The resistance of low density lipoprotein to oxidation promoted by copper and its use as an index of antioxidant therapy.

The measurement ex vivo of the resistance of low density lipoprotein (LDL) to oxidation promoted by copper is now being used in surveys of human populations at risk of developing atherosclerosis. However, it is not known whether a relationship between LDL oxidisability measured in this way and the development of atherosclerotic lesions exists. Using Watanabe rabbits as a model of the disease, we have found that dietary supplementation with the antioxidants, probucol and alpha-tocopherol, increased the resistance of LDL isolated from small volumes of plasma to oxidation. The antioxidant effects of probucol incorporated into LDL through dietary supplementation were greater than when incorporated ex vivo. When dietary supplementation was extended to a period of three months, the well established anti-atherosclerotic effects of probucol were confirmed and a highly significant relationship between the probucol content of the LDL particle and the extent of the atherosclerotic lesion in the aorta emerged. These results suggest that the assessment of the resistance of LDL isolated from plasma to oxidation promoted by copper may reflect the response of the arterial atherosclerotic process to antioxidant therapy.

The adventitia and atherogenesis: removal initiates intimal proliferation in the rabbit which regresses on generation of a 'neoadventitia'.

Removal of the carotid artery adventitia from rabbits induced the formation of an intimal hyperplastic lesion. In rabbits fed a normal diet, the lesion (measured as the intimal:medial ratio) was maximal by day 14 (0.456 +/- 0.079, n = 5, P < 0.01) and thereafter, regressed towards control dimensions (0.037 +/- 0.003, n = 14) by day 28 (0.080 +/- 0.025, n = 7, P = 0.14). In rabbits fed a high cholesterol diet, the lesion was again maximal by day 14 (0.376 +/- 0.056, n = 8, P < 0.01). Although some regression was seen, the lesion persisted to day 42 (0.272 +/- 0.052, n = 8, P < 0.01). Electron microscopy and immunocytochemistry showed two types of lesion, (a) smooth muscle cell predominant on normal diet and, (b) macrophage predominant on high cholesterol diet. Smooth muscle cell predominant lesions underwent almost complete regression, whereas macrophage predominant lesions persisted. We propose that lesion formation may be initiated following the development of arterial wall hypoxia, secondary to excision of the adventitial vasa vasorum. Furthermore, we have devised a novel method to restore a highly vascular 'neoadventitia' to an artery whose adventitia has previously been removed, using loosely placed PVC tubing. We suggest this 'neoadventitia' was able to inhibit the formation of an intimal hyperplastic lesion and to promote regression of an already established lesion by restoring arterial wall oxygenation.

Evaluation of inhibitors of eicosanoid synthesis in leukocytes: possible pitfall of using the calcium ionophore A23187 to stimulate 5' lipoxygenase.

The effect on arachidonate metabolism of two compounds (BW755C and benoxaprofen) which have been reported to inhibit 5' lipoxygenase in leukocytes has been evaluated in human polymorphonuclear leukocytes (PMN) stimulated with the calcium ionophore A23187 and serum-treated zymosan (STZ). The syntheses of leukotriene B4 (LTB4) and thromboxane B2 (TXB2) from endogenous substrate were determined by specific radioimmunoassays as indicators of 5' lipoxygenase and cyclo-oxygenase activity in the PMN respectively. Benoxaprofen inhibited the synthesis of leukotriene B4 by human PMN stimulated with the calcium ionophore A23187, but it was approximately 5 times less potent than BW755C. However, benoxaprofen (IC50 1.6 X 10(-4)M) was approximately 100 times less potent than BW755C (IC50 1.7 X 10(-6)M) at inhibiting leukotriene B4 synthesis induced by serum-treated zymosan. Both drugs inhibited thromboxane synthesis by leukocytes stimulated with A23187 or serum-treated zymosan at similar concentrations (approximately 5 X 10(-6)M). The data obtained using STZ as stimulus are consistent with previous in vivo studies and indicate that benoxaprofen is a relatively selective inhibitor of cyclo-oxygenase. However, this selectivity was far less apparent when A23187 was used as a stimulus to release the eicosanoids which suggests that this inhibition could be via an indirect mechanism and therefore A23187 should be used with caution as a stimulus of 5' lipoxygenase for evaluating inhibitors of eicosanoid synthesis.

Changes in the caldesmon isoform content and intimal thickening in the rabbit carotid artery induced by a silicone elastomer collar.

The presence of a silicone elastomer collar around one carotid artery of a rabbit induces thickening of the tunica intima. We used immunoblotting to study quantitatively changes in the isoforms of caldesmon, a protein implicated in the regulation of contractility in smooth muscle, while also monitoring the histological changes during 28 days after collaring. Control rabbit carotid arteries (n = 28) contained 245 +/- 6.4 nmol/g protein of the larger isoform of caldesmon (CDh) and 68.3 +/- 3.6 nmol/g protein of the smaller isoform (CD1). Four days after collaring, intimal thickening was slight, but 44% of arterial CDh had been lost; this loss of CDh was therefore from the tunica media. At 10 days, CDh fell to 37% of the control level. Immunofluorescence using CDh-specific antibodies showed that the CDh level was diminished but remained uniform across the wall of collared arteries. At 14 days, when intimal thickening was maximal, there was 30% more CD1 than in controls. At 28 days, the neointima had thinned, and CD1 had fallen to below control levels. Thus, CD1 levels reflected the development and regression of neointima. Changes in caldesmon isoforms showed that smooth muscle cell phenotypic changes occurred throughout the arterial wall.