Effect of an 8-week endurance training program on markers of antioxidant capacity in women.

AIM: The effects of endurance training and of exhaustive treadmill running on low density lipoprotein (LDL) oxidation in women are not clearly established. METHODS: Twenty training and 10 control persons, all not endurance trained, aged 26+/-4 and 23+/-3 years, were recruited for 8 weeks of running training 3x/week 30 min. The susceptibility of LDL to in vitro oxidation, conjugated dienes, malondialdehyde (MDA), nitric oxide (NO) and cholesterol, lipoproteins, triglycerides, apolipoprotein (apo) A-I, apo B and lipoprotein (a) were determined before and after training, at rest and after exhaustive spiroergometric exercise. The training was tailored individually at the speed of the 4 mmol/L lactate threshold. RESULTS: At rest and after treadmill running, training induced an increase in lag-time (P<0.05), a decrease in MDA (P<0.05), and lower values for cholesterol (P<0.001), LDL (P<0.01), triglycerides (P<0.05) and apo B (P<0.001), but no increase for high density lipoprotein (HDL) or apo A-I. Before training, treadmill running induced lower conjugated dienes and malondialdehyde, after training an increase for LDL and decrease for cholesterol and triglycerides, no increase for HDL or apo A-I. In the control group, all parameters remained unchanged, only NO lowered (P<0.01). CONCLUSION: Endurance training in women shows favorable effects on LDL oxidation, cholesterol, LDL-cholesterol, triglycerides and apo B.

Intimal plaque development and oxidative stress in cholesterol-induced atherosclerosis in New Zealand rabbits.

Although oxidative stress is well known in atherogenesis, the origin, nature and kinetics of free radicals involved have not been well described till now. Here, we correlated parameters of oxidative stress with cellular components during induction and stabilization of aortic intimal lesions which were induced in rabbits by feeding a cholesterol-enriched diet for 6 weeks and a normal diet for further 68 weeks. Plasma lipids, aortic plaque size and composition (macrophages, smooth muscle cells, oxidized LDL by morphometry), as well as aortic radical production (by luminol-enhanced chemiluminescence and TEMPO-9AC fluorescence) were measured after various time points. The parameters of oxidative stress were correlated with the different cellular components of the aortic plaques. The plaques increased until week 21, no significant regression was found until week 74, plasma cholesterol was maximal at week 6. Macrophages, oxidized LDL and generation of different species of free radicals were increased during plaque development, yet with different time kinetics. Whereas chemiluminescence correlated only weakly with the amount of intimal macrophages, strong correlations were found between TEMPO fluorescence and smooth muscle cells (r = 0.4778, P < 0.001) and between macrophages and oxidized LDL (r = 0.5896, P < 0.0001). Different indicators of oxidative stress were increased during plaque progression and stabilization. However, the various correlations show, that distinct types of reactive species secreted probably from macrophages and smooth muscle cells contribute to oxidative stress in the different phases of plaque development.

Prophylaxis of restenosis with (186)re-labeled stents in a rabbit model.

BACKGROUND: Intraluminal beta-irradiation has been shown to decrease neointimal proliferation after angioplasty in experimental models. The purpose of this study was to test the technical feasibility and biological effects of (186)Re-labeled stents. METHODS AND RESULTS: Thirty-four New Zealand White rabbits were fed a 0.5% cholesterol diet before balloon angioplasty and insertion of Palmaz stents in the infrarenal aorta. The animals were killed 7 weeks after stent implantation. Two of 34 animals died prematurely (aortic leak, pneumonia). Control stents (n=7) were compared with (186)Re stents (2.6 MBq [n=6], 8.1 MBq [n=5], 16.0 MBq [n=6], and 25.3 MBq [n=8]). Stent application was successful in all cases. No thrombus occlusion was observed. After 7 weeks, neointima formation was 2.2+/-0.2 mm(2) in the control group. In the treatment groups, a dose-dependent neointima reduction was detectable (0.5+/-0.5 mm(2) [2.6 MBq], 0.4+/-0.4 mm(2) [8.1 MBq], and 0 mm(2) [16.0 MBq, 25.3 MBq]). No induction of neointimal formation was observed at the edges of the stents. Radiation resulted in delayed reendothelialization. CONCLUSIONS: (186)Re stents were capable of reducing neointima formation in a dose-dependent fashion. (186)Re stents did not cause late thrombosis or neointimal induction at the stent margins in the observation period of 7 weeks.

Tc-99m-labeled endothelin derivative for imaging of experimentally induced atherosclerosis.

OBJECTIVE: to characterize the potential of an endothelin derivative labeled with technetium-99m (Tc-99m) for the imaging of experimentally induced atherosclerosis. METHODS: neointima of different cellularity and severity of stenosis was induced in 32 rabbits by balloon denudation followed by distinct dietary regimens and drug application. Angiograms and scintigrams after injection of the Tc-99m-labeled endothelin derivative were obtained. The aorta was dissected for autoradiography, sudan-III-staining, morphometry, and immunohistology. RESULTS: the lesions induced could be detected in vivo (whole body scintigram) in all the animals 15 min after the injection of the Tc-99m endothelin derivative. Autoradiography revealed a strong relationship between tracer accumulation and sudan-III-staining of lesions. Accumulation of the endothelin derivative correlated with the number of neointimal smooth muscle cells (SMC), but not with the number of medial SMC, neointimal macrophages, and neointimal area. CONCLUSIONS: the results indicate that in vivo imaging of atherosclerosis with an endothelin derivative is a feasible method of detecting and characterizing atherosclerotic arterial wall lesions at early stages.

Local intra-arterial drug delivery for prevention of restenosis: comparison of the efficiency of delivery of different radiopharmaceuticals through a porous catheter.

RATIONALE AND OBJECTIVES: Several radiopharmaceuticals were administered through a porous balloon catheter to compare the absolute amount deposited and the retention in the vessel wall. The reported efficiency of local drug delivery ranges from 0.001% to 0.1%, with poor retention after 24 hours. METHODS: An endothelin derivative (n = 6), pertechnetate (n = 6), hexamethylpropylene amineoxime (HMPAO) (n = 5), ethyl cysteinate dimer (ECD) (n = 5), and tin colloid (n = 5) were labeled with 185 MBq/mL 99m-technetium. After balloon denudation of the infrarenal aorta in 27 New Zealand White rabbits, 100 microL of each agent was administered through a porous balloon at a pressure of 4 bar. Dynamic and static whole-body scintigrams were obtained for 24 hours. The infrarenal aorta was excised and the activity calculated in a gamma counter. RESULTS: Apart from their retention in the region of local administration, the radiopharmaceuticals showed different distribution patterns. The highest regional tracer retention was observed with HMPAO. After administration of HMPAO, a significant difference between regional (vessel wall plus surrounding tissue: 14.5% of injected dose [ID]/24 hours) and local (vessel wall: 1.8% ID/24 hours) delivery was found. In contrast, ECD was eliminated quickly (local retention after 24 hours = 0% ID). The retention efficiencies were HMPAO > endothelin derivative > tin colloid > pertechnetate > ECD. CONCLUSIONS: The different physicochemical and pharmacokinetic properties of radiopharmaceuticals resulted in different delivery efficiencies after local application.

17beta-estradiol, gender independently, reduces atheroma development but not neointimal proliferation after balloon injury in the rabbit aorta.

The aim of the present study was to investigate anti-proliferative and anti-atherogenic properties of 17beta-estradiol in balloon injured female and male rabbit aortae. Thirty-two female and 32 male New Zealand White rabbits where gonadectomised. Vascular injury was performed with a balloon catheter in the lower abdominal aorta. Male and female rabbits were randomised into four groups of eight animals each. Only two of four groups received a 0.5% cholesterol-enriched diet. One cholesterol-diet group and one normal-diet group received intramuscular injections of estradiol valerate (1 mg/kg body weight/week). After 28 days, the denuded part of the abdominal aorta was excised and analysed by morphometry and immunohistochemistry. Estrogen treatment did not show an inhibitory effect on neointimal proliferation in normo-cholesterolemic male or female rabbits. A gender independent inhibitory effect of 17beta-estradiol was seen on atheroma development in cholesterol-fed female and male rabbits, while plasma total cholesterol levels were significantly reduced in male rabbits only. The 17beta-estradiol treatment was associated with a significantly decreased number of luminal endothelial cells in normo and hyper-cholesterolemic female rabbits, as evaluated by immunohistochemical staining for 'von Willebrand factor'. Staining for Ki-67-positive proliferating cells after 28 days showed a statistically significant increased proliferative activity in the neointima of hyper-cholesterolemic female rabbits. The neointimal content of macrophages increased significantly in all hyper-cholesterolemic rabbits. Under 17beta-estradiol treatment, the number of macrophages was increased in female and decreased in male rabbits by tendency. Additionally, the 'classical' vascular estrogen receptor was present in both female and male rabbit aortae without statistically significant differences. In conclusion, 17beta-estradiol did not reduce post-injury neointima formation in normo-cholesterolemic rabbits. However, in hyper-cholesterolemic rabbits, 17beta-estradiol reduced atheroma development gender independently. This effect cannot be explained by lowering of plasma cholesterol levels or endothelium-mediated pathways, and requires further investigation on, for example, antioxidative, antiproliferative or estrogen receptor mediated effects.

Oxidative stress in atherogenesis: Basic mechanisms and problems of therapy with antioxidants.

BACKGROUND: Oxidative stress is recognized as an essential mechanism of atherogenesis and plaque progression. However, the origin of increased free radical production has not yet been well described. Furthermore, therapy with antioxidants has not shown convincing results. OBJECTIVE: To consider questions concerning the impact of oxidative stress, and the effects and usefulness of antioxidants. ANIMALS AND METHODS: Atherosclerotic plaques were induced in rabbits by feeding them a cholesterol-rich diet (2%) for six weeks. Thereafter a normal diet was given up to 68 weeks. Body weight, food intake, plasma lipid concentration and antioxidative capacity were determined at various time intervals. Aortic plaque size, morphology and radical production were determined in groups of animals killed after six, 14, 21, 29, 40 and 74 weeks, and compared with values in untreated controls. Chemiluminescent methods were used to determine antioxidative capacity of plasma, generation of free radicals and redox reactivity of various antioxidants. RESULTS: Antioxidative capacity, occurrence of modified low density lipoprotein and generation of free radicals indicated oxidative stress during plaque progression; however, they showed different correlations to cellular components of the plaques. Furthermore it was shown that some antioxidants have both anti- and pro-oxidative properties. CONCLUSIONS: Oxidative stress during atherogenesis seems to correlate with different phases of plaque development and can be associated with different types of reactive species. Because plaque remodelling and stabilization may also be a phase of increased free radical generation, therapeutic antioxidants must exert specific and selective activity; in particular, whether their oxidized form acts pro-oxidatively must be determined.

Local administration of ramiprilat is less effective than oral ramipril in preventing restenosis after balloon angioplasty in an animal model.

PURPOSE: To test the hypothesis that local administration of angiotensin converting enzyme (ACE) inhibitor via a microporous balloon catheter would be more effective than oral administration of ACE inhibitor in preventing neointima formation after balloon angioplasty. MATERIALS AND METHODS: Neointima formation was induced by balloon denudation followed by 0.5% cholesterol diet in 29 New Zealand White rabbits. Directly after denudation, local administration of 1.8 mg of ramiprilat (n = 7) or saline (n = 7) with a microporous balloon catheter at a pressure of 3 atm was performed. Both groups additionally received ramipril orally (1 mg/d). Seven animals were treated exclusively with oral ramipril. The control group was fed a 0.5% cholesterol diet and given no medication (n = 8). Six weeks after intervention, the animals were killed and morphometric and immunohistologic analyses were performed. RESULTS: Oral administration of ramipril resulted in a significant reduction of placque area (-66%, P < .05). Oral and local administration of the ACE inhibitor was followed by a nonsignificant reduction of the neointimal area (-17%). Local administration of saline combined with oral ramipril failed to prevent neointimal formation (reduction of 6%, NS). CONCLUSION: Oral administration of ramipril resulted in a significant reduction of neointimal proliferation in New Zealand White rabbits. The possible benefit of an additional administration of local ramiprilat was diminished by an excessive neointimal hyperplasia, which was most likely caused by the inherent vessel trauma with use of the microporous balloon catheter.

Effect of 17-beta estradiol on pre-existing atherosclerotic lesions: role of the endothelium.

The atheroprotective effects of estrogen during the process of atherogenesis is well documented, whereas limited information is available about the effect of estrogen on pre-existing atherosclerotic lesions. After bilateral ovariectomy, 24 New Zealand White rabbits were randomized into three groups of eight animals each and subsequently fed a 0.5% cholesterol diet. In group I, the vessels were excised at day 84, whereas in group II, the cholesterol diet was continued for a total of 168 days. In group III, the animals were first fed with a cholesterol diet for 84 days; in the second phase of the experiment, the cholesterol diet was continued for a further 84 days with a combined estrogen treatment (1 mg estradiol valerate per kg body weight per week intramuscularly). At the end of the experiment, the proximal aortic arch, right carotid artery, thoracical aorta and abdominal aorta of each animal were excised and prepared for histological and immunohistological examination. By day 168, morphometrical analysis displayed a significantly lower plaque development under estrogen therapy in the carotid artery (0.08+/-0.18 mm(2) vs. 0.60+/-0.39 mm(2)), the thoracic aorta (0.56+/-0.94 mm(2) vs. 3.63+/-2.06 mm(2)), and in the abdominal aorta (0.55+/-0.70 mm(2) vs. 1.71+/-1.05 mm(2)) in comparison with the corresponding 168 day control group. However, estrogen treatment has failed to reduce further atherosclerotic plaque development in the aortic arch (9.42+/-1.79 mm(2) vs. 11. 64+/-3.29 mm(2)). Immunohistological detection of the 'anti-human factor VIII related antigen', i.e. the 'von Willebrand factor' (vWF), showed a significantly lower number of luminal cells positive for vWF in the aortic arch in the 84-day cholesterol group, compared with the corresponding controls of normocholesterolemic rabbits (65. 9+/-12.4% vs. 83.1+/-6.2%; P<0.05). Estradiol was able to inhibit the further progression of atherosclerosis when moderate vessel wall alterations were present, whereas pre-existing severe atherosclerosis was associated with a failure of the anti-atherosclerotic estrogen action. As suggested by the in situ detection of vWF as a morphological marker for endothelial cells, an intact endothelial layer might play an important role in mediating the beneficial effect of estrogen in the process of atherosclerosis.

Aortic plaque size and endometrial response in cholesterol-fed rabbits treated with estrogen plus continuous or sequential progestin.

ERT is associated with a reduced incidence of coronary risk and cardiac events in postmenopausal women, but increases the risk of endometrial hyperplasia and carcinoma. Combined estrogen and progestin therapy protects the endometrium; however, its effects on heart disease risk factors are not completely known. In our study, 56 ovariectomized New Zealand White rabbits in 7 groups received a 0.5% cholesterol diet for 12 weeks. Controls were not treated with hormones. All other animals received (per kilogram body weight per week) intramuscular injections of either 0.3 mg estrogen (estradiol valerate) alone, 8.3 mg progestin (hydroxyprogesterone caproate) alone, estrogen and progestin continuously in 3 different dosages (0.3 and 8.3 mg; 1 and 8.3 mg; or 1 and 2.8 mg; estrogen and progestin, respectively), or 1 mg estrogen with 25 mg progestin sequentially in 2-week cycles. Eight non-ovariectomized animals served as further controls for endometrial analysis. Morphometric analysis of plaque size in the aortic arch showed that estrogen monotherapy, and the 3 combined therapies with 1 mg estrogen, significantly reduced intimal thickening (P<0.05). The application of progestin alone had no effect on plaque size. The endometrium was enlarged by 3-fold after estrogen treatment, and was decreased by half after progestin treatment, compared with control uteri (P<0.05). In all groups with combined hormone regimens, endometrial size was not significantly different from control uteri. However, these uteri showed more inflammatory reactions, especially when higher doses of hormones were given. In this animal model, doses of progestin that are able to successfully reduce the proliferative effect of estrogen on endometrium do not diminish the desirable antiatherosclerotic properties of estrogen.

Does anti-atherogenic estradiol valerate treatment cause adverse effects on liver and uterus in NZW rabbits?

The rabbit has been a widely accepted animal model for atherosclerosis research since Anitschkow first used this animal in 1913 in identifying dietary-induced hypercholesterolemia as a major risk factor for atherogenesis. Experiments with cholesterol-fed rabbits have demonstrated the beneficial effects of estrogen treatment on the development of atheroma for more than 50 y. Clinical trials have found a reduction in cardiovascular events of up to 50% in postmenopausal women receiving estrogen replacement therapy. However, metabolic conditions in rabbits, as well as physiological estrogen serum levels, differ in some aspects from those in humans. In rabbits, experimentally-induced hormone levels are about 5- to 10-fold higher than those found in untreated animals. Normal physiological estrogen levels in rabbits are not cardioprotective under dietary-induced hypercholesterolemia. We investigated whether replacement induced "hyperestrogenemia" causes adverse effects on organs other than the cardiovascular system. Twenty-nine female rabbits were divided into 4 different groups, 2 without and 2 with estrogen treatment (1 mg estradiol valerate/kg body weight/w over 12 w). Organ weights, transaminases and uterine histology were examined. In rabbits treated with estrogen, we did not see relevant adverse effects on heart, kidney and liver weights, or on liver enzymes. But there was a significant increase in spleen weights, as well as notable changes in the endometrium with moderate inflammation. These findings indicate that the dosage of estrogen commonly used for atherosclerosis research does not cause serious disorders in the major organs of cholesterol-fed rabbits.

[Plaque morphology after arterial interventions in the New Zealand white rabbit--which model of restenosis is most suitable?]. / Plaquemorphologie nach arteriellen Interventionen am Weissen Neuseelandkaninchen--Wann ist welches Restenosemodell geeignet?

PURPOSE: For evaluation of therapy for possible reduction of restenosis after PTA a suitable animal model is needed. The influence of different interventions on arterial plaque composition was analysed in New Zealand White Rabbits. MATERIAL AND METHODS: The following interventions were performed in the infrarenal aorta of New Zealand White Rabbits (n = 42): a) Balloon denudation (BD) with and b) without 0.5% cholesterol diet (CD), c) application of a Wiktor stent, d) CF without BD, and e) control group, 6 weeks after intervention morphometry and histology were performed. RESULTS: After BD the stenosis rate measured 26 +/- 18%, additional CD after prior BD increased the stenoses rate by 2.5 times up to 61.1%. After stent implantation there was only a thin neointimal layer (89 +/- 68 microns) around the stent wires. CONCLUSIONS: Neither implantation of stents nor single CD are suitable as restenosis models. BD with and without CD was followed by a distinct neointima formation with different cellular composition. The New Zealand White Rabbit constitutes an acceptable model for contemporary research in arteriosclerosis.

Gender-specific differences in the effects of testosterone and estrogen on the development of atherosclerosis in rabbits.

The aim of the present study was to investigate whether there are gender-specific differences in the effects of testosterone and estrogen on the process of atherogenesis. Thirty-two castrated male and 32 ovariectomized female rabbits were separated into 4 study groups of 8 males and 8 females each and received postoperatively a 0.5% cholesterol diet for 12 weeks. During this period either no hormones, estradiol (1 mg/kg body wt/week), testosterone (25 mg/kg body wt/week IMM), or estrogen combined with testosterone in above dosages were administered. Computerized morphometric analysis of the intimal thickening in the proximal aortic arch showed a significant inhibitory effect of estrogen in female and of testosterone in male animals (P < .05). In the group with combined treatment, the plaque size in both sexes was smaller than in the animals of the control group (P < .05). These differences were independent of changes in plasma lipid parameters. The incorporation of 5'-bromo-2'-deoxyuridine, associated with cell proliferation, into cells of the neointima was not significantly affected by the different hormone application regimens in males. In females, the incorporation rate was significantly lowered in the estrogen treated group compared with the control group (P < .05). Due to the observed differences in the sex specific atheroprotective effects of testosterone and estrogen, these data suggest that complex hormone interactions, which are independent of changes in plasma lipids, may play an important role in the process of atherogenesis.

Post-prandial lipaemia after a moderate fat challenge in normolipidaemic men with and without coronary artery disease.

BACKGROUND: The role of triglycerides as a risk factor for coronary artery disease (CAD) is controversial. In prospective studies, which have reported discrepant results, triglycerides have generally been measured with subjects in the fasting state. Taking into account the fact that the average person spends up to 18 h per day in the post-prandial state, fasting triglyceride levels alone are inadequate to describe the actual effective concentrations, metabolism and atherogenicity of triglyceride-rich lipoproteins. Therefore, investigations of the dynamic post-prandial triglyceride metabolism of patients with CAD in comparison with healthy controls are necessary. METHODS: We studied the post-prandial metabolism of lipids in 99 men: 50 male patients with CAD confirmed by angiography and 49 matched healthy men. After an overnight fast of 12 h, the subjects (aged 40-60 years) ate a standardized oral fat load (3.2 MJ; 49 g fat = 58% of the total energy content). Blood samples for lipid analyses were drawn immediately prior to and hourly during the 6 h period after ingestion of the meal. RESULTS: As required by the study design, fasting triglyceride and total cholesterol levels did not differ between patients and controls; however, high-density lipoprotein (HDL), HDL2 and HDL3 cholesterol levels were significantly lower in the CAD patients. The highest post-prandial triglyceride serum concentrations were observed 3 h after ingestion of the oral fat load both in cases and in controls. Generally, CAD patients had slightly higher triglyceride values than did controls and, at the 5 h measurement point, this difference was significant. CONCLUSIONS: The results suggest that there are differences in triglyceride metabolism between patients with CAD and healthy controls after a challenge with a moderate amount of fat. These differences can best be observed in the degradation phase of post-prandial lipaemia.

Inhibition of the protective effect of estrogen by progesterone in experimental atherosclerosis.

The aim of the present study was to determine the effect of progesterone on the action of estrogen in the development of atherosclerosis. A total of 48 female New Zealand white (NZW) rabbits were ovariectomized. The animals were separated into 6 groups of 8 animals each and received subsequently a 0.5% cholesterol diet for 12 weeks. During this cholesterol feeding period, either estradiol (1 mg/kg body weight (BW)/week), progesterone (25 mg/kg BW/week), or combined estradiol/progesterone (in above dosages) was administered intramuscularly in each group (n = 8 each) of ovariectomized rabbits. One additional group of 8 animals received a combined estrogen/ progesterone regimen, but with progesterone at one third of the above mentioned dosage. In another 8 rabbits, progesterone was reduced to one ninth of the maximum dosage above, whereas estrogen was kept the same, at 1 mg/kg BW/week. Eight ovariectomized animals served as the control group and received no hormone treatment. After 12 weeks, the animals were sacrificed and the proximal aortic arch was removed for further histological examination. An inhibitory effect of estrogen of intimal thickening was found, in comparison to the control group (intimal area: 0.7 +/- 0.5 mm2 vs. 3.7 +/- 2.5 mm2, P < 0.01), whereas progesterone alone did not show a significant effect on intimal plaque size (intimal area: 4.0 +/- 2.3 mm2). In combination with progesterone (high dose), estrogen was not able to reduce intimal atherosclerosis (intimal area: 3.4 +/- 2.4 mm2). However, the beneficial effect of estrogen was not affected by progesterone, when this was reduced respectively to one third (intimal area: 0.8 +/- 0.7 mm2), or to one ninth of the highest dosage (intimal area: 0.6 +/- 0.4 mm2). Interestingly, these differences in atherosclerotic plaque development were observed without significant changes in plasma cholesterol concentrations by the administered hormones. In conclusion, progesterone was dose-dependently able to completely inhibit the beneficial effect of estrogen in experimental atherosclerosis, suggesting that progesterone exerts a direct inhibitory effect on the athero-protective action of estrogen. In the context of recently published data, the present work confirms the importance of the 'non-lipid-mediated', anti-atherosclerotic effect of estrogen, probably due to an interaction with six hormone receptors in vascular smooth muscle cells (VSMC).