Influence of 0.1 or 0.2% cholesterol-enriched diets on the induction of atherosclerosis and aorta reactivity in vitro.

Current knowledge of atherogenesis is largely based on animal models of hypercholesterolemia, which rarely show changes similar to the lesions described in humans. We studied the influence of two low cholesterol-enriched diets on the development of anatomopathologic lesions and on the reactivity of the isolated aorta in rabbits. Compared with controls (rabbits fed a normal diet), a 0.1% cholesterol-enriched diet over a 6- or 9-month period produced increases of the 5-hydroxytryptamine (5-HT)-induced contractile responses, as well as a decreases in acetylcholine (ACh)-induced relaxing response (endothelium-dependent, through the production of NO). Noradrenaline (NA)-induced contractions and relaxations elicited by sodium nitroprusside (SNP; endothelium independent) were not significantly modified. Because at 6 months, significant anatomopathologic intimal early lesions were not found, functional endothelial changes can explain such findings. There was a defect in NO synthesis, release, or diffusion; 5 HT, but not NA, may be responsible for inducing NO production. In 0.2% cholesterol-fed rabbits at 4 and 12 weeks, increases of 5-HT- and NA-induced contractile responses were found. In both cases, there was a decrease of ACh-induced relaxing effect, whereas responses to SNP remained unchanged. Intimal early and advanced lesions were present at both the 4- and 12-week periods. These data suggest abnormalities of the NO system. The effects obtained with NA may be explained by a possible decrease of catechol-O-methyltransferase (COMT) or monoamine oxidase (MAO) activities or both or by decreased amine uptake. The extent to which NA may induce NO production is small, because changes in NA-induced contractions are verified only in the presence of significant alterations in the endothelium. The use of a 0.2% cholesterol diet for a short time may induce atherosclerotic lesions, whereas the 0.1% cholesterol diet for a 9-month period, besides being closer to the human diet, allows the detection of functional abnormalities before the evidence of structural lesions.

Antioxidant treatment of experimental diabetic retinopathy in rats with nicanartine.

In order to study the contribution of oxidant stress to the pathogenesis of experimental diabetic retinopathy, male streptozotocin diabetic Lewis rats were treated with the antioxidant and lipid-lowering compound nicanartine (80 mg/kg; n = 8, blood glucose level 16.7 +/- 3.9 mmol/l; HbA1 11.8 +/- 1.6%) by oral supplementation for 6 months and compared with untreated diabetic (n = 6; blood glucose 18.1 +/- 1.4 mmol/l; HbA1 11.5 +/- 1.5%) and untreated, non-diabetic rats (n = 8; blood glucose 4.0 +/- 0.6 mmol/l; HbA1 4.8 +/- 0.9%). Diabetic retinopathy was evaluated by computer-assisted quantitative morphometry including measurement of autofluorescent advanced glycated end-products and immunohistochemistry for heme oxygenase I. Antioxidant treatment did not inhibit the 3.1-fold increase of retinal advanced glycation end products, while the expression of heme oxygenase I in both vascular and glial structures was markedly reduced. Chronic hyperglycaemia led to a 37.3% increase in endothelial cells (p < 0.001 vs normal controls) and a 26.6% reduction in pericyte numbers (p < 0.001 vs controls). Both abnormalities were significantly ameliorated by nicanartine (p < 0.001 vs diabetic controls). No effect was observed on the formation of acellular capillaries or microaneurysms. These data indicate that antioxidant therapy with nicanartine is of limited benefit in diabetic retinopathy, at least in the rodent model of streptozotocin-induced diabetes.

Influence of native and modified lipoproteins on migration of mouse peritoneal macrophages and the effect of the antioxidants vitamin E and Probucol.

Macrophage-derived foam cells are a key component of the atherosclerotic plaque. Stimulation of the migration of foam cells back to the blood potentially offers an approach to plaque regression. However, information on the natural and synthetic factors influencing macrophage migration remains limited. The aim of the present study was to determine the effects of different lipoproteins and antioxidants on the migration of mouse peritoneal macrophages and macrophage-derived foam cells. The migration of cells was determined in vitro by using a Boyden chamber. Cellular content of cholesterol and cholesteryl esters was measured by HPLC. The native lipoproteins, VLDL, LDL, and HDL, and the chemically modified lipoproteins acetylated LDL (acLDL) and oxidized LDL (oxLDL), all acted as chemoattractants for macrophages that had not been lipid-loaded. The antioxidants vitamin E and Probucol had chemoattractive and chemokinetic effects on the migration of unloaded mouse peritoneal macrophages. Foam cells (lipid-laden macrophages) showed decreased mobility with increased cellular lipid content. However, as macrophages accumulated higher amounts of cholesterol and cholesteryl esters, the additional decrease of cell migration was relatively minor. Simultaneous incubation of macrophages with acLDL and vitamin E or Probucol increased migration. The content of cellular cholesterol and cholesteryl esters was not changed when acLDL-treated cells were exposed at the same time to vitamin E, but cholesteryl ester content was increased in the presence of Probucol. Overall, the findings indicate that the antioxidants vitamin E and Probucol have a potential anti-atherogenic action in stimulating migration of lipid-laden macrophages without major impairment of the ability of the cells to accumulate and metabolize modified LDL.

Effects of the new antioxidant nicanartine in an experimental model of atherosclerosis.

Transmural direct current (DC) stimulation of rabbit carotid arteries for 4 weeks was used for induction of atherosclerotic lesions. Ten animals received nicanartine (5-(3,5-di-tert-butyl)-4-hydroxyphenyl-1-(3-pyridyl)-2-oxapentane CAS 150443-71-3, Mrz 3/124) which reveals antioxidative as well as cholesterol-lowering properties supplemented to the diet containing 0.1% cholesterol. Controls were 10 rabbits without drug. Effects on plaque growth were determined by comparing the thickness of the DC induced intimal lesions in both groups. Furthermore, using non-stimulated segments of carotid arteries vasoprotecting effects were characterized by measuring H2O2-enhanced contractility of KCI-induced contraction as well as relaxation caused by acetylcholine induced liberation of endothelial derived relaxing factors. The results show decreased plaque growth during DC stimulation, diminished effectivity of H2O2 on contractility and improved endothelial function in the drug treated group. Since plasma cholesterol was only marginally increased under these feeding conditions, the plaque-reducing effect is most probably due to the antioxidative properties of nicanartine. Similar effects on neointima formation were also shown for other antioxidants.

Confirmation of efficacy of etofibrate against peripheral atherosclerosis in non-human primates which model human lesion types I-VII.

In dyslipidemic or hyperlipidemic patients etofibrate (CAS 31637-97-5, active principle of Lipo-Merz-retard) improves plasma lipoprotein profiles by reducing low density lipoprotein cholesterol and triglycerides. Experimentally, it also promotes fibrinolysis and thrombolysis and reduces the susceptibility of lipoproteins to oxidative stress. In order to investigate the possible efficacy of etofibrate on atherosclerosis, a study in African Green Monkeys was performed. To accelerate atherogenesis, balanced groups of adult male Vervetes (Cercopithecus aethiops) were fed an atherogenic diet, with and without etofibrate, while negative controls received a prudent diet. Total dietary risk exposure was 38 months, with etofibrate treatment during the final 27 months. The etofibrate dose achieved plasma concentrations of clofibric acid comparable to the one achieved clinically. Necropsy demonstrated lesions equivalent to human atherosclerosis types I-VII, which were compared between treatments both macroscopically and microscopically. Peripheral atherosclerosis was significantly less frequent after etofibrate treatment than in positive controls. In aortas, etofibrate probably ameliorated atherogenesis, as defined by proliferation of smooth muscle and foam cells, and accumulation of cholesterol crystals. Effective reduction of plasma cholesterol by etofibrate was confirmed. In conclusion, anti-atherogenic efficacy of etofibrate was demonstrated in a non-human primate model of accelerated atherogenesis. The results on peripheral atherosclerosis confirm the preliminary clinical data in patients suffering from peripheral vascular occlusion.

Influence of experimental hypercholesterolemia on the monoamine oxidase activity in rabbit arteries.

Using a model of experimental atherogenesis in New Zealand rabbits we found a lower noradrenaline level in the aorta than in the femoral artery. The activity of monoamine oxidase was decreased in the femoral artery and increased in the aorta of the cholesterol-fed animals when compared with controls.

Low-density lipoprotein susceptibility to in vitro oxidation in healthy smokers and nonsmokers.

We analyzed the susceptibility of low-density lipoproteins (LDL) to oxidation in 17 healthy smokers (43.3 +/- 16.8 pack-years) and 19 healthy nonsmokers, matched for age (smokers: 52 +/- 7 years; nonsmokers: 53 +/- 7 years), gender, and relative body mass. Cholesterol, triglycerides, LDL cholesterol, HDL cholesterol, and apolipoprotein (apo) B were not different between smokers and nonsmokers; apo A-I was slightly lower in smokers (one-tailed P = 0.066). To study whether LDL from smokers were prone to in vitro oxidation than LDL from nonsmokers, we measured the time kinetics of diene formation and the production of malondialdehyde during oxidation of LDL in vitro. In smokers and nonsmokers, respectively, the mean (+/-SD) lag times (tinh) of diene formation were 111 +/- 26 and 100 +/- 27 min, the peak rates of diene formation (Vmax) were 5.99 +/- 2.34 and 6.34 +/- 2.30 mmol x min-1 x g-1, and the amounts of dienes produced during the propagation phase (dmax) were 250 +/- 264 and 248 +/- 56 mmol x g-1. Neither the malondialdehyde content of LDL (measured as thiobarbituric acid-reactive substances) before oxidation nor the amount of malondialdehyde generated during oxidation (smokers: 57.0 +/- 14.2 micromol x g-1; nonsmokers: 63.2 +/- 15.2 micromol x g-1 indicated any statistically significant effect of smoking. When nonsmokers and smokers were considered together, the amount of malondialdehyde generated during oxidation correlated with age (nonparametric rs = 0.405), body mass index (r2 = 0.573), and concentrations of apo B (rs = 0.480), cholesterol (rs = 0.448), triglycerides (rs = 0.436), and LDL cholesterol (rs = 0.398). Our data show that smoking is not associated with increased oxidizability of LDL in healthy men and women at ages 42-63 years.

Nicanartine improves in vitro resistance of lipoproteins to oxidation.

PURPOSE: The aim of this study is to investigate the plasma protein binding of nicanartine and to measure its antioxidant effect on lipoproteins. METHODS: The blood binding was studied with an erythrocyte partitioning method and the lipoprotein oxidation with the conjugated dienes method. RESULTS: Nicanartine was 24.7% LDL (low density lipoprotein)-bound and 29.2% AAG(alphal-acid glycoprotein)-bound. Nicanartine delayed but did not stop the oxidation of the three density classes of lipoprotein HDL (high density lipoprotein), LDL, VLDL (very low density lipoprotein). The addition of AAG to LDL in the conjugated dienes method decreased the nicanartine fraction bound to LDL and decreased its antioxidant effect. The decrease of nicanartine LDL-bound fraction and the decrease in antioxidant effect were not parallel. CONCLUSIONS: This suggested that (a) AAG-bound nicanartine dissociated to equilibrate the decrease in LDL-bound nicanartine consumed by oxidation, or (b) the oxidation conditions could involve chemical modifications of nicanartine and therefore modify its affinity for AAG.

Etofibrate suppresses neointima formation of the ballooned common carotid artery of rats.

The inhibition of neointima formation by drugs is a major goal to prevent restenosis following angioplasty. In the present study, the effect of etofibrate on blood lipids and vessels wall was investigated using a balloon injury rat model. Two weeks after ballooning the common carotid artery neointima formation was quantified by morphometric measurement of the neointimal area and cellularity in vessel cross sections, and by fluorometric evaluation of the DNA content. Etofibrate (160 mg/kg/day) had no effect on plasma triglyceride levels, but reduced serum cholesterol by about 25%. The injury-induced increase of both the neointimal area and the DNA-content was significantly inhibited by 47% (P < 0.005) and 34% (P < 0.05), respectively, in the drug-treated animals in comparison to the untreated control rats. The ratio of neointima and media was significantly (P < 0.001) reduced from 152.9 +/- 11.6% (controls) to 82.84 +/- 12.59% in the etofibrate-treated group. The cellularity (numerical profile and volume density of nuclei) in the neointima was similar in both groups. In conclusion, injury-induced neointima formation is reduced in etofibrate-treated animals, which could be due to an inhibition of smooth muscle cell proliferation.

Etofibrate treatment alters low density lipoprotein susceptibility to lipid peroxidation.

The effect of the lipid lowering drug etofibrate was investigated on lipid peroxidation as well as on cholesterol level. Rabbits were given a 0.1% cholesterol containing diet. Total cholesterol, LDL-cholesterol, triglycerides and lipid peroxidation, expressed as thiobarbituric acid reactive products, were determined. Treatment with etofibrate led to a marked decrease in total cholesterol and LDL-cholesterol. Furthermore, Cu(2+)-induced lipid peroxide formation was reduced in etofibrate treated rabbits. These results could be confirmed in a human study when patients with moderate hypercholesterolaemia were treated with etofibrate (2 x 500 mg/day) for a period of eight weeks. It could be shown that the onset of lipid peroxidation was remarkably increased, an effect which was completely reversible. Thus, etofibrate is effective not only in lowering plasma cholesterol but also in rendering LDL less susceptible to oxidation.

Differential effect of nicotinic acid derivatives on smooth muscle and endothelial cell proliferation.

The pathogenesis of atherosclerosis is a multifactorial process. A possible anti-atherosclerotic drug should therefore interfere with different targets that are important during the development of an atherosclerotic lesion. Two of the early events are the activated migration and proliferation of arterial smooth muscle cells. Here we investigated in several in vivo and in vitro experiments the effect of two nicotinic acid derivatives L44 and L44-0, on smooth muscle cell migration and proliferation. Balloon catheter de-endothelialization was used as an animal model for intimal lesion formation. Migration was subsequently quantified in vitro using the explant outgrowth technique. Subcultured smooth muscle and endothelial cells were used to test the effect of the drugs on proliferation. Time-lapse video microscopy was applied to differentiate between smooth muscle cell migration and proliferation on the level of individual cells. We showed that L44 and L44-0 are very effective in decreasing smooth muscle cell proliferation and migration. Endothelial cell proliferation, important to re-establish endothelial integrity was, however, not affected.

[Migration of monocytes and granulocytes in co-culture with endothelial cells]. / Migration von Monozyten und Granulozyten in Co-Kultur mit Endothelzellen.

The interaction of leucocytes and endothelial cells was analyzed in a co-culture system using time-lapse video microscopy. Isolated granulocytes or monocytes/macrophages were added to confluent endothelial cell cultures. The migratory behavior of the blood cells was subsequently analyzed using video direct visualization and morphometry. Using this technology the active migration of blood cells through the confluent endothelial layer in both directions could be shown. While granulocytes migrated several times through the endothelium in both directions, monocytes migrated from the top of the endothelial layer underneath the endothelial cells and remained there. The migratory speed of granulocytes was with 1166 microns/h about twice as fast as the speed of monocytes with 608 microns/h. The differentiation between the migratory speed of granulocytes on top or underneath the endothelial cells revealed a marked acceleration underneath the endothelial cells with a speed of 1519 microns/h compared to 671 microns/h on top of the endothelium. These results led to speculation that special extracellular matrix components produced by the endothelial cells activate the migratory behavior of blood cells.

The so-called anticalmodulins fluphenazine, calmidazolium, and compound 48/80 inhibit the Ca2+- transport system of the endoplasmic reticulum.

We present evidence that a Ca2+- transport system of the endoplasmic reticulum with the "mitotic" Ca2+- ATPase as an essential component is another target for the anticalmodulin drugs fluphenazine, calmidazolium, and compound 48/80. Furthermore we show by affinity chromatography that there is a direct interaction between the solubilized Ca2+- ATPase and fluphenazine. Since the Ca2+- uptake system as well as the solubilized Ca2+- ATPase are calmodulin- free, the effect of fluphenazine, calmidazolium and compound 48/80 may be understood as a result of the interaction between these drugs and the Ca2+- ATPase. We propose that there are calmodulin- like sequences in the molecule of the Ca2+- ATPase. The inhibitory effect of these three drugs can be then explained by their recognition of the calmodulin- like structures.

Cell cycle specific variations in transport capacity of an isolated Ca2+-transport system.

From sea urchin eggs as well as from mammalian cells a Ca2+-transporting system is described in its properties. One of its main components is the "mitotic" Ca2+-ATPase. If its activity is studied during the cell cycle of fertilized sea urchin eggs, fluctuations of the Ca2+-uptake capacity are found with a maximum in every cell cycle at mitosis. Additionally, only in the first cycle after fertilization, another activity increase occurs at the time of spermaster formation. This system, then, seems to qualify for one of the main regulators of the mitotic process.