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Thrombosis Research 125 (2010) 246–252
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
The effect of the long term aspirin administration on the progress of atherosclerosis in apoE-/- LDLR-/- double knockout mouse Y. Yamamoto a, T. Yamashita a,⁎, F. Kitagawa a, K. Sakamoto a, J.C. Giddings b, J. Yamamoto a a b
Laboratory of Physiology, Faculty of Nutrition, and Cooperative Research Center of Life Sciences, Kobe Gakuin University, Kobe, Japan Department of Haematology, Wales College of Medicine, Cardiff University, Cardiff, UK
a r t i c l e
i n f o
Article history: Received 26 March 2009 Received in revised form 14 October 2009 Accepted 9 November 2009 Available online 16 December 2009 Keywords: Aspirin Atherosclerosis Nitric oxide Endothelial function
a b s t r a c t We have investigated the effects of differential aspirin doses on atherogenesis. Aspirin was given to homozygous, apoE-/- and LDLR-/- double deficient mice for 12 weeks. The development of arteriosclerosis was determined morphologically by image analysis and endothelial cell function was assessed by measurement of peripheral nitric oxide (NO). Methods: ApoE-/- LDLR-/-double knockout mice were bred and maintained with a high fat diet containing aspirin (4 and 40 mg/kg B.W. /day) for twelve weeks. The development of arteriosclerosis was monitored by estimating the total area of atherosclerotic lesions in the entire aorta. Acetylcholine-induced NO release was measured in vivo using electrochemical sensors. The expression of eNOS on the endothelial surface was determined by immuno-staining. Plasma prostaglandin F1α (PGF1α), serum thromboxian B2 (TXB2) and total cholesterol were measured using enzymatic assay. Bleeding time was measured by tail cut method. Results: Arteriosclerosis in the 4 mg/kg/day aspirin group was decreased significantly compared with the placebo group, but not in the 40 mg/kg/day aspirin group. Acetylcholine-induced NO release was significantly depressed in the 40 mg/kg/day aspirin group. Immunochemical analysis with anti-eNOS antibody supported these findings. In the 4 mg/kg/day aspirin group, the severe suppression of PGI2 production was not confirmed in spite of decreasing TXB2 production, but not in the 40 mg/kg/day aspirin group. Conclusion: Our results suggest that endothelial dysfunction with low dose aspirin improved, reduced progression of atherosclerosis in apoE-/- and LDLR-/- double deficient mice and provides a pathophysiological basis for the beneficial effects of aspirin in atherosclerosis, and low doses appeared to be more efficient than high doses. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Current therapy for arteriosclerosis in high risk patients centres on the use of lipid lowering drugs such as 3-hydroxy-3-methyl-glutarylCoA (HMG-CoA) reductase inhibitors (Statins) [1]. These lower the level of circulating cholesterol, which is widely known to play a fundamental role in lipid metabolism and foam cell formation in atherosclerotic plaques. In addition, inflammation in the arterial wall appears be an important factor in atherosclerosis [2,3] and more recent studies have indicated that upregulation of the adhesion molecules VCAM-1, ICAM-1, and E-selectin in endothelial cells by inflammatory cytokines through nuclear factor kappa B activation is implicated in formation and progression of atherosclerotic plaque [4]. ⁎ Corresponding author. Laboratory of Physiology, Faculty of Nutrition, Kobe Gakuin University, 518, Arise, Igawadani-Cho, Nishiku, Kobe, Japan 651-2180. Tel.: + 81 78 974 1551; fax: + 81 78 974 5689. E-mail address: [email protected] (T. Yamashita). 0049-3848/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2009.11.008
Large-scale clinical trials have shown that aspirin is an effective treatment for acute and secondary prevention of cerebrovascular disease and cardiovascular disease and may also be useful for primary prevention in coronary artery disease [5]. Aspirin is a major inhibitor of platelet function and its mode of action depends on the inhibition of cyclooxygenase enzymes (COX-1 and COX-2) which are involved in the production of prostanoids (prostaglandin, PGE2 and thromboxane, TXA2) and other mediators of inflammation and pain. Indeed, in a recent study, aspirin seemed to be most effective at reducing cardiovascular events in individuals with an elevated C-reactive protein (CRP). The use of aspirin in clinical practice, therefore, is based on both anti-platelet and anti-inflammatory mechanisms. However, the effect of aspirin on the progression of atherosclerosis and its attendant endothelial function is still unclear. Double-homozygous apoE-/- and LDLR-/- deficient mice provide a useful model of atherogenesis [6]. In the present study we have investigated the effects aspirin on the development of arteriosclerosis and endothelial function in these genetically susceptible mice.
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2. Material & methods 2.1. Experimental animal Double-homozygous apoE-/- deficient and LDLR-/- deficient mice (DKO mice, 129 X C57BL/6J back-ground) were obtained originally from Jackson Laboratory (Bar Harbor, Maine, USA). Subsequently, the animals were bred locally by sibling mating. C57BL/6 mice aged 1013 weeks were obtained from SLC Co. Ltd. (Hamamatsu, Japan). All animals were maintained in Kobe Gakuin University in air-conditioned rooms (22.5 ± 0.50 °C , and 50 ± 5% humidity) with a 12-h light and dark cycle. Male mice were used in the present study. Animals had free access to diet and drinking water. Experimental diets were commenced from six weeks until l6 weeks of age. All procedures were conducted in compliance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Science of the Physiological Society of Japan. 2.2. Experiment protocol DKO and C57BL/6 mice were maintained with a standard solid chow (CE-2) for six weeks prior to experimentation. Subsequently, experimental diets containing aspirin (Sigma-Aldrich Chemie GmbH, Germany) or placebo were administered for twelve weeks. A control group of C57BL/6 mice was given the same feed as the Placebo group. Arteriosclerosis, endothelial function and plasma prostaglandin F1α concentration were assessed as described below. 2.3. Preparation of experimental diets
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(Image-Pro Plus, Media Cybernetics, USA , Roper Japan,). The whole area (W) of the dissected blood vessel was calculated and the atherosclerotic regions identified by Oil Red O staining. The area (R) stained positively with oil red O was quantified and the ratio [(R÷W) x100] was used as an index of atherosclerosis development. Furthermore, the heart was perfused with PBS and then with 10% PBS-buffered formaldehyde. The heart and aorta including the aortic root were cut transversely and embedded in paraffin. The aortic root in the heart was sectioned proximally to distally in 10 µm starting from the semilunar valves. To investigate the cross-sectional area of the oil red O-stained lesions in the aortic root, processing and staining were carried out as described above. 2.5. Measurement of Nitric oxide (NO) Peripheral NO release was measured using electrochemical sensors as previously described [7,8]. Briefly, the working electrodes were introduced through a femoral arteriole, and positioned at close proximity to the branch point of the abdominal aorta and the common iliac artery, without preventing of blood flow. The sensitivity and selectivity of the electrodes were confirmed each time before use using S-nitroso-N-acetyl-DL-penicillamine (SNAP, sigma). The electrodes were immersed in phosphate-buffered saline (PBS, pH 7.4) in a small chamber and were calibrated using a graded series of SNAP concentrations, from 1 × 10- 3 M to 1 × 10- 5 M. 2.6. Immunochemistry
Experimental diets were prepared from a common standard feed (AIN93G) using the following purified materials: milk casein, f3-corn starch, sucrose, cellulose powder, mineral mixture, vitamin mixture (Oriental Yeast Co. Ltd., Tokyo, Japan), L-cystine, cholesterol (Nacalai Tesque Inc., Kyoto, Japan), and choline bitartrate, tert-butylhydroquinone (Wako Pure Chemical Industries Co. Ltd., Tokyo, Japan). The composition the experimental diet was made 40% with respect to lipid, 40% with respect to carbohydrates and 20% with respect to protein. Cholesterol was added at 0.05% (w/w). Two concentrations of aspirin (low dose, 4 mg/kg body weight, and high dose, 40 mg/kg body weight) were added to this mixture. Powder diets were solidified by adding agar (0.6%) and were stored at -30 °C until required.
The mouse heart was exposed by dissection and a wing-shaped needle inserted in the left ventricle. The blood vessels were then was washed thoroughly with buffer (10 mM PBS, pH7.4) by infusion in situ for approximately three minutes. The target blood vessel was excised, fixed with 4% Paraformaldehyde Phosphate Buffer Solution (Wako Purification Industry incorporated) soaked in Optimal Cutting Temperature (O.C.T.) compound (Tissue-Tek and SAKURA), and frozen using the dry ice and acetone or liquid nitrogen. Frozen sections were cut at 6 μm thickness using a cryostat, placed on poly-L-lysine–coated microscope slides (Muto Pure Chemicals, Tokyo, Japan), wrapped in aluminum foil and stored at -80 °C. Sections were examined by immunoperoxidase staining with anti-eNOS antibody ( Lab Vision Corporation) using the streptavidin /biotinylated horseradish peroxidase method (LSAB2 kit; DAKO) as described previously [9].
2.4. Measurement of atherosclerotic burden
2.7. Measurement of total cholesterol
The development of arteriosclerosis was assessed by estimating the extent of atherosclerotic regions as a percent of the entire surface area of the aorta as previously described [6]. Briefly, hearts were exposed by abdominal incision and 50 mL phosphoric acid buffer isotonic sodium chloride solution (phosphate buffered saline, PBS and pH7.4) infused through an indwelling 20G butterfly needle (Top Kasei Co. Ltd., Tokyo, Japan) followed by 10% neutral-buffered formalin (Nacalai Tesque Inc.). In addition, the major blood vessels were washed with PBS and fixed with 10% neutral buffered formalin solution by reflux through a femoral artery. The aortic arch was carefully dissected from connective tissue and minor branching blood vessels. The extracted vessels were kept in 10% neutral buffered formalin solution until processed. The vessels were then incised along the longitudinal plane, inverted on a black rubber sheet and held in place using stainless steel pins. The tissue was washed with distilled water for 30 seconds, treated with 60% isopropyl alcohol for 1 minute, and stained with oil red O stain solution at 37 °C for 15 minutes. Finally, the tissue was washed with 60% isopropyl alcohol and distilled water. The dyed specimens were photographed using a digital still camera (COOLPIX 880 Nikon Co. Ltd., Tokyo, Japan). Images were transferred to a personal computer and analyzed using image analysis software
Anticoagulated whole blood was collected from the abdominal aorta of mice under Nembutal anesthesia using Microtainer tubes (EDTA K2; Becton Dickinson). Plasma was obtained after centrifugation at 3000 rpm for 15 min at 4 °C). Plasma total cholesterol level was determined enzymatically by using Wako reagents (Wako Pure Chemical Co) and Biomek 1000 (Beckman Coulter Inc, USA). 2.8. Bleeding time Tail-cut bleeding time was measured in all groups as a platelet function in vivo assay, as described previously [10]. Briefly, the terminal 5 mm of the tails of mice were severed and immediately immersed in 1.5-ml tubs containing phosphate―buffered saline (PBS) at 37 °C, and the time to cessation of bleeding for 20 s was determined with a stopwatch. In each group, tail-cut bleeding times were performed after 16 week feeding. 2.9. Assay of prostaglandin F1α and TXB2 Whole blood was drawn from abdominal aorta of mice under Nembutal anesthesia and collected in (1) tubes containing EDTA 2K
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(Becton Dickinson) for 6-keto PG F1α (a stable metabolite of PGI2) assay, (2) tubes with no additive for measurements of serumTXB2 (a stable metabolite of TXA2). Plasma was obtained after centrifugation at 3000 rpm for 15 min at 4 °C, and blood obtained in serum tubes were allowed to clot at room temperature followed by centrifugation for 10 min at 1000 × g. The both of plasma and serum were transferred to eppendorf-tubes and stored at -80 °C until analysis. Assay of 6-keto PG F1α: Plasma was immediately the prostanoidsrich fraction was extracted as soon as possible. In outline, plasma was acidified with 1 N HCl to pH 3.0, then passed through a Sep-Pak C18 column (Waters Associates, Milford, MA, USA) pretreated with methanol and distilled water. After washing with distilled water, 10% ethanol and hexane, the prostanoids-rich fraction was eluted with methyl formate and evaporated under nitrogen gas. The residue was dissolved in assay buffer, and levels of 6-keto PGF1a were measured with a commercial enzyme immunoassay (EIA) kit according to the manufacturers' instructions (Cayman Chemical, Ann Arbor, Michigan, USA). Assay of TXB2: Serum levels of TXB2 were measured with a commercial enzyme immunoassay (EIA) kit according to the manufacturers' instructions (Cayman Chemical, Ann Arbor, Michigan, USA). Amount of TXB2 present in serum was calculated with the use of a data analysis tool developed by Cayman Chemical.
Table 2 Total cholesterol level in each group.
Total cholesterol (mmol/L)
C57BL6
Placebo
ASA4 mg/kg/day
ASA40 mg/kg/day
3.6 ± 0.3**
19.9 ± 1.0
18.7 ± 2.1
20.0 ± 1.7
Plasma cholesterol level (mmol/L) after 12 weeks feeding **: p = b0.001 C57BL6 vs Placebo, ASA4 mg/kg/day group, ASA40 mg/kg /day group.
C57BL6 group, there was no significant difference statistically in each group (Table 2). 3.4. Development of Atherosclerosis The development of atherosclerosis was evaluated at 18 weeks in each group (Fig. 1). The extent of atherosclerosis in the placebo group was (a) 23.0 ± 1.8%; 12.6 ± 1.4%; in the 4 mg/kg aspirin group (b) and 22.1 ± 1.0%. in the 40 mg/kg aspirin group (c). The differences were statistically significant between the placebo group and 4 mg/kg aspirin group. The differences were not statistically significant between placebo group and the 40 mg/kg aspirin group. Typical images of dissected blood vessels and aortic root in each group are illustrated in Fig. 2.
2.10. Statistical Analysis
3.5. Acetylcholine induced nitric oxide (NO) release
Results were expressed as mean ± SEM. Normal distribution was examined, and all data showed the normal distribution. Comparisons among groups were made by using 1-way factorial ANOVA, followed by the Fisher PLSD test, Dunnett's multiple comparison post-test. Differences between means were considered significant at p b 0.05.
The results of acetylcholine-induced (20 μg/kg body weight) NO release in each group are shown in Fig. 3. NO release was 29.3 ± 3.3 nM in the a placebo group (b); 45.2 ± 4.3 nM in the 4 mg/kg aspirin group (c) and 15.6 ± 2.1 nM. in the 40 mg/kg aspirin. group: The differences between the 40 mg/kg aspirin group and the Placebo and 4 mg/kg aspirin groups were statistically significant.
3. Results 3.6. Immunohistochemistry 3.1. Body Weight and Food intake No significant differences were observed in the body weights of animals in any of the groups before or after the experimental diets. 3.2. Bleeding time
The expression of eNOS in the vascular endothelium in DKO mice was examined by immunohistochemistry of cross-sections of the left brachiocephalic trunk. Surface eNOS was specifically observed in the placebo group and the aspirin groups, but not in the control group (Fig. 4). Furthermore, in particular, in 4 mg ASA group the expression of eNOS was also observed in subendothelial (Fig. 4 C, D).
Results are shown in Table 1. The both of Low dose ASA group and high dose ASA group had a longer bleeding time after a tail cut than placebo group (P = 0.0486 and P b 0.001, respectively) (Table 1). The prolongation of bleeding time in high dose ASA group was considered to be statistically significant compared with low dose ASA group (P = 0.0136). 3.3. Total Cholesterol The total cholesterol levels in each group rose progressively over time to exceed 18 mmol/L, compared to a mean value of 3.6 ± 0.3 mmol/L in C57BL6 mice after 12 weeks feeding. Except for
Table 1 Bleeding time.
Bleeding time (second)
Placebo
ASA4 mg/kg/day
ASA40 mg/kg/day
90 ± 8
150 ± 16*
235 ± 36**
Bleeding time from a tail cut according to the mice (six mice in each group) after 12 weeks feeding. *: Placebo vs ASA4 mg/kg/day P = 0.0486; **: Placebo vs ASA40 mg/kg/day P b 0.001; ASA4mg/kg/day vs ASA40 mg/kg/day: P = 0.0136.
Fig. 1. The effect of ASA dose on the progression of atherosclerosis after 12 weeks feeding in apoE-/- LDLR-/- double knockout mice (The entire aortic tree ) n = 8 ∼ 11 in each group **: P b 0.001 a) Placebo group b) 4 mg/kg/day aspirin group c) 40 mg/kg/day aspirin group.
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Fig. 2. Typical images of atherosclerotic regions identified by Oil Red O staining in each group of apoE-/- LDLR-/- double knockout and control mice. (A): Entire aorta (B): Cross section of Aortic roots, a: C57BL6 (control mice) group; b: Placebo group; c: Aspirin 4mg/kg/day group; d: Aspirin 40mg/kg/day group.
3.7. The levels of 6-keto-PGF1α and TXB2 The results of plasma 6-keto-PGF1α measurements in each group are shown in the Fig. 5A. 6-keto-PGF1α concentration was 805.0 ± 150.0 pM in the C57BL6 control group; 1772.5 ± 300.0pM in the Placebo group; 1385.0 ± 293.2pM in the 4 m g/kg ASA group and 552.5 ± 143.6pM in the 40 m g/kg ASA group. The Placebo and the 4 mg/kg ASA group results were significantly higher than the control group. In contrast, the 40 mg/kg ASA group results were significantly lower than the 4 mg/kg aspirin and placebo groups. The results of serum TXB2 measurements in each group are shown in the Fig. 5B. TXB2 concentration was 107.5 ± 7.7 pg/ml in the C57BL6 control group; 140.4 ± 9.6 pg/ml in the Placebo group; 15.1 ± 0.9 pg/ml in the 4 m g/kg ASA group; 12.5 ± 0.9 pg/ml in the 40 m g/kg ASA group. Serum TXB2 lever in ASA groups was significantly decreased by ASA administration compared with placebo and C57BL6 group. However,
it was not confirmed significant difference between 4 mg ASA group and 40 mg ASA group. 4. Discussion The present studies were designed to address two hypotheses. First, we determined the effect of low dose aspirin on the progression of atherosclerosis in apoE-/- and LDLR-/- double deficient mice (DKO mice), and second, its effect on the endothelial function. Long term Low dose aspirin (4.0 mg/kg B.W. /day) promoted a significant retardation of atherosclerotic lesion development in the DKO mice, which was evident in the entire aortic tree (40%) and the regions of aortic root. The results of bleeding time indicate that the inhibition of platelet function in low dose aspirin group was not severed than high dose aspirin group. Recent reports have shown that this dosage preferentially inhibits platelet-derived TxA2 over endothelial-derived
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Fig. 3. The effect of aspirin dose on the acetylcholine induced NO release in vivo after 12 weeks feeding in apoE-/- LDLR-/- double knockout and control mice. n = 8 ∼ 11 in each group *:p b 0.05; **: p b 0.01. a: C57BL6 group; b: Placebo group; c: Aspirin 4 mg/ kg/day group; d: Aspirin 40 mg/kg/day group.
PGI2 [11]. This differential effect has theoretical advantages in that unaffected PGI2 production could promote the antithrombotic and anti-inflammatory properties of the endothelium. The present results support this, because in low dose ASA group, the severe suppression of PGI2 production was not confirmed in spite of decreasing TXB2 production, but not in high dose ASA group. Hyperlipemia is well established risk factor for atherosclerosis and coronary heart disease. The apoE-/- LDLR-/- double knockout mice used in the present investigations characteristically display a marked lipid metabolic disorder, develop severe hyperlipemia and are prone to atherosclerosis in the relatively short term [12]. The total cholesterol level in each of our groups rose progressively over time to exceed 18 mmol/L after 12 weeks feeding. It is not generally noted that aspirin lowers the level of total cholesterol like a statin. In this study, it was not recognized difference in the both of aspirin group. The lowering of cholesterol level is essential in the prevention of atherosclerotic development. However the lowering of cholesterol level was not
confirmed in ASA groups. Further studies are necessary to investigate the relationship between the level of cholesterol and the development of atherosclerosis. Atherosclerosis is a complex and multifactorial disease, and inflammation is involved in its pathogenesis and evolution [13]. According to recent reports, increased levels of several cytokines have been reported in human and murine atherosclerosis [14], and evidence for their role in the disease has been provided [15]. Activated NF-B is present in atherosclerotic lesions both in human [16] and in apolipoprotein E–deficient mice [17]. So, in our case, it predicts the up regulation of these cytokines. Recent data suggest that COX-1 may be induced and may be responsible for prostaglandin formation at sites of inflammation [18,19]. Studies with low-dose aspirin suggest that platelet COX is also a major source of the increased TXA2 biosynthesis seen in patients with atherosclerosis, possibly reflecting enhanced platelet activity [20]. Suppression of TX production may be an important mechanism for the observed effect of aspirin in this study. However, it is also reported that despite complete, elimination of the COX-1 gene expression significantly accelerated early atherosclerotic lesion formation in both LDLR–/– and apoE–/– recipient mice [21]. The role of TXA2 is controversial. Furthermore, Clinical or basic reports regarded with aspirin are complicated by the contribution of COX isoforms. The major finding of this study is that long term low dose aspirin only promotes acetylcholine-induced peripheral release of nitric oxide release, which supported upregulation of eNOS expression in the vascular endothelial cell in DKO mice by immunohistochemistry. To the best of our knowledge, our study is the first demonstration of acetylcholine-induced peripheral release of nitric oxide directly in DKO mice with atherosclerosis. Acetylcholine, a commonly used probe for testing endothelial function in humans, causes endothelium-dependent smooth muscle vasodilation, which is believed to be caused largely by release of nitric oxide and endothelium-derived hyperpolarizing factor [22,23]. Endothelium can also produce contracting factors that are mainly cyclooxygenase-dependent prostanoids or oxygen free radicals, which destroy NO and thus reduce its availability. Endothelial-derived nitric oxide and prostacyclin, are believed to show vasodilation, profilation of smooth muscle cell,
Fig. 4. Immunohistologic findings in each group. (A) eNOS staining in placebo group, (B) pseudo colour of (A), (C) eNOS staining in aspirin 4 mg/kg/day group, (D) pseudo colour of (C), (E) eNOS staining in aspirin 40 mg/kg/day group, (F)pseudo colour of (E), Magnification: x200.
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The controversy regarding the optimal dosage of aspirin for primary prevention of atherosclerotic events in human is not solved definitely [31,32]. The results of recent numerous trials suggest no difference in clinical effectiveness concerning cardiovascular risk reduction between high and low doses of aspirin [33], and furthermore cast doubt on primary prevention of cardiovascular events [34]. There is room for reconsideration in conventional guideline [35] with regard to the long-term daily dosage of aspirin. It seems that a variety of clinical settings in each study complicates the interpretation of the result more. However, it is finally hoped that numerous clinical studies and basic studies provide a more reliable guideline in the future. Although our data supports the hypothesis that low dose aspirin is enough to effectively suppress TXA2 production in platelets [36], there are different problems involved in applying the results of animal models to humans [37]. Our present data are therefore limited to experimental observations in the animal without intervention. From current observations, it is demonstrated that an unbalance of constrictor and dilator force has been largely attributed to the progress of atherosclerosis. It is possible that low dose aspirin (4.0 mg/kg B.W.) reduce the activity of endothelial derived vasoconstract factor (EDCF), by interfering, mainly via COX, without impairing PGI2 production, it leads to the improvement of acetylcholine-induced NO release. In the contrary, high dose aspirin (40 mg/kg B.W.) impaired the balance of vasoactive factors through impairing PGI2 production, it leads to the endothelial dysfunction progresses in atherosclerosis. References
Fig. 5. 6-keto-PGF1α level and TXB2 level in each group of apoE-/- LDLR-/- double knockout and control mice. A: Plasma 6-keto-PGF1α level, B: Serum TXB2 lever n = 8 ∼ 11 in each group **: p b 0.01. a: C57BL6 group; b: Placebo group; c: Aspirin 4 mg/ kg/day group; d: Aspirin 40 mg/kg/day group.
attenuation of platelet aggregation. In the contrary, endothelin as known vasoconstrictor, accelerates profilation of smooth muscle cell, platelet aggregation. It has been demonstrated that cyclooxygenase inhibition with indomethacin increased acetylcholine-mediated forearm vasodilation by 39% in patients with congestive heart failure but not in normal subjects [24,25]. Thus, the progression of atherosclerosis leads to activation of the cyclooxygenase pathway, which seems to participate in the impaired endothelium-dependent vasodilation. In atherosclerosis, a clinical condition characterized by endothelial dysfunction, cyclooxygenase seems to be a source of oxidative stress. In addition, antioxidant action of aspirin as a cyclooxygenase inhibitor may be an important mechanism for the observed effect of aspirin in this study. Furthermore, aspirin including salicylic acid is known to scavenge both oxygen free radicals and hydroxyl radicals [26–28]. Superoxide anions metabolize nitric oxide to higher, biologically inactive nitrogen oxides and are believed to be the primary mechanism underlying the reduced bioavailability of nitric oxide in atherosclerosis. We previously showed that the progression of atherosclerosis was suppressed through the intake of ω3 polyunsaturated fatty acid [6] which contains EPA and DHA, widely recognized as anti-atherogenic eicosanoid in humans and animals [29]. Free radical peroxidation of polyunsaturated fatty acids present in lipoproteins produces oxidized low-density lipoproteins which play a critical role in the development of atherosclerosis [30]. The action of aspirin as a scavenger of oxidative stress, may be contribute to prevention of the development of atherosclerosis, and needs further investigations.
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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t h r o m r e s
Regular Article
The effect of the long term aspirin administration on the progress of atherosclerosis in apoE-/- LDLR-/- double knockout mouse Y. Yamamoto a, T. Yamashita a,⁎, F. Kitagawa a, K. Sakamoto a, J.C. Giddings b, J. Yamamoto a a b
Laboratory of Physiology, Faculty of Nutrition, and Cooperative Research Center of Life Sciences, Kobe Gakuin University, Kobe, Japan Department of Haematology, Wales College of Medicine, Cardiff University, Cardiff, UK
a r t i c l e
i n f o
Article history: Received 26 March 2009 Received in revised form 14 October 2009 Accepted 9 November 2009 Available online 16 December 2009 Keywords: Aspirin Atherosclerosis Nitric oxide Endothelial function
a b s t r a c t We have investigated the effects of differential aspirin doses on atherogenesis. Aspirin was given to homozygous, apoE-/- and LDLR-/- double deficient mice for 12 weeks. The development of arteriosclerosis was determined morphologically by image analysis and endothelial cell function was assessed by measurement of peripheral nitric oxide (NO). Methods: ApoE-/- LDLR-/-double knockout mice were bred and maintained with a high fat diet containing aspirin (4 and 40 mg/kg B.W. /day) for twelve weeks. The development of arteriosclerosis was monitored by estimating the total area of atherosclerotic lesions in the entire aorta. Acetylcholine-induced NO release was measured in vivo using electrochemical sensors. The expression of eNOS on the endothelial surface was determined by immuno-staining. Plasma prostaglandin F1α (PGF1α), serum thromboxian B2 (TXB2) and total cholesterol were measured using enzymatic assay. Bleeding time was measured by tail cut method. Results: Arteriosclerosis in the 4 mg/kg/day aspirin group was decreased significantly compared with the placebo group, but not in the 40 mg/kg/day aspirin group. Acetylcholine-induced NO release was significantly depressed in the 40 mg/kg/day aspirin group. Immunochemical analysis with anti-eNOS antibody supported these findings. In the 4 mg/kg/day aspirin group, the severe suppression of PGI2 production was not confirmed in spite of decreasing TXB2 production, but not in the 40 mg/kg/day aspirin group. Conclusion: Our results suggest that endothelial dysfunction with low dose aspirin improved, reduced progression of atherosclerosis in apoE-/- and LDLR-/- double deficient mice and provides a pathophysiological basis for the beneficial effects of aspirin in atherosclerosis, and low doses appeared to be more efficient than high doses. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Current therapy for arteriosclerosis in high risk patients centres on the use of lipid lowering drugs such as 3-hydroxy-3-methyl-glutarylCoA (HMG-CoA) reductase inhibitors (Statins) [1]. These lower the level of circulating cholesterol, which is widely known to play a fundamental role in lipid metabolism and foam cell formation in atherosclerotic plaques. In addition, inflammation in the arterial wall appears be an important factor in atherosclerosis [2,3] and more recent studies have indicated that upregulation of the adhesion molecules VCAM-1, ICAM-1, and E-selectin in endothelial cells by inflammatory cytokines through nuclear factor kappa B activation is implicated in formation and progression of atherosclerotic plaque [4]. ⁎ Corresponding author. Laboratory of Physiology, Faculty of Nutrition, Kobe Gakuin University, 518, Arise, Igawadani-Cho, Nishiku, Kobe, Japan 651-2180. Tel.: + 81 78 974 1551; fax: + 81 78 974 5689. E-mail address: [email protected] (T. Yamashita). 0049-3848/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2009.11.008
Large-scale clinical trials have shown that aspirin is an effective treatment for acute and secondary prevention of cerebrovascular disease and cardiovascular disease and may also be useful for primary prevention in coronary artery disease [5]. Aspirin is a major inhibitor of platelet function and its mode of action depends on the inhibition of cyclooxygenase enzymes (COX-1 and COX-2) which are involved in the production of prostanoids (prostaglandin, PGE2 and thromboxane, TXA2) and other mediators of inflammation and pain. Indeed, in a recent study, aspirin seemed to be most effective at reducing cardiovascular events in individuals with an elevated C-reactive protein (CRP). The use of aspirin in clinical practice, therefore, is based on both anti-platelet and anti-inflammatory mechanisms. However, the effect of aspirin on the progression of atherosclerosis and its attendant endothelial function is still unclear. Double-homozygous apoE-/- and LDLR-/- deficient mice provide a useful model of atherogenesis [6]. In the present study we have investigated the effects aspirin on the development of arteriosclerosis and endothelial function in these genetically susceptible mice.
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2. Material & methods 2.1. Experimental animal Double-homozygous apoE-/- deficient and LDLR-/- deficient mice (DKO mice, 129 X C57BL/6J back-ground) were obtained originally from Jackson Laboratory (Bar Harbor, Maine, USA). Subsequently, the animals were bred locally by sibling mating. C57BL/6 mice aged 1013 weeks were obtained from SLC Co. Ltd. (Hamamatsu, Japan). All animals were maintained in Kobe Gakuin University in air-conditioned rooms (22.5 ± 0.50 °C , and 50 ± 5% humidity) with a 12-h light and dark cycle. Male mice were used in the present study. Animals had free access to diet and drinking water. Experimental diets were commenced from six weeks until l6 weeks of age. All procedures were conducted in compliance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Science of the Physiological Society of Japan. 2.2. Experiment protocol DKO and C57BL/6 mice were maintained with a standard solid chow (CE-2) for six weeks prior to experimentation. Subsequently, experimental diets containing aspirin (Sigma-Aldrich Chemie GmbH, Germany) or placebo were administered for twelve weeks. A control group of C57BL/6 mice was given the same feed as the Placebo group. Arteriosclerosis, endothelial function and plasma prostaglandin F1α concentration were assessed as described below. 2.3. Preparation of experimental diets
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(Image-Pro Plus, Media Cybernetics, USA , Roper Japan,). The whole area (W) of the dissected blood vessel was calculated and the atherosclerotic regions identified by Oil Red O staining. The area (R) stained positively with oil red O was quantified and the ratio [(R÷W) x100] was used as an index of atherosclerosis development. Furthermore, the heart was perfused with PBS and then with 10% PBS-buffered formaldehyde. The heart and aorta including the aortic root were cut transversely and embedded in paraffin. The aortic root in the heart was sectioned proximally to distally in 10 µm starting from the semilunar valves. To investigate the cross-sectional area of the oil red O-stained lesions in the aortic root, processing and staining were carried out as described above. 2.5. Measurement of Nitric oxide (NO) Peripheral NO release was measured using electrochemical sensors as previously described [7,8]. Briefly, the working electrodes were introduced through a femoral arteriole, and positioned at close proximity to the branch point of the abdominal aorta and the common iliac artery, without preventing of blood flow. The sensitivity and selectivity of the electrodes were confirmed each time before use using S-nitroso-N-acetyl-DL-penicillamine (SNAP, sigma). The electrodes were immersed in phosphate-buffered saline (PBS, pH 7.4) in a small chamber and were calibrated using a graded series of SNAP concentrations, from 1 × 10- 3 M to 1 × 10- 5 M. 2.6. Immunochemistry
Experimental diets were prepared from a common standard feed (AIN93G) using the following purified materials: milk casein, f3-corn starch, sucrose, cellulose powder, mineral mixture, vitamin mixture (Oriental Yeast Co. Ltd., Tokyo, Japan), L-cystine, cholesterol (Nacalai Tesque Inc., Kyoto, Japan), and choline bitartrate, tert-butylhydroquinone (Wako Pure Chemical Industries Co. Ltd., Tokyo, Japan). The composition the experimental diet was made 40% with respect to lipid, 40% with respect to carbohydrates and 20% with respect to protein. Cholesterol was added at 0.05% (w/w). Two concentrations of aspirin (low dose, 4 mg/kg body weight, and high dose, 40 mg/kg body weight) were added to this mixture. Powder diets were solidified by adding agar (0.6%) and were stored at -30 °C until required.
The mouse heart was exposed by dissection and a wing-shaped needle inserted in the left ventricle. The blood vessels were then was washed thoroughly with buffer (10 mM PBS, pH7.4) by infusion in situ for approximately three minutes. The target blood vessel was excised, fixed with 4% Paraformaldehyde Phosphate Buffer Solution (Wako Purification Industry incorporated) soaked in Optimal Cutting Temperature (O.C.T.) compound (Tissue-Tek and SAKURA), and frozen using the dry ice and acetone or liquid nitrogen. Frozen sections were cut at 6 μm thickness using a cryostat, placed on poly-L-lysine–coated microscope slides (Muto Pure Chemicals, Tokyo, Japan), wrapped in aluminum foil and stored at -80 °C. Sections were examined by immunoperoxidase staining with anti-eNOS antibody ( Lab Vision Corporation) using the streptavidin /biotinylated horseradish peroxidase method (LSAB2 kit; DAKO) as described previously [9].
2.4. Measurement of atherosclerotic burden
2.7. Measurement of total cholesterol
The development of arteriosclerosis was assessed by estimating the extent of atherosclerotic regions as a percent of the entire surface area of the aorta as previously described [6]. Briefly, hearts were exposed by abdominal incision and 50 mL phosphoric acid buffer isotonic sodium chloride solution (phosphate buffered saline, PBS and pH7.4) infused through an indwelling 20G butterfly needle (Top Kasei Co. Ltd., Tokyo, Japan) followed by 10% neutral-buffered formalin (Nacalai Tesque Inc.). In addition, the major blood vessels were washed with PBS and fixed with 10% neutral buffered formalin solution by reflux through a femoral artery. The aortic arch was carefully dissected from connective tissue and minor branching blood vessels. The extracted vessels were kept in 10% neutral buffered formalin solution until processed. The vessels were then incised along the longitudinal plane, inverted on a black rubber sheet and held in place using stainless steel pins. The tissue was washed with distilled water for 30 seconds, treated with 60% isopropyl alcohol for 1 minute, and stained with oil red O stain solution at 37 °C for 15 minutes. Finally, the tissue was washed with 60% isopropyl alcohol and distilled water. The dyed specimens were photographed using a digital still camera (COOLPIX 880 Nikon Co. Ltd., Tokyo, Japan). Images were transferred to a personal computer and analyzed using image analysis software
Anticoagulated whole blood was collected from the abdominal aorta of mice under Nembutal anesthesia using Microtainer tubes (EDTA K2; Becton Dickinson). Plasma was obtained after centrifugation at 3000 rpm for 15 min at 4 °C). Plasma total cholesterol level was determined enzymatically by using Wako reagents (Wako Pure Chemical Co) and Biomek 1000 (Beckman Coulter Inc, USA). 2.8. Bleeding time Tail-cut bleeding time was measured in all groups as a platelet function in vivo assay, as described previously [10]. Briefly, the terminal 5 mm of the tails of mice were severed and immediately immersed in 1.5-ml tubs containing phosphate―buffered saline (PBS) at 37 °C, and the time to cessation of bleeding for 20 s was determined with a stopwatch. In each group, tail-cut bleeding times were performed after 16 week feeding. 2.9. Assay of prostaglandin F1α and TXB2 Whole blood was drawn from abdominal aorta of mice under Nembutal anesthesia and collected in (1) tubes containing EDTA 2K
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(Becton Dickinson) for 6-keto PG F1α (a stable metabolite of PGI2) assay, (2) tubes with no additive for measurements of serumTXB2 (a stable metabolite of TXA2). Plasma was obtained after centrifugation at 3000 rpm for 15 min at 4 °C, and blood obtained in serum tubes were allowed to clot at room temperature followed by centrifugation for 10 min at 1000 × g. The both of plasma and serum were transferred to eppendorf-tubes and stored at -80 °C until analysis. Assay of 6-keto PG F1α: Plasma was immediately the prostanoidsrich fraction was extracted as soon as possible. In outline, plasma was acidified with 1 N HCl to pH 3.0, then passed through a Sep-Pak C18 column (Waters Associates, Milford, MA, USA) pretreated with methanol and distilled water. After washing with distilled water, 10% ethanol and hexane, the prostanoids-rich fraction was eluted with methyl formate and evaporated under nitrogen gas. The residue was dissolved in assay buffer, and levels of 6-keto PGF1a were measured with a commercial enzyme immunoassay (EIA) kit according to the manufacturers' instructions (Cayman Chemical, Ann Arbor, Michigan, USA). Assay of TXB2: Serum levels of TXB2 were measured with a commercial enzyme immunoassay (EIA) kit according to the manufacturers' instructions (Cayman Chemical, Ann Arbor, Michigan, USA). Amount of TXB2 present in serum was calculated with the use of a data analysis tool developed by Cayman Chemical.
Table 2 Total cholesterol level in each group.
Total cholesterol (mmol/L)
C57BL6
Placebo
ASA4 mg/kg/day
ASA40 mg/kg/day
3.6 ± 0.3**
19.9 ± 1.0
18.7 ± 2.1
20.0 ± 1.7
Plasma cholesterol level (mmol/L) after 12 weeks feeding **: p = b0.001 C57BL6 vs Placebo, ASA4 mg/kg/day group, ASA40 mg/kg /day group.
C57BL6 group, there was no significant difference statistically in each group (Table 2). 3.4. Development of Atherosclerosis The development of atherosclerosis was evaluated at 18 weeks in each group (Fig. 1). The extent of atherosclerosis in the placebo group was (a) 23.0 ± 1.8%; 12.6 ± 1.4%; in the 4 mg/kg aspirin group (b) and 22.1 ± 1.0%. in the 40 mg/kg aspirin group (c). The differences were statistically significant between the placebo group and 4 mg/kg aspirin group. The differences were not statistically significant between placebo group and the 40 mg/kg aspirin group. Typical images of dissected blood vessels and aortic root in each group are illustrated in Fig. 2.
2.10. Statistical Analysis
3.5. Acetylcholine induced nitric oxide (NO) release
Results were expressed as mean ± SEM. Normal distribution was examined, and all data showed the normal distribution. Comparisons among groups were made by using 1-way factorial ANOVA, followed by the Fisher PLSD test, Dunnett's multiple comparison post-test. Differences between means were considered significant at p b 0.05.
The results of acetylcholine-induced (20 μg/kg body weight) NO release in each group are shown in Fig. 3. NO release was 29.3 ± 3.3 nM in the a placebo group (b); 45.2 ± 4.3 nM in the 4 mg/kg aspirin group (c) and 15.6 ± 2.1 nM. in the 40 mg/kg aspirin. group: The differences between the 40 mg/kg aspirin group and the Placebo and 4 mg/kg aspirin groups were statistically significant.
3. Results 3.6. Immunohistochemistry 3.1. Body Weight and Food intake No significant differences were observed in the body weights of animals in any of the groups before or after the experimental diets. 3.2. Bleeding time
The expression of eNOS in the vascular endothelium in DKO mice was examined by immunohistochemistry of cross-sections of the left brachiocephalic trunk. Surface eNOS was specifically observed in the placebo group and the aspirin groups, but not in the control group (Fig. 4). Furthermore, in particular, in 4 mg ASA group the expression of eNOS was also observed in subendothelial (Fig. 4 C, D).
Results are shown in Table 1. The both of Low dose ASA group and high dose ASA group had a longer bleeding time after a tail cut than placebo group (P = 0.0486 and P b 0.001, respectively) (Table 1). The prolongation of bleeding time in high dose ASA group was considered to be statistically significant compared with low dose ASA group (P = 0.0136). 3.3. Total Cholesterol The total cholesterol levels in each group rose progressively over time to exceed 18 mmol/L, compared to a mean value of 3.6 ± 0.3 mmol/L in C57BL6 mice after 12 weeks feeding. Except for
Table 1 Bleeding time.
Bleeding time (second)
Placebo
ASA4 mg/kg/day
ASA40 mg/kg/day
90 ± 8
150 ± 16*
235 ± 36**
Bleeding time from a tail cut according to the mice (six mice in each group) after 12 weeks feeding. *: Placebo vs ASA4 mg/kg/day P = 0.0486; **: Placebo vs ASA40 mg/kg/day P b 0.001; ASA4mg/kg/day vs ASA40 mg/kg/day: P = 0.0136.
Fig. 1. The effect of ASA dose on the progression of atherosclerosis after 12 weeks feeding in apoE-/- LDLR-/- double knockout mice (The entire aortic tree ) n = 8 ∼ 11 in each group **: P b 0.001 a) Placebo group b) 4 mg/kg/day aspirin group c) 40 mg/kg/day aspirin group.
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Fig. 2. Typical images of atherosclerotic regions identified by Oil Red O staining in each group of apoE-/- LDLR-/- double knockout and control mice. (A): Entire aorta (B): Cross section of Aortic roots, a: C57BL6 (control mice) group; b: Placebo group; c: Aspirin 4mg/kg/day group; d: Aspirin 40mg/kg/day group.
3.7. The levels of 6-keto-PGF1α and TXB2 The results of plasma 6-keto-PGF1α measurements in each group are shown in the Fig. 5A. 6-keto-PGF1α concentration was 805.0 ± 150.0 pM in the C57BL6 control group; 1772.5 ± 300.0pM in the Placebo group; 1385.0 ± 293.2pM in the 4 m g/kg ASA group and 552.5 ± 143.6pM in the 40 m g/kg ASA group. The Placebo and the 4 mg/kg ASA group results were significantly higher than the control group. In contrast, the 40 mg/kg ASA group results were significantly lower than the 4 mg/kg aspirin and placebo groups. The results of serum TXB2 measurements in each group are shown in the Fig. 5B. TXB2 concentration was 107.5 ± 7.7 pg/ml in the C57BL6 control group; 140.4 ± 9.6 pg/ml in the Placebo group; 15.1 ± 0.9 pg/ml in the 4 m g/kg ASA group; 12.5 ± 0.9 pg/ml in the 40 m g/kg ASA group. Serum TXB2 lever in ASA groups was significantly decreased by ASA administration compared with placebo and C57BL6 group. However,
it was not confirmed significant difference between 4 mg ASA group and 40 mg ASA group. 4. Discussion The present studies were designed to address two hypotheses. First, we determined the effect of low dose aspirin on the progression of atherosclerosis in apoE-/- and LDLR-/- double deficient mice (DKO mice), and second, its effect on the endothelial function. Long term Low dose aspirin (4.0 mg/kg B.W. /day) promoted a significant retardation of atherosclerotic lesion development in the DKO mice, which was evident in the entire aortic tree (40%) and the regions of aortic root. The results of bleeding time indicate that the inhibition of platelet function in low dose aspirin group was not severed than high dose aspirin group. Recent reports have shown that this dosage preferentially inhibits platelet-derived TxA2 over endothelial-derived
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Fig. 3. The effect of aspirin dose on the acetylcholine induced NO release in vivo after 12 weeks feeding in apoE-/- LDLR-/- double knockout and control mice. n = 8 ∼ 11 in each group *:p b 0.05; **: p b 0.01. a: C57BL6 group; b: Placebo group; c: Aspirin 4 mg/ kg/day group; d: Aspirin 40 mg/kg/day group.
PGI2 [11]. This differential effect has theoretical advantages in that unaffected PGI2 production could promote the antithrombotic and anti-inflammatory properties of the endothelium. The present results support this, because in low dose ASA group, the severe suppression of PGI2 production was not confirmed in spite of decreasing TXB2 production, but not in high dose ASA group. Hyperlipemia is well established risk factor for atherosclerosis and coronary heart disease. The apoE-/- LDLR-/- double knockout mice used in the present investigations characteristically display a marked lipid metabolic disorder, develop severe hyperlipemia and are prone to atherosclerosis in the relatively short term [12]. The total cholesterol level in each of our groups rose progressively over time to exceed 18 mmol/L after 12 weeks feeding. It is not generally noted that aspirin lowers the level of total cholesterol like a statin. In this study, it was not recognized difference in the both of aspirin group. The lowering of cholesterol level is essential in the prevention of atherosclerotic development. However the lowering of cholesterol level was not
confirmed in ASA groups. Further studies are necessary to investigate the relationship between the level of cholesterol and the development of atherosclerosis. Atherosclerosis is a complex and multifactorial disease, and inflammation is involved in its pathogenesis and evolution [13]. According to recent reports, increased levels of several cytokines have been reported in human and murine atherosclerosis [14], and evidence for their role in the disease has been provided [15]. Activated NF-B is present in atherosclerotic lesions both in human [16] and in apolipoprotein E–deficient mice [17]. So, in our case, it predicts the up regulation of these cytokines. Recent data suggest that COX-1 may be induced and may be responsible for prostaglandin formation at sites of inflammation [18,19]. Studies with low-dose aspirin suggest that platelet COX is also a major source of the increased TXA2 biosynthesis seen in patients with atherosclerosis, possibly reflecting enhanced platelet activity [20]. Suppression of TX production may be an important mechanism for the observed effect of aspirin in this study. However, it is also reported that despite complete, elimination of the COX-1 gene expression significantly accelerated early atherosclerotic lesion formation in both LDLR–/– and apoE–/– recipient mice [21]. The role of TXA2 is controversial. Furthermore, Clinical or basic reports regarded with aspirin are complicated by the contribution of COX isoforms. The major finding of this study is that long term low dose aspirin only promotes acetylcholine-induced peripheral release of nitric oxide release, which supported upregulation of eNOS expression in the vascular endothelial cell in DKO mice by immunohistochemistry. To the best of our knowledge, our study is the first demonstration of acetylcholine-induced peripheral release of nitric oxide directly in DKO mice with atherosclerosis. Acetylcholine, a commonly used probe for testing endothelial function in humans, causes endothelium-dependent smooth muscle vasodilation, which is believed to be caused largely by release of nitric oxide and endothelium-derived hyperpolarizing factor [22,23]. Endothelium can also produce contracting factors that are mainly cyclooxygenase-dependent prostanoids or oxygen free radicals, which destroy NO and thus reduce its availability. Endothelial-derived nitric oxide and prostacyclin, are believed to show vasodilation, profilation of smooth muscle cell,
Fig. 4. Immunohistologic findings in each group. (A) eNOS staining in placebo group, (B) pseudo colour of (A), (C) eNOS staining in aspirin 4 mg/kg/day group, (D) pseudo colour of (C), (E) eNOS staining in aspirin 40 mg/kg/day group, (F)pseudo colour of (E), Magnification: x200.
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The controversy regarding the optimal dosage of aspirin for primary prevention of atherosclerotic events in human is not solved definitely [31,32]. The results of recent numerous trials suggest no difference in clinical effectiveness concerning cardiovascular risk reduction between high and low doses of aspirin [33], and furthermore cast doubt on primary prevention of cardiovascular events [34]. There is room for reconsideration in conventional guideline [35] with regard to the long-term daily dosage of aspirin. It seems that a variety of clinical settings in each study complicates the interpretation of the result more. However, it is finally hoped that numerous clinical studies and basic studies provide a more reliable guideline in the future. Although our data supports the hypothesis that low dose aspirin is enough to effectively suppress TXA2 production in platelets [36], there are different problems involved in applying the results of animal models to humans [37]. Our present data are therefore limited to experimental observations in the animal without intervention. From current observations, it is demonstrated that an unbalance of constrictor and dilator force has been largely attributed to the progress of atherosclerosis. It is possible that low dose aspirin (4.0 mg/kg B.W.) reduce the activity of endothelial derived vasoconstract factor (EDCF), by interfering, mainly via COX, without impairing PGI2 production, it leads to the improvement of acetylcholine-induced NO release. In the contrary, high dose aspirin (40 mg/kg B.W.) impaired the balance of vasoactive factors through impairing PGI2 production, it leads to the endothelial dysfunction progresses in atherosclerosis. References
Fig. 5. 6-keto-PGF1α level and TXB2 level in each group of apoE-/- LDLR-/- double knockout and control mice. A: Plasma 6-keto-PGF1α level, B: Serum TXB2 lever n = 8 ∼ 11 in each group **: p b 0.01. a: C57BL6 group; b: Placebo group; c: Aspirin 4 mg/ kg/day group; d: Aspirin 40 mg/kg/day group.
attenuation of platelet aggregation. In the contrary, endothelin as known vasoconstrictor, accelerates profilation of smooth muscle cell, platelet aggregation. It has been demonstrated that cyclooxygenase inhibition with indomethacin increased acetylcholine-mediated forearm vasodilation by 39% in patients with congestive heart failure but not in normal subjects [24,25]. Thus, the progression of atherosclerosis leads to activation of the cyclooxygenase pathway, which seems to participate in the impaired endothelium-dependent vasodilation. In atherosclerosis, a clinical condition characterized by endothelial dysfunction, cyclooxygenase seems to be a source of oxidative stress. In addition, antioxidant action of aspirin as a cyclooxygenase inhibitor may be an important mechanism for the observed effect of aspirin in this study. Furthermore, aspirin including salicylic acid is known to scavenge both oxygen free radicals and hydroxyl radicals [26–28]. Superoxide anions metabolize nitric oxide to higher, biologically inactive nitrogen oxides and are believed to be the primary mechanism underlying the reduced bioavailability of nitric oxide in atherosclerosis. We previously showed that the progression of atherosclerosis was suppressed through the intake of ω3 polyunsaturated fatty acid [6] which contains EPA and DHA, widely recognized as anti-atherogenic eicosanoid in humans and animals [29]. Free radical peroxidation of polyunsaturated fatty acids present in lipoproteins produces oxidized low-density lipoproteins which play a critical role in the development of atherosclerosis [30]. The action of aspirin as a scavenger of oxidative stress, may be contribute to prevention of the development of atherosclerosis, and needs further investigations.
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