Purpose: Exercise is known to reduce cardiovascular mortality. However, the precise mechanisms are still unknown. Because atherosclerotic plaque destabilization and rupture leads to dramatic cardiovascular events, stabilization of plaque might be regarded as an important goal of an exercise preventive therapy. The present study examined the plaque-stabilizing effect of long-term exercise in experimental atherosclerosis using apolipoprotein E-deficient mice (ApoE−/−).
Methods: ApoE−/− mice were subjected to 6 months of swimming exercise. A group of sedentary animals were used as controls. Morphometry and characteristics of atherosclerotic plaque stability were assessed in aortic sinus by immunohistochemistry. Aortic levels of total protein kinase Akt (protein kinase B), phosphorylated Akt at Ser473 (p-Akt), total endothelial nitric oxide synthase (eNOS), and phosphorylated eNOS at Ser1177 (p-eNOS) were assessed by Western blotting.
Results: Exercised mice developed a more stable plaque phenotype as shown by decreased macrophage and increased smooth muscle cell content. Protein expressions of Akt, p-Akt, eNOS, and p-eNOS were not modulated by exercise.
Conclusions: Long-term exercise promotes plaque stability in ApoE−/− mice. The Akt-mediated eNOS phosphorylation pathway seems not to be the primary molecular mechanism.
1EA-4267/2SBP, University of Franche-Comté, Besançon, FRANCE; 2Exercise Prevention Innovation and Technico-Sporting Watching Department, Besançon, FRANCE; 3Service of Vascular Medicine, Department of Internal Medicine, Lausanne University Hospital, CHUV, Lausanne, SWITZERLAND; and 4INSERM U866, Faculty of Medicine, University of Bourgogne, Dijon, FRANCE
Address for correspondence: Maxime Pellegrin, Ph.D., Service of Vascular Medicine, CHUV, Av. Pierre Decker 5, 1011 Lausanne, Switzerland; E-mail: Maxime.Pellegrin@chuv.ch.
Submitted for publication December 2008.
Accepted for publication April 2009.
Atherosclerosis, a complex multifactorial chronic disease, represents a major health burden in modern society. It is now widely recognized that atherosclerotic plaque destabilization and rupture leads to dramatic clinical events such as unstable angina, acute myocardial infarction or stroke. For these reasons, identification of novel therapies contributing to plaque stabilization represents a continuing challenge for the medical community.
Regular exercise has been broadly accepted as a deterrent for atherosclerosis and cardiovascular disease (1-3,9). In particular, exercise has been shown to reduce progression of atherosclerosis in patients with CAD (24). Similarly, we and others have shown that regular exercise exerts a beneficial effect on atherosclerosis extension in a variety of animal models (19,23,26,29). Experimental as well as observational data indicate that exercise reduces adiposity, blood pressure, diabetes incidence, dyslipidemia, and inflammation (1,2). Moreover, it enhances insulin sensitivity, glycemic control, fibrinolysis, and endothelial function (1,2). Although these favorable effects on atherosclerotic and thrombotic-related risk factors may slow the progression of atherosclerosis, there are too insignificant to account for the training-induced reduction in mortality in patients with cardiovascular risk or with established clinical atherosclerosis. Because plaque destabilization is a predictor of fatal atherothrombotic events, stabilization of plaque might be regarded as a primary target of an exercise preventive therapy. However, there is no report investigating whether exercise may modulate plaque composition promoting plaque stability.
In the present study, we examined the plaque-stabilizing effect of long-term exercise in experimental atherosclerosis using apolipoprotein E-deficient mice (ApoE−/−) mice.
MATERIALS AND METHODS
Male ApoE−/− mice (10-11 wk old at the beginning of the experiment) were obtained from Charles River Laboratories (L'Arbresle, France). During the experimental protocol, all animals were fed a lipid-rich Western-type diet containing 0.2% cholesterol (UAR, Epinay-sur-orge, France). Mice were randomly divided into sedentary (ApoE−/− S; n = 8) and exercise groups (ApoE−/− X; n = 8). The housing and care of the animals and all experimental procedures were approved by the local Institutional Animal Committee and were conducted in accordance with the policy statement of the American College of Sports Medicine on research with experimental animals.
The swimming exercise training protocol was adapted from a previously published procedure (29). Briefly, mice in the exercise group were trained 50 min·d−1, 5 d·wk−1 for 24 wk in water at 35-36°C. Mice were allowed to swim in groups of four. The animals were progressively familiarized with swimming during the first week, beginning with a 10-min training period on day 1 and ending with a 50-min training period on day 5, the swimming time increased for 10 min every day. During weeks 2 to 24, mice swam continuously 50 min·d−1. All training sessions took place during the early morning hours (8:00-10:00 a.m.). After all the training, wet animals were carefully dried and placed in a warm environment to avoid additional cold physiological stress and health problems.
Quantification and morphology of plaques.
The heart was fixed in formol, dehydrated, and paraffin-embedded. Serial sections (3 μm thick) obtained between the appearance and disappearance of the aortic valve were analyzed by light microscopy for the identification and quantification of plaques after staining with Movats pentachrome. Pictures of the three middle sections of the biggest atherosclerotic plaque from each mouse were taken with a digital camera (Coolpix; Nikon, Düsseldorf, Germany) and were used for atherosclerotic evaluation. The preceding and following 3-μm-thick sections were used for immunohistochemistry. An independent investigator blinded to the study protocol evaluated each section under a light microscope using the morphometry software Qwin software (Leica Systems, Wetzler, Germany). The following characteristic plaque features were evaluated: 1) quantification of fibrous cap thickness, 2) quantification of α-smooth muscle (α-SM) actin content, 3) quantification of central core (a large core was defined as occupying >50% of total surface plaque), 4) presence of media degeneration (invasion of media by plaque components, media thinning, elastic laminae rupture, and media atrophy), 5) presence of layering/foam cells above or adjacent to the central core, and 6) plaque macrophage content quantification (22). Plaque area at the aortic valve level was quantified in square micrometers by computerized planimetry using the Qwin software (Leica Systems). The average of the measured five sections per sample was used for analysis.
Fixed and paraffin-embedded sections were stained with a biotinylated mouse monoclonal immunoglobulin G 2a α-SM actin antibody or with a rat monoclonal Mac-2 antibody (Cedarlane, Hornby, Ontario, Canada). Staining of sections with specific antibodies was followed by treatment with streptavidin-peroxidase complex (Dako, Glostrup, Denmark). Peroxidase activity was revealed with 3-amino-9-ethylcarbazole (Sigma-Aldrich, St. Louis, MO). Slides were counterstained with hematoxylin (for α-SM actin). Appropriate controls with mouse or rat immunoglobulin G with irrelevant specificities at the concentrations of the primary antibodies were included. Samples were observed with a photomicroscope, and pictures were acquired with a high-sensitivity color digital camera (Leica DC Camera; Leica Systems). α-SM actin and Mac-2-positive areas were quantified in each sample by the Qwin morphometry software (22).
Protein extraction and Western blot analysis.
The expression of total eNOS (endothelial Nitric Oxide Synthase), eNOS phosphorylation at Ser1177 (p-eNOS), total Akt (also called protein kinase B), Akt phosphorylation at Ser473 (p-Akt), platelet endothelial cell adhesion molecule 1 (PECAM-1), p47phox, and p67phox were assessed by Western blotting. Total cellular proteins were extracted from mouse aorta in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8, 0.1% sodium dodecyl sulfate (SDS), 0.5% Na desoxycholate) containing a mixture of protease inhibitors (Sigma-Aldrich) and phosphatase inhibitors (2 mM Na orthovanadate). After 30 min of incubation at 4°C in lysis buffer, tissue and cell debris were eliminated by 30 min of centrifugation at 10,000g, and the supernatant was collected. Protein concentrations were measured by using a Bio-Rad protein assay (Bio-Rad, Marnes-la-Coquette, France). Protein, 30-50 μg, was incubated in loading buffer (125 mM Tris-HCl, pH 6.8, 10% β-mercaptoethanol, 4.6% SDS, 20% glycerol, and 0.003% bromophenol blue), boiled for 3 min, separated by SDS-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidine difluoride membrane (PerkinElmer, Salem, MA). After blocking nonspecific binding sites overnight with 5% nonfat milk in phosphate-buffered saline-0.1% Tween 20 (TPBS), membranes were incubated for 2 h at room temperature with various primary antibodies: rabbit polyclonal antibodies directed against total eNOS diluted 1:200 (Santa Cruz Biotechnology, Santa Cruz, CA), against p-eNOS diluted 1:200 (Santa Cruz Biotechnology), against β-actin diluted 1:200 (Santa Cruz Biotechnology), against p67phox diluted 1:200 (Santa Cruz Biotechnology), against p47phox diluted 1:200 (Santa Cruz Biotechnology), mouse monoclonal antibodies directed against total Akt diluted 1:1000 (Upstate Biotechnology, Lake Placid, NY), against p-Akt diluted 1:1000 (Upstate Biotechnology), and against PECAM-1 diluted 1:200 (Santa Cruz Biotechnology); all antibodies were diluted in TPBS. After three washes in TPBS, membranes were incubated with horseradish peroxidase-conjugated goat antimouse (Santa Cruz Biotechnology) or antirabbit antibody (Interchim, Montluçon, France) for 1 h at room temperature and washed three times in TPBS. Autoradiography of immunoblots was performed using an enhanced chemoluminescence detection kit (Santa Cruz Biotechnology). The band intensities were quantified by densitometry using a Bio-Rad image analysis system (Quantity one; Bio-Rad) and normalized against the β-actin protein. Each experiment was repeated three times with each animal.
Blood samples from 4-h fasting mice were collected in heparin-containing test tubes. Plasmas were obtained by slow-speed centrifugation and stored at −80°C until analysis. All assays were performed on a Victor 1420 Multilabel Counter (Wallac-PerkinElmer Life Sciences). Commercially available kits were used for the determination of phospholipids (Wako, Neuss, Germany), total cholesterol (Diasys, Holzhein, Germany), and triglycerides (Diasys) in plasma. For HDL- and non-HDL-cholesterol determination, 10 μL of plasma were mixed with 190 μL of potassium bromide solution adjusted to a density of 1.07 g·mL−1. After a 5-h 100,000 rpm run in a TLA120 rotor in a TLX ultracentrifuge (Beckman, Palo Alto, CA), HDL-cholesterol content was assayed in the supernatant (100 μL), and non-HDL-cholesterol content was measured in the infranatant (100 μL).
Muscle oxidative enzyme activity.
Citrate synthase activity, a marker of mitochondrial content, was measured in gastrocnemius muscle homogenates using spectrophotometric method as previously described (29).
Data were expressed as mean ± SEM. Student's t-test was used to assess significant differences. Statistical analysis of plaque morphology was carried out using the χ2 test; if more than 20% of the expected frequencies were less than 5, we used the Fisher exact test to evaluate the exact P value. Probability value of <0.05 was considered to be statistically significant.
Efficacy of the swimming exercise program.
Body weights and relative heart weights at the end of the swimming program are shown in Table 1. In accordance with the well-described body mass and cardiac adaptations induced by endurance training, the 24-wk swimming exercise decreased mice weight and increased the relative heart weight (P < 0.05). The training program resulted in a significantly improved skeletal muscle oxidative capacity (P < 0.05) as evidenced by increased citrate synthase activity (Table 1). These results suggest that exercised mice exhibit physiological effects of exercise training.
Swimming did not cause significant changes in plasma total cholesterol, HDL-cholesterol, non-HDL-cholesterol, triglycerides, and phospholipids levels (Table 1).
Effect of swimming exercise on size and stability of plaques.
Quantification of atherosclerotic lesion areas in aortic roots showed that a 24-wk swimming exercise significantly decreased the progression of atherosclerotic lesions by ≍30% (P < 0.05; Fig. 1).
In addition to the effect of swimming exercise on lesion size, we also assessed characteristics of plaque stability. The surface of central lipid core and signs of media degeneration were similar in exercised compared with sedentary mice (Table 2). The presence of a fibrous cap ensures a certain degree of stability, whereas its loss is associated with plaque rupture. In addition, mixed multiple cell layers at different stages are suggested to be the consequence of previous clinically silent ruptures and after de novo plaque growth. Swimming exercise was associated with a slight, but not significant, increase in fibrous cap thickness and a decrease in layering (Table 2). Plaque-stabilizing changes in lesions include reduced macrophage and increased smooth muscle cells (SMC) content (30). As shown in Figures 2A and B, macrophage content, determined by Mac-2 immunostaining, was considerably decreased in lesions from exercised mice (P < 0.05) compared with sedentary ones. Long-term exercise significantly increased SMC plaque content by ≍58% as determined by α-SM actin staining (Figs. 2C and D).
Effect of swimming exercise on eNOS, p-eNOS-Ser1177, Akt, p-Akt-Ser473, and PECAM-1 expression.
Exercise-induced increase of vascular eNOS expression and of eNOS-Ser1177 phosphorylation through the protein kinase Akt might be an important and potentially vasoprotective effect of exercise training (12). To investigate the underlying molecular mechanisms of long-term exercise in our mouse model, we analyzed the aortic contents of eNOS, p-eNOS on Ser1177, Akt, and p-Akt on Ser473 in mice by Western blotting. The expression of total eNOS in exercised mice did not differ significantly from that of sedentary animals (Fig. 3A). There was no significant difference in the protein levels of eNOS phosphorylation at Ser1177 among the sedentary and exercised groups (Fig. 3B). Along the same line, protein levels of Akt and Akt phosphorylation at Ser473 were similar in sedentary and exercised ApoE−/− mice (Figs. 3C and D).
PECAM-1 has been shown to modulate Akt and eNOS activation in endothelial cells in response to shear stress (7). Therefore, we quantified the protein level of PECAM-1. We found that exercise did not modulate PECAM-1 expression in ApoE−/− mice (data not shown).
Effect of swimming exercise on expression of pro-oxidant nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase subunits p47phox and p67phox.
We next investigated whether the observed antiatherosclerotic effect of long-term exercise was mediated by changes in pro-oxidant NAD(P)H oxidase subunit's protein levels. For this purpose, we assessed the protein expression of p47phox and p67phox subunits of the NAD(P)H oxidase. Translocation of the cytosolic regulatory subunits p47phox and p67phox to the plasma membrane is a prerequisite for NADPH oxidase activation, the major source of reactive oxygen species (ROS) in the circulatory system (33). Western blot analysis revealed that the expression level of p47phox and p67phox subunits remain unchanged between exercised and sedentary mice (Fig. 4).
Our results show for the first time, in a mouse model of atherosclerosis, that long-term exercise can stabilize atherosclerotic plaques.
Regular exercise is known to reduce cardiovascular mortality in humans with cardiovascular risk factors (13,21) or with CAD (35). Despite the established role of exercise in primary and secondary prevention of CAD, the mechanism responsible for this mortality benefit have not yet been clearly defined. Here, we hypothesized that long-term exercise may directly alter plaque composition and increase plaque stability. Our results showed that a 6-month exercise retards further progression of atherosclerosis in sedentary ApoE−/− mice fed a lipid-rich diet independently of blood lipid modifications. Reduction of atherosclerosis progression secondary to exercise has been previously shown in ApoE−/− mice (19,26,29). However, this effect was observed only in response to medium-term exercise (6-9 wk) (19,26,29). Our data provide first the evidence that long-term exercise is equally effective to prevent atherosclerosis extension. More importantly, exercised mice developed a more stable plaque phenotype as shown by decreased macrophage and increased SM cell. This suggests that the improvement of plaque stability with exercise, if confirmed in clinical studies, might be a key pathophysiologic mechanism contributing to exercise-mediated reduction in morbidity and mortality in patients with cardiovascular risk because atherosclerotic plaque destabilization and rupture leads to dramatic cardiovascular events as above mentioned. Reduction of macrophage plaque content, also shown in ApoE−/− mice undergoing an 8-wk swimming exercise (26), indicates that the vasculoprotective effect of exercise, and in particular swimming, may be mediated, at least in part, by a reduction in inflammation. In support of this notion, a clinical study recently showed that exercise training reduces circulating inflammatory markers in subjects at risk for coronary events (25). Furthermore, we show herein that swimming exercise induces increased SM cell plaque content. Augmented SM cell content favors plaque stabilization because SM cells produce the extracellular matrix, which is responsible for the plaque's mechanical strength. On the contrary, loss of SM cell can be detrimental for plaque's stability (18). Our data suggest that swimming exercise may promote the migration of SM cells from medial to intimal region or may inhibit SM cell apoptosis, leading to increased SM cell number and improved plaque stability. Clearly, further studies are needed to address this point.
To elucidate the molecular mechanism involved in the exercise-induced plaque stabilization, we investigated the Akt-dependent eNOS phosphorylation pathway. Exercise is a powerful stimulus to increase blood flow and shear stress in vascular beds, which, in turn, up-regulates eNOS expression leading to increased vascular nitric oxide production, a known antiatherogenic molecule (36). Exercise-mediated increase of eNOS expression and eNOS-Ser1177 phosphorylation through protein kinase Akt (protein kinase B) is most likely an important vasoprotective mechanism of exercise. In fact, Hambrecht et al. (12) have recently demonstrated in patients with CAD that 4 wk of regular aerobic exercise improves vascular function through shear stress-induced/Akt-dependent eNOS phosphorylation on Ser1177. More recently, studies have also shown that 8-10 wk of swim training stimulates this pathway in myocardium of healthy rats (14,37). However, to our knowledge, there is no report investigating whether exercise may modulate this protective pathway in experimental atherosclerosis. Interestingly, our results indicate that a 6-month swimming program did not change the expression of Akt, eNOS, and phosphorylations of Ser473-Akt and Ser1177-eNOS in the aorta of ApoE−/− mice. It is well recognized that short- and medium-term exercises increase vascular eNOS protein levels in various animal models (8,10,19,34). The lack of adaptation in arterial eNOS levels observed in response to long-term exercise reported here is not novel because, also in previous experiments, no change in eNOS protein expression was found after 3 to 5 months of regular exercise (20,28,31). This confirms a recent hypothesis suggesting that exercise-induced elevations in eNOS protein expression may be a transient molecular adaptation that occurred in the early phase of exercise and may not persist within months of training (11). Likewise, our results showed for the first time that long-term exercise did not increase posttranscriptional modification of eNOS as evidenced by the absence of up-regulation of eNOS phosphorylation on Ser1177. Thus, it seems that the Akt-dependent eNOS phosphorylation pathway is not the primary molecular mechanism contributing to the beneficial effects of long-term exercise on plaque stability in our mouse model. Alternative mechanisms should also be considered.
We next addressed the possible modulation of pro-oxidant NAD(P)H oxidase subunits p47phox and p67phox in response to exercise in our experimental model. NAD(P)H oxidase subunits p47phox and p67phox represent a major source of ROS and oxidative stress in the vasculature (33). We found that the expression of p47phox and p67phox subunits in the aorta of our exercised ApoE−/− mice was not different from that of sedentary animals. On the contrary, Laufs et al. (19) showed that the expression of these two subunits was reduced after 6 wk of voluntary cage wheel exercise in C57BL/6 mice. Differences in mouse models and exercise duration could explain these discrepancies. In accordance with our study, a recent clinical trial showed that exercise did not modify the degree of systemic oxidative stress (evaluated by measuring plasma 8-isoprostane) in humans (16). Thus, modulation of pro-oxidant p47phox and p67phox NAD(P)H oxidase subunits may not account for the antiatherosclerotic benefits of long-term exercise in ApoE−/− mice.
Forced swimming is a commonly used protocol for exercise in mice. It is generally recognized that swimming causes physical and psychological stresses. Swimming has been shown to induce corticosterone (4,15,27) and catecholamine (32) rise in rodents' plasma. However, this has not been a universal finding, and reports of unchanged and reduced corticosterone levels in response to swimming have also been published (6,17). Most importantly, it seems that only acute swimming exercise induces stress in mice. Indeed, previous studies showed that acute swimming (until exhaustion) significantly increased levels of corticosterone compared with sedentary controls, whereas continuing exercise during several weeks decreased corticosterone levels in mice (5,6). Similarly, we have previously shown no difference in terms of plasma norepinephrine levels in ApoE−/− mice undergoing a 6-wk swimming exercise compared with sedentary ones (unpublished data). Therefore, we speculate that the impact of swimming-related stress in our experiments may be negligible in comparison with the overall effect of swimming in the cardiovascular system. Finally, we must keep in mind that swimming exercise in mice is forced, whereas aquatic exercise in humans is voluntary. Further studies incorporating voluntary exercise protocols will be useful in translating mice data to possible therapies for patients with cardiovascular risk or with established clinical atherosclerosis.
In conclusion, results reported here emphasize for the first time the pivotal role of long-term exercise in atherosclerosis plaque stabilization in ApoE−/− mice. These findings may explain the clinical benefits of long-term exercise in terms of cardiovascular event protection. The Akt-dependent eNOS phosphorylation pathway does not seem to be the primary molecular mechanism responsible for the plaque-stabilizing effect of long-term exercise.
Conflict of interest: The authors report no conflicts.
The authors thank Dominique Paris, Chantal Ferniot, and Dr. Isabelle Jallat-Dalloz for excellent technical assistance. Results of the present study do not constitute endorsement by ACSM.
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Keywords:©2009The American College of Sports Medicine
ATHEROSCLEROSIS; SWIMMING EXERCISE; PLAQUE STABILITY; AKT-MEDIATED ENOS PHOSPHORYLATION