Dietary cholesterol-induced changes of protein kinase C and the effect of vitamin E in rabbit aortic smooth muscle cells

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Atherosclerosis 126(1996)253-263

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Received 27 November 1995;revised 6 May 1996;accepted 23 May 1996

Abstract The changes occurring in smooth muscle cells during the development of atherosclerosis in rabbits fed 2’% cholesterol and the effect of vitamin E treatment were investigated. Ex-vivo smooth muscle cells obtained from the aorta of cholesterol-fed rabbits exhibited a 2-fold increase of protein kinase C expression and activity. The cholesterol induced changes in protein kinase C were equally present in the membrane bound and cytosolic fraction of the enzyme. The amount of a control protein a-actin was not affected in smooth muscle cell by the high cholesterol diet treatment, indicating that protein kinase C increase was specific. The increase of protein kinase C expression and activity was not significantly affected by vitamin E treatment although a constant trend was noted. The data are discussed in the light of previous smooth muscle cell in vitro experiments. Keywords: Atherosclerosis; Vitamin E; Protein kinase C; Smooth muscle cells

1. Introduction Proliferation of smooth muscle cells that migrate from arterial media into the subendothelial space, is a central event in the onset of atherosclerosis both in humans and animals [1–5]. Epidemi-

* Corresponding author. Tel.: + 90216 4144733;fax: + 90 2164181047.

ological, clinical, genetic and animal studies have indicated that hypercholesterolemia, with elevated levels of cholesterol-rich low density lipoprotein, is one of the most important risk factors for the disease [4–6]; cholesterol, accumulating in the atherosclerotic lesions, originates primarily in plasma lipoproteins, including low density lipoproteins. Among the factors which have been found to retard the development of atherosclerosis is the intake of food with a sufficient amount

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of antioxidant vitamins, in particular vitamin E. An inverse association between serum antioxidant vitamins and coronary heart disease mortality has been demonstrated in epidemiological studies [711]. In previous studies we have shown that RRRa-tocopherol, which is the biologically most active form of vitamin E, inhibits proliferation of vascular smooth muscle cells by modulating protein kinase C activity in cell cultures. RRR-cz-tocopherol also inhibits low density lipoprotein (LDL) stimulated smooth muscle cell proliferation and protein kinase C activity [12,13]. Previous studies on the effect of cholesterol-rich diet on the development of atherosclerosis in rabbits, have shown that, in this animal model, atherosclerosis can be easily induced by a diet containing 1–20/0cholesterol [14,15]. Since vascular smooth muscle cell proliferation is a key process underlying the formation of atherosclerosis, the inhibition of smooth muscle cell proliferation by RRR-u-tocopherol may have important implications in the prevention of atherosclerosis. To better understand the mechanism by which proliferation is induced by cholesterol we have investigated the changes occurring in protein kinase C in smooth muscle cells, during the development of atherosclerosis and the effect of vitamin E treatment.

2. Materials

and methods

Vitamin E (Ephynal) was obtained from Roche Products (Istanbul, Turkey). The kit for measurement of protein kinase C activity was obtained from Upstate Biotechnology, Lake Placid NY, USA. The anti-protein kinase C antibody was from Boehringer. Smooth muscle cell anti a-actin antibody was from Sigma. Enhanced Chemiluminescence (ECL) Western blotting detection system was supplied by Amersham International, Amersham, UK. All other chemicals used were of the purest grade commercially available. 2.1. Experimental design Twenty four male albino rabbits of 2-6 months of age were divided into 3 groups. Control rabbits

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were fed with 100 g per day of rabbit chow. The second group was fed with 100 g per day of rabbit chow containing 2°/0 cholesterol. This amount has been shown in previous studies (cf. [16-23]) to induce atherosclerotic lesions in rabbit and rat. The rabbits in the third group were fed with 100 g per day of rabbit chow containing 2% cholesterol and received injections of 50 mgjkg of vitamin E intramuscularly on alternate rear legs, once daily. After 8 weeks, the rabbits were anaesthetised, plasma samples were taken and thoracic aortas were removed. Samples from thoracic aorta were taken for light microscopic, scanning and transmission electron microscopic evaluation. In the remaining aorta, the adventitia was stripped off by blunt dissection, the vessel wall was split longitudinally and the endothelial layer was removed by scraping with a scalpel. The aorta strips (media) were frozen in —70°C until they were homogenised. 2.2. Microscopic examination The samples, fixed in 4 glutaraldehyde in 0.15 M cacodylate buffer, pH 7.3, were postfixed in IYo osmium tetroxide for 2 h, dehydrated and embedded in Epon 812. Semi-thin sections were cut, stained with Azur B and examined by light microscopy. Ultra-thin sections were cut and prestained with uranyl acetate and Reynolds citrate. The specimens were examined using a Jeol EM 1200 transmission electron microscope (TEM). For scanning electron microscopy (SEM), aortic segments were fixed overnight at 4°C with 2.5°/0 glutaraldehyde in 0.15 M cacodylate buffer pH 7.3, postfixed in osmium tetroxide for 1 h, followed by washing in cacodylate buffer, dehydrated through graded alcohols, with 2 changes of amy lacetate for 10 rein, dried using a Biorad E 3000 critical Point Drying System. The specimens were mounted on stubs and coated with gold plate with a Biorad sputter coater. The specimens were examined using a Jeol JMS-5200 SEM [24–26]. 2.3. Preparation of smooth muscle cell fractions for protein kinase C measurements Medias were minced, homogenised in buffer A

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plasma level of lipids and vitamin E hThe values

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(20 mM Tris, pH 7.5, 1 mM DTT, 5mM EDTA, 10 mM EGTA, 10Yo glycerol, 1 mM phenylmethylsulfonylfloride) on ice and centrifuged at 500 x g for 5 min to sediment the nuclei and tissue debris. Supernatants were centrifuged again at 100000 x g for 60 min to obtain cytosolic fractions. Pellets were stirred in buffer A including O.lYOTriton X-1OO for 40 min and then centrifuged at 100000 x g for 60 min and the soluble membrane fraction was collected [27]. Protein content was determined by the method of Lowry et al. [28]. Protein kinase C activities of the different fractions were measured by using the protein kinase C assay kit from Upstate Biotechnology. 2.4. Chemical methods Plasma cholesterol and triglyceride levels were measured enzymatically using an autoanalyser. Vitamin E levels were measured by reverse phase HPLC [29]. 2.5. Western blot analysis Smooth muscle cell homogenate fractions were resolved by sodium dodecylsulfate/8% polyacrylamide gel electrophoresis and transferred to PVDF transfer membranes (Du Pent, NEN Rlesearch Products, Brussels). Blots were saturated in 5°/0 non fat dry milk in Trisbuffered saline for 1 h at room temperature and incubated overnight with anti-protein kinase C or smooth muscle cell anti cz-actin antibodies in phosphate buffer saline (150 mM NaCl in 10 mM phosphate buffer, pH 7.2) containing 1°/0 bovine serum albumin and O.lO/O

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NaN3. Enhanced Chemiluminescence was used for detection (Amersham International, UK).

3, Results The values of cholesterol, triglyceride and vitamin E levels of the 3 animal groups (control, cholesterol and cholesterol plus vitamin E) are shown in Table 1. The cholesterol rich diet induced a 12-fold increase in plasma cholesterol level without significant change in the triglyceride level. The administration, together with the cholesterol rich diet, of 50 mglkg i.m. vitamin E did not modify significantly the level of plasma cholesterol and triglyceride. The level of vitamin E in the control group was slightly lower than that of the cholesterol fed group, suggesting that a high fat diet may increase the absorption of vitamin E. In the vitamin E treated group a 12-fold increase of the plasma level of vitamin E was found. The aorta of each different animal was studied by light, scanning and transmission electron microscopy. Fig. 1 shows typical examples of the light microscopy pictures of rabbit aortas from the 3 different groups. Relative to the control (A), in the cholesterol group (B, C) the endothelial cell integrity was impaired and the intima contained areas of vacuolation associated with the presence of typical foam cells. However, in the cholesterol plus vitamin E treated group (D) no lipid accumulation and foam cell formation was visible, similar to the control group. The scanning electron micrographs taken from typical areas of the rabbit aorta from the

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3 groups are given in Fig. 2. It should be stressed that an accurate analysis of the entire surface of the aortas was made and that the photographs are truly representative of the overall picture. The control aorta (A) showed a smooth surface contrary to that of a typical aorta area of a cholesterol fed rabbit (B) showing a marked globular appearance and evident ruptures of the innermost membrane layer. The globular aspect can be interpreted as due to the large number of accumulated underlying lipid filled macrophages or foam cells while the membrane ruptures are due to damage of the endothelial cells. Scanning electron micrographs of the cholesterol plus vitamin E group (C) showed a pattern very similar to the control

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aortas and without evidence of those lesions observable in the cholesterol group. In Fig. 3(A) a typical smooth muscle cell of a control aorta is shown by transmission electron microscopy. The cytoplasm of smooth muscle cells from the control is dominated by microfilament bundles. In the cytoplasm of a smooth muscle cell from the cholesterol group (B) microflament bundles are less visible and cell organelles are visible which is a typical change in atherosclerosis. A transmission electron micrograph of a smooth muscle cell from the cholesterol plus vitamin E group (C) is very similar to the control. Since, following its activation, protein kinase C migrates from the cytosol to the membrane comwe have measured its activity partment,

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both in cytosol and membrane fractions in rabbit smooth muscle cells. Protein kinase C activity was found to exist both in the soluble and membrane fractions in the aortas of all rabbit groups (Fig. 4). Both cytosolic and membrane bound protein kinase C activities in cholesterol and cholesterol plus vitamin E group were significantly higher than their corresponding control group values (P< 0.01). In no case was a significant difference visible between the cytosolic and membrane distribution of protein kinase C among the different groups (P >0.01, Fig. 5). Therefore activity was evaluated as total protein kinase C activity. Total protein kinase C activities of cholesterol and cholesterol plus vitamin E groups are significantly higher than the control group (P< 0.01, Fig. 4). The diminution of protein kinase C activity produced by vitamin E was not significant, although constantly found in all animals. To examine the effect of cholesterol and cholesterol plus vitamin E treatment on protein kinase C we also evaluated the expression of

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protein kinase C in the thoracic aorta smooth muscle cells by using a Western blot technique (Fig. 6). It was found that the expression of protein kinase C was increased in the cholesterol treated group (f’ < 0.01). In the cholesterol plus vitamin E group it was also expressed more than in the controls but slightly less than in the cholesterol treated group. Although this data was not statistically significant a diminution was constantly observed in all animals. In order to understand if the cholesterol-rich diet increased specifically protein kinase C expression or if it was responsible for a general increase in protein synthesis, smooth muscle cell a-actin expression was also analysed. In this case no difference was found in the expression of smooth muscle cell u-actin among the different groups (P> 0.05, Fig. 7).

4. Discussion Lesions of atherosclerosis begin as a consequence of damage to the endothelial and smooth

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muscle cells of the artery wall cells [2,30]. Evidence to support an important role of oxidative modification in mediating the atherogenicity of LDL has been provided by the groups of Steinberg and Esterbauer (for reviews see [31,32]). In vivo smooth muscle cells exist in a condition resembling the in vitro quiescent state. Following the above described events, vascular smooth muscle proliferation may initiate, caused by the release of cytokines (IL-1, TNF-a) growth regulatory molecules (PDGF-A and -B, TGFfl) and other locally liberated factors and has been assumed to be important in the development of ,~(,,)

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atherosclerosis [1,2,27,30,33,34]. Several groups have reported that antioxidant vitamins, and especially vitamin E, have an important antiatherogenic role [7-11]. Two questions have been asked in this study. (1) Does hypercholesterolemia effect smooth muscle cells, at the level of protein kinase C, a key enzyme in the control of proliferation? (for recent reviews see [35,36]). (2) Is vitamin E, an antioxidant, capable of protecting against the molecular modifications induced by hypercholesterolemia at the level of smooth muscle cells? These questions have been prompted by the in vitro studies, show-

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ing that RRR-a-tocopherol is able to inhibit smooth muscle cell proliferation and protein kinase C activity [12,35,37–39] and that smooth muscle cell proliferation can be caused in vitro by LDL [13,40]. In the present study the rabbits were made atherosclerotic by using a cholesterol-rich diet (20 glkg), commonly used to produce atherosclerosis. A high cholesterol supplementation was preferred to lower cholesterol containing diets (5–10 g/kg). This decision turned out to have the advantage of maximizing the atherosclerotic result and diminishing the treatment time. Moreover the reported diminution of plasma cholesterol associated with vitamin E supplementation was not visible under these conditions. However the very high cholesterol supplementation may also be the reason for the lack of a statistically significant effect of vitamin E. It has been also shown that high plasma cholesterol is associated with the development of atherosclerosis as previously reported [14,41,42]. The statistically significant augmentation of plasma vitamin E concentration caused by the cholesterol-rich diet is consistent with its increased absorption and possibly with a larger LDL/VLDL plasma level, responsible for the binding of vitamin E. The administration of vitamin E produces a large increase in its plasma concentration, but no significant diminution of the cholesterol level. Following hypercholesterolemia atherosclerosis developed, with evident morphological changes, which were documented by light, electron transmission and scanning microscopy. Vitamin E was able to protect efficiently against the lesions. At a molecular level protein kinase C expression and activity were significantly stimulated by hypercholesterolemia. The phenomenon was specific, a-actin not being affected under the same conditions. The change in protein kinase C was not due to a redistribution of the enzyme between cytosol and membranes, but to an increase of the protein level, as detected by protein kinase C-specific antibodies. The importance of protein kinase C in smooth muscle cell proliferation can be inferred

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from inhibitor studies. Staurosporine, calphostin C and heparin, by affecting some stage of the signal transduction cascade [43–46], can inhibit smooth muscle cell growth. Age-related changes in vascular smooth muscle cell proliferation and alteration of protein kinase C activity and of its isoforms have been suggested to be involved in the increased susceptibility to atherosclerosis [27,33,34,47]. In previous in vitro studies LDL induced a stimulation of protein kinase C activity and proliferation, which was due to an increase in activity of the existing enzyme and not to an increased expression of protein. It appears thus that the in vitro [13,40] and in vivo systems behave differently. This discrepancy between the two systems is the object of current studies. The effect of vitamin E on protein kinase C expression in vivo was not statistically significant. This phenomenon is again in apparent contradiction to previous in vitro studies showing that RRR-a-tocopherol inhibits smooth muscle cell growth and protein kinase C activity. They have also shown that protein kinase C activity was lower in quiescent cells and higher in the late GI phase. Moreover inhibition of protein kinase C by specific inhibitors or by RRR-a-tocopherol was sufficient to control smooth muscle cell proliferation [48]. The lack of effect of vitamin E in vivo may be related to the very high cholesterol supplemented diet, to sequestration of vitamin E in VLDL resulting in lower tissue concentration (see Ref. [49]. It may also be due to the difference between the in vitro and in vivo systems: the latter being characterised by an increase in expression of protein kinase C, the former of activity. It should be noted that protection by vitamin E against the onset of cholesterol induced atherosclerosis in the rabbit occurs at a morphological level, without a significant change at the level of protein kinase C expression. This questions the apparently causal role of protein kinase C expression in the onset of atherosclerosis and its protection by vitamin E. However the existence of multiple members of the protein kinase C family with different functions and sensitivity to vitamin

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Acknowledgements [

This study was supported by F. Hoffmann La Roche A.G and by a UNESCO-MCBN grant. [

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