comments). New Engl. J. Med. 328, 1444 – 1449. Stein, Y., et al., 1989. Lack of effect of probucol on atheroma formation in cholesterol-fed rabbits kept at comparable plasma cholesterol levels. Atherosclerosis 75, 145 – 155. Steinberg, D., 1995. Clinical trials of antioxidants in atherosclerosis: are we doing the right thing. Lancet 346, 36 – 38. Stephens, N.G., et al., 1996. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet 347, 781 – 786.
Effect of vitamin E on the development of atherosclerosis Nesrin Kartal O8 zer a,*, Angelo Azzi b a
Department of Biochemistry, Faculty of Medicine, Marmara Uni6ersity, 81326 Haydarpasa, Istanbul, Turkey b Institute of Biochemistry and Molecular Biology, Uni6ersity of Bern, 3012 Bern, Switzerland Accepted 14 September 1999
Abstract The development of atherosclerosis is a multifactorial process in which both elevated plasma cholesterol levels and proliferation of smooth muscle cells play a central role. Numerous studies have suggested the involvement of oxidative processes in the pathogenesis of atherosclerosis and especially of oxidised low density lipoproteins. Some epidemiological studies have shown an association between high dietary intake or high serum concentrations of vitamin E and lower rates of ischemic heart disease. Recently, the Cambridge Heart Antioxidant Study (CHAOS) reported strong protection by high vitamin E doses against the risk of fatal and non fatal myocardial infarction. Here we have shown that incubation of vascular smooth muscle cells in the presence of a-tocopherol resulted in inhibition of cell proliferation and protein kinase C activity. Since b-tocopherol and probucol are not inhibitory, the effect of a-tocopherol is considered due to a non-oxidant mechanism. In order to understand the protective role of a-tocopherol against atherosclerosis in vivo the following rabbit studies were carried out. Atherosclerosis was induced by a vitamin E poor diet containing 2% cholesterol in a group of rabbit. The other groups had 2% cholesterol in the diet plus 50 mg/kg vitamin E i.m. or 1% probucol or 50 mg/kg vitamin E plus 1% probucol. After 4 weeks, aortas were removed and analysed by microscopy for atherosclerotic lesions. Samples of the media were analysed for protein kinase C activity. The aortas of cholesterol-fed rabbits showed typical atherosclerotic lesions, detected by microscopic examination, their media smooth muscle cells exhibited an increase in protein kinase C activity. Vitamin E fully prevented cholesterol induced atherosclerotic lesions and the induction of protein kinase C activity while probucol was not effective. These results show that the protective effect of vitamin E against hypercholesterolemic atherosclerosis is not produced by an other antioxidant such as probucol and, therefore, may not be linked to the antioxidant properties of this vitamin. The effects observed at the level of smooth muscle cells in vitro and ex-vivo suggests an involvement of signal transduction events in the protective effect of vitamin E against atherosclerosis. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vitamin E; Probucol; Protein kinase C; Vascular smooth muscle cell; Atherosclerosis
1. Introduction * Corresponding author. Tel.: + 90-216-4144733; fax: +90216-4181047. E-mail address: [email protected] (N.K. O8 zer).
The development of atherosclerosis is a multifactorial process in which both elevated plasma
0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 0 ) 0 0 2 0 9 - 2
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cholesterol levels and proliferation of smooth muscle cells play a central role. Numerous studies have suggested the involvement of oxidative processes in the pathogenesis of atherosclerosis and especially of oxidised low-density lipoprotein (Raines and Ross, 1993; Ross, 1993; Steinberg, 1995). Some epidemiological studies have shown an association between high dietary intake and high serum concentrations of vitamin E and lower rates of ischemic heart disease (Gey, 1990; Gey et al., 1993; Rimm et al., 1993; Stampfer et al., 1993). The Cambridge Heart Antioxidant Study (CHAOS) reported strong protection by high vitamin E doses against the risk of fatal and non fatal myocardial infarction (Stephens et al., 1996). One of the mechanisms of a-tocopherol action is to protect low-density lipoproteins from oxidation by peroxy-radicals (Carew et al., 1987; Esterbauer et al., 1992). Smooth muscle cell proliferation is controlled by growth factor receptors, which, via a cascade of kinases and phosphatases, produce activation and expression of proteins necessary for the progression and completion of the cell cycle (Muller et al., 1993). As a part of the main signal transduction path initiated by growth factors or hormones (Azzi et al., 1992), protein kinase C has a complex activation mechanism superimposed to a permissive phosphorylation (Bornfeldt et al., 1994; Dutil et al., 1994; Newton, 1995; Bornancin and Parker, 1996). Cell culture studies have shown that a-tocopherol brings about inhibition of smooth muscle cell proliferation. The latter event takes place via inhibition of protein kinase C activity. a-Tocopherol also inhibits low density lipoprotein stimulated smooth muscle cell proliferation and protein kinase C activity (O8 zer et al., 1993). Moreover, increased protein kinase C activity and expression in atherosclerotic rabbits was also shown (Sirikci et al., 1996). The following cell culture and animal studies were carried out to investigate the mechanism of a-tocopherol action on smooth muscle cell proliferation and on the protection against atherosclerosis; the effect of a-tocopherol was also compared with that of b-tocopherol and probucol.
2. Materials and methods Tissue culture materials were purchased from Falcon Labware (Becton Dickinson) and growth media and serum for cell culture obtained from Gibco Laboratories. [g-32P]ATP was from Amersham International. D-a-Tocopherol and D-b-tocopherol were generous gifts from Hofmann La Roche (Basel). The peptide FKKSFKL (custom synthesised by Dr Rolli, Institut fu¨r Klinische Immunologie, Inselspital Bern) was used as a substrate for the PKC assay. Vitamin E-poor rabbit diet and vitamin E (Ephynal) was kindly donated from Hoffman La Roche (Basel, Switzerland and Istanbul, Turkey), and probucol was a gift from Marion Merrell Dow Pharmaceuticals, Switzerland. The peptide PLSRTLSVAAKK, used as a substrate for the protein kinase C assay, was synthesised by Dr C. Servis, Epalinges (Switzerland). The kit for measurement of protein kinase C activity was obtained from Upstate Biotechnology, Lake Placid, NY, USA. All other chemicals used were of the purest grade commercially available.
2.1. Cell culture studies The cell line used in this work, A7r5 rat aortic smooth muscle cells was obtained from the American Type Culture Collection. The cells were grown in Dulbecco’s modified minimum Eagle medium. Following 48 h serum deprivation the cells were restimulated to growth by addition of 10% fetal calf serum (FCS) in the presence or absence of D-a, D-b-tocopherols and probucol. The cells were detached by trypsinization and counted in a hemocytometer in triplicate. The viability of the cells was assessed by the trypan blue dye exclusion method. PKC activity was measured in permeabilized smooth muscle cells according to the procedure of Alexander et al. (1990). Briefly the cells after 48 h serum deprivation were incubated for 7 h in the presence or absence of 50 mM D-a-tocopherol, D-b-tocopherol and probucol and for the last 2 h in the presence (when specified) of 100 nM phorbol myristate acetate (PMA). Afterwards they were washed and assayed for PKC activity by
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adding [g-32P]ATP (250 mM), peptide substrate (100 mM) and streptolysin-O (0.6 I.U.). After 10 min incubation at 37°C the reaction was stopped by 100 ml trichloroacetic acid and the samples centrifuged for 5 min and spotted on P81 ion exchange chromotography paper and the radioactivity counted. The background phosphorylation, measured in the absence of substrate, was subtracted.
Fig. 1. Differential effects of a-tocopherol and b-tocopherol on smooth muscle cell proliferation and protein kinase C activity. (A) Quiescent cells are restimulated to grow with FCS (10%) in the presence of a-tocopherol and/or b-tocopherol (50 mM). After 48 h restimulation cells were counted with a hemocytometer. Viability was greater than 95%. (B) Quiescent cells are restimulated to grow with FCS (10%) in the presence of a-tocopherol and/or b-tocopherol (50 mM). After 7 h restimulation the cells are permeabilized and protein kinase C is measured as described in Section 2. The basal kinase activity is subtracted in all samples and only the PMA-stimulated activity is shown.
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2.2. Animal experiments Thirty male New Zealand albino rabbits of 2–4 months of age were used in this study. They were fed 100 g per day of a diet poor in vitamin E. Probucol and cholesterol were added to the diet as diethyl ether solution. The control diet was treated with the same amount of pure solvent. All diets were dried of the solvent before use. The concentrations of cholesterol, vitamin E and probucol used were based on previous literature reports (Stein et al., 1989; Ferns et al., 1992; Bocan et al., 1993; Mantha et al., 1993; Konneh et al., 1995). The rabbits were randomly assigned to one of the following six groups. One group of rabbits was only fed with the diet, without additions or treatments. The second group received injections of 50 mg/kg vitamin E intramuscularly on alternate rear legs, once daily. The diet of the third group contained 2% cholesterol. The diet of the fourth group 2% cholesterol and the rabbits received injections of 50 mg/kg vitamin E per day. The fifth group of rabbits received 1% probucol in addition to 2% cholesterol in the daily diet. The sixth group had 2% cholesterol and 1% probucol in the diet and was injected with 50 mg/kg vitamin E per day. After 4 weeks plasma samples were taken and thoracic aortas were removed. Samples were taken from the media of thoracic aortas to measure smooth muscle cell protein kinase C activity by using a non-radioactive kit (Upstate Biotechnology). The plasma cholesterol level were determined using an automated enzymatic technique. Vitamin E levels were measured by reverse phase high-pressure liquid chromatography (Nierenberg and Nann, 1992).
3. Results The effect of a-tocopherol on smooth muscle cell proliferation and protein kinase C is shown in Fig. 1. a-Tocopherol, at concentrations of 50 mM, inhibits rat A7r5 smooth muscle cell proliferation, while b-tocopherol is ineffective. When a-tocopherol and b-tocopherol are added together, no inhibition of cell growth is seen (Fig. 1A). Both
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Fig. 2. Effect of probucol on smooth muscle cell proliferation and protein kinase C activity. Quiescent cells are restimulated to grow with FCS (10%) in the presence of the indicated amounts of probucol. Cell proliferation and protein kinase C activity were measured as indicated in Section 2.
Table 1 Effect of cholesterol, probucol and vitamin E treatment on their plasma levels in rabbitsa Group
Cholesterol (mM)
Vitamin E (mM)
Control Vitamin E Cholesterol Cholesterol +vitamin E Cholesterol +probucol Cholesterol +probucol +vitamin E
1.0 9 0.5 0.9 90.1 21.1 910.7* 12.4 96.1*
5.2 9 2.4 50.7 9 10.2* 14.69 6.5** 216.29 70.2*
13.7 9 6.6*
12.49 4.5**
11.69 3.5*
254.79 64.0*
a The plasma level of cholesterol and vitamin E have been measured in all the five rabbits of the six diet groups. The numbers (mean9 S.D.) represent the plasma values measured after 1 month of diet. * PB0.01. ** Not statistically significant.
compounds are transported equally in cells and do not compete with each other for the uptake. The prevention by b-tocopherol of the growth inhibition by a-tocopherol suggests that a site-directed event is the basis of a-tocopherol inhibition rather than a general radical scavenging reaction. In smooth muscle cells permeabilized with streptolysin-O, to permit the entry of a peptide substrate (Fig. 1B), a-tocopherol inhibits protein kinase C activity, whereas b-tocopherol is ineffective. When both are present b-tocopherol prevents the inhibitory effect of a-tocopherol. Fig. 2 addresses the question whether or not probucol, a potent lipophilic antioxidant, exerts an effect on smooth muscle cell proliferation and protein kinase C activity. Probucol (10–50 mM) does not inhibit smooth muscle cell proliferation and protein kinase C activity. The following animal experiments addresses the question if vitamin E can prevent development of atherosclerosis in vivo and whether probucol can mimic the effect of vitamin E. Cholesterol and vitamin E plasma concentrations of the six animal groups are shown in Table 1. The 2% cholesterol diet supplementation for 4 weeks resulted in an approximately 20-fold increase of plasma cholesterol. After additional supplementation with vitamin E or probucol, plasma cholesterol increased 12- and 13-fold, respectively, relative to control. Plasma vitamin E concentrations were higher in the cholesterol fed rabbits. In this study (Table 2) in the total absence of vitamin E in the diet, the activity of smooth muscle cell protein kinase C was 8.4 D-absorbance units/min per mg protein. After vitamin E treatment an approximately 50% reduction in protein kinase C activity was seen. With cholesterol supplementation the activity of protein kinase C increased to 10.2 D-absorbance units/min per mg protein. Vitamin E treatment was able in this case to reduce the values for protein kinase C activity to those measured in the absence of cholesterol (4.5 D-absorbance units/ min per mg protein). Smooth muscle cells from probucol plus cholesterol treated rabbits showed a protein kinase C activity lower than that in the presence of cholesterol alone, although the data were not statistically significant. Instead, when the rabbits were also treated with vitamin E the
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protein kinase C value decreased to 5.2 D-absorbance units/min per mg protein and reached significance relative to the cholesterol fed rabbit group.
4. Discussion An early event in the development of atherosclerosis is the intimal deposition of lipids, including cholesterol. This deposition arises from a combination of extra- and intra-cellular processes and has been shown, in some animal models, to be inhibited by antioxidants (Esterbauer et al., 1992). We have observed a specific inhibition of proliferation in vitro of smooth muscle cells by a-tocopherol at physiological concentrations. Of particular relevance was the finding of an antiproliferative effect of a-tocopherol on rat and human
Table 2 Protein kinase C activity from smooth muscle cell homogenates obtained from differently treated rabbitsa Group
Total protein kinase C activity (D-absorbance/min per mg protein)
Control Vitamin E Cholesterol Cholesterol +vitamin E Cholesterol +probucol Cholesterol +probucol +vitamin E
8.49 1.1 4.5 92.5 10.2 9 2.4 4.5 91.0
B0.02c
6.89 1.8
NSc
5.2 91.4
B0.04c
P
B0.01b
a Aortic medias were minced, homogenised and nuclei sedimented by centrifugation. Supernatants were centrifuged again at 100 000×g to obtain cytosolic fractions. Pellets were used for preparation of membrane fractions. Protein kinase C activity was measured in both fractions. Since they did not show significant membrane/cytosol distribution changes, only the values of total protein kinase C activity are reported. Results are expressed as mean 9S.D. (n= 5). NS, not statistically significant. b Referres to the control group. c Referres to the cholesterol group.
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smooth muscle cells. Proliferation of these cells is fundamental in the onset of accelerated atherosclerosis, after cardiac transplantation and restenosis. Multiple pathways are involved in the proliferative response of smooth muscle cell to an extracellular stimulus, which has rendered it difficult to identify precise targets for antiproliferative agents. One of the important elements in signal transduction cascades is protein kinase C and its inhibition appears to be sufficient to cause inhibition of smooth muscle cell proliferation in vitro. The inhibition of smooth muscle cell proliferation in vitro by a-tocopherol at physiological concentrations may explain the finding that in vivo smooth muscle cell only proliferate under stress situations (Clowes and Schwartz, 1985; Raines and Ross, 1993). A local or generalised diminution of a-tocopherol concentration, caused by dietary or oxidative factors, can lead to cell growth stimulation and atherosclerosis progress. b-Tocopherol, an antioxidant almost as potent as a-tocopherol, not only does not show any effect at the level of cell proliferation or protein kinase C activity, but rather it prevents the effects of a-tocopherol. The oxidised product of a-tocopherol, a-tocopherylquinone, is not inhibitory of smooth muscle cell proliferation (Azzi et al., 1995).Thus, it is legitimate to conclude that the mechanism of action of a-tocopherol, as a regulator of smooth muscle cell proliferation, is not due to its antioxidant properties. The data discussed above speak for the existence of an a-tocopherolbinding protein, reacting with a-tocopherol as an agonist and b-tocopherol as an antagonist. b-Tocopherol exhibits only a minor effect on cell growth or protein kinase C activity, despite the fact that it is an antioxidant almost as potent as a-tocopherol. Moreover, since b-tocopherol prevents the effects of a-tocopherol it seems logical to conclude that the mechanism of action of a-tocopherol as a regulator of smooth muscle cell proliferation cannot be associated with its antioxidant properties. The inhibition by a-tocopherol and the lack of inhibition by b-tocopherol of cell proliferation and protein kinase C activity shows that the mechanism involved is not related to the
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radical scavenging properties of these two molecules, which are essentially equal (Pryor et al., 1993). Moreover, the above results can be interpreted in terms of the existence of a common intermediate, a putative a-tocopherol-binding protein, able to bind a- and b-tocopherol with similar affinity, a-tocopherol acting as an agonist and b-tocopherol as an antagonist. By comparing a-tocopherol with analogous compounds exhibiting similar antioxidant properties such as b-tocopherol or probucol it was concluded that a-tocopherol exerts its action independently of its free-radical scavenger capacity and most probably by interacting with a yet not characterised receptor molecule in smooth muscle cells (Boscoboinik et al., 1991, 1994, 1995). Several groups have reported that antioxidant vitamins and especially vitamin E have an important anti-atherogenic role (Stahelin et al., 1992; Gey et al., 1993; Rimm et al., 1993; Stampfer et al., 1993; Steinberg 1995). The question posed in this study has been if cholesterol and vitamin E can regulate molecular events at the level of signal transduction. Since the importance of protein kinase C in smooth muscle cell proliferation was reported in many studies (Castellot et al., 1989; Matsumoto and Sasaki, 1989; Chatelain et al., 1993; Newby et al., 1995) and its proliferation is inhibited by a-tocopherol (Boscoboinik et al., 1991, 1995), the activity in the rabbit aorta smooth muscle cells was measured. The present in vivo data for cholesterol stimulation and vitamin E inhibition of protein kinase C activity agree with previous results both obtained in vitro (Boscoboinik et al., 1991; O8 zer et al., 1993; Boscoboinik et al., 1995; O8 zer et al., 1995;) and in vivo (Sirikci et al., 1996). It appears that probucol does not act as a protective agent against atherosclerotic plaque formation in the absence of vitamin E. It also appears that among the molecular events that may be responsible for the protection by vitamin E against atherosclerotic plaque formation, inhibition of smooth muscle protein kinase C may play a crucial role. In summary, these studies provide a molecular interpretation to the epidemiological information linking a decrease of plasma a-tocopherol with an increased risk of ischemic heart disease. a-Tocopherol effects are
indicative of a site-directed recognition mechanism such as the binding of a-tocopherol to a ‘receptor protein’. This would be followed by a series of events including activation (or expression) of a protein phosphatase, dephosphorylation of protein kinase C, inhibition of its activity and finally by affecting gene transcription inhibition of cell proliferation.
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