Alan Daugherty

Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions.

Oxidatively modified lipoproteins have been implicated in atherogenesis, but the mechanisms that promote oxidation in vivo have not been identified. Myeloperoxidase, a heme protein secreted by activated macrophages, generates reactive intermediates that oxidize lipoproteins in vitro. To explore the potential role of myeloperoxidase in the development of atherosclerosis, we determined whether the enzyme was present in surgically excised human vascular tissue. In detergent extracts of atherosclerotic arteries subjected to Western blotting, a rabbit polyclonal antibody monospecific for myeloperoxidase detected a 56-kD protein, the predicted molecular mass of the heavy subunit. Both the immunoreactive protein and authentic myeloperoxidase bound to a lectin-affinity column; after elution with methyl mannoside their apparent molecular masses were indistinguishable by nondenaturing size-exclusion chromatography. Peroxidase activity in detergent extracts of atherosclerotic lesions likewise bound to a lectin column and eluted with methyl mannoside. Moreover, eluted peroxidase generated the cytotoxic oxidant hypochlorous acid (HOCl), indicating that enzymatically active myeloperoxidase was present in lesions. Patterns of immunostaining of arterial tissue with antihuman myeloperoxidase antibodies were similar to those produced by an antimacrophage antibody, and were especially prominent in the shoulder region of transitional lesions. Intense foci of myeloperoxidase immunostaining also appeared adjacent to cholesterol clefts in lipid-rich regions of advanced atherosclerotic lesions. These findings identify myeloperoxidase as a component of human vascular lesions. Because this heme protein can generate reactive species that damage lipids and proteins, myeloperoxidase may contribute to atherogenesis by catalyzing oxidative reactions in the vascular wall.

Attenuation of diet-induced atherosclerosis in rabbits with a highly selective 15-lipoxygenase inhibitor lacking significant antioxidant properties.

15‐Lipoxygenase (15‐LO) has been implicated in the pathogenesis of atherosclerosis because of its localization in lesions and the many biological activities exhibited by its products. To provide further evidence for a role of 15‐LO, the effects of PD 146176 on the development of atherosclerosis in cholesterol‐fed rabbits were assessed. This novel drug is a specific inhibitor of the enzyme in vitro and lacks significant non specific antioxidant properties. PD 146176 inhibited rabbit reticulocyte 15‐LO through a mixed noncompetitive mode with a Ki of 197 nm. The drug had minimal effects on either copper or 2,2′‐azobis(2‐amidinopropane)hydrochloride (ABAP) induced oxidation of LDL except at concentrations 2 orders higher than the Ki. Control New Zealand rabbits were fed a high‐fat diet containing 0.25% wt./wt. cholesterol; treated animals received inhibitor in this diet (175 mg kg−1, b.i.d.). Plasma concentrations of inhibitor were similar to the estimated Ki (197 nm). During the 12 week study, there were no significant differences in weight gain, haematocrit, plasma total cholesterol concentrations, or distribution of lipoprotein cholesterol. The drug plasma concentrations achieved in vivo did not inhibit low‐density lipoprotein (LDL) oxidation in vitro. Furthermore, LDL isolated from PD 146176‐treated animals was as susceptible as that from controls to oxidation ex vivo by either copper or ABAP. PD 146176 was very effective in suppressing atherogenesis, especially in the aortic arch where lesion coverage diminished from 15±4 to 0% (P<0.02); esterified cholesterol content was reduced from 2.1±0.7 to 0 μg mg−1 (P<0.02) in this region. Immunostainable lipid‐laden macrophages present in aortic intima of control animals were totally absent in the drug‐treated group. Results of these studies are consistent with a role for 15‐LO in atherogenesis.

Apolipoprotein E-containing High Density Lipoprotein Promotes Neurite Outgrowth and Is a Ligand for the Low Density Lipoprotein Receptor-related Protein

Presence of the ε4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimers disease (AD), although the mechanism(s) by which it confers this risk is unknown. ApoE may play a direct role in AD neuropathology by modulating neuronal structure. We previously showed that apoE3-containing β-very low density lipoprotein (β-VLDL) can stimulate neurite outgrowth to a significantly greater extent than apoE4-enriched β-VLDL in a central nervous system-derived neuronal cell line and that this effect is mediated by interaction with the low density lipoprotein receptor-related protein (LRP). To determine whether similar differences exist when apoE is associated with other lipoprotein particles, the effects of high density lipoprotein (HDL) derived from plasma and cerebrospinal fluid were defined. ApoE3-enriched HDL significantly enhanced neurite outgrowth as compared with apoE4-enriched HDL, and the majority of this stimulation was blocked in the presence of the receptor-associated protein or a neutralizing antibody to LRP. We also found that cholesterol esterification in the presence of apoE-containing plasma HDL was attenuated in fibroblasts lacking LRP. Therefore, apoE-containing HDL can serve as an LRP ligand, and apoE isoform-specific effects on neurite outgrowth are observed when HDL is the carrier particle.

Lymphocyte Populations in Atherosclerotic Lesions of ApoE −/− and LDL Receptor −/− Mice Decreasing Density With Disease Progression

Lymphocytes are prominent components of human atherosclerotic lesions, but their presence in murine models of disease has not been confirmed. Lymphocyte subpopulations have been identified in apoE -/- and LDL receptor -/- mice fed a cholesterol-enriched diet for up to 3 months. ApoE -/- mice had higher serum cholesterol concentrations than did LDL receptor -/- mice during most of the feeding period, primarily due to large increases in VLDL concentrations. Total area of atherosclerotic lesions was greater at all times in apoE -/- than LDL receptor -/- mice (lesion area after 3 months on cholesterol-enriched diet: apoE -/-, 993 +/- 193 and LDL receptor -/-, 560 +/- 131 microns2 x 10(3), mean +/- SEM, n = 6 in each group). Lesions in apoE -/- mice contained larger macrophage-rich necrotic cores and more calcification than did those in LDL receptor -/- mice. Immunocytochemical analyses of tissue sections of ascending aortas performed with monoclonal antibodies to T and B lymphocytes and macrophages revealed that T lymphocytes immunoreactive for Thy 1.2, CD5, CD4, and CD8 were observed in lesions from both strains, but no B lymphocytes were detected. The density of Thy 1.2+ T lymphocytes in lesions was greatest at 1 month (apoE -/-, 98 +/- 23 and LDL receptor -/-, 201 +/- 40 lymphocytes/mm2, n = 6 in each group), decreasing in apoE -/- mice to 12 +/- 3 and in LDL receptor -/- mice to 51 +/- 20 lymphocytes/mm2 at 3 months. The presence of T lymphocytes in murine atherosclerotic lesions makes these animals potentially useful for studying the involvement of the immune system in atherogenesis.

Beta-carotene inhibits atherosclerosis in hypercholesterolemic rabbits.

Oxidatively damaged LDL may be of central importance in atherogenesis. Epidemiological evidence suggests that high dietary intakes of beta-carotene and vitamin E decreases the risk for atherosclerotic vascular disease, raising the possibility that lipid-soluble antioxidants slow vascular disease by protecting LDL from oxidation. To test this hypothesis, we fed male New Zealand White rabbits a high-cholesterol diet or the same diet supplemented with either 1% probucol, 0.01% vitamin E, 0.01% all-trans beta-carotene, or 0.01% 9-cis beta-carotene; then we assessed both the susceptibility of LDL to oxidation ex vivo and the extent of aortic atherosclerosis. As in earlier studies, probucol protected LDL from oxidation and inhibited lesion formation. In contrast, vitamin E modestly inhibited LDL oxidation but did not prevent atherosclerosis. While beta-carotene had no effect on LDL oxidation ex vivo, the all-trans isomer inhibited lesion formation to the same degree as probucol. Moreover, all-trans beta-carotene was undetectable in LDL isolated from rabbits fed the compound, although tissue levels of retinyl palmitate were increased. The effect of all-trans beta-carotene on atherogenesis can thus be separated from the resistance of LDL to oxidation, indicating that other mechanisms may account for the ability of this compound to prevent vascular disease. Our results suggest that metabolites derived from all-trans beta-carotene inhibit atherosclerosis in hypercholesterolemic rabbits, possibly via stereospecific interactions with retinoic acid receptors in the artery wall.

Probucol attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits.

1 Probucol was administered to rabbits fed a cholesterol‐enriched (2% wt/wt) diet to determine potential anti‐atherogenic effects in a preparation in which the disease process is due to elevated plasma concentrations of cholesterol ester‐rich very low density lipoproteins (CER‐VLDL). 2 Probucol was supplemented to the diet at 1% wt/wt which resulted in plasma concentrations rising steadily to 53 ± 8 μg ml−1 after 14 days, with no significant changes during continued administration. Dietary consumption and body weight gains were comparable in the drug‐treated and control groups during the observation period. 3 Probucol treatment did not significantly affect plasma concentrations of total cholesterol, unesterified cholesterol, triglycerides or phospholipids. 4 The concentration of CER‐VLDL in plasma and its physicochemical characteristics were not significantly changed during administration of probucol. CER‐VLDL from both control and probucol‐treated animals was a potent stimulant of the augmentation of the intracellular incorporation of [3H]‐oleate into cholesteryl‐[3H]‐oleate in cultured macrophages. 5 Despite the lack of effect of probucol on concentrations of plasma lipids and the cell interaction characteristics of CER‐VLDL, administration of the drug markedly decreased the extent of intimal aortic surface area covered by grossly discernible atherosclerotic lesions from 55.6 ± 11.8% to 11.6 ± 1.9% in thoracic sections, and from 49.1 ± 10.2% to 7.2 ± 0.4% in abdominal sections. Furthermore, probucol treatment significantly reduced the deposition of total cholesterol in vascular tissue. 6 Probucol reduced the extent of aortic atherosclerosis produced by diet‐induced hypercholesterolemia in rabbits. This reduction occurred in the absence of any significant change in the characteristics of plasma lipoproteins that were determined. These results indicate that either there is a role of oxidation in the disease process of this animal model of atherosclerosis or that probucol is acting via a presently undefined mechanism.

A specific 15-lipoxygenase inhibitor limits the progression and monocyte–macrophage enrichment of hypercholesterolemia-induced atherosclerosis in the rabbit

Oxidant signalling and lipoprotein oxidation may play important roles in atherosclerotic lesion development. Given coincident localization of 15-lipoxygenase (15-LO), stereospecific products of 15-LO and epitopes of modified LDL in atherosclerotic lesions, we hypothesized that inhibition of 15-LO by PD146176, an inhibitor of 15-LO with an IC50 in cells or isolated enzyme of 0.5-0.8 microM, may limit atherosclerotic lesion development through regulation of monocyte-macrophage enrichment. Rabbits exposed to chronic endothelial denudation of the iliac-femoral artery were meal-fed a 0.25% cholesterol (C), 3% peanut oil (PNO), 3% coconut oil (CNO) diet twice daily with and without 175 mg/kg PD146176 for 12 weeks. In a second study, atherosclerotic lesions were pre-established in rabbits through chronic endothelial denudation and meal-fed a 0.5% C, 3% PNO, 3% CNO diet for 9 weeks and a 0% C/fat diet for 6 weeks prior to an 8 week administration of PD146176 at 175 mg/kg, q.d. Plasma total and lipoprotein cholesterol exposure were similar in control and PD146176-treated animals in both studies but PD146176 increased plasma triglyceride exposure 2- to 4-fold. Plasma PD146176 concentrations ranged from 99 to 214 ng/ml at 2 h post-dose. In the progression study, the iliac-femoral monocyte-macrophage area was reduced 71%, cross-sectional lesion area was unchanged and cholesteryl ester (CE) content was reduced 63%. In the regression study, size and macrophage content of iliac-femoral, fibrous plaque-like lesions were decreased 34%, CE content was reduced 19% and gross extent of thoracic aortic lesions were reduced 41%. We conclude that PD146176 can limit monocyte macrophage enrichment of atherosclerotic lesions and can attenuate development of fibrofoamy and fibrous plaque lesions in the absence of changes in plasma total or lipoprotein cholesterol concentrations.

Isolation of low density lipoprotein from atherosclerotic vascular tissue of Watanabe heritable hyperlipidemic rabbits.

Atherogenic properties of low density lipoproteins (LDL) in vivo may reflect modification of lipoproteins associated with endothelial translocation and exposure to extracellular matrix and interstitial fluid. To examine whether modifications of LDL occur in vivo, lipoproteins were isolated from plasma and vascular tissue of Watanabe heritable hyperlipidemic (WHHL) rabbits. LDL was extracted from vascular tissue (LDL-V) by homogenization in the presence of a sodium carbonate buffer. Control experiments demonstrated that modifications did not occur under the preparative conditions used to release LDL from tissue. LDL-V contained less esterified cholesterol, but more cholesterol esters, than did LDL from plasma (LDL-P). The diameters of both LDL-V and LDL-P followed gaussian distributions, but LDL-V particles were smaller (20.3 +/- 0.1 and 26.3 +/- 0.1 nm). Mild lipid peroxidation was evident in LDL-V. The sphingomyelin content was increased in LDL-V, with less phosphatidylcholine than in LDL-P. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that apolipoprotein B was depleted in LDL-V, but Western blot analyses identified lower molecular weight proteins antigenically related to apolipoprotein B. LDL-V markedly stimulated cholesterol esterification in mouse peritoneal macrophages and also in rabbit alveolar macrophages, a cell type that did not respond to acetylated LDL. LDL-V was not recognized by cultured rabbit skin fibroblasts. Thus, LDL isolated from atherosclerotic vascular tissue in vivo was modified in a fashion that could confer atherogenic properties reflected by augmentation of cholesterol esterification in macrophages in vitro.

Enhanced development of atherosclerosis in cholesterol-fed rabbits by suppression of cell-mediated immunity.

T lymphocytes are present in atherosclerotic lesions, but the role of this cell type in the disease process has not been determined. To determine whether cell-mediated immunity influences atherogenesis, New Zealand White rabbits fed a cholesterol-supplemented diet (0.5% wt/wt) were treated with cyclosporin A (n = 20) or vehicle alone (n = 16) for 12 wk. The dose of cyclosporin A was adjusted so that a blood concentration between 100 and 200 ng/ml was maintained to achieve a selective action T-lymphocytes. Effectiveness of immunosuppression in cyclosporin A-treated rabbits was confirmed by allogeneic skin graft survival. Cyclosporin A administration did not affect total plasma lipid concentrations, body weight, or renal function. Percentage of aortic intimal area covered with atherosclerotic lesions was increased significantly by immunosuppression in both the arch region (75 +/- 3% [mean +/- SEM] compared with 60 +/- 5% in controls; P < 0.01) and the thoracic region (47 +/- 7% vs 27 +/- 6%; P = 0.04). Enhanced atherogenesis was not associated with diminished numbers of T lymphocytes in lesions, changes in T lymphocyte subtype, or any discernible change in cellular composition. Humoral immune responses to oxidized LDL were similar in the two groups: serum titres of autoantibodies against malondialdehyde-modified LDL were equivalent. These data demonstrate that cyclosporin A-induced suppression of cell-mediated immunity increased the development of macrophage-rich atherosclerotic lesions in cholesterol-fed rabbits.

The effects of probucol on the progression of atherosclerosis in mature Watanabe heritable hyperlipidaemic rabbits.

1 Probucol was administered to mature Watanabe heritable hyperlipidaemic (WHHL) rabbits (≅9 months old). Groups of WHHL rabbits were randomly selected and treated as follows: Group 1 killed at 9 months (n = 9); Group II placed on sham‐treated diet at 9 months and followed for 6 months (n = 8); Group III placed on probucol at 9 months and followed for 6 months (n = 8). Probucol was administered by mixing 1% wt/wt drug with standard laboratory diet. 2 Plasma concentrations of probucol increased to 93 ± 11 μg ml−1 in Group III during the initial 2 weeks and increased further to 149 ± 24 μg ml−1 at the end of the treatment period. 3 Plasma concentrations of total cholesterol, unesterified cholesterol and phospholipids were significantly reduced overall by probucol, while triglycerides were not affected. 4 No statistically significant differences were observed in the presence of oxidized products in low density lipoproteins (LDL) isolated from plasma of controls compared to probucol‐treated rabbits. However, LDL from probucol‐treated animals was resistant to oxidation in the presence of Cu2+ (3 μm). 5 Group I had aortic atherosclerosis covering 70 ± 5% of intimal area of thoracic aortae, that increased to 91 ± 3% in Group II. This was associated with cholesterol contents of aortae increasing from 1.4 ± 0.2 μg mg−1 in Group I to 2.7 ± 0.3 μg mg−1 in Group II. Probucol administration did not produce a statistically significant reduction of atherosclerotic lesion area (78 ± 7%). However, probucol treatment reduced cholesterol content to 1.9 ± 0.3 μg mg−1 (P < 0.01). Collagen content of aortae was not affected by probucol treatment. 6 Thus, while probucol did not promote regression, the drug did retard the continued deposition of cholesterol esters into atherosclerotic lesions of mature WHHL rabbits.