Michel Prost

Impaired Homocysteine Metabolism and Atherothrombotic Disease

Based on recent retrospective, prospective, and experimental studies, mild to moderate elevation of fasting or postmethionine-load plasma homocysteine is accepted as an independent risk factor for cardiovascular disease and thrombosis in both men and women. Hyperhomocysteinemia results from an inhibition of the remethylation pathway or from an inhibition or a saturation of the transsulfuration pathway of homocysteine metabolism. The involvement of a high dietary intake of methionine-rich animal proteins has not yet been investigated and cannot be ruled out. However, folate deficiency, either associated or not associated with the thermolabile mutation of the N5,10-methylenetetrahydrofolate reductase, and vitamin B6 deficiency, perhaps associated with cystathionine β-synthase defects or with methionine excess, are believed to be major determinants of the increased risk of cardiovascular disease related to hyperhomocysteinemia. Recent experimental studies have suggested that moderately elevated homocysteine levels are a causal risk factor for atherothrombotic disease because they affect both the vascular wall structure and the blood coagulation system. The oxidant stress that results from impaired homocysteine metabolism, which modifies the intracellular redox status, might play a central role in the molecular mechanisms underlying moderate hyperhomocysteinemia-mediated vascular disorders. Because folate supplementation can efficiently reduce plasma homocysteine levels, both in the fasting state and after methionine loading, results from further prospective cohort studies and from on-going interventional trials will determine whether homocysteine-lowering therapies can contribute to the prevention and reduction of cardiovascular risk. Additionally, these studies will provide unequivocal arguments for the independent and causal relationship between hyperhomocysteinemia and atherothrombotic disease.

Pro-thrombotic effects of a folic acid deficient diet in rat platelets and macrophages related to elevated homocysteine and decreased n-3 polyunsaturated fatty acids

Folic acid deficiency represents a vitamin deficiency that may be due either to an inadequacy of the dietary supply or to an increased requirement. It leads to a number of abnormalities including hematological, neurological and cardiovascular disorders. In this study, we investigated whether folic acid deficiency would influence platelet and macrophage activities. For 6 weeks, rats were fed a test diet containing a low amount of folic acid (250 mu g/kg) by comparison with a control diet (750 mu g/kg). We found 40 and 32 percent reductions (P < 0.05) of plasma and erythrocyte folates, respectively in the tested group. Peritoneal macrophages of the folic acid deficient animals exhibited greater (20 x) tissue factor (TF) activity than in the controls. We also found that folate depletion significantly enhanced the thrombin- and ADP-induced platelet aggregation (+64 and + 13 percent, respectively). Moreover, the results of incubations with radiolabeled arachidonic acid indicated that platelets of folic acid deficient animals incorporated more labeling than controls did. When stimulated with thrombin, the mobilization of arachidonate from platelet phospholipids and its subsequent formation of cyclooxygenase and lipoxygenase metabolites were enhanced in the deficient animals. In particular, thromboxane biosynthesis was markedly increased. The analysis of the plasma fatty acid composition showed a decrease in the plasma unsaturation index related to a marked fall of long chain (n-3) fatty acids which was also observed in platelets. These data suggested the occurrence of an oxidative stress in folic acid deficient animals which was confirmed by increases in plasma lipid peroxidation products (more than +20 percent) and an enhanced susceptibility of erythrocytes to free radicals (+23 percent). Altogether these data suggested that folic acid deficiency altered the circulating and cellular fatty acid composition and thus influenced the balance of the platelet eicosanoid synthesis. In addition, total homocysteine and glutathione concentrations were highly increased in plasma from folate-depleted rats. From these results, we conclude that folate deficiency can potentiate the coagulation pathway mediated by the macrophage TF as well as the platelet activation process. It is suggested that these dysfunctions might be related to the loss of (n-3) polyunsaturated fatty acids. The latter could result from an increased lipid peroxidation triggered by the folic acid deficiency-induced hyperhomocysteinemia.

Folic Acid Deficiency Enhances Oral Contraceptive-Induced Platelet Hyperactivity

In previous studies conducted in female rats and in women, oral contraceptives (OC) were found to induce a platelet hyperactivity that was related to an oxidative stress. Because cases of megaloblastic anemia have been reported to occur in women taking OC, these treatments are suspected of depleting folate stores. In the study presented herein, which was conducted in rats, we sought to determine the influence of dietary folic acid deficiency (FD) on the thrombogenicity of OC. Animals were fed for 6 weeks with either a folic acid-deficient diet (250 micrograms/kg folic acid) or a control diet (750 micrograms/kg). One-half of the animals in each group were treated with OC (ethinyl estradiol plus lynestrenol). FD and OC individually potentiated platelet aggregation in response to thrombin and ADP and the release and metabolism of arachidonic acid, in particular, the biosynthesis of thromboxane. These platelet activities were further enhanced in animals given both the folic acid-deficient diet and the OC treatment. In addition, FD enhanced the pro-oxidant state in OC-treated rats characterized by (1) a fall in platelet and plasma n-3 fatty acids, (2) an increase in plasma lipid peroxidation products such as conjugated dienes, lipid peroxides, and thiobarbituric reactive substances, (3) a rise in ex vivo erythrocyte susceptibility to free radicals. Moreover, we found that OC treatment led to a reduction of plasma and erythrocyte folate concentrations associated with a moderate hyperhomocysteinemia. Under our experimental conditions, we did not find significant synergistic effects between OC and FD. We propose that, although the untoward effects associated with the OC treatment may not primarily be dependent on FD, the folic acid deficiency magnified OC-induced oxidative stress, which resulted in platelet hyperactivity by elevating the pro-oxidant homocysteine plasma concentration. Despite the limitations of this animal model, the data of the present study suggest that in addition to cigarette smoking, inadequate folic acid intake might predispose those taking OC to vascular thrombosis.

Catechin inhibits platelet hyperactivity induced by an acute iron load in vivo

Reactive oxygen species and platelets are thought to be involved in the pathogenesis of cardiovascular disease. Epidemiological data have indicated that high consumption of fruits and vegetables is associated with a lower incidence of vascular events. Polyphenols were proposed to provide such a protection. In the present study performed in rats, we investigated the influence of (+)-catechin (Cat), a polyphenol identified in tea, cocoa, and red wine, on an acute iron load-induced model of platelet hyperactivity. We found that platelet function was significantly enhanced in iron-loaded rats. These changes were associated with impairment of the antioxidative defense including ex vivo free radical-induced hemolysis. Pretreatment with Cat (10 mg/kg, ip, 4 d) normalized biomarkers of antioxidative status and platelet hyperactivity. The benefits of Cat treatment were only observed in iron-loaded animals and not in control animals. In light of the known antioxidant properties of Cat (or its metabolites), we suggest that oxidative injury-induced modification of platelet calcium homeostasis may have explained the iron load-induced platelet hyperactivity. The protective effect of Cat appears to work probably through normalization of the antioxidative status.

Induction of oxiapoptophagy on 158N murine oligodendrocytes treated by 7-ketocholesterol-, 7β-hydroxycholesterol-, or 24(S)-hydroxycholesterol: Protective effects of α-tocopherol and docosahexaenoic acid (DHA; C22:6 n-3).

In demyelinating or non-demyelinating neurodegenerative diseases, increased levels of 7-ketocholesterol (7KC), 7β-hydroxycholesterol (7β-OHC) and 24(S)-hydroxycholesterol (24S-OHC) can be observed in brain lesions. In 158N murine oligodendrocytes, 7KC triggers a complex mode of cell death defined as oxiapoptophagy, involving simultaneous oxidative stress, apoptosis and autophagy. In these cells, 7KC as well as 7β-OHC and 24S-OHC induce a decrease of cell proliferation evaluated by phase contrast microscopy, an alteration of mitochondrial activity quantified with the MTT test, an overproduction of reactive oxygen species revealed by staining with dihydroethidium and dihydrorhodamine 123, caspase-3 activation, PARP degradation, reduced expression of Bcl-2, and condensation and/or fragmentation of the nuclei which are typical criteria of oxidative stress and apoptosis. Moreover, 7KC, 7β-OHC and 24S-OHC promote conversion of microtubule-associated protein light chain 3 (LC3-I) to LC3-II which is a characteristic of autophagy. Consequently, 7β-OHC and 24S-OHC, similarly to 7KC, can be considered as potent inducers of oxiapoptophagy. Furthermore, the different cytotoxic effects associated with 7KC, 7β-OHC and 24S-OHC-induced oxiapoptophagy are attenuated by vitamin E (VitE, α-tocopherol) and DHA which enhances VitE protective effects. In 158N murine oligodendrocytes, our data support the concept that oxiapoptophagy, which can be inhibited by VitE and DHA, could be a particular mode of cell death elicited by cytotoxic oxysterols.

Folate Deficiencies and Cardiovascular Pathologies

Abstract Although folates are widely distributed in foods, folate deficiencies may be more frequent than expected because their true availability may be impaired due to their lability under various food cooking and processing conditions. Folate deficiency is frequently observed in elderly people, smokers, alcoholics and oral contraceptive users. It is also associated with the mutation leading to the thermolabile variant of N5, 10-methylenetetrahydrofolate reductase which is observed in about 10 % of the population. In addition to the essential role of the intracellular pool of polyglutamates in de novo biosynthesis of deoxyribonucleotides which allow cell growth and division, the reduced and methylated form of folate, N5-methyltetrahydrofolate, is required for the remethylation of homocysteine to methionine. By inhibiting this remethylation pathway, folate deficiency induces homocysteine efflux into the circulation. Many studies have shown a negative correlation between plasma folate, particularly N5-methyltetrahydrofolate, and circulating homocysteine levels. In addition, folate deficiency is a major cause of hyperhomocysteinemia which is fully recognised as an independent risk factor for atherothrombosis. Epidemiological and recent experimental studies have demonstrated that folate deficiency might increase the risk of cardiovascular disease by increasing circulating homocysteine levels. Thus, the clinical efficiency of folate supplementation, especially N5-methyltetrahydrofolate, in reducing homocysteine-dependent cardiovascular risk should be evaluated.

Assessment of antioxidant activity of natural extracts

Abstract The aim of this study was investigate and compare the antioxidant activity of extracts from Labiatae and Oleaceae cell cultures and a natural Verbenaceae extract on pig whole blood. The antioxidant activitiy was assessed using the KRL biological test. Extract obtained from Ajuga reptans (Labiatae) cell cultures and titrated at 50% of phenylpropanoids expressed as teupolioside (T), extract obtained from Siringa vulgaris (Oleaceae) cell cultures and titrated at 50% of phenylpropanoids expressed as verbascoside (V), V and T 50/50 mixture and a natural Verbenaceae extract (NE) titrated at 5% of phenylpropanoids, expressed as verbascoside, were tested for their ability to protects pig red blood cells from 2,2’azobis (2-amidinopropane) dihydrochloride (AAPH) induced hemolysis. The protective effect of the compounds was dose-dependent at concentrations of 0.1 to 1g/L. The active principle in the NE shows an antioxidant activity 2.27 times higher (P<0.01) than the cell culture extract V and 5.11 times higher (P<0.01) than the cell culture extract T. Taking into account the total phenylpropanoids content of cell cultures and natural extract, these results clearly indicate a greater antioxidant activity for the natural Verbaneceae extract.

Protective Effects of α-Tocopherol, γ-Tocopherol and Oleic Acid, Three Compounds of Olive Oils, and No Effect of Trolox, on 7-Ketocholesterol-Induced Mitochondrial and Peroxisomal Dysfunction in Microglial BV-2 Cells

Lipid peroxidation products, such as 7-ketocholesterol (7KC), may be increased in the body fluids and tissues of patients with neurodegenerative diseases and trigger microglial dysfunction involved in neurodegeneration. It is therefore important to identify synthetic and natural molecules able to impair the toxic effects of 7KC. We determined the impact of 7KC on murine microglial BV-2 cells, especially its ability to trigger mitochondrial and peroxisomal dysfunction, and evaluated the protective effects of α- and γ-tocopherol, Trolox, and oleic acid (OA). Multiple complementary chemical assays, flow cytometric and biochemical methods were used to evaluate the antioxidant and cytoprotective properties of these molecules. According to various complementary assays to estimate antioxidant activity, only α-, and γ-tocopherol, and Trolox had antioxidant properties. However, only α-tocopherol, γ-tocopherol and OA were able to impair 7KC-induced loss of mitochondrial transmembrane potential, which is associated with increased permeability to propidium iodide, an indicator of cell death. In addition, α-and γ-tocopherol, and OA were able to prevent the decrease in Abcd3 protein levels, which allows the measurement of peroxisomal mass, and in mRNA levels of Abcd1 and Abcd2, which encode for two transporters involved in peroxisomal β-oxidation. Thus, 7KC-induced side effects are associated with mitochondrial and peroxisomal dysfunction which can be inversed by natural compounds, thus supporting the hypothesis that the composition of the diet can act on the function of organelles involved in neurodegenerative diseases.

Oil-enriched, ultra-small nanostructured lipid carriers (usNLC): a novel delivery system based on flip-flop structure.

For the development of ultra-small NLC (usNLC) the determination of the required HLB (hydrophilic lipophilic balance) was found to be a suitable method, i.e., usNLC with a size below 50 nm were obtained by this method. Loading with 5% (w/w) coenzyme Q10 (Q10) led to usNLC with a size of about 85 nm. In comparison to classical NLC with a size of 230 nm and a nanoemulsion with similar size, the Q10 loaded usNLC show a higher release, a higher antioxidant capacity, and a better skin penetration for Q10. The reason for this is a flip-flop core-shell structure of the lipid matrix, i.e., the oil with dissolved active is surrounding the solid lipid based core. As the flip-flop structure was probably achieved by admixing high contents of liquid lipid, oil enriched usNLC might represent a novel and promising carrier system for the improved delivery of lipophilic actives.

Cytoprotective Activities of Milk Thistle Seed Oil Used in Traditional Tunisian Medicine on 7-Ketocholesterol and 24S-Hydroxycholesterol-Induced Toxicity on 158N Murine Oligodendrocytes

The Asteraceae family is economically very important, because many of these plants are grown mainly for their food value, such as lettuce (Lactuca), chicory (Cichorium), and sunflower (Heliantus aminus). One of the typical properties of this family, which includes milk thistle (Sylibum marianum), is the richness of the oil in various compounds (flavonoids, alkaloids, tocopherols, and unsaturated fatty acids). Currently, and for the coming decades, age-related diseases, including neurodegenerative diseases, are a major public health problem. Preventing their appearance or opposing their evolution is a major objective. In this context, the cytoprotective activities of milk thistle seed oil produced in Tunisia were studied on the 158N model using 7-ketocholesterol (7KC) and 24S-hydroxycholesterol (24S) as cytotoxic agents. 7KC and 24S were used because they can be increased in the brain and body fluids of patients with major age-related neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases. In order to evaluate the cytoprotective properties of milk thistle seed oil, complementary techniques of microscopy, flow cytometry, and biochemistry were used. The chemical composition of milk thistle seed oil has also been determined by various chromatography techniques. Milk thistle seed oils from different area of Tunisia are rich in tocopherols and are strongly antioxidant according to various biochemical tests (KRL (Kit Radicaux Libres), FRAP (Ferric Reducing Antioxidant Power), and DPPH (2,2-diphenyl-1-picrylhydrazyl)). The main fatty acids are linoleic acid (C18:2 n-6) and oleic acid (C18:1 n-9). The main polyphenols identified are homovanillic acid, p-coumaric acid, quercetin, and apigenin, with a predominance of vanillic acid. On 158N cells, milk thistle seed oil attenuates the cytotoxicity of 7KC and 24S including: loss of cell adhesion, increased plasma membrane permeability, mitochondrial dysfunction, overproduction of reactive oxygen species, induction of apoptosis, and autophagy. The attenuation of the cytotoxicity of 7KC and 24S observed with the milk thistle seed oil is in the order of that observed with α-tocopherol used as a positive control. In the presence of nigella seed oil, considered potentially cytotoxic, no cytoprotective effects were observed. Given the chemical characteristics, antioxidant properties, and cytoprotective activities of milk thistle seed oil, our results highlight the potential benefit of this oil for human health.