Effect of vitamin E on the development of atherosclerosis.

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 alpha-tocopherol resulted in inhibition of cell proliferation and protein kinase C activity. Since beta-tocopherol and probucol are not inhibitory, the effect of alpha-tocopherol is considered due to a non-oxidant mechanism. In order to understand the protective role of alpha-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.

Homocysteine induces DNA synthesis and proliferation of vascular smooth muscle cells by a hydrogen peroxide-independent mechanism.

Elevated plasma levels of homocysteine have been identified as an important and independent risk factor for cerebral, coronary, and peripheral atherosclerosis, although the mechanisms are unclear. Homocysteine has been shown to promote cell proliferation and induction of the gene transcription factor c-fos in vascular smooth muscle cells. Earlier reports have suggested that homocysteine exert its effect via hydrogen peroxide (H2O2) produced during its metabolism. To evaluate the contribution of homocysteine to the pathogenesis of vascular diseases, we examined whether the effect of homocysteine on vascular smooth muscle cell growth is mediated by H2O2. We observed that 1.0 mM homocysteine induces DNA synthesis by 1.5-fold and proliferation of vascular smooth muscle cells two-fold in the presence of peroxide scavenging enzyme, catalase (2,600 U/ml). Our results suggest that homocysteine induces smooth muscle cell growth by an H2O2-independent pathway and that the effects of homocysteine may sum together with the known initiating events produced by oxidative stress and accelerate the progression of atherosclerosis.

alpha-Tocopherol specifically inactivates cellular protein kinase C alpha by changing its phosphorylation state.

The mechanism of protein kinase C (PKC) regulation by alpha-tocopherol has been investigated in smooth-muscle cells. Treatment of rat aortic A7r5 smooth-muscle cells with alpha-tocopherol resulted in a time- and dose-dependent inhibition of PKC. The inhibition was not related to a direct interaction of alpha-tocopherol with the enzyme nor with a diminution of its expression. Western analysis demonstrated the presence of PKCalpha, beta, delta, epsilon, zeta and micro isoforms in these cells. Autophosphorylation and kinase activities of the different isoforms have shown that only PKCalpha was inhibited by alpha-tocopherol. The inhibitory effects were not mimicked by beta-tocopherol, an analogue of alpha-tocopherol with similar antioxidant properties. The inhibition of PKCalpha by alpha-tocopherol has been found to be associated with its dephosphorylation. Moreover the finding of an activation of protein phosphatase type 2A in vitro by alpha-tocopherol suggests that this enzyme might be responsible for the observed dephosphorylation and subsequent deactivation of PKCalpha. It is therefore proposed that PKCalpha inhibition by alpha-tocopherol is linked to the activation of a protein phosphatase, which in turn dephosphorylates PKCalpha and inhibits its activity.

Measurement of reactive oxygen species by chemiluminescence in diet-induced atherosclerosis: protective roles of vitamin E and probucol on different radical species.

We have investigated the effects of a high-cholesterol diet on the production of different reactive oxygen species in rabbit aortic rings and evaluated the protective effects of vitamin E and probucol in preventing peroxidative changes. Twenty-five male albino rabbits were divided into five groups. Control rabbits were fed a vitamin E-poor rabbit chow. Rabbits in the second group were given a vitamin E-poor diet supplemented with 2% cholesterol. Other groups received either 50 mg/kg vitamin E, 1% probucol, or both, in addition to 2% cholesterol for 4 weeks. Reactive oxygen species formation in aortic rings was measured by enhanced chemiluminescence using luminol and lucigenin. (The results were given as cpm/mg wet weight.) Further differentiation of radical species involved in luminol-enhanced chemiluminescence was performed using sodium azide and L-nitroarginine, a selective inhibitor of nitric oxide production. Our results indicated that cholesterol feeding increased lucigenin and luminol chemiluminescence, where the contribution of free radicals inhibited by sodium azide (radicals originating from endothelial cells or from phagocytes) were 53% and peroxynitrite 24%. Both vitamin E and probucol were effective as scavengers of free radicals, but the effect of vitamin E was more pronounced. In conclusion, the present study demonstrated excessive generation of reactive oxygen species within the atherosclerotic vessel. Peroxidative changes could be prevented by vitamin E and probucol treatment, but vitamin E seemed to be more efficient.

RRR-alpha-tocopherol regulation of gene transcription in response to the cell oxidant status.

RRR-alpha-Tocopherol, but not RRR-beta-tocopherol, negative regulates proliferation of vascular smooth muscle cells at physiological concentrations. At the same concentrations RRR-alpha-tocopherol inhibits protein kinase C activity, whereas RRR-beta-tocopherol is ineffective. Furthermore, RRR-beta-tocopherol prevents the inhibition of cell growth and of protein kinase C activity caused by RRR-alpha-tocopherol. The negative regulation by RRR-alpha-tocopherol of protein kinase C activity appears to be the cause of smooth muscle cell growth inhibition. RRR-alpha-Tocopherol does not act by binding to protein kinase C directly but presumably by preventing protein kinase C activation. A second RRR-alpha-tocopherol effect has been found at the level of AP 1, the latter becoming activated by RRR-alpha-tocopherol under condition of protein kinase C inhibition or down regulation. AP-1 inhibition by RRR-alpha-tocopherol is seen, however, under condition of protein kinase C stimulation. Compositional changes of AP-1 have been found to be at the basis of the RRR-alpha-tocopherol effects. RRR-beta-tocopherol, provided with similar antioxidant properties, not only it does not affect AP 1 but it prevents the effects of RRR-alpha-tocopherol. Moreover, it has been observed that RRR-alpha-tocopherol is able to affect TRE regulated gene transcription. It is concluded that RRR-alpha-tocopherol acts specifically in vascular smooth muscle cells, by controlling a signal transduction pathway leading to cell proliferation by a non-antioxidant mechanism.

Molecular basis of alpha-tocopherol control of smooth muscle cell proliferation.

Rat and human vascular smooth muscle cell proliferation is specifically sensitive to alpha-tocopherol, but not beta-tocopherol. The former, but not the latter, is capable of limiting proliferation and inhibiting protein kinase C activity in a dose-dependent manner. The phenomenon occurs at concentrations in the range 10-50 microM. beta-tocopherol addition together with alpha-tocopherol, prevents both cell growth and protein kinase C inhibition. alpha-tocopherol increases de novo synthesis of protein kinase C molecules. The enzyme specific activity, however, is diminished, due to a decreased phosphorylation of protein kinase C, occurring in the presence of alpha-tocopherol. Experiments with protein kinase C isoform-specific inhibitors and precipitating antibodies show that the only isoform affected by alpha-tocopherol is protein kinase C-alpha. The effect of alpha-tocopherol is prevented by okadaic acid indicating a phosphatase of the PP2A type as responsible for protein kinase C-alpha dephosphorylation produced in the presence of alpha-tocopherol. At a gene level alpha-tocopherol but not beta-tocopherol induces a transient activation of alpha-tropomyosin gene transcription and protein expression. It is proposed that, by inhibiting protein kinase C activity via an activation of a phosphatase PP2A, alpha-tocopherol controls smooth muscle cell proliferation through changes in gene expression.

Effect of vitamin E and probucol on dietary cholesterol-induced atherosclerosis in rabbits.

The preventive effect of vitamin E and Probucol against atherosclerosis in rabbits were compared. Atherosclerosis was induced by a 2% cholesterol-containing vitamin E-poor diet (5-10 ppm). Six groups of five rabbits each were studied. Group I (control) was fed on a vitamin E-poor diet. The other groups had the following supplements: group II, 50 mg/kg vitamin E i.m.; group III, 2% cholesterol; group IV, 2% cholesterol plus 50 mg/kg vitamin E i.m., group V, 2% cholesterol plus 1% Probucol; group VI, 2% cholesterol + 1% Probucol plus 50 mg/kg vitamin E i.m. After 4 weeks, aortas were removed and analyzed by light and scanning electron microscopy for atherosclerotic lesions. Samples of the media were analyzed 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. Probucol was not effective in preventing either cholesterol-induced atherosclerotic lesions or the induction of protein kinase C activity. 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 ex vivo suggest an involvement of signal transduction events in the protective effect of vitamin E against atherosclerosis.

Signalling functions of alpha-tocopherol in smooth muscle cells.

alpha-Tocopherol but not beta-tocopherol, activates protein phosphatase 2A, decreases protein kinase C activity and attenuates smooth muscle cell proliferation at physiological concentrations. beta-Tocopherol prevents the effects of alpha-tocopherol. Inhibition of protein kinase C alpha, but not of the other isoforms, by the inhibitor Gö6976 prevents the effect of alpha-tocopherol. Protein kinase C alpha, immunoprecipitated from alpha-tocopherol treated cells, is less phosphorylated and inactive. It is proposed that the specific activation of protein phosphatase 2A by alpha-tocopherol results in dephosphorylation and inactivation of protein kinase C alpha. Finally, this cascade of events leads to smooth muscle cell proliferation inhibition.

Alpha-tocopherol as a modulator of smooth muscle cell proliferation.

The effects of alpha-tocopherol and beta-tocopherol have been studied in rat and human aortic smooth muscle cells. Alpha-tocopherol, but not beta-tocopherol, inhibited smooth muscle cell proliferation and protein kinase C in a dose-dependent manner, at concentrations ranging from 10 to 50 microM. Beta-tocopherol added simultaneously with alpha-tocopherol prevented both proliferation and protein kinase C inhibition. Protein kinase C inhibition was cell cycle-dependent and it was prevented by okadaic acid, a protein phosphatase inhibitor. Protein kinase C activity measured from aortas of cholesterol-fed rabbits was also inhibited by alpha-tocopherol. By using protein kinase C (PKC) isoform-specific inhibitors and immunoprecipitation reactions it was found that PKC-alpha was selectively inhibited by alpha-tocopherol. Further, an activation of protein phosphatase 2A by alpha-tocopherol was found, which caused PKC-alpha dephosphorylation and inhibition. Ultimately, this cascade of events at the level of cell signal transduction leads to the inhibition of smooth muscle cell proliferation.

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

The changes occuring 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 alpha-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.

Vitamin E: a sensor and an information transducer of the cell oxidation state.

We studied the effects of RRR-alpha-tocopherol and RRR-beta-tocopherol in smooth muscle cells from rat (line A7r5) and human aortas. RRR-alpha-Tocopherol, but not RRR-beta-tocopherol, inhibited smooth muscle cell proliferation in a dose-dependent manner at concentrations in the range from 10 to 50 mumol/L. RRR-beta-Tocopherol added simultaneously with RRR-alpha-tocopherol prevented growth inhibition. The earliest event brought about by RRR-alpha-tocopherol in the signal transduction cascade controlling receptor-mediated cell growth was the activation of the transcription factor AP-1. RRR-beta-tocopherol alone was without effect but in combination with RRR-alpha-tocopherol prevented the AP-1 activating effect of the latter. Protein kinase C was inhibited by RRR-alpha-tocopherol and not by RRR-beta-tocopherol, which also in this case prevented the effect of RRR-alpha-tocopherol. Calyculin A, a protein phosphatase inhibitor, prevented the effect of RRR-alpha-tocopherol on protein kinase C. The data can be rationalized by a model in which a tocopherol-binding protein discriminates between RRR-alpha-tocopherol and RRR-beta-tocopherol and initiates a cascade of events at the level of cell signal transduction that leads to the inhibition of cell proliferation.

Tocopherols and 6-hydroxy-chroman-2-carbonitrile derivatives inhibit vascular smooth muscle cell proliferation by a nonantioxidant mechanism.

The effects of two groups of similar compounds, a series of tocopherols and one of 6-hydroxy-chroman-2-carbonitrile, have been studied in vascular smooth muscle cells. A poor correlation has been found between antiproliferative and antioxidant properties of these molecules. D-alpha-Tocopherol inhibits cell proliferation, while D-alpha-tocopherylquinone has been found neither to inhibit nor to activate. D-beta-Tocopherol, a poor inhibitor of smooth muscle cell proliferation, has been shown to be capable of preventing and reversing the inhibition by D-alpha-tocopherol. It is concluded that the tocopherols and carbonitrile derivatives tested here appear to inhibit smooth muscle cell proliferation by a nonantioxidant mechanism. The competition between D-alpha-tocopherol and D-beta-tocopherol suggests the existence of a common binding site for the two molecules.

New roles of low density lipoproteins and vitamin E in the pathogenesis of atherosclerosis.

Accumulation of oxidized low density lipoproteins in macrophages and smooth muscle cells causes foam cell formation, an initial step in atherosclerosis. Active oxygen species are considered important in the pathogenesis of the disease. Antioxidants, such as tocopherols and tocotrienols have been considered to prevent the deleterious effects of active oxygen species. We found native low density lipoproteins can stimulate directly smooth muscle cell proliferation, it is associated with an increase of protein kinase C activity. d-alpha-Tocopherol, biologically most active form of vitamin E, inhibits both cell proliferation and protein kinase C activity. The effect of d-alpha-tocopherol is not related to its radical scavenging properties. Transforming growth factor-beta secreted by smooth muscle cells as growth inhibitor. Low density lipoproteins decrease the release of transforming growth factor-beta from smooth muscle cells thus activating growth. d-alpha-Tocopherol activates the cellular release of transforming growth factor-beta. These new aspects explain the important role of low density lipoproteins and vitamin E in increasing and decreasing the risk of atherosclerosis, respectively.

d-alpha-Tocopherol inhibits low density lipoprotein induced proliferation and protein kinase C activity in vascular smooth muscle cells.

Native and malondialdehyde modified low density lipoproteins have been shown to stimulate smooth muscle cell proliferation (A7r5) in vitro. The stimulation is associated with an increase of protein kinase C activity. d-alpha-Tocopherol, at physiological concentrations, has been found to inhibit both protein kinase C activity and cell proliferation.

d-alpha-tocopherol control of cell proliferation.

Uncontrolled cell growth is at the basis of neoplastic proliferation and arteriosclerotic lesions. In vitro proliferation of vascular smooth muscle cells, Balb c/3T3 fibroblasts, retinal neuroepithelial cells and neuroblastoma cells is inhibited by d-alpha-tocopherol. On the contrary Chinese hamster ovary cells, osteosarcoma cells and macrophages are not sensitive. PDGF-BB activated proliferation is highly d-alpha-tocopherol sensitive while lysophosphatidic acid induced growth is poorly inhibited. d-beta-Tocopherol, an analogue of d-alpha-tocopherol, with similar antioxidant properties, does not inhibit proliferation. Protein kinase C activity is inhibited by d-alpha-tocopherol but not by d-beta-tocopherol, suggesting a central role of this enzyme in the control of cell proliferation by d-alpha-tocopherol. Activation of the transcription activation complex AP-1 (but not NFKB) is prevented by d-alpha-tocopherol and not by d-beta-tocopherol.

Effect of chronic halothane exposure on lipid peroxidation, osmotic fragility and morphology of rat erythrocytes.

Effect of chronic halothane exposure on hepatic and erythrocyte lipid peroxidation and erythrocyte osmotic fragility and morphology were determined in rats exposed to 0.4% halothane, 8 h per day for 40 days. Hepatic lipid peroxidation was increased in the halothane-treated group compared to controls. Lipid peroxidation was not increased by halothane exposure in erythrocytes without hydrogen peroxide, but after peroxide supplementation lipid peroxidation increased more in the erythrocytes of halothane-exposed rats than in control rats. We have observed significant morphological changes in erythrocytes from halothane-treated rats. In addition, erythrocytes of halothane-treated rats were more fragile in saline solutions compared to those of controls. Our results suggest that chronic halothane exposure is not only hepatotoxic but also affects erythrocyte membrane structure and stability.

Plasma membrane potential of lymphocytes from ataxia telangiectasia patients.

The plasma membrane potential of lymphocytes prepared from ataxia telangiectasia (AT) patients and normal subjects was assessed using the optical indicator bis-(3-phenyl-5-oxoisoxazol-4-yl) pentamethineoxonol (oxonol-V). AT lymphocytes had a potential of -46 +/- 9 mV and normal lymphocytes had a potential of -63 +/- 4 mV. The intracellular cation content (Na+ and K+) of AT and normal lymphocytes was similar. AT and normal lymphocytes were both depolarized by extracellular K+ and to a similar extent. This study indicates that one feature characterizing ataxia telangiectasia is a modification of the ability of the lymphocyte cell membrane to sustain a normal membrane potential.

Lymphocyte gamma-glutamyltranspeptidase activity in ataxia-telangiectasia.

gamma-Glutamyltranspeptidase (GGTP) is a membrane-bound enzyme, that catalyzes gamma-glutamyl transfer from gamma-glutamyl compounds to amino acid and peptide acceptors. One of the most important clinical findings about ataxia-telangiectasia (A-T), a multisystemic and autosomal-recessive disease, is dysfunction of the immune system. In this study, the activity of GGTP was determined in the lymphocytes from patients with A-T. Lymphocyte GGTP activity in A-T patients was found to be significantly lower than that of control lymphocytes (P less than 0.001). This change may be due to the abnormality in the membrane of lymphocytes of A-T patients.

Studies on lymphocyte cell surface in ataxia-telangiectasia.

Lymphocyte surface proteins of patients with ataxia-telangiectasia were separated by polyacrylamide gel electrophoresis and compared by autoradiography. The patients lacked one of the two main bands (Band I at the origin). The second main band (Band II) was absent in some cases. All patients had one or two additional bands of smaller molecular weight than Band II except one case who had no band detectable. In the patients, alkaline phosphatase, total ATPase and Mg2+. ATPase were increased but 5'-nucleotidase was normal. The results suggest abnormality in the plasma membranes of the patients' lymphocytes.