Johannes Pill

Apoptosis caused by oxidized LDL is manganese superoxide dismutase and p53 dependent

Oxidized low density lipoprotein (oxLDL) induces apoptosis in human macrophages (MΦ), a significant feature in atherogenesis. We found that induction of apoptosis in MΦ by oxLDL, C2‐ceramide, tumor necrosis factor α (TNF‐α), and hydrogen peroxide (H2O2) was associated with enhanced expression of manganese superoxide dis‐mutase (MnSOD) and p53. Treatment of cells with p53 or MnSOD antisense oligonucleotides prior to stimulation with oxLDL, C2‐ceramide, TNF‐α, or H2O2 caused an inhibition of the expression of the respective protein together with a marked reduction of apoptosis. Exposure to N‐acetylcysteine before treatment with oxLDL, C2‐ceramide, TNF‐α, or H2O2 reversed a decrease in cellular glutathione concentrations as well as the enhanced production of p53 and MnSOD mRNA and protein. In apoptotic macrophages of human atherosclerotic plaques, colocalization of MnSOD and p53 immunoreactivity was found. These results indicate that in oxLDL‐induced apoptosis, a concomitant induction of p53 and MnSOD is critical, and suggest that it is at least in part due to an enhancement of the sphingomyelin/ ceramide pathway.—Kinscherf, R., Claus, R., Wagner, M., Gehrke, C., Kamencic, H., Hou, D., Nauen, O., Schmiedt, W., Kovacs, G., Pill, J., Metz, J., Deigner, H.‐P. Apoptosis caused by oxidized LDL is manganese superoxide dismutase and p53 dependent. FASEB J. 12, 461–467 (1998)

The effects of BM 15.766, an inhibitor of 7-dehydrocholesterol δ7-reductase∗, on cholesterol biosynthesis in primary rat hepatocytes

The effect of the piperazine derivative BM 15.766 (4-(2-[1-(4-chlorocinnamyl)piperazin-4-yl]ethyl]-benzoic acid) on the biosynthesis of sterols was investigated in adult rat hepatocytes in primary monolayer culture. The substance led to a dose-dependent reduction of cholesterol in the serum of various species of animals such as rat, dog and marmoset. BM 15.766 showed a dose-dependent action on the incorporation of 14C-acetate in neutral, nonsaponifiable lipids. The inhibition of the overall incorporation was 10-12% (10(-5) M). No inhibition was observed in the hepatocytes over the entire dose range of 10(-8) M to 2 X 10(-5) M, while the release of the neutral lipids from the hepatocytes into the culture medium was reduced by up to 40%. The biosynthesis of cholesterol could be reduced by more than 90%. Simultaneously, 7-dehydrocholesterol levels rose in the cells and, to a less marked extent, in the medium. This can be interpreted as an indication that 7-dehydrocholesterol is incorporated into the cell membrane, which results in a lower release of 7-dehydrocholesterol into the medium in comparison with controls. The site of attack is the inhibition of the delta 5.7-sterol delta 7-reductase. The formation of desmosterol and cholestatrienol as well as other 7-dehydrocholesterol precursors could also be demonstrated. After longer incubation, there was an additional accumulation of squalene and lanosterol with simultaneous reduction of 7-dehydrocholesterol by BM 15.766, whereas the total 14C-acetate incorporation in neutral lipids was increased.

BM 17.0744: A Structurally New Antidiabetic Compound With Insulin-Sensitizing and Lipid-Lowering Activity

BM 17.0744 (2,2-dichloro-12-(p-chlorophenyl)-dodecanoic acid) is a substance from a group of omega-substituted alkyl carboxylic acids with the general formula, ring-spacer-carboxylic acid. With BM 17.0744-a compound structurally unrelated to thiazolidinediones--antihyperglycemic and antihyperinsulinemic potency has been demonstrated in various animal models of type II diabetes. The antidiabetic effect is independent of the genetic background of the disease, gender, and animal species. The 24-hour blood glucose profile was dose- and time-dependently improved in ob/ob mice after a single and fourth oral administration of 0.3, 1, and 3 mg/kg/d. A dose-dependent reduction of hyperglycemia (10%, 15%, 28%, and 66%) was found in db/db mice after the fifth oral administration of 3, 10, 30, and 100 mg/kg/d. Hyperinsulinemia was reduced dose-dependently in yellow KK mice by 1%, 24%, 34%, and 66% after the fifth oral administration of 0.3, 1, 3, and 10 mg/kg/d. Overall glucose metabolism was predominantly higher in euglycemic-hyperinsulinemic clamp studies in obese fa/fa rats pretreated for 14 days with 10 mg/kg/d BM 17.0744. The data in diabetic and insulin-resistant animals suggest an improvement of insulin action that is supported by enhancement of insulin effects in vitro. There is no evidence of a risk for hypoglycemia in diabetic and metabolically healthy animals. Triglyceride (TG) and cholesterol were reduced in the serum of metabolically healthy rats, as well as serum lipids in db/db mice, which suggests this effect is independent of amelioration of the diabetic status. Lipid-lowering effects in diabetic and healthy animals show an additional property of BM 17.0744. Because of its antidiabetic and lipid-lowering potency, the substance is of great interest in treating the metabolic syndrome. Lipid decreases in rats are associated with a dose-dependent increase in carnitine acetyltransferase activity in the liver to about 100-fold (12.5 mg/kg/d). This together with hepatomegaly in small rodents may indicate peroxisomal proliferation, a phenomenon considered species-specific. Its relevance for humans is well documented for other classes of compounds including fibrates. Specific side effects of insulin sensitizers of the thiazolidinedione type, such as an increase in body weight and heart weight, could not be observed after 4-week oral application of BM 17.0744 in rats. In general, BM 17.0744 was well tolerated in the pharmacological dose range in all species tested.

ω-Substituted alkyl carboxylic acids as antidiabetic and lipid-lowering agents

In screening experiments certain ω-substituted alkyl carboxylic acids, were found to produce an increase in insulin-stimulated 14C-acetate incorporation into triglycerides, which may indicate an improvement in the action of insulin. Antidiabetic and lipid-lowering properties in genetically diabetic ob/ob mice demonstrated the in vivo relevance of the insulin-potentiating effects seen in vitro. The chemical structures of the ω-substituted alkyl carboxylic acids with insulin-potentiating effects correspond to the general formula ring-spacer-COOH. A close structure-activity relationship was observed. The most potent compound in ob/ob mice was 3e, which normalized blood glucose as well as hyperinsulinaemia and lowered serum triglycerides and cholesterol by 52% and 37%, respectively. On the basis of these results, ω-substituted alkyl carboxylic acids are interesting as a new class of oral antidiabetic agents with insulin-sensitizing and lipid-lowering activity.

EFFECT OF ALTERATIONS OF BLOOD CHOLESTEROL LEVELS ON MACROPHAGES IN THE MYOCARDIUM OF NEW ZEALAND WHITE RABBITS

We investigated the effect of alterations of blood cholesterol levels on macrophages (mΦ) in the myocardium of New Zealand White (NZW) rabbits. Three groups of NZW rabbits were used: controls, rabbits fed a 0.5% cholesterol‐enriched diet (CH‐D) for 96 days, and rabbits fed a 0.5% CH‐D for 96 days followed by normal chow for 4 months. Immunohistochemical analysis by mAbs directed against mΦ (RAM‐11) and Mn superoxide dismutase (MnSOD) were quantified by computer‐assisted morphometry. Using cultured human and rabbit mΦ, a cross‐reaction of the human MnSOD mAbs was found as well as the predominant localization of MnSOD‐immunoreactivity (IR) in mitochondria, In group 1, only a very few RAM‐11‐immunoreactive (ir) mΦ occurred in the interstitial space of the myocardium. In group II blood cholesterol levels significantly increased in parallel with the numbers of mΦ, which often contained lipid droplets (foam cells). Although blood cholesterol concentrations regressed about 10‐fold in group III, mΦ in the myocardium were found to be reduced only about 20%. Most mΦ were also MnSOD‐ir. In atherosclerotic coronary arteries RAM‐11‐IR was located in mΦ and also extracellularly, whereas MnSOD‐IR was found only in mΦ. Drastically induced MnSOD in the mitochondria of mΦ is suggested as an indicator of increased oxidative stress caused by in vitro conditions or by phagocytosis of low‐density lipoprotein in vivo. Elevation of the cholesterol level leads to a long‐term increase and its regression results in a delayed reduction of such mΦ, which seem to play a key role in the atherogenesis of the coronary arteries as well. J. Leukoc. Biol. 62: 719–725; 1997.

Differences in the response of Sprague-Dawley and Lewis rats to bezafibrate: the hypolipidemic effect and the induction of peroxisomal enzymes.

The effects of bezafibrate administered at 10 and 50 mg/kg/day for 7 days to male Sprague-Dawley (SD) and Lewis rats were investigated in order to determine the interrelation between the changes in serum and hepatic lipid contents and activities of selected peroxisomal, microsomal and mitochondrial enzymes in the two rat strains. In both strains, bezafibrate effectively reduced serum and hepatic lipids, increased the liver weight, induced a proliferation of peroxisomes, and selectively elevated the activities of carnitine acetyltransferase and of the enzymes of the peroxisomal Β-oxidation system. Moreover, immunoblotting revealed that the drug specifically enhanced the concentration of only those peroxisomal enzymes involved in fatty acid Β-oxidation. The data obtained demonstrate that although the responses initiated by bezafibrate are qualitatively similar in both strains, they differ in their magnitude in a dose-dependent manner, with the Lewis strain exhibiting a more pronounced response than the SD rats. These results show that dose-dependent strain differences as well as the generally known species differences should be taken into account in pharmacological and toxicological evaluations of fibrates in rodents. Furthermore, generalization and extrapolation from rodent studies should be treated with great caution.

Hypercholesterolemia increases manganese superoxide dismutase immunoreactive macrophages in myocardium

The effect of hypercholesterolemia on manganese superoxide dismutase (MnSOD)-containing macrophages was investigated in male New Zealand white rabbits. Macrophages from control animals, which were marked with the RAM-11 antibody, demonstrated co-localization with MnSOD immunoreactivity, e.g. in the peri- and paravascular space within the myocardium, but not in the bone marrow. In rabbits fed a 0.5% cholesterol-enriched diet for 42 days, a significant increase (P<0.01) of MnSOD-immunoreactive macrophages within the myocardium was found concomitant to the drastic elevation of serum cholesterol level. In the bone marrow, MnSOD immunoreactivity did not change after cholesterol feeding. Thus in cholesterol-fed rabbits, the increase of MnSOD-containing macrophages seems to parallel that of lipoproteins. MnSOD is considered as being protective against the cytotoxic effects of those superoxide anions, possibly generated in macrophages, which are involved in the metabolism of modified lipoproteins.

Atherosclerosis in the aorta of hypercholesterolemic rabbits and the influence of daltroban

These studies have examined aortic atherogenesis in cholesterol-fed rabbits and have correlated the effects of daltroban to the pathomechanism of the vessel wall lesions. After feeding a 0.5% cholesterol-enriched diet for 96 d atherosclerotic alterations were seen, which exhibited a proximo-distal pattern, to which the branching of the aorta contributed considerably. Depending on their localization and size a varying cellular constitution of the plaques was obvious. Large plaques, which were mainly seen in the aortic arch and the proximal descending thoracic aorta, consisted of numerous proliferating cells, masses of fibrillar ground substance, clusters of foam cells, and rarely contained cholesterol crystals and necroses. Emerging plaques mainly found in distal thoracic and abdominal aorta imposed as fatty streaks. Daltroban treatment, used in a clinically relevant doses of 10 mg/kg b. wt. per day, reduced extension and protrusional area of plaques to about 40%, which was evaluated using a newly developed computerized morphormetric method, in association with significant reductions in free cholesterol content within the aorta. The results suggest that daltroban inhibits the progression of atherosclerosis in cholesterol-fed rabbits. This effect may be related to its antagonistic interaction with the thromboxane A2 receptor and also to an inhibition of the cholesterol metabolism.

In vitro test system for compounds affecting cholesterol pathway: Studies in primary rat liver cell cultures

Rat liver cells derived from male and female animals in primary monolayer cultures were investigated for suitability as a test system for xenobiotics affecting the cholesterol pathway. An appropriate mode of extraction and separation of newly formed cholesterol and precursors is described. This system can be widely applied.Rat liver cells from females in oestrus cycle had a higher synthesis rate of cholesterol than those from males. The disadvantages related to the cycle phases make male rats more appropriate donor animals for the test system developed. The altered in vitro cholesterol synthesis is relevant to that in vivo.The extraction of newly synthesized cholesterol and its precursors by means of columns packed with large-pore kieselgur is precise and time saving. The modified separation by thin-layer chromatography on silica gel layers impregnated with silver nitrate enables direct separation from the extract and is sufficient to recognize cholesterol and its precursors.The method in this form is suitable for processing a large number of specimens e.g. for screening.

Pharmacological inhibition of atherosclerosis in diet-induced and genetically hypercholesterolemic rabbits

Daltroban verminderte die Atherosklerose bei Kaninchen mit sowohl einer futterinduzierten als auch einer genetischen Hypercholesterinamie. Dies zeigt, das die antiatherosklerotische Wirkung von Daltroban unabhangig von der Art der Hypercholesterinamie ist. Unter beiden Versuchsbedingungen ist die Plattchenaggregation unter Daltroban deutlich gehemmt. Dies und die Reduktion der Cholesterylesterakkumulation auf zellularer Ebene [11] konnten zum antiatherosklerotischen Effekt von Daltroban beitragen.