Oxygen-derived free radicals contribute to baroreceptor dysfunction in atherosclerotic rabbits

The goal of the present study was to determine whether oxygen-derived free radicals contribute to baroreceptor dysfunction in atherosclerosis. Baroreceptor activity was measured from the carotid sinus nerve during pressure ramps in isolated carotid sinuses of anesthetized rabbits. Rabbits fed a 0.5% to 1.0% cholesterol diet for 7.9 +/- 0.4 months (mean +/- SE; range, 5.5 to 10) developed atherosclerotic lesions in the carotid sinuses. Maximum baroreceptor activity measured at 140 mm Hg and the slope of the pressure-activity curve were reduced in atherosclerotic (n = 15) compared with normal (n = 13) rabbits (425 +/- 34 versus 721 +/- 30 spikes per second and 6.2 +/- 0.6 versus 10.8 +/- 0.8 spikes per second per mm Hg, respectively, P < .05). The level of activity was inversely related to plasma cholesterol concentration (r = .86, P < .001) and total cholesterol load (plasma concentration x duration of diet, r = .92). Mean arterial pressure was normal in both groups. Exposure of the carotid sinus to the free-radical scavengers superoxide dismutase (SOD) and catalase significantly increased maximum baroreceptor activity by 25 +/- 4% in atherosclerotic rabbits (n = 6) but caused only small and irreversible changes in activity in normal rabbits (n = 8). Catalase alone but not SOD also increased baroreceptor activity in atherosclerotic rabbits (n = 7). Exposure of the carotid sinus of normal rabbits to exogenous free radicals generated from the reaction between xanthine and xanthine oxidase inhibited baroreceptor activity in a dose-dependent and reversible manner (n = 8, P < .05). The inhibition of activity was attenuated by SOD and catalase but was not attenuated by the inhibitor of hydroxyl radical formation, deferoxamine. Neither restoration of baroreceptor activity in atherosclerotic rabbits by catalase nor inhibition of activity by xanthine/xanthine oxidase could be explained by changes in the carotid pressure-diameter relation or prostacyclin formation. These results indicate that oxidant stress inhibits baroreceptor activity and that endogenous oxyradicals produced in atherosclerotic carotid sinuses contribute to baroreceptor dysfunction.     

Gender differences in intima-media permeability to low-density lipoprotein at atherosclerosis-prone aortic sites in rabbits. Lack of effect of 17 beta-estradiol

Premenopausal women are protected from coronary heart disease, and premenopausal nonhuman primates are protected from atherosclerosis, the underlying cause of coronary heart disease. Estrogen is thought to account for this protection in females, and part of this protection is independent of the effects on risk factors, including lipoprotein levels. This study considered the hypothesis that reduced intima-media permeability to low-density lipoproteins (LDL) may account for the protection from atherosclerosis and coronary heart disease in premenopausal females and that this effect might be mediated by estrogen. Intima-media permeability to LDL was determined in male and female rabbits made hypercholesterolemic by feeding them 0.5% cholesterol for 8 days. The diet of half of the female rabbits was supplemented with 17 beta-estradiol (4 mg/d) during cholesterol feeding and the preceding 4 weeks. Estrogen treatment in the female rabbits did not influence the intima-media permeability to LDL. However, intima-media permeability to LDL for branch sites of the abdominal aorta and aortic arch (regions highly susceptible to atherosclerosis) was 43% and 38% lower, respectively, in male rabbits than in female rabbits: (2.93 +/- 0.39 microL/h/g, (n = 8), vs 6.28 +/- 0.86 microL/h/g, (n = 16), P < .001, and 4.69 +/- 0.28 microL/h/g, (n = 8) vs 7.57 +/- 0.75 microL/h/g, (n = 16), P < .02). In contrast, intima-media permeability to LDL in 7 of 8 aortic sites relatively resistant to atherosclerosis did not differ between male and female rabbits. These data suggest that the protection from atherosclerosis associated with female sex and estrogen is mediated by mechanism(s) other than reduction in intima-media permeability to LDL.     

beta-VLDL hypercholesterolemia relative to LDL hypercholesterolemia is associated with higher levels of oxidized lipoproteins and a more rapid progression of coronary atherosclerosis in rabbits

The accumulation of the oxidized apolipoprotein, apoB-100, containing lipoproteins in the arterial wall and the progression of coronary atherosclerotic lesions in rabbits with beta-VLDL and LDL hypercholesterolemia was compared. In New Zealand White (NZW) rabbits on a 0.125% cholesterol diet, LDL cholesterol levels increased from 14 +/- 1 mg/dL (mean +/- SEM; n = 9) to 170 +/- 34 mg/dL (n = 10, P = .0002). On 0.5% cholesterol, LDL cholesterol levels were similar, but beta-VLDL cholesterol levels increased from 60 +/- 4 mg/dL (n = 10) to 550 +/- 75 mg/dL (n = 8; P < .0001). In Watanabe heritable hyperlipidemic (WHHL) rabbits, LDL cholesterol levels were 2.3-fold higher (n = 13; P < .0001) than in NZW rabbits on 0.5% cholesterol, whereas their beta-VLDL cholesterol levels were 3.7-fold lower (P < .0001), resulting in similar total cholesterol levels. At 2 months, mean intimal areas of lesions in the coronary arteries of NZW rabbits on 0.125% cholesterol were 0.13 +/- 0.045 mm2 (n = 4; mean +/- SEM) and were 5.8-fold, (n = 4; P = .016) and 2.0-fold (n = 6; P = NS versus 0.125% cholesterol and P = .014 versus 0.5% cholesterol) higher in NZW rabbits on 0.5% cholesterol and in WHHL rabbits, respectively. At 5 months, mean intimal areas were 0.47 +/- 0.088 mm2 (n = 6) in NZW rabbits on 0.125% cholesterol and were 4.5-fold (n = 4; P = .0001) and 2.0-fold (n = 7; P = .012 and P = .0019) higher in rabbits on 0.5% cholesterol and in WHHL rabbits, respectively. Levels of oxidized apoB-100 containing lipoproteins (both beta-VLDL and LDL) in the lesions correlated with mean intimal area (r = .88; n = 31; P < .0001) of those lesions and with the plasma levels of total beta-VLDL/LDL (r = .72; P < .0001). Levels of oxidized apoB-100 containing lipoproteins in the arterial wall correlate with progression of hypercholesterolemia-induced coronary atherosclerotic lesions. Plasma levels of beta-VLDL relative to similar increases in LDL result in a more pronounced accumulation of oxidized apoB-100 containing lipoproteins in the arterial wall and in the plasma and a more rapid progression of coronary atherosclerosis.     

Preservation of endogenous antioxidant activity and inhibition of lipid peroxidation as common mechanisms of antiatherosclerotic effects of vitamin E, lovastatin and amlodipine

OBJECTIVES: We sought to document the common mechanisms of the antiatherogenic effects of the cholesterol-lowering hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin, the dihydropyridine Ca2+ blocker amlodipine and the antioxidant vitamin E. BACKGROUND: Vitamin E, HMG-CoA reductase inhibitors and Ca2+ blockers each inhibit atherosclerosis in hypercholesterolemic animals. METHODS: New Zealand White rabbits were fed regular chow (Group A), chow with 1% cholesterol (Group B), 1% cholesterol diet plus lovastatin (Group C), 1% cholesterol diet plus vitamin E (Group D) or 1% cholesterol diet plus amlodipine (Group E) for 12 weeks. The extent of aortic atherosclerosis was measured by planimetry of the sudanophilic area. Malondialdehyde (MDA) and superoxide dismutase (SOD) in blood were measured as indexes of lipid peroxidation and antioxidant activity, respectively. RESULTS: Group A rabbits showed no atherosclerosis, whereas Group B rabbits had 17.4 +/- 9.3% (mean +/- SD) of the aorta covered with atherosclerosis, and Groups C, D and E rabbits had significantly less atherosclerosis. Plasma SOD activity was lower in Group B than in Group A (6.9 +/- 1.1 vs. 12.8 +/- 1.5 U/ml, p < 0.01) and was preserved in the groups given lovastatin, vitamin E or amlodipine with a high cholesterol diet. The serum MDA level was higher in Group B rabbits than Group A rabbits (12.1 +/- 2.6 vs. 1.2 +/- 0.1 nmol/ml, p < 0.01) and increased minimally in rabbits given lovastatin, vitamin E or amlodipine with a high cholesterol diet. In in vitro experiments, both lovastatin and amlodipine preserved SOD activity and reduced the oxidizability of low density lipoproteins by rabbit leukocytes. CONCLUSIONS: This study suggests that a reduction in lipid peroxidation and preservation of SOD may be common mechanisms of antiatherosclerotic effects of lovastatin, vitamin E and amlodipine.     

Induction of early atherosclerosis in LDL-receptor-deficient mice immunized with beta2-glycoprotein I

BACKGROUND: Immunization with beta2-glycoprotein I (beta2GPI), the probable target of autoimmune anticardiolipin antibodies, results in experimental antiphospholipid syndrome in different mouse strains. The present study was undertaken to evaluate the effect of beta2GPI immunization on the progression of atherosclerosis. METHODS AND RESULTS: In the first experiment, 3 groups of LDL receptor-deficient (LDL-RD) mice (n=15 per group) were immunized with either beta2GPI or ovalbumin or were not immunized and were fed a chow diet for 12 weeks. In a second experiment, 3 groups of LDL-RD mice (n=10 per group) were immunized similarly and fed an atherogenic diet for 6 weeks. All beta2GPI-immunized mice developed high titers of anti-beta2GPI antibodies as well as a specific lymph node proliferation to beta2GPI. The average cholesterol levels did not differ between the mice fed similar diets, regardless of the immunization protocol. Atherosclerosis was enhanced in the beta2GPI-immunized mice (mean aortic lesion, 26 000+/-5700 microm2) in comparison with their ovalbumin-immunized (mean, 3000+/-1099 microm2; P<0.01) and nonimmunized (mean, 2250+/-700 microm2; P<0.01) littermates. The average lesion size in the beta2GPI-immunized mice fed an atherogenic diet (mean, 98 000+/-8305 microm2) was larger than the ovalbumin-immunized mice (mean, 81 250+/-12 933 microm2; P=NS) or the nonimmunized controls (mean, 75 625+/-7281 microm2; P=NS). The atherosclerotic plaques in the beta2GPI-immunized mice appeared to be more mature, and denser infiltration of CD4 lymphocytes was present in the subendothelium of the aortic sinuses from this group of mice. CONCLUSIONS: The results of the present study provide the first direct evidence for the proatherogenic effect of ss2GPI immunization and establish a new model for immune-mediated atherosclerosis.     

The loss of pacing-induced preconditioning in atherosclerotic rabbits: role of hypercholesterolaemia

A brief rapid pacing has been shown to protect rabbit heart against global myocardial ischaemia induced by subsequent longer pacing. We studied whether pacing-induced preconditioning was reproducible in experimental hypercholesterolaemia. In conscious rabbits with an implanted right ventricular electrode and left ventricular polyethylene catheters, pacing of 500 bpm over 20 min induced an intracavitary ST-segment elevation of 3.2 +/- 0.41 mV, shortened ventricular effective refractory period and increased left ventricular end-diastolic pressure from prepacing 105 +/- 3.9 ms and 4.0 +/- 0.93 mmHg to post-pacing 62 +/- 6.4 ms and 27.9 +/- 7.2 mmHg, respectively. A 10-min preconditioning pacing followed by a 5-min interval markedly attenuated these test pacing-induced ischaemic changes. Rabbits were fed a cholesterol-enriched diet over 4, 8 and 12 weeks, responded to a 5- or 10-min pacing with ischaemic changes of the same degree as did controls to a 10- or 20-min pacing, respectively. A 4-week diet elevated total serum cholesterol from 1.7 +/- 0.4 to 24.1 +/- 2.9 mmol/l without apparent atherosclerotic lesions in the thoracic aorta assessed by Oil-Red O staining and planimetry, but it abolished protection induced by a 5-min preconditioning pacing. A 12-week diet increased serum cholesterol and lesion surface area to 26.9 +/- 3.2 mmol/l and 89.6 +/- 6.4%, respectively, and continued to block preconditioning. When these animals were refed normal chow over additional 6 weeks, serum cholesterol level dropped to 2.6 +/- 0.80 mmol/l with no change in atherosclerotic lesions, the preconditioning effect, however, recovered. We conclude that hypercholesterolaemia blocks preconditioning irrespective of the development of atherosclerosis.     

Quantitative analyses of lesion areas of coronary atherosclerosis in cholesterol-fed rabbits

A simple method to quantitatively evaluate atherosclerosis in the rabbit coronary arteries by measuring macroscopic lesion areas (%) was attempted in the present study. Sixteen rabbits were fed a 0.5% cholesterol diet for 15 weeks and then 9 rabbits were sacrificed whereas the remaining 7 rabbits were maintained for further 9 weeks on a normal chow (at week 24). The left circumflex coronary arteries (LCX) were excised from the rabbit hearts under stereoscopic observation. The prepared arterial strips of LCX were 38.7 +/- 7.1 mm long and all of them reached the cardiac apex from the orifice. At week 15, the lesion area in LCX was negligible (3.2 +/- 0.4%) whereas the aortic lesions significantly developed (50.0 +/- 7.6%). At week 24, atherosclerotic lesions in both LCX and aortas increased to 32.8 +/- 9.2% and 85.9 +/- 5.6%, respectively. This is the first report that determined the luminal surface areas of atherosclerotic lesions in rabbit coronary arteries. This method may be more practical and useful for quantitative evaluation of coronary atherosclerosis in a large number of rabbits than histological observations of serial sections of rabbit hearts.     

Hepatic and adrenal changes in rabbits associated with hyperlipidemia caused by a semi-synthetic diet

Several investigators have reported that feeding a semi-synthetic diet of casein and dextrose to New Zealand White (NZW) rabbits will increase total serum cholesterol concentration, principally through an increase in the beta-lipoprotein fractions, thereby creating a useful model for atherosclerosis research. Although there is evidence to suggest that the dextrose/casein diet alters low-density lipoprotein receptor and bile acid clearance of cholesterol, the underlying mechanism is not completely understood. The effects of the diet on the overall physiology of the rabbit have received little attention. In this study feeding a diet of casein and dextrose of male NZW rabbits for 4 weeks resulted in changes in the serum lipid concentrations. During that time the rabbits fed the dextrose/casein diet gained less weight than did control rabbits. In the test diet rabbits, liver aspartate and alanine transaminase activities were increased from baseline values of 27 +/- 2 U/L and 89 +/- 9 U/L respectively to 112 +/- 21 U/L and 281 +/- 34 U/L respectively, then returned to the high end of the reference range. Necropsy findings included hepatomegaly caused by vacuolar hepatopathy in 19 or 20 experimental rabbits; rabbits fed the control diet had no hepatic lesions. Ultrastructural analysis revealed that enlargement of the liver cells was due to glycogen deposition. Adrenal glands from animals fed the experimental diet had a minimal change in the size of the adrenocortical cells consisting of slight ballooning and rarefaction of the cytoplasm. In a second study the level of dietary fiber was doubled. This resulted in a three-fold increase in lipid concentrations, compared with the fivefold increase in the first study. The liver enzyme activities were increased to the same extent as in the first study. Histologic changes were comparable to those in the first study. The activity of hepatic cholesterol 7alpha-hydroxylase was 3.7 +/- 0.4 pmol/min/mg of protein, compared with the control value of 7.7 +/- 1.1 pmol/min/mg of protein (P < 0.05) in the second study. The improved rate of weight gain and the lesser increase in total serum cholesterol concentration in the second study with increased dietary fiber suggest that two separate activities may be involved. Although the level of dietary fiber may be related to weight gain and total serum cholesterol values, the relation to the decrease in liver transaminase activities in study 1 was probably coincidental. It appears that the dextrose/casein diet causes decreased activity of hepatic cholesterol 7alpha-hydroxylase, which could cause a decrease in the biliary excretion of cholesterol.     

Respiratory infection in lipid-fed rabbits enhances sudanophilia and the expression of VCAM-1

The pathogenesis of atherosclerosis has been related to infection of the arterial wall, but it is not clear whether this occurs before or after the development of lipid-containing lesions. Respiratory bacterial infection increases the expression of vascular cell adhesion molecule-1 (VCAM-1). We therefore examined whether a similar infection would enhance atherosclerosis in New Zealand White rabbits fed chow supplemented by 15% (w/w) egg yolk for 50 days. Rabbits with naturally acquired respiratory infection by Pasteurella multocida, pathogen-free (SPF) animals infected by P. multocida in the laboratory, and age-matched SPF rabbits maintained in a disease-free environment were used. Endothelial cells expressing VCAM-1 in the aorta between intercostal arteries 3 and 5 were identified using anti-VCAM-1 (Rb1/9) and an alkaline-phosphatase-linked secondary antibody and quantified in H?utchen preparations. The remainder of the aorta was stained with Sudan IV to show lipid deposition. The expression of VCAM-1 (mean +/- SEM per 10,000 cells) was 22 +/- 8 (n = 5) in the lipid-fed SPF rabbits, significantly different from that in the lipid-fed rabbits with naturally occurring infection (190 +/- 51 (n = 5)) or from rabbits infected in the laboratory (106 +/- 25 (n = 5)). The extent of Sudanophilia was significantly greater in the naturally infected rabbits (8.3 +/- 1.2%) or infected SPF rabbits (10.3 +/- 1.8%) than in the SPF rabbits (2.7 +/- 0.8%; P < 0.05). Antibiotic treatment in naturally infected rabbits reduced the number of cells expressing VCAM-1 and the extent of the Sudanophilia to baseline levels. Thus, Sudanophilia is enhanced by bacterial infection in rabbits fed egg yolk and is associated with a significant increase in VCAM-1.     

Carvedilol, a new beta adrenoreceptor blocker and free radical scavenger, attenuates myocardial ischemia-reperfusion injury in hypercholesterolemic rabbits

Oxygen-derived free radicals play a critical role in atherogenesis and reperfusion injury. The present experiment evaluated the effects of carvedilol, a new beta adrenoreceptor blocker with potent free radical-scavenging activity, on myocardial ischemia and reperfusion injury in a hypercholesterolemic rabbit model. New Zealand rabbits were fed a normal diet, a high-cholesterol diet, or a high-cholesterol diet supplemented with 1200 ppm carvedilol or propranolol. Eight weeks later, the rabbits were subjected to 60 min of myocardial ischemia followed by 60 min of reperfusion. The nontreated cholesterol-fed animals experienced greater cardiac damage after ischemia and reperfusion than rabbits fed a normal diet (necrosis 51% +/- 4% vs. 28% +/- 3% in the normal-diet group, P < .01). In addition, nontreated cholesterol-fed rabbits showed a significantly decreased vasorelaxant response to ACh in U-46619-precontracted aortic rings (56% +/- 5% vs 90% +/- 3% in the control group, P < .001). Treatment with propranolol neither preserved endothelial function after cholesterol feeding nor reduced neutrophil accumulation in ischemic-reperfused myocardial tissue. Propranolol treatment did significantly decrease HR, pressure-rate index and infarct size (necrosis 33% +/- 4%). Despite their having essentially identical effects on HR and pressure-rate index, carvedilol exerted more profound cardiac protective effects than propranolol (necrosis 19% +/- 3%). Moreover, carvedilol treatment significantly preserved aortic endothelial function and markedly reduced neutrophil accumulation in ischemic-reperfused myocardial tissue. These results indicate that in addition to its beta blocking activity, the antioxidant and endothelial protective activities of carvedilol contributed significantly to its cardiac protective effects after ischemia and reperfusion.     

Effects of two low-fat diets, high and low in polyunsaturated fatty acids, on plasma lipid peroxides and serum vitamin E levels in free-living hypercholesterolaemic men

Diet enriched with polyunsaturated fat may increase the susceptibility of LDL to oxidation. Therefore the effects of two low-fat diets on plasma lipid peroxides in free-living mildly hypercholesterolaemic men (n = 37) were investigated in a randomized single-blind 28-week study. Composition of the diets were (1) American Heart Association (AHA) type 32/10:8:8 (indicating percentages of energy from total fat/saturated fat:monoenes:polyenes in actual diet); (2) low-fat 30/12:8:3. The subjects kept 3-day dietary records five times during the study to estimate the intake of nutrients. Plasma lipid peroxides were measured photometrically as the thiobarbituric-acid reactive substances (TBARS). Levels of serum vitamin E during the study were also determined. Mean change (+/- SD) in serum low density lipoprotein (LDL) cholesterol was similar in both groups (-0.32 +/- 0.76 vs -0.32 +/- 0.87 mmol/l) (AHA type vs low-fat). Level of TBARS decreased (P < 0.05) during the AHA type diet (-8.4 +/- 37.1%) (mean +/- SD) and increased (P = 0.228) during the low-fat diet (+8.7 +/- 27.0%) from 0 to 6 months. The mean intake of total active tocopherols was greater (14.7 +/- 3.7 mg) during the AHA type diet compared to the low-fat diet (7.8 +/- 2.1 mg). Serum vitamin E to LDL cholesterol ratio increased from 8.9 +/- 2.9 to 9.6 +/- 2.4 nmol/mmol (0 vs 6 months) (P = 0.07) during the AHA type diet and from 8.6 +/- 2.6 to 9.3 +/- 2.4 nmol/mmol (P = 0.159) during the low-fat diet.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrasonographic bone velocity in pregnancy: a longitudinal study

1. Sympathetic neurotransmission and noradrenaline content of the tail artery of Donryu rats fed for 2 months with a cholesterol-supplemented diet enriched with 4% cholesterol, 1% cholic acid, 0.5% thiouracil (CCT), were examined. 2. Total serum cholesterol level of CCT fed rats (7.05 +/- 1.77 mg ml(-1), n = 8) was significantly greater than lab-chow fed controls (2.58 +/- 0.32 mg ml(-1), n = 8). Low density lipoprotein level was also significantly increased in CCT-fed (1.79 +/- 0.26 mg ml(-1), n = 8) compared with control fed rats (1.35 +/- 0.25 mg ml(-1), n = 8) but plasma levels of triglyceride and high density lipoproteins did not differ significantly between the two groups. 3. Contractile responses of the arterial rings to transmural nerve stimulation (65 V, 0.1 ms, 4-64 Hz, 1 s), were markedly attenuated in the CCT fed animals compared with the controls. This reduction involved the noradrenergic rather than purinergic component of sympathetic transmission. 4. Vasoconstrictor responses to exogenous noradrenaline (0.01-300 microM) and adenosine 5'-triphosphate (0.3-1000 microM) were unaffected by CCT diet, indicating prejunctional alteration of sympathetic neurotransmission during CCT-induced hyperlipidaemia. 5. The noradrenaline content of the tail arteries of CCT fed animals (2.64 +/- 0.36 ng mg(-1), n = 6) was significantly lower than that of controls (3.82 +/- 0.32 ng mg(-1), n = 6). 6. These findings show that chronic treatment of Donryu rats with a cholesterol-supplemented diet led to altered levels of circulating lipid fractions accompanied by attenuated sympathetic noradrenergic neurotransmission and reduced noradrenaline content of the rat tail artery.     

Effects of bifemelane hydrochloride on atherosclerosis in aged rats fed low-calcium diets

The preventative effects of bifemelane (4-(o-benzylphenoxy)-N-methylbutylamine hydrochloride) on atherosclerosis in aged rats fed low-calcium diets were investigated. Male 18-month-old Wistar rats were maintained for 90 days on the following: (A) standard diet (n = 7), (B) low calcium, low magnesium, high aluminium diet (n = 8), (C) standard diet plus oral intubation with 10 mg bifemelane/kg daily (n = 6), (D) low calcium and magnesium, high aluminium diet plus oral intubation with 10 mg bifemelane/kg daily (n = 6). All groups were give these diets and water ad lib for 90 days, after which blood samples were taken from the abdominal aorta and samples of aorta were examined for atherosclerotic changes. The serum concentrations of the following were determined: calcium, magnesium, zinc, aluminium, inorganic phosphorus, cholesterol, glutamate-oxaloacetate transaminase, glutamate-pyruvate transaminase, lactate dehydrogenase, cholinesterase, creatine phosphokinase, blood urea nitrogen and N-terminal parathyroid hormone. The only significant differences between the groups in serum chemistry were reduced concentrations of cholinesterase and magnesium in groups B and D, increased aluminium in group B, and increased N-terminal parathyroid hormone in groups B and D. In groups C and D the atherosclerosis was much improved compared with that in groups A and B. It appears that bifemelane largely prevents atherosclerosis caused by calcium deposition in the arteries of rats fed low-calcium diets, due to its effect in maintaining magnesium and calcium in bones.     

Enhanced monocyte tissue factor response after experimental balloon angioplasty in hypercholesterolemic rabbit: inhibition with dietary L-arginine

BACKGROUND: There is evidence that tissue factor (TF) is a major contributor to the thrombogenicity of a ruptured atherosclerotic plaque. Nitric oxide (NO) has antiatherogenic and antithrombotic properties. We investigated whether L-arginine (L-arg), the endogenous precursor of NO, might affect the ability of monocytes to produce TF. METHODS AND RESULTS: We studied TF expression in 18 rabbits with atherosclerosis induced by bilateral iliac damage and 10 weeks of a 2% cholesterol diet. Six weeks after the initiation of the diet, an angioplasty was performed. After angioplasty, the surviving rabbits (n=15) were randomized to receive L-arg (2.25%) supplementation in drinking water (L-arg group, n=8) or no treatment (untreated group, n=7). TF expression was evaluated in mononuclear cells from arterial blood in the presence and absence of endotoxin stimulation. Monocyte TF expression, as assessed with an amidolytic assay, did not differ significantly before or after the induction of atherosclerotic lesions (87+/-15 versus 70+/-12 mU of TF/1000 monocytes, P=NS). Endotoxin-stimulated TF activity increased significantly 4 weeks after angioplasty (138+/-22 versus 70+/-12 mU of TF/1000 monocytes, P=0.02). This increase was blunted by L-arg (43+/-16 mU of TF/1000 monocytes, P=0.01). CONCLUSIONS: This study demonstrates that angioplasty-induced plaque rupture is associated with a marked increase in monocyte TF response that is blunted by the oral administration of L-arg. This suggests that the documented antithrombotic properties of NO may be related in part to an inhibitory effect on monocyte TF response.     

Impairment of endothelium-dependent vasorelaxation in experimental atherosclerosis is dependent on gender

OBJECTIVE: Nitric oxide (NO) has been suggested to have antiatherosclerotic effects. It has also been demonstrated that there is a greater basal release of endothelium derived relaxing factor (EDRF) in female as compared to male rabbit aorta, which also might have beneficial effects in atherosclerosis. We thus sought to determine if gender influences the severity of atherosclerosis. METHODS: We studied 18 female and 18 male New Zealand White rabbits that were randomly divided in two groups of 9 animals each and fed either a standard or a cholesterol diet (0.75%) for 15 weeks. RESULTS: In cholesterol-fed rabbits the percentage of atherosclerotic lesions in the aorta was identical in females and males and was inversely correlated with the maximal aortic relaxation to acetylcholine as assessed in organ chamber experiments (females: P < 0.0008, males: P < 0.0002). Importantly, the cholesterol diet induced a significantly (P < 0.025) more severe impairment of maximal vasorelaxation to acetylcholine in males from 78.4 +/- 1.2% to 29.4 +/- 10.2%) compared to females (from 84.4 +/- 1.2% to 60.7 +/- 8.5%). Both male gender (P < 0.0001) and the extent of impairment of endothelium-dependent relaxation (P < 0.0002) were associated with a reduced aortic sensitivity to S-nitroso-N-acetyl-D,L-penicillamine, which releases NO into the organ bath. In contrast, the aortic sensitivity to the organic nitrates pentaerythritol tetranitrate and isosorbide 5-nitrate, which release NO after enzymatic metabolization within the smooth muscle, was not reduced. CONCLUSIONS: These results suggest that the impairment of endothelium-dependent vasorelaxation induced by atherosclerosis is dependent on gender. This may be due to a greater degradation of extracellular NO in the vessel wall of males.     

Centrosomes with striated rootlets in rabbit zygotes

The purpose of this study was to determine if the calcium entry antagonist felodipine inhibited intimal lesion formation in hypercholesterolemic rabbits, and to determine if this was due to an effect upon monocyte and/or endothelial determinants of this interaction. Twenty-three male New Zealand White rabbits received the following treatment regimen for 10 weeks: normal chow (NP, n = 3); normal chow with felodipine infusion (NF, n = 6); 0.5% cholesterol chow (CP, n = 7); or 0.5% cholesterol chow and felodipine infusion (CF, n = 7). After 10 weeks blood was collected for biochemical measurements and mononuclear cell binding assays, and thoracic aortae were harvested for vascular reactivity studies and histomorphometry. In the animals receiving normal chow, felodipine did not significantly affect blood pressure, plasma cholesterol levels, binding studies, vascular reactivity, or structure; therefore these animals were analyzed as one group (N). Plasma cholesterol levels were significantly elevated in groups receiving the 0.5% cholesterol diet (N, 29 +/- 3 mg/dl; CP, 1221 +/- 73 mg/dl; CF, 979 +/- 108 mg/dl). High-density lipoprotein cholesterol was not different between the groups (25 +/- 4 vs 23 +/- 4 vs 27 +/- 4 mg/dl; N vs CF vs CP respectively; p = NS). Cholesterol feeding markedly augmented the adhesiveness of mononuclear cells, as demonstrated by a 250% increase in cell binding. Felodipine did not alter the adhesiveness of mononuclear cells in hypercholesterolemic animals. Cholesterol feeding significantly impaired endothelium-dependent relaxations. Endothelium-dependent relaxations were restored by felodipine treatment as reflected by the maximal responses to acetylcholine (40 +/- 7% vs 58 +/- 4% vs 67 +/- 5%; CP vs CF vs N respectively). The improvement in endothelium-dependent relaxation in the felodipine-treated animals was associated with a 2.2-fold reduction in lesion surface area of the thoracic aorta (8.2 +/- 6.3% vs 18.2 +/- 9.5%; CF vs CP; p < 0.01). Moreover, the intima/media ratio reflecting lesion thickness was substantially reduced by felodipine treatment (0.05 +/- 0.02 vs 0.20 +/- 0.07; CF vs CP; p = 0.006). Ex vivo studies revealed that felodipine inhibited the adhesiveness of vascular endothelium, but not mononuclear cells, derived from hypercholesterolemic animals. Low-dose felodipine appears to inhibit monocyte-endothelial interaction, as indicated by a reduction in the formation of lesions in hypercholesterolemic animals. This effect is not due to an alteration in adhesiveness of mononuclear cells. The salutary effect of felodipine is associated with an increase in vascular nitric oxide activity which may reduce endothelial adhesiveness.     

Chronic inhibition of nitric oxide production accelerates neointima formation and impairs endothelial function in hypercholesterolemic rabbits

To determine if endogenous local levels of nitric oxide (NO) modulate atherogenesis, we studied the effect of inhibiting NO with NG-nitro-L-arginine methyl ester (L-NAME) on early neointima formation in cholesterol-fed rabbits. Male rabbits were fed for 5 weeks with a 0.5% cholesterol diet alone or treated in addition during the last 4 weeks with L-NAME (12 mg/kg per day SC) via osmotic minipump. Endothelial cell function was assessed in isolated aortic rings by vascular reactivity and levels of cyclic GMP. In L-NAME-treated rabbits there was inhibition of endothelium-dependent relaxations to acetylcholine and the calcium ionophore A23187 as well as impaired cyclic GMP accumulation in response to acetylcholine. Neointima formation in the ascending thoracic aorta was assessed by determining media and intima cross-sectional areas with computerized image analysis. Compared with rabbits that consumed the cholesterol diet alone, L-NAME-treated rabbits had significant increases in lesion area (0.29 +/- 0.04 versus 0.15 +/- 0.03 mm2) and in lesion/media ratio (0.06 +/- 0.01 versus 0.03 +/- 0.01). Plasma levels of cholesterol and fluorescent lipid peroxide products were unchanged, suggesting no difference in cholesterol metabolism or oxidation. Because arterial blood pressure was not altered by L-NAME treatment, the increased atherogenesis could not be attributed to an increase in blood pressure. These results indicated that local inhibition of NO accelerates early neointima formation possibly because of modulating monocyte recruitment or foam cell lipid accumulation.     

Development of cerebellar somatosensory evoked potentials in the rat

The effect of radio and microwave radiation on dietary atherosclerosis of rabbits was tested. 16 New Zealand white rabbits were exposed to microwave (2.45 GHz) radiation at a power density of 20--30 mW/cm2 for 4 h a day, 5 days a week for 8 to 10 weeks. Irradiated animals had serum cholesterol concentrations, aortic wall cholesterol concentrations and percentage of intimal surface involved in atherosclerotic lesions which were not different from age and weight matched controls. Continuous radio frequency irradiation (1 MHz) for 8 to 11 weeks with a field strength of 30 V/cm also failed to change these indices of atherogenesis. We conclude that under the conditions of these experiments radio and microwave irradiation do not affect the course of diet induced atherogenesis.