Atherogenesis In Cholesterol-fed Rabbits Research Articles

P6204Oral vaccination of Lactococcus lactis expressing Ling Zhi 8 protein prevents nonalcoholic fatty liver and early atherogenesis in cholesterol-fed rabbits

Abstract Background Atherosclerosis is an inflammatory disease characterized by lipid deposits in the subendothelial space leading to severe inflammation. Nonalcoholic fatty liver disease (NAFLD) shares several risk factors with atherosclerosis. Epidemiology studies have indicated that NAFLD may be an independent risk factor for atherosclerosis. Anti-inflammation therapy by inhibiting IL-1β led to a significantly lower cardiovascular event rate in recent clinical trial (CANTOS trial). Ling Zhi 8 (LZ8) is an immunomodulatory protein that possesses a broad range of pharmacological properties, including anti-inflammatory activities. Methods and findings In this study, we developed an oral vaccination of Lactococcus lactis expressing LZ8 protein in a nisin-controlled gene expression system and investigated its anti-inflammation properties. Experimental rabbits received commercial rabbit chow supplemented with 2% cholesterol for 5 weeks and recombinant LZ8 L. lactis vaccine once a day on weekdays. The expression of IL-1β in the aorta (Figure A) was significantly suppressed after LZ8 vaccination. Moreover, in hematoxylin and eosin staining of the aorta, the intima-medial thickness was decreased, and foam cells were significantly reduced in the sub-endothelial space (Figure C). LZ8 also inhibited the expression of IL-1β in the liver (Figure B), decreased fat droplet deposits and infiltration of inflammatory cells (Figure D), and improved liver function by decreasing liver enzymes. Figure 1 Conclusions Our results suggest that LZ8 could be used as a therapeutic tool to improve both atherosclerosis and NAFLD due to its anti-inflammatory effect. Acknowledgement/Funding TCVGH-1067317C, TCVGH-1063108C

Investigations on the Significance of Inductable Cyclooxygenase in Atherogenesis in Cholesterol-Fed Rabbits

Atherosclerosis is the most prevalent disease in middle and later years of human life. Heart attacks and strokes are among the most frequent causes of death. New approaches in therapy are the use of NSAID’s (nonsteroidal antiphlogistical drugs) of the new generation (selective inhibitors of COX-2) and DHEA (Dehydroepiandrostenon). The key enzyme of prostaglandin synthesis, the COX-2 isoenzyme, is predominantly found in inflammatory tissue. Out of this results a possible importance of COX-2-inhibitors in prophylaxis of atherosclerosis. Our study intended to examine the significance of COX-2 and the COX-2-formed prostanoids, as well as the significance of COX-2-independent iso-prostanes on atherosclerosis. For that purpose we tested the effect of the selective COX-2-inhibitor Celecoxib on rabbits fed with cholesterol and compared this with the effect of the steroid hormone DHEA in 4 groups: healthy control, cholesterol-fed control, DHEA-group and Celecoxib group. In the test prostanoids, nitrate and nitrite were measured by gas chromatograpy/tandem-mass-spectrometry (GC-MS/MS) in 24-hours-collected urine. Additionally we measured cholesterol and triglycerides in plasma. The aortas of the examinated animals were measured optically using a planimetrical method. The measurement of prostanoids, isoprostanes, nitrate and nitrite showed considerable variations and particulary significant differences (p < 0.01) even in the initial values. By the treatment with Celecoxib the rate of atherosclerosis was reduced in a highly significant way in comparison to the cholesterol group and the DHEA-group. Consequently the test demonstrated a significant role of COX-2 in the development of atherogenesis.

Addition of dietary fat to cholesterol in the diets of LDL receptor knockout mice: effects on plasma insulin, lipoproteins, and atherosclerosis

The factors underlying cardiovascular risk in patients with diabetes have not been clearly elucidated. Efforts to study this in mice have been hindered because the usual atherogenic diets that contain fat and cholesterol also lead to obesity and insulin resistance. We compared plasma glucose, insulin, and atherosclerotic lesion formation in LDL receptor knockout (Ldlr(-/-)) mice fed diets with varying fat and cholesterol content that induced similar lipoprotein profiles. Ldlr(-/-) mice fed a high-fat diet developed obesity, mild hyperglycemia, hyperinsulinemia, and hypertriglyceridemia. Quantitative and qualitative assessments of atherosclerosis were unchanged in diabetic Ldlr(-/-) mice fed a high-fat diet compared with lean nondiabetic control mice after 20 weeks of diet. Although one group of mice fed diets for 40 weeks had larger lesions at the aortic root, this was associated with a more atherogenic lipoprotein profile. The presence of a human aldose reductase transgene had no effect on atherosclerosis in fat-fed Ldlr(-/-) mice with mild diabetes. Our data suggest that when lipoprotein profiles are similar, addition of fat to a cholesterol-rich diet does not increase atherosclerotic lesion formation in Ldlr(-/-) mice.

The Nine Lives of ACAT Inhibitors

Atherosclerosis is the product of excessive lipid accumulation and inflammation in the artery wall. The lipid that tops every list of suspects is cholesterol, which is the primary lipid in low-density lipoproteins (LDL). Cholesterol exists either as a simple molecule or as cholesterol esters, in which the hydroxyl group is linked to a fatty acyl moiety. In cells, cholesterol esters are synthesized in an intracellular reaction catalyzed by acyl coenzyme A (CoA):cholesterol acyltransferase (ACAT) enzymes.1,2 Owing to the discoveries of cholesterol esters in arterial lesions in 19103 and of ACAT activity in the mid 1900s,4 inhibiting ACAT has been considered as a strategy for preventing or treating atherosclerosis. Over the past 25 years, interest in ACAT inhibitors has waxed and waned as new studies advance knowledge in the field. Prominently reported and disappointing results from a recent human trial of an ACAT inhibitor5 dampened enthusiasm for this potential therapy. However, a study by Bell et al in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology 6 revives the idea of targeting ACAT enzymes and highlights a key unanswered question: Can ACAT2-specific inhibitors lower plasma cholesterol and treat or prevent atherosclerosis? See page 1814 After the discovery of the ACAT reaction, two rationales for inhibiting ACAT emerged. One, in retrospect, was perhaps overly simplistic: blocking cholesterol esterification in macrophages would diminish macrophage “foam cell” formation and thereby decrease atherosclerotic lesion development. The other was to decrease hepatic and intestinal cholesterol ester formation, resulting in decreases in plasma levels …

NTE-122, an Acyl-CoA:Cholesterol Acyltransferase Inhibitor, Prevents the Progression of Atherogenesis in Cholesterol-Fed Rabbits

The cholesterol-lowering and anti-atherosclerotic effects of NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylaminophenyl)ureido]methyl]cyclohexane), an acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, were evaluated in 1% cholesterol diet-fed rabbits. NTE-122 (1, 3 and 10 mg/kg per day) lowered the total cholesterol levels in both plasma and liver dose-dependently (by 99% and 94% at 10 mg/kg per day, respectively). In the aortic wall of the rabbits given NTE-122, the atherosclerotic lesion area in both aortic arch and thoracic aorta were dose-dependently reduced (by 100% at 10 mg/kg per day), and the total cholesterol content in aortic arch was also lowered significantly at more than 3 mg/kg per day. These results suggest that NTE-122 is capable of exhibiting anti-atherosclerotic effects.

3.P.157 Role of the thymus in experimental atherogenesis in cholesterol-fed rabbits: Immunocytochemical analysis of cellular components

3.P.157 Role of the thymus in experimental atherogenesis in cholesterol-fed rabbits: Immunocytochemical analysis of cellular components

Effect of exogenous hyperinsulinaemia on atherogenesis in cholesterol-fed rabbits

To examine the hypothesis that hyperinsulinaemia promotes atherosclerosis, cholesterol-fed rabbits were injected subcutaneously with 6 i.u. of human insulin (n = 16) or placebo (n = 20) daily for 24 weeks; injection of insulin resulted in hyperinsulinaemia for up to 16 h after injection. Compared to placebo rabbits, insulin-treated rabbits had higher levels of insulin antibodies in plasma, similar levels of intermediate density, low density and high density lipoprotein cholesterol and similar activities of hepatic and lipoprotein lipase in post-heparin plasma, but lower levels of plasma C-peptide, blood glucose, postprandial plasma triglycerides, plasma cholesterol and very low density lipoprotein cholesterol. On univariate analysis, with and without adjustment for differences in plasma cholesterol levels between the two groups, there were no significant differences in extent or severity of atherosclerosis between insulin and placebo rabbits. Furthermore, after combining the results from all the rabbits to examine plasma insulin levels and the other variables mentioned above as predictors of atherosclerosis severity, plasma insulin level was not a predictor, on univariate or multiple linear regression analysis; the first ranked independent predictors were postprandial intermediate density lipoprotein cholesterol in the arch, and postprandial plasma triglyceride in both the thoracic and abdominal aorta. These results suggest that exogenous hyperinsulinaemia does not promote atherogenesis in cholesterol-fed rabbits, but that postprandial levels of intermediate density lipoprotein cholesterol or plasma triglycerides may be involved in atherogenesis.

Exogenous hyperinsulinemia and atherogenesis in cholesterol-fed rabbits

Exogenous hyperinsulinemia and atherogenesis in cholesterol-fed rabbits

Alacepril, an ACE inhibitor, reduced atherosclerosis as well as suppressing atherogenesis in cholesterol-fed rabbits; use of high-resolution miniature transesophageal echocardiography

Alacepril, an ACE inhibitor, reduced atherosclerosis as well as suppressing atherogenesis in cholesterol-fed rabbits; use of high-resolution miniature transesophageal echocardiography

Role of the thymus in experimental atherogenesis in cholesterol-fed rabbits

Role of the thymus in experimental atherogenesis in cholesterol-fed rabbits

SIM 6080, A New Calcium Antagonist, Reduces Aortic Atherosclerosis in Cholesterol-Fed Rabbits

SIM 6080 is a new calcium antagonist, structurally related to diphenylalkylamines, which combines transmembrane and intracellular calcium antagonist activities. In the present study we investigated the effect of SIM 6080 on atherogenesis in cholesterol-fed rabbits. Subcutaneous administration of the compound at 0.33, 1, and 3 mg kg -1/bid for 60 days neither affected plasma lipids nor blood pressure. However at 1 and 3 mg kg -1/bid SIM 6080 reduced in a dosedependent manner both the area of the aorta covered by plaques and aortic cholesterol content. Determination of SIM 6080 plasma and aortic content indicated that the compound could concentrate up to 10 times in the arterial tissue. In vitro studies demonstrated that at concentrations similar to those observed in the aorta this compound may stimulate rabbit βVLDL catabolism by smooth muscle cells in an homologous system suggesting that the up-regulation of LDL-receptors in the aorta may contribute to the antiatherosclerotic properties of SIM 6080.

Differential effects of renin-angiotensin system blockade on atherogenesis in cholesterol-fed rabbits.

To investigate the mechanism by which angiotensin-converting enzyme (ACE) inhibition attenuates atherogenesis, we have studied the effects of a non-sulfhydryl ACE inhibitor, enalapril, and an angiotensin receptor antagonist, SC-51316, in cholesterol-fed rabbits. After 3 mo of enalapril treatment (10 mg/kg per d, p.o.) the percent plaque areas in the thoracic aortas of treated animals were significantly reduced (controls: 86.8 +/- 3.5%; treated: 31.1 +/- 8%, P < 0.001). Aortic cholesterol content was also reduced (controls: 31.4 +/- 3.2 mg/g tissue; treated: 7.4 +/- 1.8 mg/g, P < 0.001). Enalapril had no significant effect on plasma lipid levels or conscious blood pressure. In a second study, the angiotensin II receptor antagonist SC-51316 was administered at a dose equivalent to enalapril at blocking angiotensin pressor effects in vivo (30 mg/kg per d, p.o.). Evaluation after 3 mo indicated no significant attenuation of aortic atherosclerosis. These results demonstrate that: (a) enalapril attenuates atherogenesis without affecting either blood pressure or plasma lipid levels; (b) antioxidant activity, found with sulfhydryl-containing ACE inhibitors, is not necessary for reducing plaque formation; and (c) the attenuation of atherogenesis by ACE inhibition may not be due to blockade of the renin-angiotensin system. Alternatively, one must consider the multiple effects of ACE inhibition on other hormone systems, such as bradykinin, or the possibility that alternate angiotensin II receptors may be involved in atherosclerosis.

Effect of testosterone on atherogenesis in cholesterol-fed rabbits with similar plasma cholesterol levels

The aim was to examine the effect of testosterone on atherogenesis in cholesterol-fed, castrated male rabbits not mediated via different plasma cholesterol levels. The rabbits in the testosterone group ( n = 19) and in the placebo group ( n = 17) were injected intramuscularly twice weekly for 17 weeks with 25 mg testosterone enantate and placebo, respectively, reaching plasma testosterone levels of 50–150 nmol/l in the testosterone group. No effect of testosterone on liver function or body weight was detected, but at week 15 mean blood pressure was 75 ± 2 mmHg (mean ± S.E.) in the testosterone group compared with 69 ± 2 mmHg in the placebo group ( P < 0.05). To reduce variation in plasma cholesterol between the two groups, the amount of cholesterol fed to each rabbit was adjusted on the basis of weekly determinations of plasma cholesterol; the mean plasma cholesterol levels during the 17 weeks were 20.9 ± 1.0 and 20.4 ± 0.9 mmol/l for the placebo and testosterone groups. In the intima-inner medias of the aortic arch, the thoracic and the abdominal aorta there were no consistent significant differences in aortic cholesterol content, expressed either as nmol/cm 2 or nmol/mg protein, between the two groups. However, the aortic cholesterol content tended to be lower in the testosterone group than in the placebo group. These findings suggest that in cholesterol-fed, castrated male rabbits, testosterone does not promote atherogenesis by an effect directly on the arterial wall.

Does vibration cause poststenotic dilatation in vivo and influence atherogenesis in cholesterol-fed rabbits?

Arterial post-stenotic dilatation (PSD) is a fusiform swelling immediately down-stream to a stenosis. It is characterized by the presence of turbulent blood flow and wall vibration which has been claimed by others to be causal by producing structural weakening. We tested the hypothesis that vibration causes PSD in vivo by attaching electromagnetic and pneumatic vibrators to the aortic wall in chronic rabbits. We also observed whether mechanical vibration of the aorta in vivo influenced the distribution of oil-red-O lesions during one percent dietary cholesterol feeding. Low mass vibration gauges were developed to measure the vibration. Electromechanical vibrators having a ceramic magnet slug within a coil supplied with 50 Hz were glued to the aorta of chronic rabbits and the vibration maintained for an average of 8 weeks. Despite greater amounts of energy imparted to the wall there was no dilatation or difference in oil-red-O staining from the controls. Five weeks vibration at 100 Hz and an amplitude equal to the normal diameter pulse also produced no dilatation. We conclude that vibration does not cause PSD in vivo and suggest that its cause is likely to involve the vascular muscle stimulated by the effect of turbulent flow on the endothelium.

Atherosclerosis in the aorta of hypercholesterolemic rabbits and the influence of daltroban

These studies have examined aortic atherogenesis in cholesterol-fed rabbits and have correlated the effects of daltroban to the pathomechanism of the vessel wall lesions. After feeding a 0.5% cholesterol-enriched diet for 96 d atherosclerotic alterations were seen, which exhibited a proximo-distal pattern, to which the branching of the aorta contributed considerably. Depending on their localization and size a varying cellular constitution of the plaques was obvious. Large plaques, which were mainly seen in the aortic arch and the proximal descending thoracic aorta, consisted of numerous proliferating cells, masses of fibrillar ground substance, clusters of foam cells, and rarely contained cholesterol crystals and necroses. Emerging plaques mainly found in distal thoracic and abdominal aorta imposed as fatty streaks. Daltroban treatment, used in a clinically relevant doses of 10 mg/kg b. wt. per day, reduced extension and protrusional area of plaques to about 40%, which was evaluated using a newly developed computerized morphormetric method, in association with significant reductions in free cholesterol content within the aorta. The results suggest that daltroban inhibits the progression of atherosclerosis in cholesterol-fed rabbits. This effect may be related to its antagonistic interaction with the thromboxane A2 receptor and also to an inhibition of the cholesterol metabolism.

Evidence for increased aortic plasma membrane calcium transport caused by experimental atherosclerosis in rabbits.

Several lines of evidence, including the reported ability of calcium channel blockers to prevent atherogenesis in cholesterol-fed rabbits, suggest that calcium mediates one or more of the pathologic changes in atherosclerosis. Moreover, it has long been known that calcium accumulates in atherosclerotic blood vessels. To test the hypothesis that a substantial fraction of this accumulated calcium is intracellular and to identify possible causes of this accumulation, calcium fluxes and contents were determined in aortic segments from cholesterol-fed rabbits and age-matched controls. A new method, based on 45Ca efflux experiments and computer-assisted kinetic analysis, was used to measure intracellular and extracellular calcium contents (nmol calcium/g wet wt tissue) and fluxes. Total intracellular calcium increased from 269 +/- 11.6 to 1,300 +/- 352 nmol/g in cholesterol-fed animals compared with controls (p less than 0.01). This change was sufficient to account for the observed increase in total tissue calcium from 4,190 +/- 211 to 5,240 +/- 477 nmol/g (p less than 0.05). Thus, the fraction of tissue calcium that is intracellular increased significantly from 0.065 +/- 0.006 to 0.223 +/- 0.048 (p less than 0.01) in experimental atherosclerosis. In addition, the data were quantitatively consistent with the hypothesis that these changes are brought about by a 4.8-fold increase in the plasma membrane calcium permeability of aortic smooth muscle cells. These results provide evidence that increased intracellular calcium is a possible mediator of cholesterol-induced atherogenesis.

Effect of verapamil on accumulation of free and esterified cholesterol in the thoracic aorta of cholesterol-fed rabbits.

In order to investigate the effect of the calcium antagonist verapamil on atherogenesis in cholesterol-fed rabbits, 3 groups of 11 animals were fed a 2% cholesterol-enriched diet for 10 weeks. One group received verapamil in a daily dose of 16 mg/kg orally plus 2 mg/kg subcutaneously. This dosage resulted in plasma concentrations of verapamil in the same range as the usual therapeutic levels in humans. Another group received verapamil in a daily dose of 8 mg/kg orally and 0.5 mg/kg subcutaneously. The third group received placebo capsules orally and isotonic saline subcutaneously. Total cholesterol concentrations in plasma over the 10 weeks were 37 +/- 4, 42 +/- 4 and 45 +/- 3 mM (mean +/- SE) in the high verapamil-, in the low verapamil- and in the placebo group, respectively. These values were not significantly different. The distribution of cholesterol between HDL, LDL and VLDL in plasma was similar in the 3 groups. The high verapamil group had a significantly (P less than 0.05) lower concentration of cholesterol in the thoracic aorta than the placebo group (29 +/- 5 vs 43 +/- 9 mumol/g wet weight). The low verapamil group and the placebo group had the same aortic cholesterol concentrations. Neither dosage of verapamil affected the permeability of the aortic endothelium to plasma lipoproteins and albumin, as measured by use of radioactive tracers at the end of the experiment.

No effect of nifedipine on atherogenesis in cholesterol-fed rabbits.

It was recently reported that the calcium antagonist nifedipine suppresses aortic cholesterol accumulation in cholesterol-fed rabbits without reducing hypercholesterolemia. We extended this study on plasma lipoprotein levels and aortic influx of cholesteryl ester. We gave 40 mg per day of nifedipine orally to 17 rabbits fed a 2% cholesterol diet for 8 weeks. For the same period of time 15 control rabbits received placebo capsules and the same diet. During the study, total cholesterol, HDL, LDL, and VLDL cholesterol concentrations in plasma were not significantly different in the experimental and control animals. At the end of the study we found no difference in the two groups in accumulation of cholesterol in the intima media of the proximal thoracic aorta, the distal thoracic aorta, and the corresponding media layers. Furthermore, aortic influx of free cholesterol, cholesteryl ester, and albumin from plasma measured by radioactive tracers was not significantly affected by nifedipine.

Calcium antagonists — New aspects and problems unsolved

Vasodilators called calcium antagonists were initially developed as anti-anginal drugs. However, at present, they are also used in the treatment of hypertension, some sort of arrhythmias, heart failure, peripheral vascular disease, cerebrovasospasm, etc. Quite recently, calcium antagonists have been shown to prevent atherogenesis in cholesterol-fed rabbits and arterial calcinosis produced by aging or various interventions in rats. Calcium antagonists have also been shown to potentiate cytotoxicity of some anti-tumor agents in vitro and in vivo. These predict a new era for calcium antagonists not only as therapeutic agents but as research objectives.

Suppression of atherogenesis in cholesterol-fed rabbit treated with nifedipine.

We tested the effects of nifedipine, a calcium antagonist, on atherogenesis in rabbits fed a 2% cholesterol diet. The drug was given orally, 40 mg/dl, and control rabbits received placebo. Nifedipine was well tolerated, and evoked only transient, moderate reductions in arterial pressure. Plasma total cholesterol after 8 wk before killing the rabbits was similar in the placebo and nifedipine-treated groups, averaging 1,903 +/- 138 (n = 13) and 1,848 +/- 121 mg/dl (n = 13; mean +/- SE; P greater than 0.8). In placebo-treated rabbits, aortic lesions stainable with Sudan IV covered 40 +/- 5% of the intimal surface, and the cholesterol concentration in aortic tissue was 47 +/- 5 mg/g protein. Corresponding values for the aortas from nifedipine-treated rabbits were significantly lower and averaged 17 +/- 3% (P less than 0.001) and 29 +/- 2 mg/g protein (P less than 0.001). We conclude that nifedipine suppressed atherogenesis without reducing hypercholesterolemia.