Christer Westerlund

Dissociation of atherogenesis from aortic accumulation of lipid hydro(pero)xides in Watanabe heritable hyperlipidemic rabbits

Antioxidants can inhibit atherosclerosis, but it is unclear how inhibition of intimal lipid oxidation relates to atherogenesis. Here we tested the effect of probucol and its metabolite bisphenol on aortic lipid (per)oxidation and atherogenesis in Watanabe heritable hyperlipidemic (WHHL) rabbits. LDL and aortas from rabbits fed probucol contained bisphenol at concentrations comparable to those in bisphenol-treated animals. Bisphenol treatment increased plasma cholesterol slightly, and plasma and aortic alpha-tocopherol more substantially; these parameters were unaffected by probucol. Bisphenol and probucol treatment both enhanced the resistance of circulating LDL to peroxyl radical-induced lipid peroxidation; this was due to bisphenol, not probucol. Only probucol enhanced LDLs resistance to Cu(2+)-induced oxidation. Both bisphenol and probucol treatment strongly inhibited aortic accumulation of hydroperoxides and hydroxides of cholesteryl esters and triglycerides [LO(O)H]. Despite this, however, probucol had a modestly significant effect on the extent of lesion formation; bisphenol had no inhibitory effect. In addition, the extent of atherosclerosis did not correlate with amounts of aortic LO(O)H present, but, as expected, it did correlate with aortic alpha-tocopherol and cholesterol. Together, these results suggest that aortic accumulation of LO(O)H is not required for, nor is alpha-tocopherol depleted during, the initiation and progression of atherogenesis in WHHL rabbits.

Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice

Antioxidants can inhibit atherosclerosis in animals, though it is not clear whether this is due to the inhibition of aortic lipoprotein lipid (per)oxidation. Coantioxidants inhibit radical‐induced, tocopherol‐mediated peroxidation of lipids in lipoproteins through elimination of tocopheroxyl radical. Here we tested the effect of the bisphenolic probucol metabolite and coantioxidant H 212/43 on atherogenesis in apolipoprotein E and low density lipoprotein (LDL) receptor gene double knockout (apoE‐/‐;LDLr‐/‐) mice, and how this related to aortic lipid (per)oxidation measured by specific HPLC analyses. Dietary supplementation with H 212/43 resulted in circulating drug levels of ∼200 µM, increased plasma total cholesterol slightly and decreased plasma and aortic α‐tocopherol significantly relative to age‐matched control mice. Treatment with H 212/43 increased the antioxidant capacity of plasma, as indicated by prolonged inhibition of peroxyl radical‐induced, ex vivo lipid peroxidation. Aortic tissue from control apoE‐/‐;LDLr‐/‐ mice contained lipid hydro(pero)xides and substantial atherosclerotic lesions, both of which were decreased strongly by supplementation of the animals with H 212/43. The results show that a coantioxidant effectively inhibits in vivo lipid peroxidation and atherosclerosis in apoE‐/‐;LDLr‐/‐ mice, consistent with though not proving a causal relationship between aortic lipoprotein lipid oxidation and atherosclerosis in this model of the disease.—Witting, P. K., Pettersson, K., Östlund‐Lindqvist, A.‐M., Westerlund, C., Westin Eriksson, A., Stocker, R. Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice. FASEB J. 13, 667–675 (1999)

Characterization of novel indenoindoles. Part I. Structure-activity relationships in different model systems of lipid peroxidation.

Structure-activity relationships are presented for some representative compounds from a novel series of potent inhibitors of lipid peroxidation. The compounds are indenoindole derivatives with oxidation potentials in organic solvents of between 0.2 and 1.5 V. Two of these compounds, cis-5,5a,6,10b-tetrahydro-9-methoxy-7-methylindeno[2,1-b]indole (H 290/51) with an oxidation potential of 0.32 V and cis-4b,5,9b,10- tetrahydro-8-methoxy-6-methylindeno[1,2-b]indole (H 290/30) with an oxidation potential of 0.30 V, have been tested more extensively and compared with reference compounds in several pharmacological models of lipid peroxidation. The inhibitory potencies (pIC50) of the compounds in respect to Fe/Ascorbate-induced production of thiobarbituric acid-reactive substances (TBARS) in a suspension of purified soybean lecithin were calculated. These data are 8.2 for H 290/51; 8.0 for H 290/30; 5.6 for vitamin E; and 6.6 for butylated hydroxytoluene (BHT). In isolated rat renal tissue subjected to hypoxia and reoxygenation, the potency for inhibition of TBARS formation is 6.9 for H 290/51, 6.9 for H 290/30, and <5 for vitamin E. In oxidative modification of low-density lipoproteins (LDL) induced by mouse peritoneal macrophages, the corresponding pIC50 values for TBARS inhibition for each compound are: 8.7, 8.3, <5, and 6.9, respectively. It is concluded that the synthetic indenoindoles are potent antioxidants. The results suggest that indenoindoles such as H 290/51 and H 290/30 could be useful as therapeutic agents in pathophysiological situations where lipid peroxidation plays an important role.

Characterisation of novel indenoindoles. Part II. Redox-recycling with ascorbate

In the accompanying paper it was shown that the new antioxidant, H 290/51 (cis-5,5a,6,10b-tetrahydro-9-methoxy-7-methylindenol[2,1-b]indole) , is a powerful antioxidant in several pharmacological models of lipid peroxidation and could be useful as a therapeutic agent in pathophysiological situations where lipid peroxidation plays an important role. In the present study, we characterised H 290/51 as an inhibitor of peroxidation of pure methyl linoleate. H 290/51 almost completely inhibited peroxidation induced by a lipid-soluble initiator at 37 degrees C during the induction period, both in an aqueous solution of micelles in the presence of detergents and in a homogeneous ethanol solution. In both systems, the time of the induction period was linearly related to the concentration of H 290/51. In the ethanol solution, ascorbic acid had a sparing effect on H 290/51, indicating effective interference with radical chain propagation. In aqueous solution with micelles of methyl linoleate made with the nonionic detergents Triton X-100 or Lubrol PX, ascorbic acid did not inhibit peroxidation. However, in these micelles, H 290/51 showed a concentration-dependent extension of the induction period by ascorbic acid, suggesting recycling. In the presence of the zwitterionic detergent CHAPS, although a clear induction period is seen with H 290/51, no recycling by ascorbic acid was found. The ability of H 290/51 to recycle in aqueous solutions, thus depends on the micellar composition.

Preparation of cis-5-methoxy-and-7-methoxy-1-acetoxy-1,2,3,4,4a,10a- hexahydro-9(10H)-phenanthrenone. An epoxide-arene reaction involving a selective 1,2-alkyl shift rearrangement.

The preparation of cis-1α-acetoxy-7-methoxy-1,2,3,4,4a,10a-hexahydro-9(10H)- phenanthrenone 5 was accomplished starting from 6-methoxy-1-tetralone. Reduction of 7-methoxy-1,2,3, 4,9,10-hexahydro-1-oxo-phenanthrene 8, acetylation and subsequent oxidation delivered 5. Application of an analogus procedure to the preparation of cis-1β-acetoxy-5-methoxy-1,2,3,4,,4a,10a-hexahydro-9(10H)- phenanthrenone 6 was not feasible. A more elaborate route was developed for the synthesis of compound 6, where an epoxide-arene reaction involving a 1,2-alkyl shift rearrangement, constituted a highly selective key transformation. The compounds 5 and 6 were prepared. A route was developed for the synthesis of compound 6, where an epoxide-arene reaction involving a 1,2-alkyl shift rearrangement, constituted a highly selective key transformation.

Binding of a dihydropyridine felodipine-analogue to calmodulin and related calcium-binding proteins

A dihydropyridine-affinity column was prepared by coupling a physiologically active and vasoselective amino-derivative of felodipine to divinylsulfone-activated Trisacryl GF2000. Calmodulin (CaM) as well as the homologous calcium-binding proteins skeletal and cardiac Troponin C (sTnC and cTnC) and S100b bound to this resin in a calcium-dependent manner. In contrast, other homologous proteins such as parvalbumin and the intestinal calcium-binding protein did not bind. Competition studies showed that CaM had a higher affinity for the felodipine-column than sTnC or cTnC. Through studies with a series of proteolytic fragments of CaM and sTnC, it was found that the felodipine binding site is located in the amino-terminal domain of the protein. These results illustrate the utility of affinity-chromatography for the study of dihydropyridine-binding proteins.

The potential of antioxidants to prevent atherosclerosis development and its clinical manifestations.

Atherosclerosis is the disease underlying most cardiovascular events, including ischemic heart disease. The disease is initiated when young, but develops slowly, and clinical complications are normally manifested later on in life. Many risk factors for accelerated development are known, including male sex, a family history of cardiovascular disease, hyperlipidemia, hypertension and smoking.

A 62 kDa protein is photo affinity labelled by [3H]felodipine in vascular smooth muscle, but not in cardiac and skeletal muscle

Irradiation of a cytosolic fraction from vascular smooth muscle in the presence of [3H]felodipine resulted in the labelling of a protein with an apparent molecular weight of 62 kDa. The labelling was seen on UV-irradiation at 360 nm, but not at 254, 278 or at wavelengths above 410 nm. The photolabelling was enhanced in the absence of oxygen. In cytosolic fractions prepared from porcine liver, cardiac and skeletal muscle no photoaffinity labelling of proteins between 90 and 45 kDa could be demonstrated. The results suggest that felodipine is a photoaffinity ligand and that felodipine binds to a soluble protein present in vascular smooth muscle but not in the other tissues tested.