Cholesterol-fed Group Research Articles

Efficacy of oat bran (Avena sativa L.) in comparison with atorvastatin in treatment of hypercholesterolemia in albino rat liver.

Introduction: The present study deals with the effect of oat bran (Avena sativa L.) in the treatment of hypercholesterolemia in comparison with a hypocholesterolemic drug, atorvastatin, on hypercholesterolemic liver in male albino rats. Material and Methods: For this purpose four groups of rats (each containing 6 rats) were used. The first group was used as a control, the second was cholesterol-fed group with cholesterol (0.5% w/w) for 6 weeks. The third group was oats-fed hypercholesterolemic rats on oat supplemented diet (20% w/w) for 4 weeks and the forth group was atorvastatin-treated hypercholesterolemic rats orally at a dose of 0.18 mg/Kg body weight/day for 4 weeks. Results: The biochemical results revealed a significant increase in serum LDL-C and a significant decrease in HDL-C level. In addition the activity of AST was increased in cholesterol-fed rats. Meanwhile, the treatment with oat soluble fiber or atorvastatin drug improved the above mentioned parameters. The histopathological examination of liver sections of cholesterol-fed group showed accumulation of lipid. Hepatocytes showed ballooning degeneration and manifested clear necrotic signs. Inflammatory cellular infiltration was found around the blood vessels, mild fibrosis near the portal blood vessels. However, liver impairment was reduced markedly in the liver of oat soluble fiber fed rats rather than atorvastatin drug treated rats. Conclusion: The present study, however, confirms that the cereal grain oat may have potent beneficial health effects in reducing LDL cholesterol and should be included in the prudent diet of individuals with hyperlipidemia.

Efficacy of oat bran (Avena sativa L.) in comparison with atorvastatin in treatment of hypercholesterolemia in albino rat liver.

Introduction: The present study deals with the effect of oat bran (Avena sativa L.) in the treatment of hypercholesterolemia in comparison with a hypocholesterolemic drug, atorvastatin, on hypercholesterolemic liver in male albino rats. Material and Methods: For this purpose four groups of rats (each containing 6 rats) were used. The first group was used as a control, the second was cholesterol-fed group with cholesterol (0.5% w/w) for 6 weeks. The third group was oats-fed hypercholesterolemic rats on oat supplemented diet (20% w/w) for 4 weeks and the forth group was atorvastatin-treated hypercholesterolemic rats orally at a dose of 0.18 mg/Kg body weight/day for 4 weeks. Results: The biochemical results revealed a significant increase in serum LDL-C and a significant decrease in HDL-C level. In addition the activity of AST was increased in cholesterol-fed rats. Meanwhile, the treatment with oat soluble fiber or atorvastatin drug improved the above mentioned parameters. The histopathological examination of liver sections of cholesterol-fed group showed accumulation of lipid. Hepatocytes showed ballooning degeneration and manifested clear necrotic signs. Inflammatory cellular infiltration was found around the blood vessels, mild fibrosis near the portal blood vessels. However, liver impairment was reduced markedly in the liver of oat soluble fiber fed rats rather than atorvastatin drug treated rats. Conclusion: The present study, however, confirms that the cereal grain oat may have potent beneficial health effects in reducing LDL cholesterol and should be included in the prudent diet of individuals with hyperlipidemia.

Effects of aspirin on atherosclerosis and the cyclooxygenase-2 expression in atherosclerotic rabbits

Atherosclerosis is a complex vascular inflammatory disease. Aspirin is a mainstay in the prevention of vascular complications of atherosclerosis. In this study, the effectiveness of aspirin in suppressing atherosclerosis and the inflammation process was evaluated in rabbits fed with a high fat diet. Eighteen male New Zealand rabbits were randomly divided into 3 groups: control group, untreated cholesterol-fed group, aspirin treated cholesterol-fed group, which were fed for 12 weeks. After 12 weeks, the aorta was harvested for pathologic morphology observation. Immunohistochemical analysis of cyclooxygenase-2 (COX-2), macrophage and vascular smooth muscle cell (VSMC) was performed. The statistical analysis was performed by the statistical program SPSS10.0. The aorta plaque/intima size (P/I) by pathologic morphology observation was 0%, (59.6 +/- 13.7)% and (36.3 +/- 16.5)% in the control, untreated cholesterol-fed group and aspirin treated group, respectively. The maximum plaque thickness, the degree of artery stenosis and the proportion of the intimal circumference occupied by atheroma of the 3 groups were significantly different from each other (P < 0.01). The expression of COX-2 and macrophage in plaque of the aspirin treated group were decreased compared with that in untreated cholesterol-fed group. However, no difference was found in the expression of VSMC between the aspirin treated and the untreated cholesterol-fed group. The mechanism of atherosclerosis suppression by aspirin in cholesterol-fed rabbits is related to the inhibition of COX-2 expression together with the reduced inflammation followed by, but not related to the hypolipidemic effects.

The Olive Constituent Oleuropein Exhibits Anti-Ischemic, Antioxidative, and Hypolipidemic Effects in Anesthetized Rabbits

Thgoal of this study was to evaluate the efficacy of the antioxidant olive constituent, oleuropein, on infarct size, oxidative damage, and the metabolic profile in rabbits subjected to ischemia. Oleuropein, 10 or 20 mg/(kg·d), was administered to 8 groups that consumed a normal or hypercholesterolemic diet for 6 wk or only the higher dose for 3 wk. Circulating levels of malondialdehyde, protein carbonyl, nitrite+nitrate, cholesterol, triglycerides, SOD activity, and the metabolic profile were measured using 1H NMR spectra. In rabbits that consumed the normal diet, the infarct size (percentage of infarct to risk areas) was reduced by the administration of 10 mg oleuropein/(kg·d) (16.1 ± 2.9%) or 20 mg oleuropein/(kg·d) for 3 wk (21.7 ± 2.2%) or for 6 wk (24.3 ± 1.3%) compared with the control group (48.05 ± 2.0%, P < 0.05). Only the higher dose of 20 mg/(kg·d) reduced the infarct size in hypercholesterolemic rabbits (34.7 ± 4.4% for 6 wk and 34.8 ± 6.1% for 3 wk) compared with the cholesterol-fed control group (52.8 ± 2.4%, P < 0.05). Oleuropein decreased the plasma lipid peroxidation product and protein carbonyl concentrations compared with the control groups, in which these factors increased relative to baseline due to ischemia and reperfusion. Furthermore, in rabbits administered oleuropein, RBC superoxide dismutase activity did not change during ischemia and reperfusion. This activity was significantly higher than in both control groups in which it was reduced by ischemia and reperfusion compared with baseline. Treatment for 6 wk with both doses of oleuropein reduced total cholesterol and triglyceride concentrations. 1H NMR spectra revealed a different profile of glycolysis metabolites in the oleuropein-treated groups compared with the controls. Oleuropein, for 3 or 6 wk, reduced the infarct size, conferred strong antioxidant protection and reduced the circulating lipids. This is the first experimental study in vivo that suggests the possibility of using an olive constituent in the treatment of ischemia.

Increased superoxide anion production is associated with early atherosclerosis and cardiovascular dysfunctions in a rabbit model

Hypercholesterolemia (HC) has been associated with impairment of vascular and myocardial functions. As HC could generate an alteration in the oxidative status, we studied the effects of a 1-month cholesterol diet on cardiovascular oxidative stress. New Zealand rabbits received cholesterol (1%) or normal chow for 1 month. At 30 days, superoxide anion levels, assessed by ESR spectroscopy, NAD(P)H oxidase (NOX) activity, and dihydroethidium (DHE) staining of aortas were higher in the cholesterol-fed (CF) group compared with control (respectively, 4.0 +/- 0.6 Arbitrary Units/mg (AU/mg) vs. 2.6 +/- 0.3, p < 0.05; 4231 +/- 433 vs. 2931 +/- 373 AU/mg, p<0.05; 21.4 +/- 1.2 vs. 12.9 +/- 1.7% fluorescence/mm2, p < 0.001). NOX gp91 phox and p67 phox expression in the aortas were higher in the CF group vs. control (1.5 +/- 0.2 vs. 0.5 +/- 0.2, p < 0.001; 0.9 +/- 0.2 vs. 0.3 +/- 0.2, p<0.05). The endothelium-dependent relaxation evaluated on the iliac arteries was higher in control than in the CF group (64.8 +/- 10.1 vs. 13.1 +/- 3.70%, p<0.001). The cardiac diastolic pressure estimated on isolated hearts was higher in the CF group than in control (21.1 +/- 4.1 vs. 10.3 +/- 1.4 mmHg, p<0.05) after 60 min of ischemia. Hypercholesterolemia induced increased levels of superoxide in the aortas and a higher expression of NOX subunits, associated with altered vasorelaxation. The increased diastolic pressure observed in hearts, consistent with a post-ischemic contractile dysfunction might be mediated by the production of superoxide.

Effects of Neem ( Azadirachta indica ) Leaf Extracts on Lipid and C-reactive Protein Concentrations in Cholesterol-fed Rats

Objectives: Aim of this study is to investigate the effects of Neem ( Azadirachta indica ) leaf extract on serum lipid parameters and C-reactive protein (CRP) concentrations. The association between CRP and serum lipid profiles in hypercholesterolemic rats is also investigated. Methods: Total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), triglyceride (TG) and CRP were measured at week 0, 2, 4, 6 and 8 in 4 groups of animals; the control group (C), cholesterol control group (CC), cholesterol-fed groups treated with Neem leaf extract at doses of 50 and 300 mg/kg/day orally (N1&N2), respectively. Results: Cholesterol feeding had resulted in a significant increase (p<0.05) in TC, LDL&TG in group CC starting from week 2 and a decrease in HDL levels (p<0.05). CRP concentrations also showed a significant increment in the cholesterol-treated rats (CC) compared to group C, N1&N2 (p<0.05). Neem extract treated, TC, LDL and TG levels remained within the normal range similar to Group C and CRP concentrations were lowered but HDL levels were not. Conclusions: It is shown in this study that the concentrations of CRP had increased in CC rats but not in rats with normal cholesterol levels. Neem extract at 50 and 300 mg/kg doses had prevented the rise of TC, LDL and TG in cholesterol-fed rats. No significant changes in CRP concentrations were noted in the Neem-treated animals. Association between CRP and serum lipid concentrations is directly proportionate. They also suggest that Neem leaf extracts at 50 and 300 mg/kg concentrations are excellent lipid-lowering agent.

Dietary acetic acid reduces serum cholesterol and triacylglycerols in rats fed a cholesterol-rich diet

To investigate the efficacy of the intake of vinegar for prevention of hyperlipidaemia, we examined the effect of dietary acetic acid, the main component of vinegar, on serum lipid values in rats fed a diet containing 1 % (w/w) cholesterol. Animals were allowed free access to a diet containing no cholesterol, a diet containing 1 % cholesterol without acetic acid, or a diet containing 1 % cholesterol with 0.3 % (w/w) acetic acid for 19 d. Then, they were killed after food deprivation for 7 h. Cholesterol feeding increased serum total cholesterol and triacylglycerol levels. Compared with the cholesterol-fed group, the cholesterol and acetic acid-fed group had significantly lower values for serum total cholesterol and triacylglycerols, liver ATP citrate lyase (ATP-CL) activity, and liver 3-hydroxy-3-methylglutaryl-CoA content as well as liver mRNA levels of sterol regulatory element binding protein-1, ATP-CL and fatty acid synthase (P<0.05). Further, the serum secretin level, liver acyl-CoA oxidase expression, and faecal bile acid content were significantly higher in the cholesterol and acetic acid-fed group than in the cholesterol-fed group (P<0.05). However, acetic acid feeding affected neither the mRNA level nor activity of cholesterol 7alpha-hydroxylase. In conclusion, dietary acetic acid reduced serum total cholesterol and triacylglycerol: first due to the inhibition of lipogenesis in liver; second due to the increment in faecal bile acid excretion in rats fed a diet containing cholesterol.

Comparison between phytoestrogens and estradiol in the preventionof atheroma in ovariectomized cholesterol-fed rabbits

Objectives There is increasing interest in the role of complementary and alternative medicines for the treatment of menopause-related problems. This study compared the preventive effect on atheroma formation of a commercially available mixed phytoestrogen concentrate with that of estradiol.Methods An ovariectomized cholesterol-fed rabbit model of atheroma formation was used. Rabbits were ovariectomized before the commencement of the 12-week treatment period. There were two control groups. Control Group 1 received isoflavone-free rabbit chow whilst Control Group 2 received 1% cholesterol-enriched isoflavone-free rabbit chow. Rabbits in Group 3 received 1% cholesterol-enriched isoflavone-free rabbit chow plus a 500 mg tablet containing a concentrated extract of Trifolium pretense (red clover). Rabbits in Group 4 received 1% cholesterol-enriched isoflavone-free rabbit chow plus a 0.5 mg tablet of oral estradiol. Atheroma formation was measured by, first, calculation of the area of atheroma on the intimal surface, and, second, measuring the cholesterol content in the aorta.Results There were no significant differences in serum cholesterol between the cholesterol-fed control Group 2 and the treatment Groups 3 and 4. However, there was significantly less staining for atheroma and significantly less cholesterol accumulation in the aorta in Group 4 (estradiol-treated) rabbits compared with either control Group 2 or Group 3 (phytoestrogen-treated) rabbits.Conclusion In this study, only estradiol was shown to have a significant protective effect against atheroma formation.

Hyperlipidemia Attenuates the Infarct Size-Limiting Effect of Ischemic Preconditioning: Role of Matrix Metalloproteinase-2 Inhibition

Hyperlipidemia attenuates the cardioprotective effect of preconditioning via unknown mechanisms. We have reported previously that in normolipidemic rats, preconditioning decreased ischemia-induced activation and release of myocardial matrix metalloproteinase (MMP)-2 into the coronary perfusate. Here, we investigated whether hyperlipidemia interferes with the cardioprotective effect of preconditioning through modulation of MMP-2. Hearts isolated from male Wistar rats fed 2% cholesterol-enriched or control chow for 9 weeks were subjected to a preconditioning protocol (three intermittent periods of ischemia/reperfusion of 5-min duration each) or a time-matched nonpreconditioning protocol. This was followed by a test ischemia/reperfusion (30-min ischemia and 120-min reperfusion) in both groups. Preconditioning decreased infarct size in the control but not the cholesterol-fed group. Cardioprotection in the preconditioned control group but not in the cholesterol-fed group was associated with an 18 +/- 3% (p < 0.05) inhibition of test ischemia/reperfusion-induced activation and release of myocardial MMP-2 into the perfusate. Myocardial protein levels of tissue inhibitors of MMPs [tissue inhibitor of metalloproteinases (TIMP)-2 and TIMP-4] were not changed in either group. A reduction of infarct size in nonpreconditioned hearts from both control and cholesterol-fed group was produced by the MMP inhibitor ilomastat at 0.25 microM, a concentration producing MMP-2 inhibition comparable with that of preconditioning in the control group. We conclude that hyperlipidemia blocks preconditioning-induced cardioprotection, hyperlipidemia abolishes preconditioning-induced inhibition of myocardial MMP-2 activation and release, preconditioning-induced inhibition of MMP-2 activation and release is not mediated by TIMPs, and pharmacological inhibition of MMPs produces cardioprotection in both normal and hyperlipidemic rats.

Possible anti‐atherogenic effect of kolaviron (a Garcinia kola seed extract) in hypercholesterolaemic rats

1. The hypolipidaemic effect of kolaviron, a mixture of Garcinia biflavonoid 1 (GB1), Garcinia biflavonoid 2 (GB2) and kolaflavanone, used in the treatment of various ailments in southern Nigeria, was investigated in rats. The ability of Questran (Bristol-Myers Squibb, Hounslow, UK), a hypolipidaemic therapeutic drug, to attenuate hypercholesterolaemia in rats was also examined. 2. In order to assess the hypolipidaemic effect of this extract in experimental animals, thiobarbituric acid-reactive substances (TBARS), cholesterol, phospholipid, low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol and triglyceride levels were determined in the plasma and liver. 3. Cholesterol administered orally to rats at a dose of 30 mg/0.3 mL five times a week for 8 consecutive weeks resulted in a significant increase (P<0.001) in the relative weight of the heart of hypercholesterolaemic animals compared with control. However, cotreatment with kolaviron and Questran ameliorated the cholesterol-induced enlargement of the heart. Kolaviron (100 and 200 mg/kg) elicited 88.5 and 87.4% reductions, respectively, in plasma cholesterol levels of pretreated animals compared with the cholesterol-fed group. In addition, kolaviron produced a significant decrease (P<0.05) in post-mitochondrial fraction (PMF) cholesterol levels in treated animals compared with untreated hypercholesterolaemic animals. Similarly, Questran significantly decreased (P<0.05) the cholesterol-induced increase in plasma cholesterol levels compared with untreated hypercholesterolaemic animals. In addition, (100 and 200 mg/kg) significantly (P<0.05) decreased plasma LDL-C levels by over 70% in treated animals compared with untreated hypercholesterolaemic animals. Similarly, kolaviron significantly decreased (P<0.05) PMF LDL-C levels by over 60% in treated animals compared with untreated hypercholesterolaemic animals. 4. The significantly (P<0.05) higher values of plasma and PMF triglycerides obtained in cholesterol-fed animals compared with control animals were unaltered following cotreatment with kolaviron and Questran. In the present study, there was a significant decrease (P<0.05) in plasma formation of malondialdehyde in kolaviron- and Questran-treated animals compared with untreated hypercholesterolaemic animals. 5. The results of the present study demonstrate that kolaviron exerts a hypocholesterolaemic effect and reduces the relative weight of the heart in cholesterol-fed animals. This reduction and the favourable lipid profile indicate a possible anti-atherogenic property of the extract.

Effects of cholesterol supplementation on antioxidant enzyme activities in rat hepatic tissues: possible implications of hepatic paraoxonase in atherogenesis

The effects of cholesterol supplementation on antioxidant enzyme activities were investigated hepatic tissue taken from Sprague Dawley rats. METHODS AND REULTS: The study involved 14 male Sprague Dawley rats: seven fed a normal laboratory diet and seven a normal diet plus cholesterol (3.6 g/kg/day) for three months, during which blood samples were obtained to measure serum cholesterol levels. At the end of the 3-month period, the livers were surgically removed in order to measure antioxidant enzyme activities (superoxide dismutase, catalase, glutathione peroxidase and paraoxonase-1). At the end of the study period, serum total cholesterol and HDL-cholesterol levels were significantly higher in the cholesterol-fed group than the control group. There were no significant between-group differences in hepatic superoxide dismutase, catalase and glutathione peroxidase activities, but there was a significant decrease in hepatic paraoxonase-1 activity in the cholesterol-fed group. Cholesterol supplementation significantly decreases paraoxonase-1 activity in rat liver tissue without changing the activities of other antioxidant enzymes. These results suggest that cholesterol significantly suppresses hepatic paraoxonase-1 synthesis. It seems that the decreased paraoxinase-1 activity in the plasma HDL-fraction of atherosclerotic patients is associated with suppressed liver synthesis. A reduction in paraoxonase-1 activity may therefore lead to the more intensive exposure of LDL to oxidant attacks.

Phytoestrogen α-Zearalanol Inhibits Atherogenesis and Improves Lipid Profile in Ovariectomized Cholesterol-Fed Rabbits

Although favorable effects of estrogen replacement therapy on atherosclerosis have been recognized, the benefit versus risk of estrogen replacement on overall cardio- vascular health remains controversial. The main adverse effect jeopardizing the clinical usage of estrogen is the increased risk of breast and endometrial cancer. Zearalenone (ZEN) is a universal endogenous hormone possessing estrogen-like effects and facilitating plant growth. alpha-Zearalanol (alpha-ZAL), a new phytoestrogen, is a reductive product of ZEN. Our preliminary evidence suggested that alpha-ZAL is anti-atherosclerotic. The aim of this study was to examine the effect of alpha-ZAL on atherosclerotic formation and serum lipid profile. Adult female nulliparous rabbits were ovariectomized or sham-operated and fed a high-cholesterol diet with different doses of alpha-ZAL or 17beta-estradiol for 12 wk. The aortic intimal atherosclerotic plaque was significantly larger in the cholesterol-fed group compared to control and sham groups. alpha-ZAL and 17beta-estradiol treatments significantly reduced plaque formation and improved serum profile of lipid (TC, TG, HDL-C, and LDL-C) and lipoprotein (ApoAl and ApoB). Both alpha-ZAL and 17beta-estradiol reconciled ovariectomy-induced uterine atrophy, although alpha-ZAL was significantly less potent than 17beta-estradiol in stimulating uterine growth. Our findings indicate that the phytoestrogen alpha-ZAL has an important anti-atherogenic property, analogous to that of estrogen.

Hyperlipidemia induced by a cholesterol-rich diet leads to enhanced peroxynitrite formation in rat hearts.

We investigated the influence of experimental hyperlipidemia on the formation of cardiac NO, superoxide, and peroxynitrite (ONOO(-)) in rat hearts. Wistar rats were fed 2% cholesterol-enriched diet or normal diet for 8 weeks. Separate groups of normal and hyperlipidemic rats were injected twice intraperitoneally with 2 x 20 micromol/kg FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), a ONOO(-) decomposition catalyst, 24 h and 1 h before isolation of the hearts. A cholesterol diet significantly decreased myocardial NO content, however, myocardial Ca(2+)-dependent and Ca(2+)-independent NO synthase activity and NO synthase protein level did not change. Myocardial superoxide formation and xanthine oxidase activity were significantly increased; however, cardiac superoxide dismutase activity did not change in the cholesterol-fed group. Dityrosine in the perfusate, a marker of cardiac ONOO(-) formation, and plasma nitrotyrosine, a marker for systemic ONOO(-) formation, were both elevated in hyperlipidemic rats. In cholesterol-fed rats, left ventricular end-diastolic pressure (LVEDP) was significantly elevated as compared to controls. Administration of FeTPPS normalized LVEDP in the cholesterol-fed group. We conclude that cholesterol-enriched diet-induced hyperlipidemia leads to an increase in cardiac ONOO(-) formation and a decrease in the bioavailability of NO which contributes to the deterioration of cardiac performance and may lead to further cardiac pathologies.

Time dependent changes of arterial distensibility induced by cholesterol and balloon injury in rabbits: an in vivo intravascular ultrasound study.

The aim of this study was to validate in vivo measurement of intravascular ultrasound (IVUS) for the analysis of structural and functional vessel wall alterations in a chronic animal model. Furthermore, the relation between functional and structural alteration of the vessel wall should be investigated. Fifteen cholesterol-fed rabbits (1%) and 15 control rabbits underwent balloon injury of the abdominal aorta. Immediately before and after balloon traumatization as well as 2 and 6 weeks later IVUS depiction of 10 aortal vessel segments was performed (n = 1,100 measurements). In vivo IVUS measurements and morphometric analysis of the neointimal area of same aortal segments showed a high correlation (n = 148, r = 0.844, p < 0.001). Plaque area determined by morphometry revealed larger areas than the evaluation by IVUS (0.162 +/- 0.138 vs. 0.130 +/- 0.126 mm2, p < 0.001). Before balloon traumatization, pulsatility of the aortal vessel segments was less in cholesterol-fed rabbits (0.067 vs. 0.090, p < 0.01) and neointimal index higher (0.003 vs. 0). Investigation using IVUS 2 and 6 weeks after balloon traumatization demonstrated a continuous loss of arterial distensibility and an increase of neointimal index, being more pronounced in the cholesterol-fed group. As demonstrated by IVUS the loss of distensibility preceded the atherosclerotic alterations. Our investigation suggests using IVUS in this animal model is a reliable setting for long-term investigation of characteristics of the vessel wall. We could demonstrate that altered function of the vessel wall precedes the structural atherosclerotic vessel wall alterations.

In vitro and in vivo (SCID mice) effects of phytosterols on the growth and dissemination of human prostate cancer PC-3 cells.

The dietary effect of phytosterols (PS) versus cholesterol on the growth and metastasis of the PC-3 human prostate cancer cells in SCID mice was studied. Also, their direct effect on the growth and migration of these cells in vitro was analysed. In the in vivo experiment, SCID mice were fed a diet containing 2% of either PS mixture or cholesterol plus 0.2% cholic acid and implanted with 2 x 10(6) tumour cells per mouse. Tumour growth was monitored for 8 weeks post inoculation. Animals fed the PS diet had tumours 40-43% smaller than those fed the cholesterol diet. Furthermore, the number of mice with lymph node and lung metastasis was almost one-half that of the cholesterol-fed group. In the in vitro studies, both beta-sitosterol and campesterol inhibited the growth of PC-3 cells by 70% and 14%, respectively, while cholesterol supplementation increased the growth by 18% when compared with controls. PS inhibited the invasion of PC-3 cells into Matrigel-coated membranes by 78% while cholesterol increased it by 43% as compared with the cells in the control media. Migration of tumour cells through 8 microm pore membranes was reduced by 60-93% when the PC-3 cells were in PS media, as compared with a 67% increase after cholesterol supplementation. PS supplementation reduced the binding of PC-3 cells to laminin by 15-38% and fibronectin by 23% while cholesterol increased binding to type IV collagen by 36%. It was concluded that PS indirectly (in vivo as a dietary supplement) and directly (in tissue culture media) inhibited the growth and metastasis of PC-3 cells. beta-Sitosterol was more effective than campesterol in offering this protection in most of the parameters studied.

Mycophenolate mofetil treatment reduces cholesterol-induced atherosclerosis in the rabbit

Immunosuppressive therapy has been shown to either improve or, more frequently, enhance the development of atherosclerosis. We tested the effect of mycophenolate mofetil (MMF), an inhibitor of nucleotide synthesis widely used in transplant therapy, in diet-induced atherosclerosis in the rabbit. Two groups ( n=10 each) of New Zealand White (NZW) rabbits were fed a 1% cholesterol diet for 12 weeks. One group received MMF (CHOL+MMF group) by gastric gavage (30 mg/kg daily) and the other group (CHOL) received the same volume of saline by the same route. There were no differences in the serum cholesterol (mean values ≥30 mmol/l in both groups after 2 weeks) or in the triglyceride, blood sugar, total protein, and albumin serum levels and weight gain in both groups of animals. The cholesterol-fed untreated rabbits had atherosclerotic plaques covering 43.9.1±SD 16.40% of their thoracic aorta and 41.9±22.59% of their abdominal aorta, while the MMF treated group had 18.5±7.17% and 17.7±9.71%, respectively ( P<0.01). The cholesterol content of the aorta (mg/g) in the cholesterol-fed untreated group was 4.61±SD 1.21 in the thoracic aorta and 4.54±2.07 in the abdominal aorta, whereas the MMF treated group had and 2.83±0.84 and 2.77±1.44, respectively ( P<0.01). Infiltrating macrophages (RAM 11 positive cells/100 nuclei) in the intimal layer of the aorta were 58.4±SD26.16 in the CHOL group and 8.5±5.51 in the CHOL+MMF group: ( P<0.001). CD18 positive cells/100 nuclei were 27.4±17.6 in the CHOL group and 5.3±3.82 in the CHOL+MMF group ( P<0.01), and the intima/media ratio was 0.66±0.11 in the CHOL group and 0.30±0.09 in the MMF treated rabbits ( P<0.001). MMF also reduced proliferating smooth muscle cells (HHF35 positive) infiltrating between the macrophages. These results indicate that MMF ameliorates importantly the atherogenic potential of a high cholesterol diet and this effect is associated with a reduction in macrophage and foam cell infiltration and smooth muscle cell proliferation and infiltration. Since chronic treatment with this drug is given routinely in various clinical conditions with relatively minor side effects, consideration may be given to its use as adjuvant therapy in artheriosclerotic cardiovascular disease.

Effects of defibrotide on aorta and brain malondialdehyde and antioxidants in cholesterol-induced atherosclerotic rabbits

The effects of a high-cholesterol diet in the presence and absence of defibrotide, a single-stranded polydeoxyribonucleotide compound, on the lipid peroxidation product malondialdehyde, endogenous antioxidant enzymes catalase, glutathione peroxidase, and the antioxidant thiol compound GSH were investigated. Forty male New Zeland white rabbits were divided into four groups each consisting of 10 rabbits. Group I received a regular rabbit chow diet and group II 1% cholesterol plus regular chow, group III was given defibrotide (60 mg/kg per day p.o. in water) and was fed with regular chow, and group IV received defibrotide plus 1% cholesterol for 9 weeks. Blood cholesterol and malondialdehyde, catalase, glutathione peroxidase, and GSH were determined before starting the experimental diet regimen (basal). After 9 weeks, the same parameters were determined in blood, aorta, and brain tissues (end -experiment). Aortic tissue was examined under a light microscope for morphological alterations indicative of atherosclerosis. The increase in serum total cholesterol was greater in group II than group IV. Plasma malondialdehyde in group II was higher than in group III. Brain malondialdehyde in group II was higher than all other groups, and aortic malondialdehyde in this group was higher than group I and III. Serum catalase activity decreased in group II and increased in group III, compared with basal values. Brain catalase activity in group I was higher than group II, and aorta catalase in group IV was higher than in group I and III. Blood glutathione peroxidase activity in group III and IV was higher than basal. GSH concentrations decreased significantly in the cholesterol-fed groups (group II and IV). Histological alterations in the cholesterol-fed groups were more pronounced in group II. The increased levels of malondialdehyde in plasma, aorta, and brain tissue of group II suggest a role of oxygen free radicals in the pathogenesis of cholesterol-induced atherosclerosis. The higher malondialdehyde values in the brain tissues of animals in group II compared with group IV suggest a protective role of defibrotide in the brain against lipid peroxidation in the oxidant stress of cholesterol-induced atherosclerosis. Increased catalase activities in the blood and aortic tissues and increased glutathione peroxidase activities in the blood of rabbits receiving defibrotide suggest an induction of these antioxidant enzyme activities by defibrotide. These results imply that anti-atherosclerotic, anti-ischemic effects of this drug may be due to the beneficial effects on the oxidant-antioxidant balance of various tissues.

Reduced platelet serotonin content in rabbits with dietary hypercholesterolemia.

Serotonin [5-hydroxytryptamine (5-HT)] has been implicated in platelet activation and vasoconstriction, two processes that contribute to arterial thrombosis in atherosclerotic diseases. In the present study, Japanese White rabbits fed 1% cholesterol for 5 weeks were used to investigate the response of hypercholesterolemic vascular arteries and platelets to 5-HT. Contractions of the thoracic aorta induced by 5-HT were comparable between the cholesterol-fed group and the age-matched control group. However, acetylcholine-induced vasodilation in arteries preconstricted with 5-HT was moderately but significantly attenuated in the cholesterol-fed rabbits. Platelet aggregation responses to 5-HT (0.1-3 micromol/l) in combination with epinephrine (5 micromol/l), adenosine diphosphate (ADP) (0.3-10 micromol/l), 9,11-dideoxy-9alpha,11alpha-methanoepoxy-prostaglandin F2alpha (U-46619) (1-30 micromol/l) or collagen (3 microg/ml) were significantly enhanced in cholesterol-fed rabbits. In contrast, platelet 5-HT content determined with a high-performance liquid chromatography-electrochemical detector (HPLC-ECD) was significantly decreased in cholesterol-fed rabbits. These results suggest a possible association among the endothelial dysfunction, platelet aggregation and platelet 5-HT content in rabbits with dietary hypercholesterolemia.

Inhibition of N-methyl-N-nitrosourea- and 7,12-dimethylbenz[a] anthracene-induced rat mammary tumorigenesis by dietary cholesterol is independent of Ha-Ras mutations.

Dietary cholesterol has previously been shown to inhibit rat mammary tumorigenesis but the mechanisms remain unclear. Uptake of serum low density lipoprotein cholesterol by tissues leads to down-regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis that catalyzes the formation of mevalonate. In addition to being a precursor of cholesterol, mevalonate is necessary for DNA synthesis and cell proliferation. Isoprenoids, also derived from mevalonate, are required for the post-translational modification of Ras proteins that are mutated in a number of carcinogen-induced rat mammary tumors. The purpose of this study, therefore, was to determine whether inhibition of tumorigenesis by cholesterol is dependent on the frequency of mutations in the Ha-ras gene. Female Sprague-Dawley rats (30/group) were given a single dose of either N-methyl-N-nitrosourea (MNU, 50 mg/kg i.p.) or 7, 12-dimethylbenz[a]anthracene (DMBA, 100 mg/kg intragastrally), carcinogens that produce tumors with either a high (MNU) or low (DMBA) frequency of Ha-ras mutations in codon 12 or 61, respectively. Rats were fed either a control AIN-93G diet or the control diet supplemented with 0.3% cholesterol for 14 weeks. Dietary cholesterol significantly decreased the final tumor incidence in rats given DMBA (83 versus 100%, P < 0.05) or MNU (53 versus 77%, P < 0.05). HMG-CoA reductase activity was higher in mammary tumors than in normal mammary glands, but the activity of this enzyme was reduced by cholesterol feeding only in mammary glands and not in tumors. Tumors induced by MNU had a high frequency of Ha-ras mutations in both the control (65%) and cholesterol-fed (68%) groups. Tumors induced by DMBA had a low frequency of Ha-ras mutations that also did not differ between the control (21%) and cholesterol-fed (18%) groups. These findings show that dietary cholesterol inhibits mammary tumorigenesis induced by either MNU or DMBA and that the inhibition is independent of the type or extent of mutations in the Ha-ras gene.

Hypercholesterolemia causes mechanical weakening of rabbit atheroma : local collagen loss as a prerequisite of plaque rupture.

Hypercholesterolemia may render atherosclerotic plaques prone to rupture. To test this hypothesis, catheters with matrix-covered balloons were implanted into the aorta of rabbits fed standard or 0. 5% cholesterol chow (n=70). In 1 month, fibrous plaques developed around the balloon. Time-dependent accumulation of cholesteryl esters and free cholesterol was detected in the plaques of the cholesterol-fed group only. The pressure needed to rupture the plaque by balloon inflation was used as an index of plaque strength. Three months after the catheter implantation, the breaking pressure was 2.1 times lower (P<0.05) in cholesterol-fed rabbits. It was accompanied by collagen loss, as measured by plaque hydroxyproline content, but not with deficiency of collagen cross-linking. Sirius red staining showed preservation of collagen originally covering the balloon and accumulation of nascent collagen in the lesions of standard chow-fed rabbits. In the cholesterol-fed group, both mature and new collagen underwent degradation predominantly in the plaque shoulders. Collagen breakdown was associated with local accumulation of foamy macrophages. Gel zymography demonstrated relative enhancement of gelatinolytic activity at 92 and 72 kDa, as well as caseinolytic activity at 57, 45, and 19 kDa in the lipid-laden plaques. Lipid accumulation in the plaque was also associated with a loss of smooth muscle cells, the cellular source of the collagen fibers. The remaining smooth muscle cells showed increased collagen synthesis, although it was insufficient to counterbalance collagen degradation and cell loss. Thus, we have obtained direct evidence that hypercholesterolemia is accompanied by enhanced local collagen degradation, which is potentially responsible for plaque weakening.