A.N. Makheja

Anti-inflammatory drugs in experimental atherosclerosis

Groups of New Zealand white male rabbits were fed atherogenic diets containing 1% cholesterol. The diets of experimental groups were supplemented additionally with either aspirin, phenylbutazone, mefenamic acid, flufenamic acid, oxyphenylbutazone or aminopyrine. Blood cholesterol and phospholipids were measured at 3--4 week intervals. After 12 weeks the animals were sacrificed and the severity of atherosclerosis in the thoracic aorta was measured. In separate experiments, rabbit platelets were incubated with each of the drugs individually and conversion of [14C]arachidonic acid to thromboxanes and related compounds was assayed. Inhibition of collagen and arachidonic acid-induced platelet aggregation by each drug was also measured. All drugs inhibited thromboxane synthesis and platelet aggregation in varying degrees with flufenamate and aspirin being most and aminopyrine least effective. The pattern of metabolite formation from [14C]arachidonate was consistent with a block in the cyclooxygenase reaction. Phenylbutazone, flufenamic acid and oxyphenylbutazone produced significant reductions in atherosclerotic plaque formation without major changes in blood cholesterol levels or blood cholesterol--phospholipid ratios. Aspirin and aminopyrine were ineffective. The results indicate that the effectiveness of anti-inflammatory drugs as inhibitors of thromboxane synthesis and platelet aggregation in vitro does not afford a sufficient predictive index of their anti-atherogenicity in vivo. The significance of these findings is discussed in terms of the possible involvement of cyclooxygenase derivatives in atherogenesis.

Differential separation of thromboxanes from prostaglandins by one and two-dimensional thin layer chromatography.

[14C]-labelled thromboxane B2 and hydroxy fatty acids were isolated using thin layer and gas chromatographic procedures from human platelets incubated with [1-14C]-arachidonic acid. A number of TLC solvent systems were evaluated for differential separation of thromboxanes and hydroxy fatty acids from prostaglandins E2, A2, D2 and F2alpha. Chromatographic properities in nine different solvent systems are tabulated. Two dimensional TLC procedures suitable for complete resolution of mixtures of these compounds on a single plate were developed. The systems were used to demonstrate conversion of [1-14C]-arachidonic acid to thromboxane B2 and prostaglandin E2 by human lung fibroblasts in tissue culture.

Anti-inflammatory drugs in experimental atherosclerosis: 7. Spontaneous atherosclerosis in WHHL rabbits and inhibition by cortisone acetate

The Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for familial hypercholesterolemia, has a deficiency in low density lipoprotein (LDL) receptor binding and exhibits elevated plasma lipoprotein levels and spontaneous atherosclerosis. Since atherosclerotic plaque formation has a number of features in common with the inflammatory process, we have investigated the effect of dietary supplementation with an anti-inflammatory steroid (cortisone acetate, 5 mg daily for 3 months) on atherosclerosis using the WHHL rabbit as a model. Atherosclerotic plaque formation in cortisone-fed animals was reduced by about 60% compared to control WHHL rabbits. Steroid administration increased circulating cholesterol levels modestly and triglycerides were increased about 6-fold. While very low density lipoprotein (VLDL)-cholesterol was increased, LDL-cholesterol levels were decreased and the particle was more triglyceride-enriched as well as less dense. Steroid-fed animals also exhibited decreased platelet aggregation and increased aortic 15-lipoxygenase activity. The histological observations showed typical fibrous plaques in aortas of both control and cortisone-fed rabbits, with intima thickened by foamy macrophages and subcellular lipoproteinaceous debris covered by a fibrous cap. These findings thus indicate that steroids reduce the rate of plaque initiation or progression but do not significantly change the histological appearance of the lesion.

THE ACTIVE PRINCIPLE IN FEVERFEW

to migraine and asthma. Collier et awl. report that extracts of feverfew inhibit synthesis of prostaglandins. A key event in platelet aggregation is the release of arachidonic acid and its conversion to the proaggregatory prostaglandin thromboxane A2. We therefore examined the effect of feverfew on thromboxane synthesis and aggregation in human platelets. Air-dried feverfew leaves were extracted with 20 volumes of phosphate buffer pH 7-4. The effect of these extracts on human platelets was tested in a standard aggregation assay system. 3

Systemic activation of 15-lipoxygenase in heart, lung, and vascular tissues by hypercholesterolemia: relationship to lipoprotein oxidation and atherogenesis

There is evidence that oxidized lipoproteins are a major contributing factor in atherosclerosis. 15-Lipoxygenase is the principal mammalian enzyme that can oxidize polysaturated fatty acids present in intact lipoproteins, and in membrane phospholipids in situ. We, and others, have reported previously that levels of the enzyme are increased in aortas of cholesterol-fed and spontaneously atherosclerotic WHHL rabbits. In the present study, rabbits were fed an atherogenic diet containing 1% cholesterol for 14 weeks, and levels of [14C]arachidonate metabolizing enzymes in the excised tissues were measured by HPLC analysis. 15-Lipoxygenase levels in heart, aortic adventitia, and lung, but not in liver, were increased up to 100-fold above controls, without major significant changes in prostaglandin endoperoxide synthases or the 5- and 12-lipoxygenases. The induced 15-lipoxygenase activity in the aortic adventitia was approximately 15 times greater than that found in the vessel wall. Hypercholesterolemia and elevated 15-lipoxygenase were associated with a 40% lowering of blood hematocrit. The hemolytic agent phenylhydrazine duplicated the effects of hypercholesterolemia on hematocrit, and induced up to 100-fold increases in 15-lipoxygenase activity in tissues within 7 days. The induced 15-lipoxygenase activities in heart and lung were 4 and 8 times greater, respectively, than in reticulocytes, previously the richest known source of the enzyme. Direct measurements of hemoglobin content also demonstrated that contaminating reticulocytes were not the source of the tissue enzyme. A similar tissue-specific activation of 15-lipoxygenase was observed in rat heart and lung, but also not in liver. It is concluded that the elevated level of 15-lipoxygenase activity previously reported in atherosclerotic aorta is symptomatic of a generalized and massive induction of the enzyme in cardio-pulmonary tissues by hypercholesterolemia, which may be related to the membrane perturbation and increased hemolysis that is induced by cholesterol feeding.

Relationship of vascular 15-lipoxygenase induction to atherosclerotic plaque formation in rabbits

Aortas from atherosclerotic rabbits have increased levels of 15-lipoxygenase, but the relationship between induction of this enzyme and the atherosclerotic process has not been defined. We found that dietary administration of cortisone acetate significantly suppressed atherosclerotic plaque formation in both Watanabe Heritable Hyperlipidemic (WHHL) and cholesterol-fed WHHL/NZW heterozygous rabbits. There was, however, no corresponding decrease in the elevated 15-lipoxygenase activity. In addition, the elevated 15-lipoxygenase activity in atherosclerotic rabbit aortas was uniformly distributed throughout the aorta, and was not preferentially localized in the lesions. These results indicate that induction of the 15-lipoxygenase is not necessarily causally related to plaque development, and that plaques are not the major source of the increased enzyme activity. However, the results confirm that hypercholesterolemia is a necessary condition for both atherosclerosis and 15-lipoxygenase induction, suggesting that perhaps the 15-lipoxygenase may represent a protective response to the hyperlipidemic stress. This possibility is supported by the finding that the induced 15-lipoxygenase converts linoleic acid, which is the predominant essential fatty acid in aorta, to 13-hydroxyoctadecadienoic acid (13-HODE). This compound is a chemorepellant factor for platelets, inhibits platelet thromboxane synthesis, and stimulates prostacyclin synthesis by endothelial cells.