Physical Inactivity Increases Oxidative Stress, Endothelial Dysfunction, and Atherosclerosis
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动脉硬化血栓血管生物学 2005年第4期
From the Klinik für Innere Medizin III (U.L., S.W., T.C., M.E., M.B., G.N.), Universit?tsklinikum des Saarlandes, Homburg/Saar, and Med. Klinik und Poliklinik II (T.M.), Johannes Gutenberg-Universit?t, Mainz, Germany.
Correspondence to Dr Ulrich Laufs, Klinik für Innere Medizin III, Universit?tsklinikum des Saarlandes, D-66421 Homburg/Saar, Germany. E-mail ulrich@laufs.com
Abstract
Objective— Sedentary lifestyle is associated with increased cardiovascular events. The underlying molecular mechanisms are incompletely understood. Reactive oxygen species (ROS) contribute to endothelial dysfunction and atherosclerosis. An important source of vascular ROS is the NADPH oxidase.
Methods and Results— C57BL6 mice were subjected to regular housing (physical inactivity) or voluntary training on running wheels (6 weeks). Inactivity increased vascular lipid peroxidation to 148±9% and upregulated superoxide release to 176±17% (L-012 chemiluminescence) and 188±29% (cytochrome C reduction assay), respectively. ROS production was predominantly increased in the endothelium and the media (dihydroethidium fluorescence). Activity of the NADPH oxidase was increased to 154±22% in the sedentary group. Rac1 GST-PAK pull-down assays showed an upregulation of rac1 activity to 161±14%. Expression levels of the subunits nox1, p47phox, and p67phox were increased. To address the significance of the antioxidative effects of running, experiments were repeated in apolipoprotein E–deficient mice treated with a high-cholesterol diet. Inactivity increased vascular superoxide production and impaired endothelium-dependent vasorelaxation. Atherosclerotic lesion formation was significantly accelerated in sedentary mice.
Conclusions— Inactivity increases vascular NADPH oxidase expression and activity and enhances vascular ROS production, which contributes to endothelial dysfunction and atherosclerosis during sedentary as opposed to physically active lifestyle.
Sedentary lifestyle predicts vascular risk. To study underlying mechanisms, mice were subjected to physical inactivity or voluntary training on running wheels. Inactivity increased vascular lipid peroxidation, superoxide release, and NADPH oxidase expression and activity. In apoE–/– mice, inactivity significantly impaired endothelium-dependent vasorelaxation and accelerated atherosclerotic lesion formation.
Key Words: physical inactivity ? exercise ? oxidative stress ? endothelial dysfunction ? atherosclerosis
Introduction
The prevalence of physical inactivity in adults is increasing.1–3 Nearly 40% of the adult population subject themselves to <10 minutes of continuous physical activity per week.3 These lifestyle changes are of special interest because physical inactivity has been proposed as an independent cardiovascular risk factor.4,5 However, the molecular mechanisms leading from sedentary lifestyle to cardiovascular events are poorly defined.6
On the other hand, prospective epidemiological data indicate that moderate (eg, walking) and vigorous exercise are associated with substantial reductions in the incidence of cardiovascular events.4,5 Physical training improves endothelial function, exercise capacity, and collateralization in patients with coronary artery disease7–9 and prevents the progression of carotid atherosclerosis.10,11 Among other beneficial effects, physical activity is associated with improved mood, body weight, blood pressure, insulin sensitivity, and hemostatic and inflammatory variables.12,13
Reactive oxygen species (ROS) play a pivotal role in the pathogenesis of endothelial dysfunction and atherosclerosis.14 However, uncertainty remains regarding the effect of exercise on ROS. Acutely, intensive exercise has been shown to increase oxidative stress because increased aerobic metabolism is a source of oxidative stress,15,16 whereas long-term moderate exercise may upregulate antioxidative enzymes and decrease indices of oxidative stress.16,17
The aim of this study was to compare the effects of physical inactivity with voluntary running. Mice were subjected to regular housing or cages equipped with running wheels. We postulated that giving the animals the opportunity of voluntary running resembles their natural habitat more closely and tested the effects of voluntary running compared with sedentary behavior on parameters of vascular oxidative stress. In addition, we used the model of cholesterol-fed apolipoprotein E–deficient (apoE–/–) mice to examine the effects of inactivity versus running on endothelial function and atherosclerotic lesion formation.
Methods
The Methods section is available in the online data supplement, available at http://atvb.ahajournals.org.
Results
Physical Inactivity Increases Vascular Oxidative Stress
The mean running distance of active mice was 4900±700 m per 24 hours. Body weight did not differ between active and inactive mice (24±0.3 versus 23±0.3 g). As a global parameter of oxidative stress, lipid peroxidation of the aortic wall was compared. Inactive mice displayed upregulation of vascular lipid hydroperoxides to 148±9% compared with active mice (n=6 per group; P<0.05; Figure 1A). Because superoxide radicals are involved in the pathogenesis of atherosclerosis, we determined aortic superoxide production by 2 different methods. As shown in Figure 1B and 1C, vascular superoxide release was significantly increased in inactive compared with active mice (176±17% as assessed by L-012 chemiluminescence assays [n=8 per group; P<0.05] and 188±29% as determined by cytochrome C reduction assays [n=4 per group; P<0.05], respectively). Because NADPH oxidase is a major source of superoxide radicals in the vascular wall, we assessed NADPH oxidase activity in aortic tissue. Figure 1D displays that NADPH oxidase activity was upregulated to 154±22% (n=6 per group; P<0.05) in the sedentary group.
Figure 1. Physical inactivity and vascular oxidative stress. Parameters of vascular oxidative stress were assessed in sedentary (inactive) and voluntarily running (active) wild-type mice. Concentrations of lipid hydroperoxides were determined in aortic homogenates (A; mean±SEM; n=6 per group; *P<0.05 vs active mice). Superoxide production in intact aortic ring preparations was assessed by L-012 chemiluminescence (B) and by the superoxide dismutase–inhibitable cytochrome C reduction assay (C; mean±SEM; n=8 and n=4 per group, respectively; *P<0.05 vs active mice). NADPH oxidase activity in aortic homogenates was measured by a lucigenin-enhanced chemiluminescence assay using NADPH (100 μmol/L) as substrate and 5 μmol/L lucigenin (D; mean±SEM; n=6 per group; *P<0.05 vs active mice).
Upregulation of NADPH Oxidase Subunits in Inactive Mice
Translocation of the cytosolic regulatory subunits p47phox, p67phox, and the small GTP-binding protein rac1 to the plasma membrane is a prerequisite for NADPH oxidase activation and subsequent ROS production. Rac1 GST-PAK pull-down assays were performed to quantitate rac1 activity. In inactive mice, rac1 activity was increased to 161±14% compared with active animals (n=6 per group; P<0.05; Figure 2A and 2B). In addition, RT-PCR analysis showed increased expression of the subunits p47phox (285±19%) and p67phox (161±23%) in sedentary mice as opposed to active mice (n=6 per group; P<0.05; Figure 2C). Similarly, membrane protein expression of p47phox (343±29%) and p67phox (180±25%) was increased in the inactive mice (n=6 per group; P<0.05), whereas no difference was observed in cytosolic protein expression of these NADPH oxidase subunits (Figure 2D). Furthermore, inactive mice displayed higher expression levels of the nox1 subunit of NADPH oxidase (181±37%; n=6 per group; P<0.05), whereas the subunits p22phox, gp91phox, and nox4 remained unaltered between the groups (Figure 2C).
Figure 2. Physical inactivity and NADPH oxidase subunits. NADPH oxidase subunits were assessed in active and inactive wild-type mice. Rac1 GTPase activity in aortic homogenates was determined by rac1 GST-PAK pull-down assays. A and B, Representative example (A) and densitometric quantification (B; mean±SEM; n=6 per group; *P<0.05 vs active mice). Aortic mRNA expression of NADPH oxidase subunits was assessed by RT-PCR (C). For quantification, mRNA expression was normalized to the expression of GAPDH (mean±SEM; n=6 per group; *P<0.05 vs active mice). Aortic membrane and cytosolic protein expression of the NADPH oxidase subunits p47phox and p67phox and the housekeeping gene ?-actin were determined by Western blot analysis (n=6 per group). D, Representative blots.
To evaluate the localization of increased ROS formation and NADPH oxidase subunit expression within the aortic wall, dihydroethidium (DHE) fluorescence microscopy and immunohistochemical stainings of aortic sections were performed. These experiments revealed that ROS production, as well as protein expression of the NADPH oxidase subunits p47phox and p67phox, was predominantly increased in the endothelium and the media of the aortic wall of inactive mice as opposed to active animals (Figure I, available online at http://atvb.ahajournals.org).
Physical Inactivity Impairs Endothelium-Dependent Vasorelaxation
To address the significance of the pro-oxidative effects of physical inactivity for vascular pathology, experiments were repeated with apoE–/– mice treated with high-cholesterol diet. Figure 3 shows that inactive apoE–/– mice developed severe endothelial dysfunction compared with wild-type animals (n=6 per group; P<0.05), as demonstrated by impaired endothelium-dependent vasorelaxation in isolated aortic segments, whereas endothelium-independent vasodilation was not altered. In contrast, voluntary running significantly improved endothelium-dependent vasorelaxation (n=8 per group; P<0.05).
Figure 3. Endothelium-dependent and -independent vasorelaxation. ApoE–/– mice were treated with a high-cholesterol diet and were in parallel subjected to voluntary exercising (active) or sedentary lifestyle (inactive). Aortic segments of these apoE–/– mice and of wild-type mice were isolated and their functional performance was assessed in organ chamber experiments. A and B, Endothelium-dependent vasorelaxation induced by carbachol (A) and endothelium-independent vasodilation induced by nitroglycerin (B) expressed in percentage of maximal phenylephrine-induced vasoconstriction (mean±SEM; n=6 to 8 per group; *P<0.05 vs inactive apoE–/– mice; #P<0.05 vs active and inactive apoE–/– mice).
Atherosclerotic Plaque Formation Is Accelerated in Sedentary ApoE–/– Mice
ApoE–/– mice developed atherosclerotic lesions in the aortic root and ascending aorta after 6 weeks of treatment with high-cholesterol diet. A representative example of an inactive and an active apoE–/– mouse is shown in Figure 4A. Histomorphometric analysis revealed that physically inactive apoE–/– mice experienced significantly accelerated atherosclerotic lesion formation in the aortic root and the ascending aorta compared with physically active mice (n=8 per group; P<0.05; Figure 4B).
Figure 4. Physical inactivity, atherosclerotic lesion formation, and oxidative stress in apoE–/– mice. Atherosclerotic lesion formation in the aortic sinus and the ascending aorta was determined by oil red-O stainings in active and inactive cholesterol-fed apoE–/– mice. Representative examples (A) and histomorphometric analysis (B; mean±SEM; n=8 per group; *P<0.05 vs active mice). Aortic superoxide production (C) as determined by L-012 chemiluminescence and corresponding NADPH oxidase activity (D) in wild-type (wt) and apoE–/– mice (mean±SEM; n=8 per group; *P<0.05 vs wild-type; #P<0.05 vs wild-type and active apoE–/–).
Physical Inactivity Increases Vascular Oxidative Stress in ApoE–/– Mice
Compared with wild-type animals, cholesterol-fed apoE–/– mice displayed 2-fold higher vascular superoxide levels, as assessed by L-012 chemiluminescence (n=8 per group; P<0.05; Figure 4C). Importantly, physical inactivity increased vascular superoxide production even further in the apoE–/– mice (188±14% compared with active apoE–/– mice; n=8 per group; P<0.05). Similarly, apoE–/– animals showed upregulation of vascular NADPH oxidase activity compared with wild-type mice, which was further increased by physical inactivity (n=8 per group; P<0.05; Figure 4D).
Physical Inactivity and Regulation of Endothelial NO Synthase
Voluntary running was associated with marked upregulation of vascular endothelial NO synthase (NOS) mRNA and protein expression and NOS activity in wild-type mice compared with inactive wild-type animals (n=4 to 8 per group; P<0.05; Figure 5A through 5C). Inactive apoE–/– mice showed a trend toward lower aortic NOS activity compared with inactive wild-type animals and active apoE–/– mice, which was not statistically significant (n=6 per group). In contrast to wild-type mice, voluntary running did not significantly increase NOS activity in apoE–/– animals (n=6 per group; Figure 5C). Treatment with NG-nitro-L-arginine methyl ester (L-NAME) inhibited NOS activity in active wild-type and apoE–/– mice (n=6 per group; P<0.05).
Figure 5. Physical inactivity and regulation of eNOS. Aortic protein (A) and mRNA expression (B) of eNOS in active and inactive wild-type mice, as assessed by Western blot analysis and RT-PCR (mean±SEM; n=4 and n=8 per group, respectively; *P<0.05 vs active mice). Aortic NOS activity (C) as determined by [3H]-arginine-citrulline conversion assays in active and inactive wild-type (wt) and ApoE–/– mice in the presence and absence of L-NAME, (mean±SEM; n=6 per group; *P<0.05 vs active wild-type; #P<0.05 vs active and inactive ApoE–/–; P<0.05 vs active and inactive wild-type).
Discussion
This study compared oxidative stress, endothelial function, and atherosclerosis between mice kept under usual conditions in regular cages with mice in identical cages supplied with a running wheel. The mice spent considerable time, mostly at night, running in and on the wheels, as reflected by the mean distance of 4.9 km per 24 hours. In contrast to previous studies, neither of the 2 groups was subjected to stress, force, or manipulation of any kind.17–20 A second major difference to previously reported experimental training protocols of limited (eg, 30 minutes per day, 5 days per week) intensive exercise (eg, swimming or motorized treadmill) is the moderate but repetitive pattern of exercise chosen voluntarily by the mice. Therefore, we propose that having the opportunity to exercise resembles the natural habitat of mice more closely than cages without running wheels. Consequently, the inactivity of regular laboratory mice has to be considered the experimental intervention in this study. This notion is supported by previous findings that survival rates decrease in sedentary as opposed to exercising rodents.21
Five independent assays were applied to assess the effect of physical inactivity on vascular oxidative stress. In wild-type mice, physical inactivity significantly increased vascular lipid peroxidation as a global marker of oxidative stress. More specific analysis of intact aortic rings showed increased superoxide production in sedentary mice, as assessed by L-012 chemiluminescence, superoxide dismutase–inhibitable cytochrome C reduction assays, and DHE staining. This finding may be important because superoxide has been shown to promote endothelial dysfunction and atherosclerosis.14 Activity of vascular NADPH oxidase was measured because this enzyme represents a major source of superoxide in the vascular wall14,22 and was found to be increased in sedentary mice. NADPH oxidase is a multicomponent enzyme complex that consists of the membrane-bound cytochrome b558, which is a heterodimer of gp91phox and p22phox in endothelial cells and nox1 and p22phox in smooth muscle cells, and the cytosolic regulatory subunits p47phox, p67phox, and rac1 GTPase. Translocation of these cytosolic regulatory subunits to the plasma membrane is a prerequisite for oxidase activation and ROS production.14,23 Rac1 GTPase has been shown to be a central regulator of NADPH oxidase–induced superoxide release in the vasculature and in the myocardium.24–26 In addition, it has been demonstrated that increased expression levels of p22phox and nox1 are associated with enhanced NADPH oxidase activity.27 Physically inactive mice displayed increased rac1 GTPase activity and enhanced mRNA and membrane protein expression of p47phox and p67phox compared with active mice. Furthermore, expression of nox1 was found to be increased in sedentary mice, whereas expression levels of the subunits p22phox, gp91phox, and nox4 were not different between the groups. Regulation of rac1 GTPase by physical activity may have implications for the cardiovascular system beyond regulation of NADPH oxidase; however, further studies are needed to address this point.
The increase of superoxide production was not limited to sedentary wild-type animals but was also observed in apoE–/– mice despite higher baseline levels of superoxide release. Vascular superoxide production and NADPH oxidase activity were significantly higher in apoE–/– mice than in wild-type animals, indicating the potential importance of oxidative stress in the pathology of atherogenesis in this animal model. In agreement with previous studies, running wild-type mice showed increased endothelial NOS (eNOS) expression.18,28 Although eNOS may uncouple under certain conditions and become a superoxide-producing enzyme,14,29 the reduction of superoxide production measured in the same samples suggests that NO production and the superoxide-scavenging properties of NO predominate under the exercising conditions of the present study. Meilhac et al found that intensive running on a motorized treadmill 5 days per week for 6 to 12 weeks increased lipid peroxidation and eNOS expression in cholesterol-fed low-density lipoprotein receptor–deficient mice.19 It is interesting to speculate that this form of intensive short-term exercise may result in uncoupling of eNOS and a pro-oxidant status, whereas a continuous pattern of running does not. This is supported by a recent study showing augmentation of endothelium-dependent vasodilation by moderate-intensity aerobic exercise through increased production of NO but increased oxidative stress after high-intensity exercise.16 In rats, administration of the eNOS inhibitor L-NAME reduced the benefits of physical training on the vessel wall after balloon injury.28 In eNOS–/– mice, moderate exercise was shown to worsen energy metabolism in oxidative skeletal muscle.30 In contrast to wild-type mice, physical exercise did not increase vascular NOS activity in apoE–/– mice. It is not clear whether the lack of eNOS responsiveness to physical activity contributes to atherogenesis in this model. The role of eNOS during atherogenesis in apoE–/– mice may be double-edged because overexpression and inhibition have been reported to accelerate lesion formation in apoE–/– mice.31,32 These data, together with our findings, show that eNOS-dependent as well as eNOS-independent mechanisms (such as regulation of NADPH oxidase) contribute to the regulation of vascular free radical load and NO bioavailability by physical activity in wild-type and apoE–/– mice. Clearly, additional studies are needed to further dissect the role of eNOS in the pathogenesis of atherosclerosis of apoE–/– mice.
The major finding of this study is the impairment of endothelial function and acceleration of atherosclerosis in inactive animals compared with mice equipped with running wheels. These results suggest that sedentary lifestyle is associated with enhanced vascular oxidative stress, which, in turn, propagates vascular dysfunction. It may be speculated that these mechanisms may contribute to the elevated cardiovascular event rates associated with physical inactivity in humans. In addition, our findings may have implication for the future design of animal studies because the animal husbandry in cages without the possibility to exercise may not reflect the desired baseline condition frequently equated with the control setting. According to the presented data, physical inactivity is a risk factor for vascular disease by promoting NADPH oxidase activity, resulting in increased vascular superoxide release and ultimately vascular dysfunction and atherosclerotic lesion formation. Physical activity is a powerful intervention to improve endothelial function and to prevent progression of atherosclerosis.
Acknowledgments
This study was supported by the Deutsche Forschungsgemeinschaft (to U.L., M.B., and G.N.) and by the European Vascular Genomics Network, a Network of Excellence granted by the European Commission (Contract LSHM-CT-2003-503254). We thank Simone J?ger and Isabel Páez-Maletz for excellent technical assistance.
References
Kimm SY, Glynn NW, Kriska AM, Barton BA, Kronsberg SS, Daniels SR, Crawford PB, Sabry ZI, Liu K. Decline in physical activity in black girls and white girls during adolescence. N Engl J Med. 2002; 347: 709–715.
Bull F. Defining physical inactivity. Lancet. 2003; 361: 258–259.
Yancey AK, Wold CM, McCarthy WJ, Weber MD, Lee B, Simon PA, Fielding JE. Physical inactivity and overweight among Los Angeles County adults. Am J Prev Med. 2004; 27: 146–152.
Hakim AA, Petrovitch H, Burchfiel CM, Ross GW, Rodriguez BL, White LR, Yano K, Curb JD, Abbott RD. Effects of walking on mortality among nonsmoking retired men. N Engl J Med. 1998; 338: 94–99.
Manson JE, Greenland P, LaCroix AZ, Stefanick ML, Mouton CP, Oberman A, Perri MG, Sheps DS, Pettinger MB, Siscovick DS. Walking compared with vigorous exercise for the prevention of cardiovascular events in women. N Engl J Med. 2002; 347: 716–725.
Lees SJ, Booth FW. Sedentary death syndrome. Can J Appl Physiol. 2004; 29: 447–460.
Hambrecht R, Wolf A, Gielen S, Linke A, Hofer J, Erbs S, Schoene N, Schuler G. Effect of exercise on coronary endothelial function in patients with coronary artery disease. N Engl J Med. 2000; 342: 454–460.
Belardinelli R, Paolini I, Cianci G, Piva R, Georgiou D, Purcaro A. Exercise training intervention after coronary angioplasty: the ETICA trial. J Am Coll Cardiol. 2001; 37: 1891–1900.
Schuler G, Hambrecht R, Schlierf G, Niebauer J, Hauer K, Neumann J, Hoberg E, Drinkmann A, Bacher F, Grunze M. Regular physical exercise and low-fat diet. Effects on progression of coronary artery disease. Circulation. 1992; 86: 1–11.
Rauramaa R, Halonen P, Vaisanen SB, Lakka TA, Schmidt-Trucksass A, Berg A, Penttila IM, Rankinen T, Bouchard C. Effects of aerobic physical exercise on inflammation and atherosclerosis in men: the DNASCO study: a six-year randomized, controlled trial. Ann Intern Med. 2004; 140: 1007–1014.
Nordstrom CK, Dwyer KM, Merz CN, Shircore A, Dwyer JH. Leisure time physical activity and early atherosclerosis: the Los Angeles Atherosclerosis Study. Am J Med. 2003; 115: 19–25.
Stewart KJ. Exercise training and the cardiovascular consequences of type 2 diabetes and hypertension: plausible mechanisms for improving cardiovascular health. J Am Med Assoc. 2002; 288: 1622–1631.
Wannamethee SG, Lowe GD, Whincup PH, Rumley A, Walker M, Lennon L. Physical activity and hemostatic and inflammatory variables in elderly men. Circulation. 2002; 105: 1785–1790.
Harrison D, Griendling KK, Landmesser U, Hornig B, Drexler H. Role of oxidative stress in atherosclerosis. Am J Cardiol. 2003; 91: 7A–11A.
Chevion S, Moran DS, Heled Y, Shani Y, Regev G, Abbou B, Berenshtein E, Stadtman ER, Epstein Y. Plasma antioxidant status and cell injury after severe physical exercise. Proc Natl Acad Sci U S A. 2003; 100: 5119–5123.
Goto C, Higashi Y, Kimura M, Noma K, Hara K, Nakagawa K, Kawamura M, Chayama K, Yoshizumi M, Nara I. Effect of different intensities of exercise on endothelium-dependent vasodilation in humans: role of endothelium-dependent nitric oxide and oxidative stress. Circulation. 2003; 108: 530–535.
Rush JW, Turk JR, Laughlin MH. Exercise training regulates SOD-1 and oxidative stress in porcine aortic endothelium. Am J Physiol Heart Circ Physiol. 2003; 284: H1378–H1387.
Kojda G, Cheng YC, Burchfield J, Harrison DG. Dysfunctional regulation of endothelial nitric oxide synthase (eNOS) expression in response to exercise in mice lacking one eNOS gene. Circulation. 2001; 103: 2839–2844.
Meilhac O, Ramachandran S, Chiang K, Santanam N, Parthasarathy S. Role of arterial wall antioxidant defense in beneficial effects of exercise on atherosclerosis in mice. Arterioscler Thromb Vasc Biol. 2001; 21: 1681–1688.
Napoli C, Williams-Ignarro S, De Nigris F, Lerman LO, Rossi L, Guarino C, Mansueto G, Di Tuoro F, Pignalosa O, De Rosa G, Sica V, Ignarro LJ. Long-term combined beneficial effects of physical training and metabolic treatment on atherosclerosis in hypercholesterolemic mice. Proc Natl Acad Sci U S A. 2004; 101: 8797–8802.
Holloszy JO, Smith EK. Effects of exercise on longevity of rats. Fed Proc. 1987; 46: 1850–1853.
Nickenig G, Harrison DG. The AT(1)-type angiotensin receptor in oxidative stress and atherogenesis: part I: oxidative stress and atherogenesis. Circulation. 2002; 105: 393–396.
Landmesser U, Cai H, Dikalov S, McCann L, Hwang J, Jo H, Holland SM, Harrison DG. Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension. 2002; 40: 511–515.
Bokoch GM, Diebold BA. Current molecular models for NADPH oxidase regulation by Rac GTPase. Blood. 2002; 100: 2692–2696.
Maack C, Kartes T, Kilter H, Sch?fers HJ, Nickenig G, B?hm M, Laufs U. Oxygen free radical release in human failing myocardium is associated with increased activity of Rac1-GTPase and represents a target for statin treatment. Circulation. 2003; 108: 1567–1574.
Wassmann S, Laufs U, Baumer AT, Muller K, Konkol C, Sauer H, B?hm M, Nickenig G. Inhibition of geranylgeranylation reduces angiotensin II-mediated free radical production in vascular smooth muscle cells: involvement of angiotensin AT1 receptor expression and Rac1 GTPase. Mol Pharmacol. 2001; 59: 646–654.
Ambasta RK, Kumar P, Griendling KK, Schmidt HH, Busse R, Brandes RP. Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J Biol Chem. 2004; 279: 45935–45941.
Indolfi C, Torella D, Coppola C, Curcio A, Rodriguez F, Bilancio A, Leccia A, Arcucci O, Falco M, Leosco D, Chiariello M. Physical training increases eNOS vascular expression and activity and reduces restenosis after balloon angioplasty or arterial stenting in rats. Circ Res. 2002; 91: 1190–1197.
Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, Harrison DG. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest. 2003; 111: 1201–1209.
Momken I, Lechene P, Ventura-Clapier R, Veksler V. Voluntary physical activity alterations in endothelial nitric oxide synthase knockout mice. Am J Physiol Heart Circ Physiol. 2004; 287: H914–H920.
Kuhlencordt PJ, Gyurko R, Han F, Scherrer-Crosbie M, Aretz TH, Hajjar R, Picard MH, Huang PL. Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase double-knockout mice. Circulation. 2001; 104: 448–454.
Ozaki M, Kawashima S, Yamashita T, Hirase T, Namiki M, Inoue N, Hirata K, Yasui H, Sakurai H, Yoshida Y, Masada M, Yokoyama M. Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deficient mice. J Clin Invest. 2002; 110: 331–340.(Ulrich Laufs; Sven Wassma)
Correspondence to Dr Ulrich Laufs, Klinik für Innere Medizin III, Universit?tsklinikum des Saarlandes, D-66421 Homburg/Saar, Germany. E-mail ulrich@laufs.com
Abstract
Objective— Sedentary lifestyle is associated with increased cardiovascular events. The underlying molecular mechanisms are incompletely understood. Reactive oxygen species (ROS) contribute to endothelial dysfunction and atherosclerosis. An important source of vascular ROS is the NADPH oxidase.
Methods and Results— C57BL6 mice were subjected to regular housing (physical inactivity) or voluntary training on running wheels (6 weeks). Inactivity increased vascular lipid peroxidation to 148±9% and upregulated superoxide release to 176±17% (L-012 chemiluminescence) and 188±29% (cytochrome C reduction assay), respectively. ROS production was predominantly increased in the endothelium and the media (dihydroethidium fluorescence). Activity of the NADPH oxidase was increased to 154±22% in the sedentary group. Rac1 GST-PAK pull-down assays showed an upregulation of rac1 activity to 161±14%. Expression levels of the subunits nox1, p47phox, and p67phox were increased. To address the significance of the antioxidative effects of running, experiments were repeated in apolipoprotein E–deficient mice treated with a high-cholesterol diet. Inactivity increased vascular superoxide production and impaired endothelium-dependent vasorelaxation. Atherosclerotic lesion formation was significantly accelerated in sedentary mice.
Conclusions— Inactivity increases vascular NADPH oxidase expression and activity and enhances vascular ROS production, which contributes to endothelial dysfunction and atherosclerosis during sedentary as opposed to physically active lifestyle.
Sedentary lifestyle predicts vascular risk. To study underlying mechanisms, mice were subjected to physical inactivity or voluntary training on running wheels. Inactivity increased vascular lipid peroxidation, superoxide release, and NADPH oxidase expression and activity. In apoE–/– mice, inactivity significantly impaired endothelium-dependent vasorelaxation and accelerated atherosclerotic lesion formation.
Key Words: physical inactivity ? exercise ? oxidative stress ? endothelial dysfunction ? atherosclerosis
Introduction
The prevalence of physical inactivity in adults is increasing.1–3 Nearly 40% of the adult population subject themselves to <10 minutes of continuous physical activity per week.3 These lifestyle changes are of special interest because physical inactivity has been proposed as an independent cardiovascular risk factor.4,5 However, the molecular mechanisms leading from sedentary lifestyle to cardiovascular events are poorly defined.6
On the other hand, prospective epidemiological data indicate that moderate (eg, walking) and vigorous exercise are associated with substantial reductions in the incidence of cardiovascular events.4,5 Physical training improves endothelial function, exercise capacity, and collateralization in patients with coronary artery disease7–9 and prevents the progression of carotid atherosclerosis.10,11 Among other beneficial effects, physical activity is associated with improved mood, body weight, blood pressure, insulin sensitivity, and hemostatic and inflammatory variables.12,13
Reactive oxygen species (ROS) play a pivotal role in the pathogenesis of endothelial dysfunction and atherosclerosis.14 However, uncertainty remains regarding the effect of exercise on ROS. Acutely, intensive exercise has been shown to increase oxidative stress because increased aerobic metabolism is a source of oxidative stress,15,16 whereas long-term moderate exercise may upregulate antioxidative enzymes and decrease indices of oxidative stress.16,17
The aim of this study was to compare the effects of physical inactivity with voluntary running. Mice were subjected to regular housing or cages equipped with running wheels. We postulated that giving the animals the opportunity of voluntary running resembles their natural habitat more closely and tested the effects of voluntary running compared with sedentary behavior on parameters of vascular oxidative stress. In addition, we used the model of cholesterol-fed apolipoprotein E–deficient (apoE–/–) mice to examine the effects of inactivity versus running on endothelial function and atherosclerotic lesion formation.
Methods
The Methods section is available in the online data supplement, available at http://atvb.ahajournals.org.
Results
Physical Inactivity Increases Vascular Oxidative Stress
The mean running distance of active mice was 4900±700 m per 24 hours. Body weight did not differ between active and inactive mice (24±0.3 versus 23±0.3 g). As a global parameter of oxidative stress, lipid peroxidation of the aortic wall was compared. Inactive mice displayed upregulation of vascular lipid hydroperoxides to 148±9% compared with active mice (n=6 per group; P<0.05; Figure 1A). Because superoxide radicals are involved in the pathogenesis of atherosclerosis, we determined aortic superoxide production by 2 different methods. As shown in Figure 1B and 1C, vascular superoxide release was significantly increased in inactive compared with active mice (176±17% as assessed by L-012 chemiluminescence assays [n=8 per group; P<0.05] and 188±29% as determined by cytochrome C reduction assays [n=4 per group; P<0.05], respectively). Because NADPH oxidase is a major source of superoxide radicals in the vascular wall, we assessed NADPH oxidase activity in aortic tissue. Figure 1D displays that NADPH oxidase activity was upregulated to 154±22% (n=6 per group; P<0.05) in the sedentary group.
Figure 1. Physical inactivity and vascular oxidative stress. Parameters of vascular oxidative stress were assessed in sedentary (inactive) and voluntarily running (active) wild-type mice. Concentrations of lipid hydroperoxides were determined in aortic homogenates (A; mean±SEM; n=6 per group; *P<0.05 vs active mice). Superoxide production in intact aortic ring preparations was assessed by L-012 chemiluminescence (B) and by the superoxide dismutase–inhibitable cytochrome C reduction assay (C; mean±SEM; n=8 and n=4 per group, respectively; *P<0.05 vs active mice). NADPH oxidase activity in aortic homogenates was measured by a lucigenin-enhanced chemiluminescence assay using NADPH (100 μmol/L) as substrate and 5 μmol/L lucigenin (D; mean±SEM; n=6 per group; *P<0.05 vs active mice).
Upregulation of NADPH Oxidase Subunits in Inactive Mice
Translocation of the cytosolic regulatory subunits p47phox, p67phox, and the small GTP-binding protein rac1 to the plasma membrane is a prerequisite for NADPH oxidase activation and subsequent ROS production. Rac1 GST-PAK pull-down assays were performed to quantitate rac1 activity. In inactive mice, rac1 activity was increased to 161±14% compared with active animals (n=6 per group; P<0.05; Figure 2A and 2B). In addition, RT-PCR analysis showed increased expression of the subunits p47phox (285±19%) and p67phox (161±23%) in sedentary mice as opposed to active mice (n=6 per group; P<0.05; Figure 2C). Similarly, membrane protein expression of p47phox (343±29%) and p67phox (180±25%) was increased in the inactive mice (n=6 per group; P<0.05), whereas no difference was observed in cytosolic protein expression of these NADPH oxidase subunits (Figure 2D). Furthermore, inactive mice displayed higher expression levels of the nox1 subunit of NADPH oxidase (181±37%; n=6 per group; P<0.05), whereas the subunits p22phox, gp91phox, and nox4 remained unaltered between the groups (Figure 2C).
Figure 2. Physical inactivity and NADPH oxidase subunits. NADPH oxidase subunits were assessed in active and inactive wild-type mice. Rac1 GTPase activity in aortic homogenates was determined by rac1 GST-PAK pull-down assays. A and B, Representative example (A) and densitometric quantification (B; mean±SEM; n=6 per group; *P<0.05 vs active mice). Aortic mRNA expression of NADPH oxidase subunits was assessed by RT-PCR (C). For quantification, mRNA expression was normalized to the expression of GAPDH (mean±SEM; n=6 per group; *P<0.05 vs active mice). Aortic membrane and cytosolic protein expression of the NADPH oxidase subunits p47phox and p67phox and the housekeeping gene ?-actin were determined by Western blot analysis (n=6 per group). D, Representative blots.
To evaluate the localization of increased ROS formation and NADPH oxidase subunit expression within the aortic wall, dihydroethidium (DHE) fluorescence microscopy and immunohistochemical stainings of aortic sections were performed. These experiments revealed that ROS production, as well as protein expression of the NADPH oxidase subunits p47phox and p67phox, was predominantly increased in the endothelium and the media of the aortic wall of inactive mice as opposed to active animals (Figure I, available online at http://atvb.ahajournals.org).
Physical Inactivity Impairs Endothelium-Dependent Vasorelaxation
To address the significance of the pro-oxidative effects of physical inactivity for vascular pathology, experiments were repeated with apoE–/– mice treated with high-cholesterol diet. Figure 3 shows that inactive apoE–/– mice developed severe endothelial dysfunction compared with wild-type animals (n=6 per group; P<0.05), as demonstrated by impaired endothelium-dependent vasorelaxation in isolated aortic segments, whereas endothelium-independent vasodilation was not altered. In contrast, voluntary running significantly improved endothelium-dependent vasorelaxation (n=8 per group; P<0.05).
Figure 3. Endothelium-dependent and -independent vasorelaxation. ApoE–/– mice were treated with a high-cholesterol diet and were in parallel subjected to voluntary exercising (active) or sedentary lifestyle (inactive). Aortic segments of these apoE–/– mice and of wild-type mice were isolated and their functional performance was assessed in organ chamber experiments. A and B, Endothelium-dependent vasorelaxation induced by carbachol (A) and endothelium-independent vasodilation induced by nitroglycerin (B) expressed in percentage of maximal phenylephrine-induced vasoconstriction (mean±SEM; n=6 to 8 per group; *P<0.05 vs inactive apoE–/– mice; #P<0.05 vs active and inactive apoE–/– mice).
Atherosclerotic Plaque Formation Is Accelerated in Sedentary ApoE–/– Mice
ApoE–/– mice developed atherosclerotic lesions in the aortic root and ascending aorta after 6 weeks of treatment with high-cholesterol diet. A representative example of an inactive and an active apoE–/– mouse is shown in Figure 4A. Histomorphometric analysis revealed that physically inactive apoE–/– mice experienced significantly accelerated atherosclerotic lesion formation in the aortic root and the ascending aorta compared with physically active mice (n=8 per group; P<0.05; Figure 4B).
Figure 4. Physical inactivity, atherosclerotic lesion formation, and oxidative stress in apoE–/– mice. Atherosclerotic lesion formation in the aortic sinus and the ascending aorta was determined by oil red-O stainings in active and inactive cholesterol-fed apoE–/– mice. Representative examples (A) and histomorphometric analysis (B; mean±SEM; n=8 per group; *P<0.05 vs active mice). Aortic superoxide production (C) as determined by L-012 chemiluminescence and corresponding NADPH oxidase activity (D) in wild-type (wt) and apoE–/– mice (mean±SEM; n=8 per group; *P<0.05 vs wild-type; #P<0.05 vs wild-type and active apoE–/–).
Physical Inactivity Increases Vascular Oxidative Stress in ApoE–/– Mice
Compared with wild-type animals, cholesterol-fed apoE–/– mice displayed 2-fold higher vascular superoxide levels, as assessed by L-012 chemiluminescence (n=8 per group; P<0.05; Figure 4C). Importantly, physical inactivity increased vascular superoxide production even further in the apoE–/– mice (188±14% compared with active apoE–/– mice; n=8 per group; P<0.05). Similarly, apoE–/– animals showed upregulation of vascular NADPH oxidase activity compared with wild-type mice, which was further increased by physical inactivity (n=8 per group; P<0.05; Figure 4D).
Physical Inactivity and Regulation of Endothelial NO Synthase
Voluntary running was associated with marked upregulation of vascular endothelial NO synthase (NOS) mRNA and protein expression and NOS activity in wild-type mice compared with inactive wild-type animals (n=4 to 8 per group; P<0.05; Figure 5A through 5C). Inactive apoE–/– mice showed a trend toward lower aortic NOS activity compared with inactive wild-type animals and active apoE–/– mice, which was not statistically significant (n=6 per group). In contrast to wild-type mice, voluntary running did not significantly increase NOS activity in apoE–/– animals (n=6 per group; Figure 5C). Treatment with NG-nitro-L-arginine methyl ester (L-NAME) inhibited NOS activity in active wild-type and apoE–/– mice (n=6 per group; P<0.05).
Figure 5. Physical inactivity and regulation of eNOS. Aortic protein (A) and mRNA expression (B) of eNOS in active and inactive wild-type mice, as assessed by Western blot analysis and RT-PCR (mean±SEM; n=4 and n=8 per group, respectively; *P<0.05 vs active mice). Aortic NOS activity (C) as determined by [3H]-arginine-citrulline conversion assays in active and inactive wild-type (wt) and ApoE–/– mice in the presence and absence of L-NAME, (mean±SEM; n=6 per group; *P<0.05 vs active wild-type; #P<0.05 vs active and inactive ApoE–/–; P<0.05 vs active and inactive wild-type).
Discussion
This study compared oxidative stress, endothelial function, and atherosclerosis between mice kept under usual conditions in regular cages with mice in identical cages supplied with a running wheel. The mice spent considerable time, mostly at night, running in and on the wheels, as reflected by the mean distance of 4.9 km per 24 hours. In contrast to previous studies, neither of the 2 groups was subjected to stress, force, or manipulation of any kind.17–20 A second major difference to previously reported experimental training protocols of limited (eg, 30 minutes per day, 5 days per week) intensive exercise (eg, swimming or motorized treadmill) is the moderate but repetitive pattern of exercise chosen voluntarily by the mice. Therefore, we propose that having the opportunity to exercise resembles the natural habitat of mice more closely than cages without running wheels. Consequently, the inactivity of regular laboratory mice has to be considered the experimental intervention in this study. This notion is supported by previous findings that survival rates decrease in sedentary as opposed to exercising rodents.21
Five independent assays were applied to assess the effect of physical inactivity on vascular oxidative stress. In wild-type mice, physical inactivity significantly increased vascular lipid peroxidation as a global marker of oxidative stress. More specific analysis of intact aortic rings showed increased superoxide production in sedentary mice, as assessed by L-012 chemiluminescence, superoxide dismutase–inhibitable cytochrome C reduction assays, and DHE staining. This finding may be important because superoxide has been shown to promote endothelial dysfunction and atherosclerosis.14 Activity of vascular NADPH oxidase was measured because this enzyme represents a major source of superoxide in the vascular wall14,22 and was found to be increased in sedentary mice. NADPH oxidase is a multicomponent enzyme complex that consists of the membrane-bound cytochrome b558, which is a heterodimer of gp91phox and p22phox in endothelial cells and nox1 and p22phox in smooth muscle cells, and the cytosolic regulatory subunits p47phox, p67phox, and rac1 GTPase. Translocation of these cytosolic regulatory subunits to the plasma membrane is a prerequisite for oxidase activation and ROS production.14,23 Rac1 GTPase has been shown to be a central regulator of NADPH oxidase–induced superoxide release in the vasculature and in the myocardium.24–26 In addition, it has been demonstrated that increased expression levels of p22phox and nox1 are associated with enhanced NADPH oxidase activity.27 Physically inactive mice displayed increased rac1 GTPase activity and enhanced mRNA and membrane protein expression of p47phox and p67phox compared with active mice. Furthermore, expression of nox1 was found to be increased in sedentary mice, whereas expression levels of the subunits p22phox, gp91phox, and nox4 were not different between the groups. Regulation of rac1 GTPase by physical activity may have implications for the cardiovascular system beyond regulation of NADPH oxidase; however, further studies are needed to address this point.
The increase of superoxide production was not limited to sedentary wild-type animals but was also observed in apoE–/– mice despite higher baseline levels of superoxide release. Vascular superoxide production and NADPH oxidase activity were significantly higher in apoE–/– mice than in wild-type animals, indicating the potential importance of oxidative stress in the pathology of atherogenesis in this animal model. In agreement with previous studies, running wild-type mice showed increased endothelial NOS (eNOS) expression.18,28 Although eNOS may uncouple under certain conditions and become a superoxide-producing enzyme,14,29 the reduction of superoxide production measured in the same samples suggests that NO production and the superoxide-scavenging properties of NO predominate under the exercising conditions of the present study. Meilhac et al found that intensive running on a motorized treadmill 5 days per week for 6 to 12 weeks increased lipid peroxidation and eNOS expression in cholesterol-fed low-density lipoprotein receptor–deficient mice.19 It is interesting to speculate that this form of intensive short-term exercise may result in uncoupling of eNOS and a pro-oxidant status, whereas a continuous pattern of running does not. This is supported by a recent study showing augmentation of endothelium-dependent vasodilation by moderate-intensity aerobic exercise through increased production of NO but increased oxidative stress after high-intensity exercise.16 In rats, administration of the eNOS inhibitor L-NAME reduced the benefits of physical training on the vessel wall after balloon injury.28 In eNOS–/– mice, moderate exercise was shown to worsen energy metabolism in oxidative skeletal muscle.30 In contrast to wild-type mice, physical exercise did not increase vascular NOS activity in apoE–/– mice. It is not clear whether the lack of eNOS responsiveness to physical activity contributes to atherogenesis in this model. The role of eNOS during atherogenesis in apoE–/– mice may be double-edged because overexpression and inhibition have been reported to accelerate lesion formation in apoE–/– mice.31,32 These data, together with our findings, show that eNOS-dependent as well as eNOS-independent mechanisms (such as regulation of NADPH oxidase) contribute to the regulation of vascular free radical load and NO bioavailability by physical activity in wild-type and apoE–/– mice. Clearly, additional studies are needed to further dissect the role of eNOS in the pathogenesis of atherosclerosis of apoE–/– mice.
The major finding of this study is the impairment of endothelial function and acceleration of atherosclerosis in inactive animals compared with mice equipped with running wheels. These results suggest that sedentary lifestyle is associated with enhanced vascular oxidative stress, which, in turn, propagates vascular dysfunction. It may be speculated that these mechanisms may contribute to the elevated cardiovascular event rates associated with physical inactivity in humans. In addition, our findings may have implication for the future design of animal studies because the animal husbandry in cages without the possibility to exercise may not reflect the desired baseline condition frequently equated with the control setting. According to the presented data, physical inactivity is a risk factor for vascular disease by promoting NADPH oxidase activity, resulting in increased vascular superoxide release and ultimately vascular dysfunction and atherosclerotic lesion formation. Physical activity is a powerful intervention to improve endothelial function and to prevent progression of atherosclerosis.
Acknowledgments
This study was supported by the Deutsche Forschungsgemeinschaft (to U.L., M.B., and G.N.) and by the European Vascular Genomics Network, a Network of Excellence granted by the European Commission (Contract LSHM-CT-2003-503254). We thank Simone J?ger and Isabel Páez-Maletz for excellent technical assistance.
References
Kimm SY, Glynn NW, Kriska AM, Barton BA, Kronsberg SS, Daniels SR, Crawford PB, Sabry ZI, Liu K. Decline in physical activity in black girls and white girls during adolescence. N Engl J Med. 2002; 347: 709–715.
Bull F. Defining physical inactivity. Lancet. 2003; 361: 258–259.
Yancey AK, Wold CM, McCarthy WJ, Weber MD, Lee B, Simon PA, Fielding JE. Physical inactivity and overweight among Los Angeles County adults. Am J Prev Med. 2004; 27: 146–152.
Hakim AA, Petrovitch H, Burchfiel CM, Ross GW, Rodriguez BL, White LR, Yano K, Curb JD, Abbott RD. Effects of walking on mortality among nonsmoking retired men. N Engl J Med. 1998; 338: 94–99.
Manson JE, Greenland P, LaCroix AZ, Stefanick ML, Mouton CP, Oberman A, Perri MG, Sheps DS, Pettinger MB, Siscovick DS. Walking compared with vigorous exercise for the prevention of cardiovascular events in women. N Engl J Med. 2002; 347: 716–725.
Lees SJ, Booth FW. Sedentary death syndrome. Can J Appl Physiol. 2004; 29: 447–460.
Hambrecht R, Wolf A, Gielen S, Linke A, Hofer J, Erbs S, Schoene N, Schuler G. Effect of exercise on coronary endothelial function in patients with coronary artery disease. N Engl J Med. 2000; 342: 454–460.
Belardinelli R, Paolini I, Cianci G, Piva R, Georgiou D, Purcaro A. Exercise training intervention after coronary angioplasty: the ETICA trial. J Am Coll Cardiol. 2001; 37: 1891–1900.
Schuler G, Hambrecht R, Schlierf G, Niebauer J, Hauer K, Neumann J, Hoberg E, Drinkmann A, Bacher F, Grunze M. Regular physical exercise and low-fat diet. Effects on progression of coronary artery disease. Circulation. 1992; 86: 1–11.
Rauramaa R, Halonen P, Vaisanen SB, Lakka TA, Schmidt-Trucksass A, Berg A, Penttila IM, Rankinen T, Bouchard C. Effects of aerobic physical exercise on inflammation and atherosclerosis in men: the DNASCO study: a six-year randomized, controlled trial. Ann Intern Med. 2004; 140: 1007–1014.
Nordstrom CK, Dwyer KM, Merz CN, Shircore A, Dwyer JH. Leisure time physical activity and early atherosclerosis: the Los Angeles Atherosclerosis Study. Am J Med. 2003; 115: 19–25.
Stewart KJ. Exercise training and the cardiovascular consequences of type 2 diabetes and hypertension: plausible mechanisms for improving cardiovascular health. J Am Med Assoc. 2002; 288: 1622–1631.
Wannamethee SG, Lowe GD, Whincup PH, Rumley A, Walker M, Lennon L. Physical activity and hemostatic and inflammatory variables in elderly men. Circulation. 2002; 105: 1785–1790.
Harrison D, Griendling KK, Landmesser U, Hornig B, Drexler H. Role of oxidative stress in atherosclerosis. Am J Cardiol. 2003; 91: 7A–11A.
Chevion S, Moran DS, Heled Y, Shani Y, Regev G, Abbou B, Berenshtein E, Stadtman ER, Epstein Y. Plasma antioxidant status and cell injury after severe physical exercise. Proc Natl Acad Sci U S A. 2003; 100: 5119–5123.
Goto C, Higashi Y, Kimura M, Noma K, Hara K, Nakagawa K, Kawamura M, Chayama K, Yoshizumi M, Nara I. Effect of different intensities of exercise on endothelium-dependent vasodilation in humans: role of endothelium-dependent nitric oxide and oxidative stress. Circulation. 2003; 108: 530–535.
Rush JW, Turk JR, Laughlin MH. Exercise training regulates SOD-1 and oxidative stress in porcine aortic endothelium. Am J Physiol Heart Circ Physiol. 2003; 284: H1378–H1387.
Kojda G, Cheng YC, Burchfield J, Harrison DG. Dysfunctional regulation of endothelial nitric oxide synthase (eNOS) expression in response to exercise in mice lacking one eNOS gene. Circulation. 2001; 103: 2839–2844.
Meilhac O, Ramachandran S, Chiang K, Santanam N, Parthasarathy S. Role of arterial wall antioxidant defense in beneficial effects of exercise on atherosclerosis in mice. Arterioscler Thromb Vasc Biol. 2001; 21: 1681–1688.
Napoli C, Williams-Ignarro S, De Nigris F, Lerman LO, Rossi L, Guarino C, Mansueto G, Di Tuoro F, Pignalosa O, De Rosa G, Sica V, Ignarro LJ. Long-term combined beneficial effects of physical training and metabolic treatment on atherosclerosis in hypercholesterolemic mice. Proc Natl Acad Sci U S A. 2004; 101: 8797–8802.
Holloszy JO, Smith EK. Effects of exercise on longevity of rats. Fed Proc. 1987; 46: 1850–1853.
Nickenig G, Harrison DG. The AT(1)-type angiotensin receptor in oxidative stress and atherogenesis: part I: oxidative stress and atherogenesis. Circulation. 2002; 105: 393–396.
Landmesser U, Cai H, Dikalov S, McCann L, Hwang J, Jo H, Holland SM, Harrison DG. Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension. 2002; 40: 511–515.
Bokoch GM, Diebold BA. Current molecular models for NADPH oxidase regulation by Rac GTPase. Blood. 2002; 100: 2692–2696.
Maack C, Kartes T, Kilter H, Sch?fers HJ, Nickenig G, B?hm M, Laufs U. Oxygen free radical release in human failing myocardium is associated with increased activity of Rac1-GTPase and represents a target for statin treatment. Circulation. 2003; 108: 1567–1574.
Wassmann S, Laufs U, Baumer AT, Muller K, Konkol C, Sauer H, B?hm M, Nickenig G. Inhibition of geranylgeranylation reduces angiotensin II-mediated free radical production in vascular smooth muscle cells: involvement of angiotensin AT1 receptor expression and Rac1 GTPase. Mol Pharmacol. 2001; 59: 646–654.
Ambasta RK, Kumar P, Griendling KK, Schmidt HH, Busse R, Brandes RP. Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J Biol Chem. 2004; 279: 45935–45941.
Indolfi C, Torella D, Coppola C, Curcio A, Rodriguez F, Bilancio A, Leccia A, Arcucci O, Falco M, Leosco D, Chiariello M. Physical training increases eNOS vascular expression and activity and reduces restenosis after balloon angioplasty or arterial stenting in rats. Circ Res. 2002; 91: 1190–1197.
Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, Harrison DG. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest. 2003; 111: 1201–1209.
Momken I, Lechene P, Ventura-Clapier R, Veksler V. Voluntary physical activity alterations in endothelial nitric oxide synthase knockout mice. Am J Physiol Heart Circ Physiol. 2004; 287: H914–H920.
Kuhlencordt PJ, Gyurko R, Han F, Scherrer-Crosbie M, Aretz TH, Hajjar R, Picard MH, Huang PL. Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase double-knockout mice. Circulation. 2001; 104: 448–454.
Ozaki M, Kawashima S, Yamashita T, Hirase T, Namiki M, Inoue N, Hirata K, Yasui H, Sakurai H, Yoshida Y, Masada M, Yokoyama M. Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deficient mice. J Clin Invest. 2002; 110: 331–340.(Ulrich Laufs; Sven Wassma)