Atherosclerosis In Hypercholesterolemic Rabbits Research Articles

Regular transient limb ischemia protects endothelial function against hypercholesterolemic damage in rabbits.

Regular transient limb ischemia (RTLI) can prevent atherosclerosis in hypercholesterolemic rabbits. As endothelial dysfunction is the initial factor leading to atherosclerosis, we investigated the effect of RTLI on endothelial function in hypercholesterolemic rabbits. We randomly allocated 15 New Zealand white rabbits to three groups, five animals per group: the hypercholesterolemic group (Group H), the sham RTLI group (Group S), and the RTLI group (Group L). All rabbits received hypercholesterolemic fodder daily. No intervention was performed on the rabbits in Group H. Rabbits in Group S were kept in hutches, with a deflated cuff applied to their left hind limb for 60 min every day. For rabbits in Group L, RTLI (six cycles of 5-min ischemia and 5-min reperfusion of the left hind limb) was applied once daily for 12 weeks. At the end of week 12, a segment of the abdominal aorta was isolated from each rabbit for in vitro measurement of the endothelium-dependent vasodilation (EDV) response to different concentrations of acetylcholine and the endothelium-independent vasodilation (EIV) response to sodium nitroprusside. The EDV response was significantly higher in Group L than in Groups S and H (p < 0.05), with no significant difference between Groups S and H (p > 0.05). There was no difference in the EIV response among the three groups. RTLI could improve the EDV response, protecting endothelial function against hypercholesterolemic damage.

Effects of Alcoholic Extract of Stem and Aerial Parts of the Alcea angulate Plant on Aorta Atherosclerotic Plaque Formation in Rabbit

Background: Atherosclerosis is the main cause of mortality in heart disease patients that its dependence on cholesterol levels has been shown. The side effects of chemical drugs have created a tendency toward medicinal plants for treatment of diseases. Objective: The aim of this study is to evaluate the effect of alcoholic extract of the Alcea angulate on the formation of atherosclerotic plaque due to hypercholesterolemia in rabbit aorta. Methods: 25 male New Zealand white rabbits were divided randomly into 5 groups. Two months respectively treated with basic diet [group 1], basic diet plus 1% cholesterol [control group, group 2], 1% cholesterol with diet intervention Alcea angular at doses of 200, 400 and 800 mg/kg of body weight [groups 3, 4 and 5]. Blood lipid profile levels were measured after 60 days. At the end of the study, the animals were scarified and lipid profile and atherosclerotic plaque formation in the aorta arteries was measured. Data using SPSS ver. 16 were analyzed. Tukey HSD test was used to compare paired groups. Results: Serum levels of total cholesterol and LDL.C of the groups which treated with the Alcea angulata was significantly lower than the control group (p<0.01). Serum levels of HDL in treated groups compared to the control group significantly increased (p<0.01). Pathology studies showed the desired effects and mitigation of extract on the progression of aorta atherosclerosis in hypercholesterolemic rabbits. Conclusion: The overall result demonstrated that use of the Alcea angulate plant reduced total cholesterol and LDL in the blood and inhibited the atherosclerotic plaque formation.

Different responses of fluvastatin to cholesterol‐induced oxidative modifications in rabbits: evidence for preventive effect against DNA damage

Hypercholesterolemia is a major risk factor for atherosclerosis and related occlusive vascular diseases. We investigated the effect of low-dose fluvastatin (2 mg kg(-1) day(-1)) on antioxidant enzyme activities [superoxide dismutase (SOD), catalase], vascular reactivity changes and oxidatively induced DNA damage in early stage of atherosclerosis in hypercholesterolemic rabbits. The animals were divided into three groups each composed of 10 rabbits. The control group received a regular rabbit chow diet, and the cholesterol group had hypercholesterolemic diet (2%, 4 weeks). The fluvastatin group was given hypercholesterolemic diet plus fluvastatin. Dietary intake of cholesterol significantly increased total cholesterol levels in rabbits (control, 0.85 ± 0.29; cholesterol, 12.04 ± 4.61; fluvastatin, 8.07 ± 2.72 mmol l(-1)). Hypercholesterolemic diet revealed discernible fatty streaks in arcus aortae. Fluvastatin significantly reduced the areas of the lesions. The diet significantly increased SOD activities in both erythrocyte and tissue. Treatment with fluvastatin normalized the increased activity of SOD in both erythrocyte and aortic tissues from the cholesterol group. Cholesterol feeding decreased the sensitivity to acetylcholine, and treatment with fluvastatin significantly restored the diminished sensitivity to acetylcholine in thoracic aortae. Cholesterol feeding caused oxidatively induced DNA damage in liver tissues determined by the increased levels of 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Fluvastatin decreased only FapyGua level in liver. In conclusion, our results may suggest that fluvastatin seems to play a protective role on high cholesterol-induced oxidative stress and DNA damage.

SCM-198 attenuates early atherosclerotic lesions in hypercholesterolemic rabbits via modulation of the inflammatory and oxidative stress pathways

ObjectiveTo investigate SCM-198 (also known as “leonurine”), a compound in Herba leonuri, as well as its effect on the progression of atherosclerosis in hypercholesterolemic rabbits and underlying mechanisms. MethodsThirty New Zealand male White rabbits (5 groups, n = 6) were fed either a normal diet or a high-cholesterol diet (1%). The rabbits on the high-cholesterol diet received treatments of low, moderate, and high doses of SCM-198 and placebo concurrently. Eight weeks later, the animals underwent ultrasonographic imaging. They were then sacrificed for further pathological and molecular biological analysis. ResultsSCM-198-treated rabbits showed a significant alleviation in the development of atherosclerosis in a dose-dependent manner. The lesions were smaller after the SCM-198 treatments. In addition, the elasticity of the arteries and hemodynamic status improved, accompanied with a decrease in smooth muscle cell migration and, macrophage infiltration, as well as the expression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) in the aortas. Marginal changes in the lipid parameters were found in the SCM-198-treated rabbits, with high-density lipid cholesterol (HDL-C) elevation and triglyceride (TG) reduction at the high dose. SCM-198 treatment dose-dependently reduced levels of serum soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), as well as the mRNA levels of VCAM-1, IL-6, TNF-α, monocyte chemoattractant protein-1 (MCP-1), iNOS and MMP-9 in the aorta. In addition, SCM-198 also dose-dependently increased the total antioxidant capacities and in parallel decreased the lipid peroxidation levels in the serum and liver. The antioxidant effects of SCM-198 were implicated by the enhanced activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and levels of glutathione (GSH) in the liver, as well as the mRNA levels of CAT, SOD-1 and GPx in the aorta. ConclusionsIn a rabbit atherosclerotic model, SCM-198 dose-dependently ameliorated the progression of atherosclerotic lesions and vascular dysfunction accompanied by the suppression of inflammatory factors and oxidative stress. These findings suggested that SCM-198 might be a potential agent for the treatment of atherosclerosis.

Regression of hypercholesterolemic atherosclerosis in rabbits by hydroalcoholic extracts of Hypericum perforatum

Regression and suppression of atherosclerotic lesion may be a realistic goal in some patients. Antioxidants and hypolipidemic agents suppress the development of hypercholesterolemic atherosclerosis and induce regression of atherosclerosis. The objectives of this study were to determine effects of Hypericum perforatum L. (HPL) on regression of atherosclerosis in hypercholesterolemic rabbits. Rabbits were assigned to four groups: Group I control diet (75 days); Group II cholesterol diet (75 days); Group III cholesterol diet (45 days) followed by regular diet (30 days); Group IV cholesterol diet (45 days) followed by regular diet and HPL (30 days). Blood samples were collected from rabbits before and after 45 days and 75 days respective for measurement of biochemical factors. At the end, aorta was removed for assessment of atherosclerotic plaques. It increases in serum total cholesterol, triglycerides (TG), low density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), apolipoproteinB (apoB), AI, high-sensitive C-reactive protein (hs-CRP), Malondialdehyde (MDA), oxidized LDL (OX-LDL), apolipoproteinA (apoA), white blood cell (WBC), fibrinogen and platelet in Group II,III, ΙV on the 45th days, comparing with Group Ι and the beginning of the study. At the end, biochemical factors serum decreased in Groups III and ΙV. There was more Biochemical factors serum and atherosclerotic lesions decreased in Groups IV compared to Group III in regression period. The reduction in atherosclerotic lesions was associated with a reduction in oxidative stress. These results suggest that regular diet following a high cholesterol diet accelerates atherosclerosis; HPL treatment prevents the progression of atherosclerosis on Group IV; prevention of progression is associated with a reduction of inflammatory factors and antioxidant mechanism may induces regression of atherosclerosis lesion. Key words: Atherosclerosis, regression, Hypericum perforatum, rabbits, oxidized low density lipoprotein (OX-LDL), C-reactive protein, white blood cell (WBC), fibrinogen, platelet.

Lipid Lowering and Antioxidant Effects of Newly Synthesized 4-[(Butylsulfinyl)methyl]-1,2-benzenediol (SMBD) in Diet-induced Hypercholesterolemic Rabbits

We investigated the effects of newly synthesized 4-[(butylsulfinyl)methyl]-1,2-benzenediol (SMBD) on the prevention of atherosclerosis in hypercholesterolemic rabbits. SMBD exhibited stronger inhibition of <TEX>$Cu^{2+}$</TEX>-induced low-density lipoprotein oxidation than that of ascorbic acid or simvastatin. Three-month-old rabbits were fed an atherogenic diet containing 0.5% cholesterol and 10% coconut oil, while other two groups were given an atherogenic diet with intravenous injection of either simvastatin or SMBD (0.33 mg/kg/day) for 4 weeks. The concentrations of plasma cholesterol and thiobarbituric acid reactive substances were significantly decreased in SMBD groups, compared to the control group. Also, aortic lipid level in the SMBD group significantly lower than that in the control group. Furthermore, compared with the control group, the SMBD group significantly inhibited the increase of aortic intimal thickness by 36% via reducing of aortic reactive oxygen species and cyclooxygenase-2 protein levels. We conclude that raised antioxidant effect of SMBD results in significant prevention against hypercholesterolemia.

Kefiran Reduces Atherosclerosis in Rabbits Fed a High Cholesterol Diet

Kefiran is an exopolysaccharide produced by Lactobacillus kefiranofaciens, and has been proposed to have many health-promoting properties. We investigated the antiatherogenic effect of kefiran on rabbits fed a high-cholesterol diet. Male New Zealand White rabbits were fed a 0.5% cholesterol diet without (control group, n = 7) or with kefiran (kefiran group, n = 8) for eight weeks. The aorta was analyzed by histochemistry and atherosclerotic lesions were quantified. Lipids and sugars in serum were measured. Foam cell formation of RAW264.7 by βVLDL derived from both groups of rabbits was also investigated. Cholesterol, triglyceride and phospholipids levels of serum and lipoprotein fractions were not significantly different between these groups. Atherosclerotic lesions of the aorta in the kefiran group were statistically lower than those of the control group, with marked differences in the abdominal aorta. T-lymphocytes were not detectable in the aorta of the kefiran group. Cholesterol contents in stools were almost identical in both groups. Cholesterol content in the liver of the kefiran group was statistically lower than in the control group. Galactose content of βVLDL derived from the kefiran group was higher, and the lipid peroxidation level was much lower than in the control group. RAW264.7 macrophages treated with βVLDL from the kefiran group showed a more spherical shape and accumulated statistically lower cholesterol than macrophages treated with βVLDL from the control group. Orally derived kefiran is absorbed in the blood. Kefiran prevents the onset and development of atherosclerosis in hypercholesterolemic rabbits by anti-inflammatory and anti-oxidant actions.

Acute effects of vinegar intake on some biochemical risk factors of atherosclerosis in hypercholesterolemic rabbits

BackgroundExaggerated postprandial spikes in blood glucose and lipids induce proportional increases in oxidative stress, which acutely trigger impairment endothelial, inflammation and increased risk of future cardiovascular events. In this research, we have investigated acute effects of vinegar intake on some of the biochemical atherosclerosis risk factors in high cholesterol fed rabbits to see if we can find a probable protective value for it.MethodsThe rabbits were randomly divided into four groups: normal diet, high cholesterol diet (%1cholesterol), %1 cholesterol with 5 ml vinegar (low dose), %1 cholesterol with 10 ml vinegar (high dose). After fasting for 12-15 hours, blood samples were taken to determine baseline values. Three hours after feeding, blood samples were collected again to investigate acute effects of vinegar intake on the measured factors.ResultsUsing high-dose vinegar with cholesterolemic diet caused significant reduce in LDL-cholesterol (LDL-C), oxidized-LDL (ox-LDL), malondialdehyde (MDA), total cholesterol (TC) and apolipoprotein B (ApoB) in comparison with hypercholesterolemic diet. Consumption low-dose vinegar with cholesterolemic diet induced a significant decrease in fibrinogen and glucose compared to hypercholesterolemic diet. Level of serum nitrite, nitrate, triacylglycerol (TAG), HDL-cholesterol (HDL-C), apolipoprotein A (ApoA), serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetate transaminase (SGOT) and C-reactive protein (CRP) were not significantly difference in low and high doses vinegar with cholesterolemic diet compared to hypercholesterolemic diet. A significant difference was observed for LDL-C, ApoB100 and TC between low and high doses vinegar.ConclusionThis study suggest that vinegar, might have some acute effects on biochemical risk factors of atherosclerosis and a probable protective value can be considered for its postprandial use.

3-(4′-Hydroxyl-3′,5′-dimethoxyphenyl)propionic Acid, an Active Principle of Kimchi, Inhibits Development of Atherosclerosis in Rabbits

The effects of 3-(4'-hydroxyl-3',5'-dimethoxyphenyl)propionic acid (HDMPPA) originating from Korean cabbage kimchi were investigated, showing an antioxidant effect on the prevention of atherosclerosis in hypercholesterolemic rabbits. Twenty-one 3-month-old rabbits were fed an atherogenic diet containing 0.5% (w/w) cholesterol and 10% (w/w) coconut oil, whereas another two groups were given an atherogenic diet with intravenous injection of either HDMPPA or simvastatin (0.33 mg/kg/day) for 4 weeks. HDMPPA inhibited the oxidative modification of low-density lipoprotein (IC 50 = 1.4 microg/mL) and increased 2,2'-diphenyl-1-picrylhydrazyl radical scavenging activity (IC 50 = 0.78 microg/mL) in a dose-dependent manner. In hypercholesterolemic rabbits, the thickness of intima of aorta of the HDMPPA group was significantly reduced (control versus HDMPPA, 42%; simvastatin, 38%) without a plasma cholesterol-lowering effect. Thiobarbituric acid reactive substance formation in the plasma of the HDMPPA group was significantly decreased compared to that of the control group. Furthermore, the generation of vascular reactive oxygen species in HDMPPA group was suppressed as the cyclooxygenase-2 protein level decreased. These findings suggest that HDMPPA prevents the development of aortic atherosclerosis in high-cholesterol-fed rabbits. The antiatherosclerotic effect of HDMPPA may be due to an antioxidative effect at a low dose without cholesterol-lowering effects.

Suppression of vascular cell adhesion molecule-1 expression by crocetin contributes to attenuation of atherosclerosis in hypercholesterolemic rabbits

To elucidate the molecular mechanism by which antioxidants alleviate atherosclerosis, we investigated the effect of crocetin, a naturally occurred carotinoid with potent antioxidant power, on vascular cell adhesion molecule-1 (VCAM-1) expression in atherosclerotic rabbits. Twenty-four male New Zealand White rabbits were allocated to three groups fed on standard diet (control group), high lipid diet (HLD group) or high lipid diet supplemented with crocetin (crocetin group), respectively. After 8 weeks of treatment, rabbits in HLD group developed severe hypercholesterolemia and atherosclerosis in aortas, together with a significantly up-regulated expression of both protein and mRNA for VCAM-1. In contrast, supplementation with crocetin resulted in markedly ameliorated atherosclerosis, coupled with a significantly decreased VCAM-1 expression, though plasma lipids level remained comparable to that of HLD group. Regression analysis revealed a positive correlation between VCAM-1 expression and the extent of atherosclerosis ( P < 0.01). In addition, immunohistochemical analysis showed an increased activation of nuclear factor kappa B (NF-κB), a redox sensitive transcription factor essential for VCAM-1 expression, in aortas from rabbits fed on high lipid diet, which was evidently suppressed by crocetin supplementation. These findings suggest that the antiatherosclerotic effect of crocetin might be attributed, at least in part, to the suppressed expression of VCAM-1, which might result from reduced NF-κB activation. This study provides a further insight into the molecular mechanism by which antioxidants attenuate atherosclerosis and suggests a potential target for the treatment of atherosclerosis with antioxidants.

Regulating Protective Immunity in Atherosclerosis

Atherosclerosis is a disease characterized by chronic inflammation. Looking back it is almost embarrassing how long it took for us to realize that the presence of these innate immune responses also was associated with activation of adaptive immunity. The first hints of an involvement of adaptive immunity in atherosclerosis came from studies performed by Goran Hansson and his coworkers about 20 years ago demonstrating the presence of activated T cells and expression of class II molecules in atherosclerotic plaques.1 This finding provoked an intense interest in the role of immunity in atherosclerosis, and it is now generally recognized that adaptive immune responses have a key role in determining the balance between disease progression and regression.2 It also focused attention on the immune system as a target for prevention and treatment of cardiovascular disease. Mice with defective adaptive immunity, such as SCID and Rag-1, develop less atherosclerosis indicating that adaptive immune responses are primarily proatherogenic. Oxidized LDL has been identified as one of the most important autoantigens in atherosclerosis.3 A large fraction of the T cells present in atherosclerotic plaques are specific for oxidized LDL,4 and oxidized LDL IgG is prevalent in the circulation of both humans and atherosclerosis-prone animals. However, studies evaluating the role of immune responses against oxidized LDL by immunizing animals with in vitro oxidized LDL unexpectedly revealed a protective effect.5,6 Although it still remains to be established whether naturally occurring autoimmune responses against oxidized LDL also have atheroprotective effects, these observations suggest the fascinating possibility of developing an immunomodulatory therapy for atherosclerosis. However, manipulating the immune system in complex diseases does not come without risk, and if the development of an immunomodulatory therapy for atherosclerosis is to become successful it will require detailed characterization of both disease-related and protective immune responses. The …

Hypocholesterolemic and antiatherosclerotic effect of flax lignan complex isolated from flaxseed

Hypercholesterolemia, low HDL-C and oxygen radicals have been implicated in the development of atherosclerosis. Lignan complex isolated from flaxseed contains secoisolariciresinol diglucoside (SDG), 3-hydroxy-3methylglutaric acid (HMGA) and cinnamic acids. SDG and cinnamic acids are antioxidants, and HMGA is a hypocholesterolemic agent. Antioxidants are known to reduce hypercholesterolemic atherosclerosis. The objectives of this study were to determine if lignan complex reduces (i) serum cholesterol, (ii) oxidative stress, and (iii) atherosclerosis in hypercholesterolemic rabbits. Rabbits were assigned to four groups: Group I, control; Group II, lignan complex control (lignan complex, 40 mg/kg body weight daily orally); Group III, 0.5% cholesterol; Group IV, 0.5% cholesterol diet + lignan complex, (40 mg/kg body weight daily orally). Blood samples were collected before (time 0) and after 1 and 2 months of experimental diets for measurement of serum triglycerides (TG), total cholesterol (TC), LDL-C, HDL-C and serum malondialdehyde (MDA), a lipid peroxidation product. At the end of the protocol, the aorta was removed for measurement of atherosclerotic plaques, MDA and aortic tissue chemiluminescence (Aortic CL), a marker of antioxidant reserve. Rabbits in Group III developed atherosclerosis (50.84 ± 6.23% of the intimal surface of the aorta was covered with atherosclerotic changes) which was associated with an increase in the serum TG, TC, LDL-C, HDL-C, MDA and aortic MDA and antioxidant reserve. Lignan complex reduced the development of atherosclerosis by 34.37% and this was associated with a decrease in serum TC by 20%, LDL-C by 14%, TC/HDL-C by 34%, serum MDA by 35% and aortic MDA by 58%. Serum HDL-C was elevated by 30% in hypercholesterolemic rabbits and by 25% in normocholesterolemic rabbits with lignan complex. Lignan complex did not affect the TC and LDL-C and serum MDA in the normocholesterolemic rabbits. However, it increased the aortic MDA in the normocholesterolemic rabbits. These results suggest that lignan complex isolated from flaxseed reduced the extent of hypercholesterolemic atherosclerosis and this effect was associated with marked decreases in oxidative stress, serum total cholesterol, LDL-C and risk ratio, and elevation of serum HDL-C. Lignan complex may, therefore, be beneficial in preventing atherosclerosis, and reducing risk factors for coronary artery disease and stroke.

Effects of flaxseed oil on serum lipids and atherosclerosis in hypercholesterolemic rabbits.

Flaxseed oil has very high content of alpha-linolenic acid (C18:3n-3, omega-3 [n-3] fatty acid). Based on the usefulness of n-3 fatty acid in fish oil against cardiovascular diseases, flaxseed oil is marketed as a health food. The n-3 fatty acid in flaxseed oil is different than that of fish oil. Indirect evidence suggests that the omega-3 fatty acid in flaxseed oil is not effective in lowering serum lipids and hypercholesterolemic atherosclerosis. The effects of flaxseed oil on serum lipids and hypercholesterolemic atherosclerosis are not known. An investigation, therefore, was made of flaxseed oil on high-cholesterol diet-induced atherosclerosis, serum lipids (triglycerides, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, risk ratio of total cholesterol to high-density lipoprotein cholesterol), serum and aortic malondialdehyde, an index of levels of reactive oxygen species, aortic chemiluminescence (a measure of antioxidant reserve), and reactive oxygen species-producing activity of white blood cell chemiluminescence in rabbits. The rabbits were assigned to four groups: Group I, regular diet (control); Group II, 5% flaxseed oil in regular diet; Group III, 0.5% cholesterol diet; Group IV, diet containing 0.5% cholesterol and 5% flaxseed oil. Blood samples were collected before and after 4 and 8 weeks of experimental diets for measurement of serum lipids, serum malondialdehyde, and white blood cell chemiluminescence. At the end of 8 weeks of the experimental diet, aortas were removed for measurement of atherosclerotic plaques, aortic malondialdehyde, and antioxidant reserve. Serum total cholesterol, triglycerides, and low-density and high-density lipoprotein cholesterol, and risk ratio of total cholesterol to high-density lipoprotein cholesterol, were elevated to a similar extent in Groups III and IV compared to Groups I and II. The extent of atherosclerosis in Groups III and IV was similar (56.74% +/- 11.14% vs 58.01% +/- 10.95%). Groups III and IV both had similar increases in serum and aortic malondialdehyde and antioxidant reserve. Reactive oxygen species-producing activity of white blood cells increased in Group III, and flaxseed oil prevented the cholesterol-induced increase in white blood cell chemiluminescence in Group IV. These results suggest that flaxseed oil does not produce an alteration in serum lipids or in the extent of hypercholesterolemic atherosclerosis; however, it decreases white blood cell chemiluminescence. The ineffectiveness of flaxseed oil was associated with its ineffectiveness in altering the levels of oxidative stress.

Regression of early events of atherosclerosis in hypercholesterolemic rabbits by prophylactic treatment with nitroderivative of acetyl salicylic acid

AbstractThe potential of a newly developed compound, nitro‐aspirin (NO‐aspirin, NCX‐4016), on regression of early atherosclerotic events in hypercholesterolemic rabbit (HypR) was examined. Hypercholesterolemia was induced by feeding rabbits 2% cholesterol‐enriched diet for 28 days. During this induction period, HypRs were divided into two groups: one was given an oral single daily dose of 56 mg/kg/day NO‐aspirin in olive oil, whereas the other received olive oil alone and served as a control. A normal group of rabbits fed plain chow diet plus olive oil was also included. At the end of the 28 days, samples from blood, aorta, heart, and coronary arteries were separated for biochemical and histopathological examination. Tissues from HypR aorta and coronaries developed major changes that include formation of large intimal plaques, endothelial gaps with marked accumulation of foam cells. Highly significant increases in serum triglycerides and malondialdehyde and a decrease in reduced glutathione (GSH) in heart and aorta were also observed. Administration of NO‐aspirin markedly improved aortic and coronary architecture to almost normal appearance. NO‐aspirin also improved the antioxidant status in aorta and heart by increasing GSH levels and ameliorating malondialdehyde accumulation. These data support prophylactic use of NO‐aspirin in regression of atherosclerosis. Drug Dev. Res. 53:237–243, 2001. © 2001 Wiley‐Liss, Inc.

The effect of chronic treatment with NO donors during intimal thickening and fatty streak formation.

Intimal thickening in arteries is considered as a site of predilection for atherosclerosis. We investigated whether oral application of the nitric oxide (NO) donors SPM-5185 (N-nitratopivaloyl-S-(N'-acetylalanyl)-cysteine ethylester, 10 mg/kg body weight/b.i.d.) and molsidomine (pro-drug of 3-morpholino-sydnonimine (SIN-1), 10 mg/kg body weight/day) can retard intimal thickening and changes in vascular reactivity induced by a silicone collar positioned around the carotid artery of rabbits. Intimal thickening was significantly inhibited by SPM-5185 (cross-sectional area 18 +/- 6 vs. 44 +/- 10 x 10(-3) mm2; P < 0.05), but not by molsidomine (28 +/- 6 vs. 35 +/- 9 x 10(-3) mm2), which is a donor of both NO and superoxide anions. In organ chamber studies collaring was associated with a decreased sensitivity to acetylcholine (ACh). SPM-5185 evoked a tendency towards normalization of the pD2 of ACh in collared arteries. We also investigated whether chronic nitric oxide (NO) treatment affected vascular reactivity and fatty streak development in the rabbit aorta. During 16 weeks rabbits received 150 g/day of a standard diet, or diets with 0.3% cholesterol, with 0.02% molsidomine (10 mg/kg body weight/day) or with the combination. The NO donor enhanced the area of fatty streaks, without affecting hypercholesterolemia. Moreover, it desensitized the smooth muscle cells of the rabbit aorta to vasodilators acting via the cytoplasmic guanylate cyclase and suppressed the capacity of the endothelial cells to release NO in response to muscarinic receptor stimulation. This suggested that chronic exposure to large quantities of NO caused a negative feedback, with selective decreases of both the endothelial capacity to generate NO and the responsiveness to vasodilators operating via cyclic GMP. In conclusion, we demonstrated that exogenous NO can decrease intimal hyperplasia in vivo. However, prolonged in vivo treatment with a donor of NO enhanced atherosclerosis in hypercholesterolemic rabbits.

Effect of Immunization With Homologous LDL and Oxidized LDL on Early Atherosclerosis in Hypercholesterolemic Rabbits

Although the existence of an immune response against modified lipoproteins in atherosclerosis has been observed in experimental animals as well as in humans, the precise pathophysiological relevance of these findings remains unclear. In this study we determined the effect of an immunization with homologous LDL and copper-oxidized LDL on the formation of atherosclerotic plaque in hypercholesterolemic rabbits. Immunizations were performed at the start of a cholesterol-rich diet and 3 weeks later. After 16 weeks, antibodies against oxidized LDL had developed in rabbits given hypercholesterolemic diet alone, but the titers were increased by twofold in rabbits immunized with oxidized LDL as well as in rabbits immunized with LDL, suggesting that the LDL had also become oxidized during the preparation and/or immunization procedure. Immunization with LDL and oxidized LDL reduced atherosclerotic lesions in the proximal aorta by 74% (P < .05) and 48% (P = NS), respectively. The cellular composition of the lesions was not affected by the immunizations. These results support the hypothesis that an immune response against modified LDL has a protective effect against the development of early atherosclerotic lesions.

Resveratrol promotes atherosclerosis in hypercholesterolemic rabbits

The hypothesis was tested that resveratrol, a compound in red wine, would inhibit atherosclerotic development in rabbits fed 0.5% cholesterol for 60 days. Rabbits were supplemented with or without oral resveratrol. During the study, body weights and food consumption were similar for the two groups. The lack of differences between liver weights and a series of serum parameters indicative of liver disease suggest that liver function was similar in the two groups. The diet produced hypercholesterolemia in both groups, but no differences in lipoprotein-cholesterol concentrations. The electrophoretic mobility of plasma low-density lipoprotein (LDL) and plasma LDL after induced oxidation also was not different between the groups. Staining of atherosclerotic lesions in the control and resveratrol-treated groups revealed that the resveratrol-treated rabbits had significantly more aortic surface area covered by atherosclerotic lesions (P < 0.02). Therefore, resveratrol promoted atherosclerotic development, rather than protect against it, by a mechanism that is independent of observed differences in gross animal health, liver function, plasma cholesterol concentrations, or LDL oxidative status.

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.

Inhibition of atherosclerosis in hypercholesterolemic rabbits by combination of nutrient antioxidants

Inhibition of atherosclerosis in hypercholesterolemic rabbits by combination of nutrient antioxidants

Long-term inhibition of NO synthesis promotes atherosclerosis in the hypercholesterolemic rabbit thoracic aorta. PGH2 does not contribute to impaired endothelium-dependent relaxation.

We examined whether prostaglandin (PG) H2, as an endothelium-dependent contracting factor, or the disturbed production of endothelium-derived relaxing factor, impairs endothelium-dependent relaxation and whether long-term inhibition of nitric oxide (NO) synthesis aggravates atherosclerosis in hypercholesterolemic rabbits. Male New Zealand White rabbits were fed one of the following diets: (1) standard chow; (2) 2% cholesterol-supplemented chow; (3) standard chow with 80 micrograms/mL N omega-nitro-L-arginine methylester (L-NAME), an NO synthetase inhibitor, in their drinking water; or (4) 2% cholesterol-supplemented chow with 80 or 160 micrograms/mL L-NAME in their drinking water. The rabbits were fed these diets for 8 or 12 weeks. Then aortic rings were obtained, and changes in isometric tension were recorded. Intimal atherosclerotic areas of the thoracic aortas were subsequently measured by planimetry. The cholesterol-supplemented diet significantly impaired endothelium-dependent aortic relaxation to acetylcholine. Pretreatment with the thromboxane A2/PGH2 receptor antagonist ONO-3708 did not reverse this impaired response. Vessels from both normocholesterolemic and hypercholesterolemic rabbits given L-NAME showed more impaired endothelium-dependent relaxation than those from their dietary counterparts not given L-NAME. Morphometric analysis revealed marked enlargement of intimal atherosclerotic areas in aortas from L-NAME-treated hypercholesterolemic rabbits compared with those from untreated hypercholesterolemic rabbits. These findings suggest that PGH2 does not contribute to impaired endothelium-dependent relaxation and that long-term administration of L-NAME promotes atherosclerosis by inhibition of NO synthesis in the hypercholesterolemic rabbit thoracic aorta.