Thomas L. Foxall

Vitamin E Inhibits Low-Density Lipoprotein–Induced Adhesion of Monocytes to Human Aortic Endothelial Cells In Vitro

Monocyte adhesion to human aortic endothelial cells (ECs) is one of the early events in the development of atherogenesis. ECs were used to investigate the role of vitamin E in human monocyte adhesion to ECs in vitro. ECs incubated with 40 to 193 mg/dL of low-density lipoprotein cholesterol (LDL) for 22 hours exhibited increasing dose-dependent adherence for untreated, isolated human monocytes (P < .05). ECs exposed to the highest dose of LDL (193 mg/dL) but pretreated with 19 mumol/L alpha-tocopherol for 24 hours showed a trend to lower adherence for monocytes compared with non-treated ECs (4.4 +/- 1.2% versus 7.6 +/- 1.9%; P = .09). This effect of vitamin E became more significant (P < .05) when ECs were exposed to a lower level of LDL (40 mg/dL) or were pretreated with a higher level of alpha-tocopherol (42 mumol/L) and then exposed to 80 mg/dL LDL. Presupplementation of ECs with 15, 19, and 37 mumol/L alpha-tocopherol significantly (P < .05) reduced monocyte adhesion by 6 +/- 1%, 37 +/- 6%, and 69 +/- 17%, respectively. Levels of soluble intercellular adhesion molecule-1 (sICAM-1), one of the adhesion molecules for monocytes, increased after incubation of ECs with LDL 80 mg/dL (4.7 +/- 0.7 versus 6.4 +/- 1.2 ng/mL, respectively; P < .05). Treatment of ECs with alpha-tocopherol (42 mumol/L) significantly reduced induction of sICAM-1 by LDL to 2.2 +/- 2.3 ng/mL. After exposure to LDL, prostaglandin I2 production by ECs was diminished, whereas presupplementation of ECs with alpha-tocopherol partially reversed the LDL effect. Production of interleukin-1 beta was not detectable when ECs were treated with alpha-tocopherol, LDL, or alpha-tocopherol followed by LDL. Our findings indicate that vitamin E has an inhibitory effect on LDL-induced production of adhesion molecules and adhesion of monocytes to ECs via its antioxidant function and/or its direct regulatory effect on sICAM-1 expression.

Comparison of the toxins of the blue-green alga Aphanizomenon flos-aquae with the Gonyaulax toxins

A toxic strain of Aphanizomenon flos-aquae (NH-1), isolated from a toxic bloom in a pond in Durham, New Hampshire, has been mass cultured in the laboratory. The toxin was extracted by ultrasonic disruption of the cells and purified by; (a) filtration through a 10 kilodalton filter, and (b) chromatography on a strong cation exchange resin column using 0.01 M, then 0.1 M, pH 5, sodium acetate buffer followed by 0.75 M hydrochloric acid. Mouse assays and fluorescence generated by hydrogen peroxide oxidation were used to monitor the fractions. Only a nonfluorescent toxic peak followed immediately by a fluorescent less-toxic peak were detected, both eluting with the hydrochloric acid fractions. The toxins were identical in behavior to neosaxitoxin and saxitoxin, respectively, when compared with elution profiles of the paralytic shellfish poisons from Gonyaulax tamarensis var. excavata and by paper electrophoretic and thin-layer chromatographic comparisons. The toxin profile appears to be different from that of a previously isolated strain of A. flos-aquae from Kezar Lake.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in hamster aortic atherosclerosis: correlation with in-situ zymography.

Atherogenesis requires extracellular matrix (ECM) alterations, a process possibly mediated by matrix-degrading metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs). The objective of this study was to examine the immunohistochemical expression patterns of MMPs-1, -2, -3 and -9 and their tissue inhibitors, TIMPs-1, -2, -3 and -4 during the three major stages of atherosclerotic lesion development in hypercholesterolemic Syrian Golden hamsters. Aortic atherosclerotic lesions (fatty streak, fibro-fatty and advanced) were histologically characterized in treated hamsters at 12, 24, and 49 weeks. The immunochemistry expression of these MMPs and TIMPs were examined in treated aortic sections with lesions and control aortic sections without lesions. MMP activity in control aortas and atherosclerotic lesions was characterized by in-situ zymography. Positive immunoreactivity for MMPs-2, -3, -9 and TIMPs-1, -2,-3, and -4 was observed in both control and atherosclerotic aortic arch segments, while MMP-1 was only observed in atherosclerotic lesions. Using in-situ zymography, we identified casein and gelatin degradation in fatty streak, fibro-fatty and advanced lesions. The immunohistochemical expression of these MMPs and TIMPs were examined in treated aortic sections with lesions and control aortic sections without lesions. In all lesion stages, substrate degradation was inhibited with 1,10-phenanthroline. Degradation of these substrates was not observed in control aortas. In addition, substrate degradation was inhibited with 1,10-phenanthroline. These findings suggested that in control segments, the net proteolytic balance was shifted in favor of MMP inhibition. Alternatively, despite the colocalization of MMPs and TIMPs in the treated segments, net proteolytic balance favored the catalytic MMPs.

Pterins of the cyanobacterium Aphanizomenon flos-aquae

The cyanobacterium Alphanizomenon flos-aquae contains various glycosides of a pterin that had previously been tentatively identified as 6-(threo-1,2-dihydroxypropyl)-pterin. The pterin is now identified as 6-(l-threo-1,2-dihydroxypropyl)pterin(6-l-threo-biopterin) by NMR and mass spectrometry, thinchromatographic comparisons with authentic samples and by optical rotation. The 6-l-threo-biopterin glycoside-1 from toxic strain NH-1 gave rise to xylose and glucose on hydrolysis, whereas its glycoside-2 from Cambridge Collection and nontoxic NH-1 strains gave mannose and glucose. This pterin may be a useful marker for certain species of cyanobacteria.

Decreased aortic early atherosclerosis in hypercholesterolemic hamsters fed oleic acid-rich TriSun oil compared to linoleic acid-rich sunflower oil

Previous studies have demonstrated that low density lipoprotein (LDL) enriched in polyunsaturated fatty acids (PUFA) are more susceptible to oxidation (ex vivo) than those containing monounsaturated fatty acids (MUFA). To test whether this observation was associated with various parameters considered to be related with the development of early aortic atherosclerosis, hamsters were fed commercial hypercholesterolemic diets (HCD) containing either the PUFA, sunflower oil (SF) or the MUFA, TriSun oil (TS) at 10% with 0.4% cholesterol (wt/wt). LDL isolated from hamsters fed TS had significantly longer lag phase (30%, P < 0.05), a decreased propagation phase (-62%, P < 0.005), and fewer conjugated dienes formed (-37%, P < 0.007) compared to hamsters fed SF. Aortic vasomotor function, measured as degree of aortic relaxation, was significantly greater in the TS vs SF-fed hamsters whether acetylcholine or the calcium ionophore A23187 was used as the endothelium-dependent agonist. As a group, the SF-fed hamsters had significantly more early atherosclerosis than hamsters fed TS (46%, P < 0.006). When animals across the two diets were pair-matched by plasma LDL-C levels, there was an 82% greater mean difference (P < 0.002) in early atherosclerosis in the SF versus the TS-fed hamsters. While there were no significant associations with plasma lipids and lipoprotein cholesterol, early atherosclerosis was significantly correlated with lag phase (r = -0.67, p < 0.02), rate of LDL conjugated diene formation (r = 0.74, p < 0.006) and maximum dienes formed (r = 0.67, p < 0.02). Compared to TS-fed animals, aortic sections from hamsters fed the SF-containing diet revealed that the cytoplasm of numerous foam cells in the subendothelial space reacted positively with the monoclonal anti-bodies MDA-2 and NA59 antibody, epitopes found on oxidized forms of LDL. The present study suggests that compared to TS, hamsters fed the SF-diet demonstrated enhanced LDL oxidative susceptibility, reduced aortic relaxation, greater early aortic atherosclerosis and accumulation of epitopes found on oxidized forms of LDL.

Effects of a Hepatic Toxin from the Cyanophyte Microcystis Aeruginosa

Microcystis aeruginosa is commonly involved in freshwater blooms and one of its toxins (microcystin) causes liver damage in birds and mammals. This study determined the site of action of microcystin and characterized the hepatic damage at the ultrastructural level. Histological changes in centrilobular regions of liver tissue were noted after i.p. administration in mice. Hepatic sinusoids expanded, parenchymal cords disintegrated, and cells lysed resulting in extensive tissue damage and subsequent death within one hour. Ultrastructural studies showed that sinusoidal epithelium and hepatocyte plasma membranes ruptured with the release of cellular components that pooled with blood. Mitochondria appeared swollen but there were no obvious distortions of organelles. Extensive vesiculation of membrane fragments was observed. Hepatic damage caused hemorrhaging into the liver where blood and cell debris accumulated and produced a significant increase in liver weight.

Effects of dietary fish oil and butterfat on serum lipids and monocyte and platelet interactions with aortic endothelial cells.

We studied effects of dietary lipids on some of the initial events in atherogenesis. Adult swine were fed low fat/low cholesterol diets, then challenged with a high cholesterol (1%, w/w) diet supplemented with 11.5% (w/w) butterfat (BF) or MaxEPA fish oil (FO). Serum lipids and monocyte and platelet adhesion to porcine aortic endothelial cells in vitro were measured during feeding of the low fat diet and at 1, 2, and 5 weeks after the dietary challenge. Total cholesterol increased significantly in animals fed the BF and FO diets, but there was no difference between the groups. Animals fed FO had total cholesterol/high-density lipoprotein cholesterol values twice those fed BF (P less than 0.01). After 2 weeks on the hypercholesterolemic diet, monocyte adhesion to endothelial cells increased in swine fed FO by 123% above those fed a low fat diet, and adhesion values remained elevated (56% above baseline value) after 5 weeks. Monocytes from swine fed BF showed increased adhesion by 87, 53, and 14% above those fed the low fat diet at 1, 2, and 5 weeks respectively. Platelet adhesion to endothelial cells decreased (P less than 0.05) after diet change and remained low. Adhesion of platelets from swine fed FO was significantly lower than those fed BF at 1 and cholesterol profile and greater monocyte adhesion to endothelial cells, conditions which in vivo may promote lesion initiation.

Unidirectional transfer in vivo of high-density lipoprotein cholesteryl esters to lower-density lipoproteins in the pig, an animal species without plasma cholesteryl ester transfer activity.

The metabolism of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesteryl esters (CE) was studied in the pig, an animal species without plasma cholesteryl ester transfer activity (CETA). In the first series of experiments, LDL and HDL from normocholesterolemic pigs were radiolabeled with cholesteryl (1-14C)oleate and intravenously administered to two groups of four normocholesterolemic pigs. Radioactive tracer in LDL remained associated with the LDL fraction, and there was no transfer of LDL-CE to HDL. The transport rate (which represents the production and disposal rate) of LDL-CE in normocholesterolemic pigs was 39 mumol CE/h/L. However, radiolabeled HDL-CE were transferred to LDL (25%), and 36% of the LDL-CE mass was derived from the HDL. The transport rate of HDL-CE was 54 mumol CE/h/L, and the flux of HDL-CE to LDL was 14 mumol CE/h/L. There was no accumulation of radiolabeled HDL-CE in very-low-density lipoprotein (VLDL), which suggests that there was no transfer to VLDL. However, this does not rule out the possibility that either the very low levels of VLDL-CE (< 0.09 mmol/L) or the rapid turnover rate of the VLDL pool might have prevented the accumulation of substantial amounts of tracer in VLDL. Therefore, in a second set of experiments, the kinetics of HDL-CE were studied in high-fat-and high-cholesterol-fed pigs with elevated VLDL-CE concentrations (1.92 mmol/L). Hypercholesterolemia was associated with increased transport rates of LDL-CE (165 mumol/h/L) and HDL-CE (78 mumol/h/L) and with an increased flux of HDL-CE to LDL (78 mumol/h/L).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of exercise on plasma lipids and LDL subclass metabolism in miniature swine.

The effects of exercise on plasma lipids and lipoproteins, high density lipoprotein (HDL) subclass cholesterol levels, and low density lipoprotein (LDL) subclass composition and metabolism were studied in Yucatan miniature swine following 2 yr of training. The exercise protocol produced significant training effects. Post-heparin lipolytic activity was also significantly increased. Although plasma cholesterol and triglycerides did not differ significantly (P = 0.08) between the exercised and control groups, multivariate analysis indicated a strong association between lipoprotein lipase (LPL) and HDL2-C (P less than 0.0001). Although HDL-C levels rose only slightly (P less than 0.09) with exercise, a significant shift was noted in the distribution of cholesterol from the HDL3 to the HDL2 fractions, perhaps mediated by the substantial increase in LPL activity. Exercise had little effect on the chemical composition of the major lipoprotein classes; however, the triglyceride content of the lighter LDL1 subclass was significantly reduced. In the more dense LDL2 subclass, exercise resulted in a significant decrease in triglycerides concomitant with a significant increase in free cholesterol levels. In contrast with the small reductions in fractional catabolic rates (FCR) in either subclass, production rates of the exercised group were reduced, which accounted for the reduction in LDL subclass pool size. These data indicate that exercise produces subtle but significant changes in lipoprotein metabolism that have been previously associated with reduced risk of atherosclerosis.

Doxazosin, an α-1 antagonist, prevents further progression of the advanced atherosclerotic lesion in hypercholesterolemic hamsters

The aim of this study was to examine the effect of doxazosin (DOX) on the further progression and regression of the advanced atherosclerotic lesion in the hypercholesterolemic hamster. Thirty-six, male F(1)B Golden Syrian hamsters, 10 weeks of age, were divided into 3 groups of 12 and fed a nonpurified hypercholesterolemic diet (HCD) containing 10% coconut oil and 0.1% cholesterol (wt/wt) for 9 months (HCD 9). One group of hamsters was euthanized at 9 months and their aortas were collected, fixed, and stored until analysis. The remaining hamsters were either maintained on the HCD for an additional 6 months (HCD 15) or fed the HCD plus 20 mg/kg/d DOX for the 6 months. At the end of the study (15 months), the DOX-treated hamsters had significantly lower plasma total cholesterol (TC) (-68%), low-density lipoprotein-cholesterol (LDL-C) (-73%), and triglycerides (TG) (-74%) compared with the HCD 15. The lumenal narrowing and intimal thickening atherosclerotic lesions were significantly less in the DOX-treated hamsters compared with the HCD 15 (-66% and -70%, respectively). These data suggest that DOX treatment prevents further progression of the advanced atherosclerotic lesion possibly by lowering plasma TC, LDL-C, and TG in hypercholesterolemic hamsters.