Gordon A.A. Ferns

Vitamin E inhibits the intimal response to balloon catheter injury in the carotid artery of the cholesterol-fed rat

We have investigated the effect of the naturally occurring, lipid-soluble antioxidant, vitamin E, on the intimal response to balloon injury in the cholesterol-fed rat. We found that in animals receiving a 0.5% vitamin E plus 1% cholesterol diet, neo-intimal thickening was reduced by 30% (P < 0.025) compared to animals receiving either cholesterol alone, or a control chow diet. In all three dietary groups, the intimal lesion consisted predominantly of smooth muscle cells, and few monocytes/macrophages (< 0.5%) could be identified by staining with the monoclonal antibody ED-1. In vitro, vitamin E inhibited platelet-derived growth factor- (PDGF) (20 ng/ml) and serum (2%)-induced mitogenesis of both adult rat thoracic aortic smooth muscle cells and an embryonic rat aortic smooth muscle cell line (A7r5), dose-dependently. These data suggest that reactive oxygen species may be involved in the intimal response to balloon catheter injury, and that antioxidants, such as vitamin E, may offer some protection against restenosis. Although the way by which it does so is unclear, one possible mechanism is by a direct inhibitory effect on the accumulation of smooth muscle cells within the developing neo-intima.

Vitamin E reverses cholesterol-induced endothelial dysfunction in the rabbit coronary circulation

Hypercholesterolaemia and atherosclerosis are associated with impaired endothelium-dependent vasodilation. In this study we have examined the effects of vitamin E on cholesterol-induced endothelial dysfunction in the rabbit coronary circulation. Rabbits were maintained for 4 or 8 weeks on one of three experimental diets: (a) control chow, (b) 1% cholesterol or (c) 1% cholesterol for the first half of the treatment period followed by 1% cholesterol + 0.2% vitamin E during the last half of the treatment. After sacrifice, vasodilator responses to acetylcholine and sodium nitroprusside in the isolated perfused heart were studied. Responses to sodium nitroprusside were similar between the groups whereas responses to acetylcholine were significantly impaired in cholesterol-fed rabbits after both 4 and 8 weeks when compared to controls. In the cholesterol + vitamin E group, responses to acetylcholine were similar to controls and significantly greater than in the group receiving cholesterol alone. These results show that both 4 and 8 weeks of cholesterol-feeding induces an endothelial dysfunction in the coronary circulation of the rabbit, and that vitamin E protects against this dysfunction. By comparing responses to acetylcholine in the 4 week cholesterol group with the 8 week cholesterol + vitamin E group it was shown that vitamin E may not only prevent further deterioration of the endothelial function in the rabbit heart, but may also reverse the adverse effects of hypercholesterolaemia.

Dietary vitamin E increases the resistance to lipoprotein oxidation and attenuates endothelial dysfunction in the cholesterol-fed rabbit

This study was conducted to determine if vitamin E could reverse or attenuate endothelial dysfunction following an atherogenic diet. Rabbits were initially fed 1% cholesterol for 4 weeks to induce endothelial dysfunction. During the next 4 weeks the rabbits were fed either 1% cholesterol +0.2% vitamin E or 1% cholesterol alone, and were then killed. Endothelium-dependent responses to acetylcholine, calcium ionophore A23187 and sodium nitroprusside (SNP) were studied in the preconstricted perfused rabbit ear. Dietary vitamin E partially reversed the impaired endothelium-dependent responses to acetylcholine associated with cholesterol feeding. The maximum decrease in perfusion pressure in response to acetylcholine was 77.8% +/- 3.6% in control animals, 35.3% +/- 2.6% in cholesterol-fed animals, and 49.1% +/- 4.7% in cholesterol+vitamin E treated animals. The response to A23187 or sodium nitroprusside did not differ between the groups. The susceptibility of rabbit beta-VLDL to oxidation was markedly decreased in the vitamin E treated animals as assessed by the formation of conjugated dienes. The formation of lipid peroxidation products were also significantly inhibited by vitamin E. These data suggest that dietary vitamin E is beneficial in reducing the oxidative injury that may lead to the impairment of nitric oxide (NO)-mediated responses in early hypercholesterolaemia.

Hyaluronan (HYAL-BV 5200) inhibits neo-intimal macrophage influx after balloon-catheter induced injury in the cholesterol-fed rabbit

Hyaluronan is a glycosaminoglycan, elaborated by several cell types, and is a major constituent of the extracellular matrix. Recent studies suggest that hyaluronan influences cell migration and proliferation. At high concentrations, it has been shown to inhibit macrophage migration in vitro. We have investigated the effects of hyaluronan administration on neo-intimal lesion development following balloon catheter injury of the common carotid artery in the cholesterol-fed New Zealand White rabbit. Hyaluronan, administered as sodium hyaluronate at the time of surgery and daily until sacrifice, 2 weeks later, reduced the absolute neo-intimal response to injury by 42% (117 +/- 16 microns to 68 +/- 11 microns; P < 0.05), and the intima-media ratio by 35% (0.91 +/- 0.10 to 0.59 +/- 0.11; P < 0.05). This was associated with a 62% reduction in intimal macrophage content (8.63 +/- 1.85% to 3.25 +/- 1.05%; P < 0.02). At the time of killing, serum cholesterol levels and weight gain were comparable between the groups of animals receiving a cholesterol diet (P > 0.05). In both groups mean serum cholesterol levels at the time of the balloon injury and killing were significantly greater than at entry (P < 0.001), and significantly higher than in a group receiving control chow (P < 0.001). These data suggest that the effect of hyaluronic acid on neo-intimal size may be mediated, in part, by an inhibition of monocyte/macrophage influx, and support the view that hyaluronan impairs monocyte migration.

Oxidative modification of low-density lipoprotein by human polymorphonuclear leucocytes to a form recognised by the lipoprotein scavenger pathway

The oxidative modification of low-density lipoprotein (LDL) results in the formation of cytotoxic and chemotactic lipids which are thought to be of importance in the development of atherosclerotic lesions. In the present study we show that polymorphonuclear leucocytes (PMNs) can modify LDL to a form which is rapidly incorporated by macrophages via a scavenger receptor pathway. Incubation of 125I-labelled LDL with PMNs in Hams F-10 medium resulted in oxidation as shown by the appearance of thiobarbituric acid-reactive substances, increased electrophoretic mobility of the LDL and increased degradation of the LDL by mouse peritoneal macrophages. The presence of the anti-oxidant butylated hydroxytoluene or the metal ion chelator, EDTA inhibited the PMN-mediated modification. The degradation of 125I-labelled PMN modified LDL by macrophages was competitively inhibited by unlabelled copper-oxidised LDL but not by native LDL, indicating that the degradation was mediated by the scavenger receptor. The oxidative modification of LDL by PMNs could be of pathophysiological importance in inflammation and in the accelerated atherosclerosis seen following cardiac reperfusion injury.

Insulin-like growth factor-I modulates monocyte adhesion to EAhy 926 endothelial cells

IGF‐I is a ubiquitous growth factor, found in platelets and elaborated by many other cell types. It is thought to be involved in several pathophysiological processes including embryonic development, angiogenesis and wound healing. We report that the adherence of human peripheral blood monocytes to an endothelial cell line (EAhy 926) is inhibited in a dose and time‐dependent manner by pre‐incubating the endothelial cells with IGF‐I (P < 0.001). Monocyte adhesion was inhibited 17.9 ± 1.9% by IGF‐I at a dose of 1000 ng/ml (P < 0.01). In contrast, IGF‐I had no significant effect on monocyte adherence to plastic. The inhibitory effects of IGF‐I were reversed by co‐incubating the endothelial cells with the nitric oxide synthase inhibitor, L‐NAME. These data suggest that the effects of IGF‐I are mediated by the release of nitric oxide from the endothelial cells.

Insulin-like growth factor binding protein-1 inhibits arterial smooth muscle cell proliferation in vitro but does not reduce the neointimal response to balloon catheter injury

The biological effects of the insulin-like growth factors (IGFs) are modulated by circulating binding proteins (BPs), including IGFBP-1. We have investigated the effects of recombinant IGFBP-1 on smooth muscle cell (SMC) proliferation in vitro using cultured rat aortic SMCs and in vivo using the ballooned rat carotid artery model. IGFBP-1 inhibited IGF-1 induced and spontaneous SMC proliferation dose-dependently. In vivo, the effective half-life of IGFBP-1 was approximately 5 h when administered by intraperitoneal injection. High peri-operative plasma levels of IGFBP-1 (mean 1780 ng/ml) were attained by giving and intravenous dose immediately prior to balloon injury in 9 rats. Animals injected with human serum albumin or saline were used as controls. In vivo cell proliferation was assessed by BrdU pulse labeling each animal prior to the termination of the experiment, 6 days after balloon injury. Absolute intimal thickness, intima-media ratio and cell proliferation indices were measured for each animal. Although IGFBP-1 inhibited SMC proliferation in vitro, high plasma concentrations of IGFBP-1 did not reduce neointimal size or cell proliferation. IGFBP-1 administration was, however, associated with a significantly greater loss of body weight (P < 0.05), indicating that the peptide had a profound metabolic effect. Our data suggest that IGF-1 does not have a major role in inducing SMC proliferation in the early phases following angioplasty.

Tissue Distribution of α-Tocopherol Following Dietary Supplementation in the Rat: Effects of Concomitant Cholesterol Feeding

Abstract Vitamin E is a potent, naturally occurring, lipid-soluble antioxidant, which is reported to be protective against several disease processes, including coronary atherosclerosis. We have measured the α-tocopherol content of the aorta, liver, skeletal muscle, and kidney of rats fed one of the following diets for 10 weeks: a normal control chow diet (i); or the same diet containing 1% cholesterol (ii); 0.5% vitamin E (iii); or 1% cholesterol plus 0.5% vitamin E (iv). The α-tocopherol content of serum and tissue extracts was measured by HPLC using γ-tocopherol as an internal standard. Tissue and serum cholesterol content was measured using a cholesterol oxidase enzyme reagent kit. In all animals receiving the 1% cholesterol diet, serum cholesterol levels increased significantly (P < 0.005). By the 10th week, mean serum α-tocopherol levels rose significantly in both groups of animals receiving dietary vitamin E supplements (P < 0.0001) compared with their respective control group. This was accompanied by a significant increase in the absolute α-tocopherol content of liver (8- to 9-fold) and aorta (3- to 4-fold). The α-tocopherol content of renal and skeletal muscle tissue was raised 1- to 2-fold in both groups of rats on vitamin E supplements, however the increase attained significance only for the renal tissue. The aortic tissue α-tocopherol/cholesterol ratio was 4-fold higher in the rats receiving concomitant 1% cholesterol plus 0.5% vitamin E compared with animals receiving 1% cholesterol alone (P < 0.02), and was 5-fold higher in the rats receiving 0.5% vitamin E compared with those receiving control chow (P < 0.01). These data suggest that dietary vitamin E supplementation results in a differential uptake of α-tocopherol, which may be dependent, in part, on selective lipoprotein particle accumulation.

Antioxidants in Restenosis Following Vascular Injury

Percutaneous transluminal coronary angioplasty (PTCA), has associated with it high a incidence of restenosis (Leimgruber et. al, 1986).