Atherosclerosis Supplements 18 (2015) 59e66 www.elsevier.com/locate/atherosclerosis
Impact of high-fat diet and voluntary running on body weight and endothelial function in LDL receptor knockout mice Heike Langbein1, Anja Hofmann1, Coy Brunssen, Winfried Goettsch, Henning Morawietz* Division of Vascular Endothelium and Microcirculation, Department of Medicine III, University Hospital Carl Gustav Carus, TU Dresden, Fetscherstr. 74, 01307 Dresden, Germany
Abstract Objective: Obesity and physical inactivity are important cardiovascular risk factors. Regular physical exercise has been shown to mediate beneficial effects in the prevention of cardiovascular diseases. However, the impact of physical exercise on endothelial function in proatherosclerotic low-density lipoprotein receptor deficient (LDLR/) mice has not been studied so far. Methods: Six-week-old male LDLR/ mice were fed a standard diet or a high-fat diet (39 kcal% fat diet) for 20 weeks. The impact of high-fat diet and voluntary running on body weight and amount of white adipose tissue was monitored. Basal tone and endothelial function was investigated in aortic rings using a Mulvany myograph. Results: LDLR/ mice on high-fat diet had increased cumulative food energy intake, but also higher physical activity compared to mice on control diet. Body weight and amount of visceral and retroperitoneal white adipose tissue of LDLR/ mice were significantly increased by high-fat diet and partially reduced by voluntary running. Endothelial function in aortae of LDLR/ mice was impaired after 20 weeks on standard and high-fat diet and could not be improved by voluntary running. Basal tone showed a trend to be increased by high-fat diet. Conclusion: Voluntary running reduced body weight and amount of white adipose tissue in LDLR/ mice. Endothelial dysfunction in LDLR/ mice could not be improved by voluntary running. In a clinical context, physical exercise alone might not have an influence on functional parameters and LDL-C levels in patients with familial hypercholesterolemia. However, physical activity in these patients may be in general beneficial and should be performed. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Atherosclerosis; Body weight; Endothelial function; Voluntary running
1. Introduction Obesity and physical inactivity are important cardiovascular risk factors [1]. In clinical studies, regular physical exercise has been shown to mediate beneficial effects in the prevention of cardiovascular diseases [2]. Increasing evidence supports an anti-atherogenic activity of regular physical exercise [3]. In this context, mouse models can be * Corresponding author. Tel.: þ49 351 458 6625; fax: þ49 351 458 6354. E-mail address: [email protected] (H. Morawietz). 1 Both the first two authors contributed equally to this study. http://dx.doi.org/10.1016/j.atherosclerosissup.2015.02.010 1567-5688/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
important tools to analyze the mechanism of cardiovascular diseases in vivo. Murine atherosclerotic models depend on generating a non-HDL based hypercholesterolemia. This is most readily accomplished by genetic ablation of LDL receptor (LDLR). Although LDL receptor deficiency in humans is rare, the absence of a functional LDLR in humans results in familial hypercholesterolemia with increased risk of cardiovascular disease [4]. The arterial lesions in human familial hypercholesterolemia have some of the characteristics of the mouse lesion, including the presence of lesions in the aortic valves and the aortic root [5]. Even while some clinical characteristics of patients with obesity and metabolic-vascular syndrome differ from
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patients with familial hypercholesterolemia, studies in the LDL receptor knockout mice model have significantly contributed to our understanding of the mechanisms of atherogenesis [6]. LDLR/- mouse develop less severe atherosclerotic lesions compared to apoE knockout mice [7], but have certain advantages like a lipoprotein profile closer to human disease. Furthermore, a variety of high-fat diets with different content of cholesterol has been used, so that studies with this model are often not precisely standardized or comparable from one laboratory to another [6]. In addition, the impact of regular physical exercise on endothelial function in the proatherosclerotic model of LDLR/mice has not been studied so far. Therefore, we analyzed the impact of feeding a standard or a high-fat diet with 39 kcal% fat for 20 weeks on body weight, fat accumulation and endothelial function in LDLR/- mice. Furthermore, we assessed the impact of normal or accelerated physical activity by voluntary running on these parameters in the LDLR/- model. 2. Methods 2.1. Mice Six-week-old male LDLR/- mice (B6.129S7-Ldlrtm1Her/ J; Jackson Laboratories, Ben Harbor, ME) were fed a standard diet (STD) (ssniff EF R/M CD88137 control, 11% fat, 24% protein, 65% carbohydrates, gross energy: 18.4 MJ/kg), or a high-fat diet (FD) (ssniff EF R/M modified from TD88137 þ cholesterol, 39% fat, 20% protein, 41% carbohydrates, gross energy: 22.1 MJ/kg) for 20 weeks. Development of body weight and food intake was determined once per week and cumulative food intake was quantified over 20 weeks feeding. All performed experiments were in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The animal research ethics committee of the Dresden University of Technology and the Regional Council (Regierungspra¨sidium) Dresden approved the animal facilities and the experiments according to institutional guidelines and German animal welfare regulations (AZ: 24D-9168.11-1/2007-9). 2.2. Voluntary running LDLR/- mice on either standard or high-fat diet had free access to a running wheel (Fig. 1A). The velocity of individual running wheels was recorded online (Fig. 1B). Individual running distance and median daily activity of LDLR/- mice in voluntary running cages was determined. In addition, separate groups of freely moving mice on either standard or high-fat diet housed in standard cages without running wheels were studied. They served as controls to imitate a more sedentary lifestyle compared to the increased voluntary physical activity in a running wheel.
2.3. Adipose tissue and heart weight After 20 weeks on standard or high-fat diet with or without voluntary running, mice were sacrificed and the weight of visceral and retroperitoneal white adipose tissue (WAT) was determined and normalized to body weight. In addition, the heart-to-body weight ratio was quantified. 2.4. Vascular function Vascular function was analysed in aortic segments using a Mulvany myograph [8]. First, the basal tone in all four experimental groups (LDLR/- on standard or high-fat diet with or without voluntary running) was measured after applying 20 mmHg distension to aortic segments. Endothelial function was analysed in aortic rings after precontraction with 0.3 mmol/L phenylephrine (PE) and subsequent relaxation in response to increasing concentrations of acetylcholine (ACh). The NO-mediated amount of ACh-induced relaxation was determined using NO synthase inhibitor L-NAME. To analyze vascular smooth muscle function, NO donor sodium nitroprusside (SNP) was added to PE-precontracted aortic segments. 2.5. Statistics Data are shown as means SEM. Statistical analysis was performed by Student’s t-test or One-Way ANOVA followed by Bonferroni’s method (Sigma Stat 3.11, Systat Software, Inc., San Jose, CA), as appropriate. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Voluntary running The voluntary running model was successfully established in our laboratory (Fig. 1A). The experimental set-up allows voluntary running of 6 mice (one per cage) in parallel. The major physical activity was between 6 p.m. and 3 a.m. reaching peak velocities of more than 25 m/min (Fig. 1B). LDLR/- mice were running an average distance of 4 km/d. In the first weeks of intervention the median activity of mice remarkably increased (Fig. 1C). After 4e5 weeks of voluntary running, daily activity reached a peak value slowly decreasing with increasing age of mice. Interestingly, LDLR/- mice on high-fat diet showed a higher median daily activity, compared to mice on standard diet (Fig. 1C). 3.2. Body weight and cumulative food intake Body weight in LDLR/- mice on high-fat diet constantly increased during feeding period, compared to age-matched LDLR/- mice on standard diet (Fig. 2A).
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Fig. 1. Voluntary running. A) Left: Experimental set-up allowing voluntary running of 6 mice (one per cage) in parallel. Right: Mouse in a running wheel. B) Representative recording of characteristic physical activity pattern of LDLR/- mice during 24 h with access to voluntary running. C) Monitoring of daily physical activity of LDLR/- mice during 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR). *P < 0.05 vs. STD þ VR.
After 20 weeks, body weight of LDLR/- mice on high-fat diet was almost doubled, compared to standard diet. Voluntary running decreased the body weight initially even in mice with standard diet. After 20 weeks on standard diet, no significant changes in body weight could be observed between groups with or without voluntary running. In contrast, voluntary running significantly reduced the body weight of LDLR/- mice on high-fat diet. However, body
weight was higher in the high-fat diet þ voluntary running group, compared to mice with or without voluntary running on standard diet (Fig. 2A). The cumulative food intake after 20 weeks showed a tendency to be higher in the groups of LDLR/- mice on high-fat diet with or without voluntary running, compared to mice on standard diet with or without voluntary running (Fig. 2B).
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Fig. 2. Impact of high-fat diet and voluntary running on body weight and cumulative food intake. A) Development of body weight of LDLR/- mice during 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR). ***P < 0.001 vs. STD and STD þ VR. #P < 0.05 vs. FD þ VR. B) Cumulative food intake after 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR).
3.3. Adipose tissue and heart weight The relative amount of visceral (Fig. 3A) and retroperitoneal (Fig. 3B) white adipose tissue (WAT) was increased in LDLR/- mice after 20 weeks of high-fat diet. Animals with access to voluntary running had decreased amounts of both types of WAT, compared to animals with less physical activity on the same diet. However, the development of WAT in response to high-fat diet could not be prevented completely by voluntary running (Fig. 3A, B). The heart-to-body weight ratio was decreased in LDLR/- mice after high-fat diet feeding, compared to standard diet. LDLR/- mice with voluntary running on high-fat diet had a decreased heart-to-body weight ratio compared to voluntary running on standard diet, but an increased heart-to-body ratio compared to mice on high-fat diet with lower physical activity (Fig. 3C). 3.4. Vascular function Vascular function was assessed in aortic rings of LDLR/- mice after standard diet, high-fat diet with or without voluntary running for 20 weeks. After precontraction with phenylephrine, endothelial function was determined by relaxation to increasing concentrations of
acetylcholine. The endothelial function in the aorta of all LDLR/- groups was impaired compared to wild-type mice. High-fat diet of 39 kcal% fat and voluntary running for 20 weeks had no significant effect on endothelial function in the aorta of LDLR/- mice (Fig. 4A). Relaxation in response to acetylcholine was completely inhibited using NOS inhibitor L-NG-Nitro arginine methyl ester (L-NAME) (data not shown). The vascular smooth muscle function in response to increasing dosages of the NO donor SNP was not different between all experimental groups studied (Fig. 4B). Interestingly, the basal tone showed a trend to be increased in the aorta of LDLR/- mice after high-fat diet (Fig. 4C). This was not affected by voluntary running. 4. Discussion The voluntary running model has the advantage of free access of mice to increased physical activity [9]. Using this approach mouse exercised much longer, compared to individual training on treadmill devices. The observed major physical activity at night is in agreement with the circadian rhythm of mice showing increased nocturnal activity. The running distance of LDLR/- (4 km/d) was shorter compared to previously published data of wild-type mice (6.5 km/d) [10]. This might partially reflect an
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Fig. 3. Impact of high-fat diet and voluntary running on white adipose tissue and heart weight. Weight of visceral white adipose tissue (WAT) (A), retroperitoneal WAT (B) and heart (C) was normalized to body weight of LDLR/- mice after 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR). ***P < 0.001 vs. STD. #P < 0.05 vs. STD þ VR.
impaired endothelial function in LDLR/- mice [11]. The daily activity using the running wheel increased in the first 4e5 weeks. This reflects an improved physical activity due to training effects in the first weeks. The slight decrease in
activity during the following weeks might be the result of increasing age and impairment of endothelial function in LDLR/- mice. Especially interesting is the higher physical activity of LDLR/- mice on high-fat diet. This might be
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Fig. 4. Effect of high-fat diet and voluntary running on vascular function. A) Endothelial function was analysed as concentrationeresponse curve to acetylcholine (ACh) in aortic rings of LDLR/- mice precontracted with phenylephrine in a Mulvany myograph after 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR). B) Determination of smooth muscle function using increasing concentrations of NO donor sodium nitroprusside (SNP) in aortic segments of LDLR/- mice after 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR). C) Basal tone was measured after applying 20 mmHg distension to aortic segments of LDLR/- mice after 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR).
the result of the observed higher energy uptake in the highfat diet group resulting in an increased physical performance after training, especially in the first weeks of voluntary running. In this context it has to be considered that LDLR/- mice display decreased susceptibility to Western-type diet-induced obesity due to increased thermogenesis [12]. The observed increase in body weight of LDLR/- mice on high-fat diet and the partial reduction by exercise are in agreement with previous studies using 30 min/day training on a treadmill for up to 12 weeks [13]. This reflects the beneficial effects of increased physical activity resulting in weight loss, even while feeding a high-fat diet resulted in higher energy uptake. Furthermore, voluntary running reduced the weight of mice on standard diet especially in the initial phase. At higher age, body weight and energy uptake were not different between LDLR/- mice on standard diet with or without voluntary running. The beneficial effects of voluntary running on body weight are accelerated after feeding a high-fat diet. This is in agreement with clinical studies showing efficient weight loss in patients combining a healthy diet with regular exercise [2]. The relative amount of visceral and retroperitoneal white adipose tissue in LDLR/- mice in our different experimental groups is in agreement with the observed changes in body weight. Therefore, we can show that the increased uptake of energy in the high-fat diet group leads to increased formation of white adipose tissue. This can be partially reduced by voluntary running and is a major benefit of increased physical activity in our model. The reduced heart-to-body weight ratio after high-fat diet might mainly reflect the increased body weight and fat deposition, which is not accompanied by cardiac hypertrophy. This is in agreement with recent own studies in wild-type mice. Feeding C57BL/6 mice a high-fat diet for 10 weeks did not increase signs of heart failure or cardiac hypertrophy (Catar et al., unpublished). A few studies have analyzed the impact of increased physical activity on metabolic and cardiovascular parameters in LDLR/- mice so far. Exercise of 30 min/day on a treadmill for up to 12 weeks reduced atherosclerotic lesion size [13]. The adverse effects of an anabolicandrogenic steroid on cardiac remodeling and lipoprotein profile could be counteracted by treadmill running [14]. Recently, regular treadmill running protected against highfat diet induced hypothalamic inflammation [15]. Low and moderate-intensity aerobic exercise (30 min daily for 8 weeks) induced similar beneficial effects on atherosclerosis and hepatic oxidative stress in LDLR/- mice [16]. More recently, voluntary running significantly reduced total and non-HDL plasma cholesterol levels and spatial memory deficits of LDLR/- mice in the object location task [17]. This task is based on the spontaneous tendency of rodents, previously exposed to two identical objects, to later explore one of the objects e replaced in a novel location e for a longer time than they explore the non-displaced object,
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and has been used for the evaluation of hippocampaldependent memories [18]. This is the first study analyzing the endothelial function in LDLR/- mice after high-fat diet in combination with voluntary running. The endothelial function of LDLR/mice was impaired, compared to wild-type mice. High-fat diet with 39 kcal% fat did not further decrease endothelial function in the thoracic aorta after 20 weeks. Voluntary running had no significant effect on endothelial function in this setting. This might reflect the severe impairment of endothelial function in these mice even under basal conditions [19]. Interesting is the trend of an increased basal tone in LDLR/- mice on high-fat diet. This might contribute to endothelial dysfunction as well. On the other hand, the type and duration of high-fat diet and exercise could affect the outcome as well. In a recent study, regular exercise or changing diet did not influence aortic valve disease progression in LDLR/- mice [20]. In addition, bile acid sequestration normalized plasma cholesterol and atherosclerosis in LDLR/- mice, while voluntary running had no additional effect [21]. Furthermore, the local type of flow might affect the susceptibility to endothelial dysfunction and atherosclerosis [22e26]. Due to the limited availability of aortic tissue and the focus on functional studies in the Mulvany myograph we could not study the underlying molecular mechanisms in detail so far. The exercise-induced modulation of endothelial NO production could play a crucial role in this context [2,27e29]. Exercise by graduated swimming lowered plasma cholesterol, increased production of nitric oxide and decreased in combination with L-arginine and antioxidants atherosclerotic lesion formation [30]. Furthermore, voluntary physical training improved cerebral endothelial NO synthase (eNOS)-dependent flowmediated dilation and wall compliance [31]. In previous studies, we found a reduced eNOS protein expression in the thoracic aorta of LDLR/- mice compared to C57BL/ 6 wild-type mice (Hofmann, Langbein et al., unpublished). This reduced capacity to generate NO further supports an endothelial dysfunction in our LDLR/mice. Therefore, the eNOS expression in the aorta of mice with or without high-fat diet in combination with long-term voluntary running will be the focus of future studies. Homozygous patients with familial hypercholesterolemia are very rare. Early diagnosis of familial hypercholesterolemia and prompt initiation of diet and lipidlowering therapy are critical. Patients with suspected familial hypercholesterolemia should be promptly referred to specialist centers for a comprehensive accelerated, premature atherosclerotic cardiovascular disease evaluation and clinical management. Lifestyle intervention and maximal statin therapy are the mainstays of treatment, often with other lipid-modifying therapy. As patients rarely achieve LDL-C targets, adjunctive lipoprotein apheresis is recommended where available [32]. Physical exercise alone
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does not have an influence on LDL-C levels in these patients. Nevertheless, physical activity in these patients may be beneficial and should be performed. In conclusion, voluntary running reduced body weight and amount of white adipose tissue in LDLR/- mice. Endothelial dysfunction in LDLR/- mice could not be improved by voluntary running. In a clinical context, physical exercise alone might not have an influence on functional parameters and LDL-C levels in patients with familial hypercholesterolemia. However, physical activity in these patients may be in general beneficial and should be performed. Conflict of interest HL, AH, CB, WG and HM do not report disclosures. Acknowledgments We are grateful to Jennifer Mittag for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (MO 1695/4-1, MO 1695/5-1 to HM), the Else Kro¨ner-Fresenius-Stiftung (2010_A105 to HM), the BMBF (Professorship of Vascular Endothelium and Microcirculation to HM), the Doktor Robert Pfleger Foundation, Bamberg, Germany (adiposity and endothelial function; LOX-1, aldosterone, mineralocorticoid receptors and vascular function to HM), and the European Section of the Aldosterone Council (ESAC) Deutschland (aldosterone, LOX-1 and adipose tissue to AH, HM). References [1] Magnussen CG, Smith KJ, Juonala M. When to prevent cardiovascular disease? As early as possible: lessons from prospective cohorts beginning in childhood. Curr Opin Cardiol 2013;28:561e8. http://dx. doi.org/10.1097/HCO.0b013e32836428f4. [2] Schuler G, Adams V, Goto Y. Role of exercise in the prevention of cardiovascular disease: results, mechanisms, and new perspectives. Eur Heart J 2013;34:1790e9. http://dx.doi.org/10.1093/eurheartj/eht111. [3] Szostak J, Laurant P. The forgotten face of regular physical exercise: a ‘natural’ anti-atherogenic activity. Clin Sci (Lond) 2011;121: 91e106. http://dx.doi.org/10.1042/CS20100520. [4] Hobbs HH, Russell DW, Brown MS, Goldstein JL. The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane protein. Annu Rev Genet 1990;24:133e70. http://dx.doi. org/10.1146/annurev.ge.24.120190.001025. [5] Bentzon JF, Falk E. Atherosclerotic lesions in mouse and man: is it the same disease? Curr Opin Lipidol 2010;21:434e40. http://dx.doi. org/10.1097/MOL.0b013e32833ded6a. [6] Getz GS, Reardon CA. Animal models of atherosclerosis. Arterioscler Thromb Vasc Biol 2012;32:1104e15. http://dx.doi.org/10. 1161/ATVBAHA.111.237693. [7] Kapourchali FR, Surendiran G, Chen L, Uitz E, Bahadori B, Moghadasian MH. Animal models of atherosclerosis. World J Clin Cases 2014;2:126e32. http://dx.doi.org/10.12998/wjcc.v2.i5.126. [8] Eichhorn B, Muller G, Leuner A, Sawamura T, Ravens U, Morawietz H. Impaired vascular function in small resistance arteries of LOX-1 overexpressing mice on high-fat diet. Cardiovasc Res 2009;82:493e502. doi:cvp089, [pii] 10.1093/cvr/cvp089.
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[9] Novak CM, Burghardt PR, Levine JA. The use of a running wheel to measure activity in rodents: relationship to energy balance, general activity, and reward. Neurosci Biobehav Rev 2012;36:1001e14. http://dx.doi.org/10.1016/j.neubiorev.2011.12.012. [10] Hayes A, Williams DA. Beneficial effects of voluntary wheel running on the properties of dystrophic mouse muscle. J Appl Phys 1985; 1996(80):670e9. [11] Steioff K, Rutten H, Busch AE, Plettenburg O, Ivashchenko Y, Lohn M. Long term Rho-kinase inhibition ameliorates endothelial dysfunction in LDL-Receptor deficient mice. Eur J Pharmacol 2005; 512:247e9. http://dx.doi.org/10.1016/j.ejphar.2005.03.001. [12] Ngai YF, Quong WL, Glier MB, Glavas MM, Babich SL, Innis SM, et al. Ldlr-/- mice display decreased susceptibility to Western-type diet-induced obesity due to increased thermogenesis. Endocrinology 2010;151:5226e36. http://dx.doi.org/10.1210/en.2010-0496. [13] 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;21:1681e8. [14] Fontana K, Oliveira HC, Leonardo MB, Mandarim-de-Lacerda CA, da Cruz-Hofling MA. Adverse effect of the anabolic-androgenic steroid mesterolone on cardiac remodelling and lipoprotein profile is attenuated by aerobicz exercise training. Int J Exp Pathol 2008;89: 358e66. http://dx.doi.org/10.1111/j.1365-2613.2008.00601.x. [15] Yi CX, Al-Massadi O, Donelan E, Lehti M, Weber J, Ress C, et al. Exercise protects against high-fat diet-induced hypothalamic inflammation. Physiol Behav 2012;106:485e90. http://dx.doi.org/10. 1016/j.physbeh.2012.03.021. [16] Teodoro BG, Natali AJ, Fernandes SA, Silva LA, Pinho RA, Matta SL, et al. Improvements of atherosclerosis and hepatic oxidative stress are independent of exercise intensity in LDLr(-/-) mice. J Atheroscler Thromb 2012;19:904e11. [17] Moreira EL, Aguiar Jr AS, de Carvalho CR, Santos DB, de Oliveira J, de Bem AF, et al. Effects of lifestyle modifications on cognitive impairments in a mouse model of hypercholesterolemia. Neurosci Lett 2013;541:193e8. http://dx.doi.org/10.1016/j.neulet.2013.02. 043. [18] Assini FL, Duzzioni M, Takahashi RN. Object location memory in mice: pharmacological validation and further evidence of hippocampal CA1 participation. Behav Brain Res 2009;204:206e11. http://dx.doi.org/10.1016/j.bbr.2009.06.005. [19] Ketonen J, Mervaala E. Effects of dietary sodium on reactive oxygen species formation and endothelial dysfunction in low-density lipoprotein receptor-deficient mice on high-fat diet. Heart Vessels 2008; 23:420e9. http://dx.doi.org/10.1007/s00380-008-1066-5. [20] Schlotter F, Matsumoto Y, Mangner N, Schuler G, Linke A, Adams V. Regular exercise or changing diet does not influence aortic valve disease progression in LDLR deficient mice. PLoS One 2012;7: e37298. http://dx.doi.org/10.1371/journal.pone.0037298.
[21] Meissner M, Wolters H, de Boer RA, Havinga R, Boverhof R, Bloks VW, et al. Bile acid sequestration normalizes plasma cholesterol and reduces atherosclerosis in hypercholesterolemic mice. No additional effect of physical activity. Atherosclerosis 2013;228: 117e23. http://dx.doi.org/10.1016/j.atherosclerosis.2013.02.021. [22] Duerrschmidt N, Stielow C, Muller G, Pagano PJ, Morawietz H. NOmediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells. J Physiol 2006;576:557e67. [23] Goettsch C, Goettsch W, Brux M, Haschke C, Brunssen C, Muller G, et al. Arterial flow reduces oxidative stress via an antioxidant response element and Oct-1 binding site within the NADPH oxidase 4 promoter in endothelial cells. Basic Res Cardiol 2011;106:551e61. http://dx.doi.org/10.1007/s00395-011-0170-3. [24] Morawietz H, Talanow R, Szibor M, Rueckschloss U, Schubert A, Bartling B, et al. Regulation of the endothelin system by shear stress in human endothelial cells. J Physiol (Lond) 2000;525:761e70. [25] Muller G, Morawietz H. Nitric oxide, NAD(P)H oxidase, and atherosclerosis. Antioxid Redox Signal 2009;11:1711e31. http://dx. doi.org/10.1089/ARS.2008.2403. [26] Warboys CM, de Luca A, Amini N, Luong L, Duckles H, Hsiao S, et al. Disturbed flow promotes endothelial senescence via a p53dependent pathway. Arterioscler Thromb Vasc Biol 2014;34:985e95. http://dx.doi.org/10.1161/ATVBAHA.114.303415. [27] Gielen S, Sandri M, Erbs S, Adams V. Exercise-induced modulation of endothelial nitric oxide production. Curr Pharm Biotechnol 2011; 12:1375e84. [28] Hintze TH, Vatner SF. Reactive dilation of large coronary arteries in conscious dogs. Circ Res 1984;54:50e7. [29] Holtz J, Forstermann U, Pohl U, Giesler M, Bassenge E. Flowdependent, endothelium-mediated dilation of epicardial coronary arteries in conscious dogs: effects of cyclooxygenase inhibition. J Cardiovasc Pharmacol 1984;6:1161e9. [30] Napoli C, Williams-Ignarro S, De Nigris F, Lerman LO, Rossi L, Guarino C, et al. Long-term combined beneficial effects of physical training and metabolic treatment on atherosclerosis in hypercholesterolemic mice. Proc Natl Acad Sci U S A 2004;101:8797e802. http://dx.doi.org/10.1073/pnas.0402734101. [31] Bolduc V, Drouin A, Gillis MA, Duquette N, Thorin-Trescases N, Frayne-Robillard I, et al. Heart rate-associated mechanical stress impairs carotid but not cerebral artery compliance in dyslipidemic atherosclerotic mice. Am J Physiol Heart Circ Physiol 2011;301: H2081e92. http://dx.doi.org/10.1152/ajpheart.00706.2011. [32] Cuchel M, Bruckert E, Ginsberg HN, Raal FJ, Santos RD, Hegele RA, et al. Homozygous familial hypercholesterolaemia: new insights and guidance for clinicians to improve detection and clinical management. A position paper from the Consensus Panel on Familial Hypercholesterolaemia of the European Atherosclerosis Society. Eur Heart J 2014;35:2146e57. http://dx.doi.org/10.1093/eurheartj/ ehu274.
Impact of high-fat diet and voluntary running on body weight and endothelial function in LDL receptor knockout mice Heike Langbein1, Anja Hofmann1, Coy Brunssen, Winfried Goettsch, Henning Morawietz* Division of Vascular Endothelium and Microcirculation, Department of Medicine III, University Hospital Carl Gustav Carus, TU Dresden, Fetscherstr. 74, 01307 Dresden, Germany
Abstract Objective: Obesity and physical inactivity are important cardiovascular risk factors. Regular physical exercise has been shown to mediate beneficial effects in the prevention of cardiovascular diseases. However, the impact of physical exercise on endothelial function in proatherosclerotic low-density lipoprotein receptor deficient (LDLR/) mice has not been studied so far. Methods: Six-week-old male LDLR/ mice were fed a standard diet or a high-fat diet (39 kcal% fat diet) for 20 weeks. The impact of high-fat diet and voluntary running on body weight and amount of white adipose tissue was monitored. Basal tone and endothelial function was investigated in aortic rings using a Mulvany myograph. Results: LDLR/ mice on high-fat diet had increased cumulative food energy intake, but also higher physical activity compared to mice on control diet. Body weight and amount of visceral and retroperitoneal white adipose tissue of LDLR/ mice were significantly increased by high-fat diet and partially reduced by voluntary running. Endothelial function in aortae of LDLR/ mice was impaired after 20 weeks on standard and high-fat diet and could not be improved by voluntary running. Basal tone showed a trend to be increased by high-fat diet. Conclusion: Voluntary running reduced body weight and amount of white adipose tissue in LDLR/ mice. Endothelial dysfunction in LDLR/ mice could not be improved by voluntary running. In a clinical context, physical exercise alone might not have an influence on functional parameters and LDL-C levels in patients with familial hypercholesterolemia. However, physical activity in these patients may be in general beneficial and should be performed. Ó 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Atherosclerosis; Body weight; Endothelial function; Voluntary running
1. Introduction Obesity and physical inactivity are important cardiovascular risk factors [1]. In clinical studies, regular physical exercise has been shown to mediate beneficial effects in the prevention of cardiovascular diseases [2]. Increasing evidence supports an anti-atherogenic activity of regular physical exercise [3]. In this context, mouse models can be * Corresponding author. Tel.: þ49 351 458 6625; fax: þ49 351 458 6354. E-mail address: [email protected] (H. Morawietz). 1 Both the first two authors contributed equally to this study. http://dx.doi.org/10.1016/j.atherosclerosissup.2015.02.010 1567-5688/Ó 2015 Elsevier Ireland Ltd. All rights reserved.
important tools to analyze the mechanism of cardiovascular diseases in vivo. Murine atherosclerotic models depend on generating a non-HDL based hypercholesterolemia. This is most readily accomplished by genetic ablation of LDL receptor (LDLR). Although LDL receptor deficiency in humans is rare, the absence of a functional LDLR in humans results in familial hypercholesterolemia with increased risk of cardiovascular disease [4]. The arterial lesions in human familial hypercholesterolemia have some of the characteristics of the mouse lesion, including the presence of lesions in the aortic valves and the aortic root [5]. Even while some clinical characteristics of patients with obesity and metabolic-vascular syndrome differ from
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patients with familial hypercholesterolemia, studies in the LDL receptor knockout mice model have significantly contributed to our understanding of the mechanisms of atherogenesis [6]. LDLR/- mouse develop less severe atherosclerotic lesions compared to apoE knockout mice [7], but have certain advantages like a lipoprotein profile closer to human disease. Furthermore, a variety of high-fat diets with different content of cholesterol has been used, so that studies with this model are often not precisely standardized or comparable from one laboratory to another [6]. In addition, the impact of regular physical exercise on endothelial function in the proatherosclerotic model of LDLR/mice has not been studied so far. Therefore, we analyzed the impact of feeding a standard or a high-fat diet with 39 kcal% fat for 20 weeks on body weight, fat accumulation and endothelial function in LDLR/- mice. Furthermore, we assessed the impact of normal or accelerated physical activity by voluntary running on these parameters in the LDLR/- model. 2. Methods 2.1. Mice Six-week-old male LDLR/- mice (B6.129S7-Ldlrtm1Her/ J; Jackson Laboratories, Ben Harbor, ME) were fed a standard diet (STD) (ssniff EF R/M CD88137 control, 11% fat, 24% protein, 65% carbohydrates, gross energy: 18.4 MJ/kg), or a high-fat diet (FD) (ssniff EF R/M modified from TD88137 þ cholesterol, 39% fat, 20% protein, 41% carbohydrates, gross energy: 22.1 MJ/kg) for 20 weeks. Development of body weight and food intake was determined once per week and cumulative food intake was quantified over 20 weeks feeding. All performed experiments were in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The animal research ethics committee of the Dresden University of Technology and the Regional Council (Regierungspra¨sidium) Dresden approved the animal facilities and the experiments according to institutional guidelines and German animal welfare regulations (AZ: 24D-9168.11-1/2007-9). 2.2. Voluntary running LDLR/- mice on either standard or high-fat diet had free access to a running wheel (Fig. 1A). The velocity of individual running wheels was recorded online (Fig. 1B). Individual running distance and median daily activity of LDLR/- mice in voluntary running cages was determined. In addition, separate groups of freely moving mice on either standard or high-fat diet housed in standard cages without running wheels were studied. They served as controls to imitate a more sedentary lifestyle compared to the increased voluntary physical activity in a running wheel.
2.3. Adipose tissue and heart weight After 20 weeks on standard or high-fat diet with or without voluntary running, mice were sacrificed and the weight of visceral and retroperitoneal white adipose tissue (WAT) was determined and normalized to body weight. In addition, the heart-to-body weight ratio was quantified. 2.4. Vascular function Vascular function was analysed in aortic segments using a Mulvany myograph [8]. First, the basal tone in all four experimental groups (LDLR/- on standard or high-fat diet with or without voluntary running) was measured after applying 20 mmHg distension to aortic segments. Endothelial function was analysed in aortic rings after precontraction with 0.3 mmol/L phenylephrine (PE) and subsequent relaxation in response to increasing concentrations of acetylcholine (ACh). The NO-mediated amount of ACh-induced relaxation was determined using NO synthase inhibitor L-NAME. To analyze vascular smooth muscle function, NO donor sodium nitroprusside (SNP) was added to PE-precontracted aortic segments. 2.5. Statistics Data are shown as means SEM. Statistical analysis was performed by Student’s t-test or One-Way ANOVA followed by Bonferroni’s method (Sigma Stat 3.11, Systat Software, Inc., San Jose, CA), as appropriate. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Voluntary running The voluntary running model was successfully established in our laboratory (Fig. 1A). The experimental set-up allows voluntary running of 6 mice (one per cage) in parallel. The major physical activity was between 6 p.m. and 3 a.m. reaching peak velocities of more than 25 m/min (Fig. 1B). LDLR/- mice were running an average distance of 4 km/d. In the first weeks of intervention the median activity of mice remarkably increased (Fig. 1C). After 4e5 weeks of voluntary running, daily activity reached a peak value slowly decreasing with increasing age of mice. Interestingly, LDLR/- mice on high-fat diet showed a higher median daily activity, compared to mice on standard diet (Fig. 1C). 3.2. Body weight and cumulative food intake Body weight in LDLR/- mice on high-fat diet constantly increased during feeding period, compared to age-matched LDLR/- mice on standard diet (Fig. 2A).
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Fig. 1. Voluntary running. A) Left: Experimental set-up allowing voluntary running of 6 mice (one per cage) in parallel. Right: Mouse in a running wheel. B) Representative recording of characteristic physical activity pattern of LDLR/- mice during 24 h with access to voluntary running. C) Monitoring of daily physical activity of LDLR/- mice during 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR). *P < 0.05 vs. STD þ VR.
After 20 weeks, body weight of LDLR/- mice on high-fat diet was almost doubled, compared to standard diet. Voluntary running decreased the body weight initially even in mice with standard diet. After 20 weeks on standard diet, no significant changes in body weight could be observed between groups with or without voluntary running. In contrast, voluntary running significantly reduced the body weight of LDLR/- mice on high-fat diet. However, body
weight was higher in the high-fat diet þ voluntary running group, compared to mice with or without voluntary running on standard diet (Fig. 2A). The cumulative food intake after 20 weeks showed a tendency to be higher in the groups of LDLR/- mice on high-fat diet with or without voluntary running, compared to mice on standard diet with or without voluntary running (Fig. 2B).
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Fig. 2. Impact of high-fat diet and voluntary running on body weight and cumulative food intake. A) Development of body weight of LDLR/- mice during 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR). ***P < 0.001 vs. STD and STD þ VR. #P < 0.05 vs. FD þ VR. B) Cumulative food intake after 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR).
3.3. Adipose tissue and heart weight The relative amount of visceral (Fig. 3A) and retroperitoneal (Fig. 3B) white adipose tissue (WAT) was increased in LDLR/- mice after 20 weeks of high-fat diet. Animals with access to voluntary running had decreased amounts of both types of WAT, compared to animals with less physical activity on the same diet. However, the development of WAT in response to high-fat diet could not be prevented completely by voluntary running (Fig. 3A, B). The heart-to-body weight ratio was decreased in LDLR/- mice after high-fat diet feeding, compared to standard diet. LDLR/- mice with voluntary running on high-fat diet had a decreased heart-to-body weight ratio compared to voluntary running on standard diet, but an increased heart-to-body ratio compared to mice on high-fat diet with lower physical activity (Fig. 3C). 3.4. Vascular function Vascular function was assessed in aortic rings of LDLR/- mice after standard diet, high-fat diet with or without voluntary running for 20 weeks. After precontraction with phenylephrine, endothelial function was determined by relaxation to increasing concentrations of
acetylcholine. The endothelial function in the aorta of all LDLR/- groups was impaired compared to wild-type mice. High-fat diet of 39 kcal% fat and voluntary running for 20 weeks had no significant effect on endothelial function in the aorta of LDLR/- mice (Fig. 4A). Relaxation in response to acetylcholine was completely inhibited using NOS inhibitor L-NG-Nitro arginine methyl ester (L-NAME) (data not shown). The vascular smooth muscle function in response to increasing dosages of the NO donor SNP was not different between all experimental groups studied (Fig. 4B). Interestingly, the basal tone showed a trend to be increased in the aorta of LDLR/- mice after high-fat diet (Fig. 4C). This was not affected by voluntary running. 4. Discussion The voluntary running model has the advantage of free access of mice to increased physical activity [9]. Using this approach mouse exercised much longer, compared to individual training on treadmill devices. The observed major physical activity at night is in agreement with the circadian rhythm of mice showing increased nocturnal activity. The running distance of LDLR/- (4 km/d) was shorter compared to previously published data of wild-type mice (6.5 km/d) [10]. This might partially reflect an
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Fig. 3. Impact of high-fat diet and voluntary running on white adipose tissue and heart weight. Weight of visceral white adipose tissue (WAT) (A), retroperitoneal WAT (B) and heart (C) was normalized to body weight of LDLR/- mice after 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR). ***P < 0.001 vs. STD. #P < 0.05 vs. STD þ VR.
impaired endothelial function in LDLR/- mice [11]. The daily activity using the running wheel increased in the first 4e5 weeks. This reflects an improved physical activity due to training effects in the first weeks. The slight decrease in
activity during the following weeks might be the result of increasing age and impairment of endothelial function in LDLR/- mice. Especially interesting is the higher physical activity of LDLR/- mice on high-fat diet. This might be
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Fig. 4. Effect of high-fat diet and voluntary running on vascular function. A) Endothelial function was analysed as concentrationeresponse curve to acetylcholine (ACh) in aortic rings of LDLR/- mice precontracted with phenylephrine in a Mulvany myograph after 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR). B) Determination of smooth muscle function using increasing concentrations of NO donor sodium nitroprusside (SNP) in aortic segments of LDLR/- mice after 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR). C) Basal tone was measured after applying 20 mmHg distension to aortic segments of LDLR/- mice after 20 weeks on standard diet (STD) or high-fat diet (FD) in combination with voluntary running (VR).
the result of the observed higher energy uptake in the highfat diet group resulting in an increased physical performance after training, especially in the first weeks of voluntary running. In this context it has to be considered that LDLR/- mice display decreased susceptibility to Western-type diet-induced obesity due to increased thermogenesis [12]. The observed increase in body weight of LDLR/- mice on high-fat diet and the partial reduction by exercise are in agreement with previous studies using 30 min/day training on a treadmill for up to 12 weeks [13]. This reflects the beneficial effects of increased physical activity resulting in weight loss, even while feeding a high-fat diet resulted in higher energy uptake. Furthermore, voluntary running reduced the weight of mice on standard diet especially in the initial phase. At higher age, body weight and energy uptake were not different between LDLR/- mice on standard diet with or without voluntary running. The beneficial effects of voluntary running on body weight are accelerated after feeding a high-fat diet. This is in agreement with clinical studies showing efficient weight loss in patients combining a healthy diet with regular exercise [2]. The relative amount of visceral and retroperitoneal white adipose tissue in LDLR/- mice in our different experimental groups is in agreement with the observed changes in body weight. Therefore, we can show that the increased uptake of energy in the high-fat diet group leads to increased formation of white adipose tissue. This can be partially reduced by voluntary running and is a major benefit of increased physical activity in our model. The reduced heart-to-body weight ratio after high-fat diet might mainly reflect the increased body weight and fat deposition, which is not accompanied by cardiac hypertrophy. This is in agreement with recent own studies in wild-type mice. Feeding C57BL/6 mice a high-fat diet for 10 weeks did not increase signs of heart failure or cardiac hypertrophy (Catar et al., unpublished). A few studies have analyzed the impact of increased physical activity on metabolic and cardiovascular parameters in LDLR/- mice so far. Exercise of 30 min/day on a treadmill for up to 12 weeks reduced atherosclerotic lesion size [13]. The adverse effects of an anabolicandrogenic steroid on cardiac remodeling and lipoprotein profile could be counteracted by treadmill running [14]. Recently, regular treadmill running protected against highfat diet induced hypothalamic inflammation [15]. Low and moderate-intensity aerobic exercise (30 min daily for 8 weeks) induced similar beneficial effects on atherosclerosis and hepatic oxidative stress in LDLR/- mice [16]. More recently, voluntary running significantly reduced total and non-HDL plasma cholesterol levels and spatial memory deficits of LDLR/- mice in the object location task [17]. This task is based on the spontaneous tendency of rodents, previously exposed to two identical objects, to later explore one of the objects e replaced in a novel location e for a longer time than they explore the non-displaced object,
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and has been used for the evaluation of hippocampaldependent memories [18]. This is the first study analyzing the endothelial function in LDLR/- mice after high-fat diet in combination with voluntary running. The endothelial function of LDLR/mice was impaired, compared to wild-type mice. High-fat diet with 39 kcal% fat did not further decrease endothelial function in the thoracic aorta after 20 weeks. Voluntary running had no significant effect on endothelial function in this setting. This might reflect the severe impairment of endothelial function in these mice even under basal conditions [19]. Interesting is the trend of an increased basal tone in LDLR/- mice on high-fat diet. This might contribute to endothelial dysfunction as well. On the other hand, the type and duration of high-fat diet and exercise could affect the outcome as well. In a recent study, regular exercise or changing diet did not influence aortic valve disease progression in LDLR/- mice [20]. In addition, bile acid sequestration normalized plasma cholesterol and atherosclerosis in LDLR/- mice, while voluntary running had no additional effect [21]. Furthermore, the local type of flow might affect the susceptibility to endothelial dysfunction and atherosclerosis [22e26]. Due to the limited availability of aortic tissue and the focus on functional studies in the Mulvany myograph we could not study the underlying molecular mechanisms in detail so far. The exercise-induced modulation of endothelial NO production could play a crucial role in this context [2,27e29]. Exercise by graduated swimming lowered plasma cholesterol, increased production of nitric oxide and decreased in combination with L-arginine and antioxidants atherosclerotic lesion formation [30]. Furthermore, voluntary physical training improved cerebral endothelial NO synthase (eNOS)-dependent flowmediated dilation and wall compliance [31]. In previous studies, we found a reduced eNOS protein expression in the thoracic aorta of LDLR/- mice compared to C57BL/ 6 wild-type mice (Hofmann, Langbein et al., unpublished). This reduced capacity to generate NO further supports an endothelial dysfunction in our LDLR/mice. Therefore, the eNOS expression in the aorta of mice with or without high-fat diet in combination with long-term voluntary running will be the focus of future studies. Homozygous patients with familial hypercholesterolemia are very rare. Early diagnosis of familial hypercholesterolemia and prompt initiation of diet and lipidlowering therapy are critical. Patients with suspected familial hypercholesterolemia should be promptly referred to specialist centers for a comprehensive accelerated, premature atherosclerotic cardiovascular disease evaluation and clinical management. Lifestyle intervention and maximal statin therapy are the mainstays of treatment, often with other lipid-modifying therapy. As patients rarely achieve LDL-C targets, adjunctive lipoprotein apheresis is recommended where available [32]. Physical exercise alone
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does not have an influence on LDL-C levels in these patients. Nevertheless, physical activity in these patients may be beneficial and should be performed. In conclusion, voluntary running reduced body weight and amount of white adipose tissue in LDLR/- mice. Endothelial dysfunction in LDLR/- mice could not be improved by voluntary running. In a clinical context, physical exercise alone might not have an influence on functional parameters and LDL-C levels in patients with familial hypercholesterolemia. However, physical activity in these patients may be in general beneficial and should be performed. Conflict of interest HL, AH, CB, WG and HM do not report disclosures. Acknowledgments We are grateful to Jennifer Mittag for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (MO 1695/4-1, MO 1695/5-1 to HM), the Else Kro¨ner-Fresenius-Stiftung (2010_A105 to HM), the BMBF (Professorship of Vascular Endothelium and Microcirculation to HM), the Doktor Robert Pfleger Foundation, Bamberg, Germany (adiposity and endothelial function; LOX-1, aldosterone, mineralocorticoid receptors and vascular function to HM), and the European Section of the Aldosterone Council (ESAC) Deutschland (aldosterone, LOX-1 and adipose tissue to AH, HM). References [1] Magnussen CG, Smith KJ, Juonala M. When to prevent cardiovascular disease? As early as possible: lessons from prospective cohorts beginning in childhood. Curr Opin Cardiol 2013;28:561e8. http://dx. doi.org/10.1097/HCO.0b013e32836428f4. [2] Schuler G, Adams V, Goto Y. Role of exercise in the prevention of cardiovascular disease: results, mechanisms, and new perspectives. Eur Heart J 2013;34:1790e9. http://dx.doi.org/10.1093/eurheartj/eht111. [3] Szostak J, Laurant P. The forgotten face of regular physical exercise: a ‘natural’ anti-atherogenic activity. Clin Sci (Lond) 2011;121: 91e106. http://dx.doi.org/10.1042/CS20100520. [4] Hobbs HH, Russell DW, Brown MS, Goldstein JL. The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane protein. Annu Rev Genet 1990;24:133e70. http://dx.doi. org/10.1146/annurev.ge.24.120190.001025. [5] Bentzon JF, Falk E. Atherosclerotic lesions in mouse and man: is it the same disease? Curr Opin Lipidol 2010;21:434e40. http://dx.doi. org/10.1097/MOL.0b013e32833ded6a. [6] Getz GS, Reardon CA. Animal models of atherosclerosis. Arterioscler Thromb Vasc Biol 2012;32:1104e15. http://dx.doi.org/10. 1161/ATVBAHA.111.237693. [7] Kapourchali FR, Surendiran G, Chen L, Uitz E, Bahadori B, Moghadasian MH. Animal models of atherosclerosis. World J Clin Cases 2014;2:126e32. http://dx.doi.org/10.12998/wjcc.v2.i5.126. [8] Eichhorn B, Muller G, Leuner A, Sawamura T, Ravens U, Morawietz H. Impaired vascular function in small resistance arteries of LOX-1 overexpressing mice on high-fat diet. Cardiovasc Res 2009;82:493e502. doi:cvp089, [pii] 10.1093/cvr/cvp089.
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[21] Meissner M, Wolters H, de Boer RA, Havinga R, Boverhof R, Bloks VW, et al. Bile acid sequestration normalizes plasma cholesterol and reduces atherosclerosis in hypercholesterolemic mice. No additional effect of physical activity. Atherosclerosis 2013;228: 117e23. http://dx.doi.org/10.1016/j.atherosclerosis.2013.02.021. [22] Duerrschmidt N, Stielow C, Muller G, Pagano PJ, Morawietz H. NOmediated regulation of NAD(P)H oxidase by laminar shear stress in human endothelial cells. J Physiol 2006;576:557e67. [23] Goettsch C, Goettsch W, Brux M, Haschke C, Brunssen C, Muller G, et al. Arterial flow reduces oxidative stress via an antioxidant response element and Oct-1 binding site within the NADPH oxidase 4 promoter in endothelial cells. Basic Res Cardiol 2011;106:551e61. http://dx.doi.org/10.1007/s00395-011-0170-3. [24] Morawietz H, Talanow R, Szibor M, Rueckschloss U, Schubert A, Bartling B, et al. Regulation of the endothelin system by shear stress in human endothelial cells. J Physiol (Lond) 2000;525:761e70. [25] Muller G, Morawietz H. Nitric oxide, NAD(P)H oxidase, and atherosclerosis. Antioxid Redox Signal 2009;11:1711e31. http://dx. doi.org/10.1089/ARS.2008.2403. [26] Warboys CM, de Luca A, Amini N, Luong L, Duckles H, Hsiao S, et al. Disturbed flow promotes endothelial senescence via a p53dependent pathway. Arterioscler Thromb Vasc Biol 2014;34:985e95. http://dx.doi.org/10.1161/ATVBAHA.114.303415. [27] Gielen S, Sandri M, Erbs S, Adams V. Exercise-induced modulation of endothelial nitric oxide production. Curr Pharm Biotechnol 2011; 12:1375e84. [28] Hintze TH, Vatner SF. Reactive dilation of large coronary arteries in conscious dogs. Circ Res 1984;54:50e7. [29] Holtz J, Forstermann U, Pohl U, Giesler M, Bassenge E. Flowdependent, endothelium-mediated dilation of epicardial coronary arteries in conscious dogs: effects of cyclooxygenase inhibition. J Cardiovasc Pharmacol 1984;6:1161e9. [30] Napoli C, Williams-Ignarro S, De Nigris F, Lerman LO, Rossi L, Guarino C, et al. Long-term combined beneficial effects of physical training and metabolic treatment on atherosclerosis in hypercholesterolemic mice. Proc Natl Acad Sci U S A 2004;101:8797e802. http://dx.doi.org/10.1073/pnas.0402734101. [31] Bolduc V, Drouin A, Gillis MA, Duquette N, Thorin-Trescases N, Frayne-Robillard I, et al. Heart rate-associated mechanical stress impairs carotid but not cerebral artery compliance in dyslipidemic atherosclerotic mice. Am J Physiol Heart Circ Physiol 2011;301: H2081e92. http://dx.doi.org/10.1152/ajpheart.00706.2011. [32] Cuchel M, Bruckert E, Ginsberg HN, Raal FJ, Santos RD, Hegele RA, et al. Homozygous familial hypercholesterolaemia: new insights and guidance for clinicians to improve detection and clinical management. A position paper from the Consensus Panel on Familial Hypercholesterolaemia of the European Atherosclerosis Society. Eur Heart J 2014;35:2146e57. http://dx.doi.org/10.1093/eurheartj/ ehu274.
