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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:471-480 (2007)
© 2007 The Gerontological Society of America

Prior Exercise Improves Age-Dependent Vascular Endothelial Growth Factor Downregulation and Angiogenesis Responses to Hind-Limb Ischemia in Old Rats

Dario Leosco, Giuseppe Rengo, Guido Iaccarino, Emma Sanzari, Luca Golino, Gabriella De Lisa, Carmela Zincarelli, Francesca Fortunato, Michele Ciccarelli, Vincenzo Cimini, Giovanna Giuseppina Altobelli, Federico Piscione, Gennaro Galasso, Bruno Trimarco, Walter J. Koch and Franco Rengo

1 Dipartimento di Medicina Clinica, Scienze Cardiovascolari ed Immunologiche, Università Federico II, Napoli, Italy.
2 Fondazione Salvatore Maugeri–Istituto di Ricerca a Carattere Scientifico, Telese (Benevento), Italy.
3 Center for Translational Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania.
4 Dipartimento di Scienze Biomorfologiche e Funzionali, Università Federico II, Napoli, Italy.

Address correspondence to Dario Leosco, MD, PhD, Dipartimento di Medicina Clinica, Scienze Cardiovascolari ed Immunologiche, Federico II University, Via Pansini 5, Edificio 2, 80131 Naples, Italy. E-mail: dleosco{at}unina.it


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Downregulation of hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor (VEGF) are shown to be involved in age-dependent impairment of angiogenesis. In this study, we explore whether prior exercise is able to affect these molecular patterns favorably and to enhance neoangiogenesis in old Wistar rats with hind-limb ischemia. At day 7 after surgery, HIF-1{alpha} and VEGF expression increased in the ischemic muscle of trained animals. Exercise increased capillary density and limb perfusion as revealed by histologic, angiographic, and dyed bead techniques. Furthermore, exercise capacity and limb trophism have significantly improved in trained aged rats. In these animals, the reduction of VEGF serum levels has reflected the comprehensive improvement in local ischemia evoked by exercise. In conclusion, prior exercise represents a valid tool to counteract age-related molecular alterations resulting in impaired angiogenesis in response to ischemia.

ANGIOGENESIS responses to tissue ischemia are impaired with aging (1). This impairment might be related to age-dependent endothelial dysfunction (2–5), which could blunt mechanisms of activation, proliferation, and migration of endothelial cells that are essential for development of new vessels (6). Downregulation of vascular endothelial growth factor (VEGF) and reduced expression and activity of hypoxia inducible factor-1 (HIF-1) are shown to be responsible for impaired angiogenesis observed with age (1,7,8). HIF-1 is a heterodimeric (HIF-1{alpha} and HIF-1ß) transcription factor involved in several physiological responses to hypoxia (9–11). It is essential for VEGF transcriptional activation, both in vitro (12) and in vivo (13). HIF-1{alpha} protein expression and HIF-1 DNA binding activity are significantly reduced in older cells (8). Furthermore, posttranscriptional mechanisms, such as HIF-1 protein stabilization, are impaired with age (8).

Exercise represents a valid tool to stimulate angiogenesis in physiologic and pathologic conditions. In trained mice, improved neoangiogenesis is associated with an increase of serum VEGF and endothelial circulating progenitor cells (14). In aged rats with hind-limb ischemia, training increases limb collateral-dependent blood flow that is associated with a significant increase of exercise tolerance (15). Interestingly, even prior exercise is able to enhance angiogenesis in experimental limb ischemia (16). In healthy humans, endurance training increases HIF-1 and VEGF messenger RNA (mRNA) muscle levels, and enhances skeletal muscle capillary density (17–19). Compared to sedentary controls, exercised patients with peripheral arterial disease show increased VEGF serum levels and circulating endothelial progenitor cells (20).

Until now, no data have been produced about the potential effect of exercise in restoring age-related impairment of HIF-1-dependent regulation of VEGF. The aim of the present study has been to evaluate whether prior exercise is able to affect this molecular pathway favorably and to improve neoangiogenesis responses in aged rats subjected to surgical hind-limb ischemia. Another aim has been to demonstrate a relationship between enhanced angiogenesis and exercise performance in trained animals. Due to the high prevalence of peripheral arterial disease in the elderly population, the results of this study support the importance of an active lifestyle in the clinical setting.


   MATERIALS AND METHODS
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animal Population, Exercise Training Protocol, and Measurements of Exercise Capacity
This study protocol was designed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health in the United States (NIH Publication No. 85-23, revised 1996) and approved by the Ethics Committee for the Use of Animals in Research of our institution. Thirty old male (24 months; weight = 470 ± 45 g) and 30 young male (4 months; weight 280 ± 18 g) Wistar rats (Charles River Laboratories, Calco, Italy) were separately housed in groups of two animals per cage, at room temperature (22–28°C) with 12-hour light/dark cycles. All animals had free access to food and water, and were acclimated to a treadmill exercise by walking 10 min/d at a speed of 10 m/min (for 2 weeks) on a type 50190 TAKRAF treadmill (Schmalkalden, Germany). Rats of both age groups were numbered from 1 to 30 (1y to 30y; 1o to 30o). Afterwards, rats from each group were assigned alternatively to exercise and sedentary protocols (i.e., 1y and 3o to exercise; 20y and 13o to sedentary). In this way, 15 old and 15 young rats of each group were assigned to a treadmill exercise training program lasting 12 weeks, consisting of 5 d/wk, 45 min/day, with a running speed of 17 m/min. This protocol produced exercise with relative intensity estimated between 70% and 85% of maximal oxygen uptake (21,22). The remaining 15 old and 15 young rats followed a 12-week sedentary protocol consisting of walking 10 min/d once a week to maintain treadmill familiarity. Maximal exercise capacity was evaluated before starting training or sedentary protocols, at the end of 12 weeks of both protocols, and 21 days after hind-limb ischemia. Each evaluation was performed twice in each rat, in separated tests, and the average score was considered for analysis. Time to exhaustion (minutes) was considered as an index of maximal exercise capacity. The protocol for the maximal exercise capacity test consisted in walking at 10 m/min for 5 minutes, then at 2 m/min increasing the speed every 2 minutes until the rat reached exhaustion. Rats were considered exhausted when they failed to stay off of a shock bar. The grade of the treadmill was set at 15°. The technique used for the evaluation of maximal exercise capacity was highly reproducible at all protocol steps (R2 = 0.75).

Rat Ischemic Hind-Limb Model and Surgical Follow-Up
On day 1 after the end of 12 weeks of training or sedentary protocols, all rats were anesthetized with a mixture of ketamine (50 mg/kg) and xylazine (0.5 mg/kg). A surgical incision was made in the skin overlying the middle portion of the left hind limb. After ligation of the proximal end of the femoral artery, the distal portion of the saphenous artery was ligated, and the artery and all side branches were dissected free and excised. Then the skin was closed with a 2.0 silk suture. On day 21 after surgery, trophism of the ischemic hind limb was evaluated in all operated animals.

Digital Angiography and Limb Blood Flow Determination by Dyed Beads Assay
On day 21 after surgery, animals were anesthetized and the left common carotid exposed as previously described (23). A flame-stretched PE 50 catheter was advanced into the abdominal aorta right before the iliac bifurcation, under fluoroscopic visualization (Advantix LCX; General Electric, Milwaukee, WI). Maximal vasodilation was obtained by nitroglycerin (20 µg i.a.). An electronically regulated injector (ACIST Medical Systems, Eden Prairie, MN) was used to deliver, at constant pressure (900 psi), 0.2 mL of contrast medium (Iomeron 400; Bracco Diagnostics, Milano, Italy). The cineframe number for thrombolysis in myocardial infarction (TIMI) frame count assessment was measured with a digital frame counter on the suitable cineviewer monitor. All angiograms were filmed at 5 frames per second and were analyzed by two blinded investigators (G.G., D.L). TIMI frame count was done starting from the first frame in which the contrast medium entered the iliac artery until the frame of full visualization of first paw artery bifurcation. Thus, the score was inversely correlated with blood flow velocity. After angiography, we injected 108 orange dyed beads diluted in 1 mL of 0.9% NaCl (Triton Technology, San Diego, CA), and then animals were killed with a lethal dose of pentobarbital. Gastrocnemius samples of the ischemic and nonischemic hind limb were collected and frozen with liquid nitrogen and stored at –80°C. Samples were then homogenized and digested, and the beads were collected and suspended in N,N-dimethylthioformamide (DMTF). The release of dye was assessed by light absorption at 450 nm. Data are expressed as the ischemic to nonischemic muscle ratio.

Histology
Tissue specimens were dissected and immediately fixed by immersion in 0.01 M phosphate-buffered saline (PBS) (pH 7.2–7.4) and formalin for at least 24 hours. They were then dehydrated by crescent alcohol concentration and embedded in paraffin; 5 µm-thick sections were processed for histochemistry. After rehydration, they were incubated with Bandeiraea simplicifolia I (BS-I) biotinylated lectin (1:50; Sigma, St. Louis, MO) overnight. BS-I specific adhesion to capillary endothelium was revealed by a secondary incubation for 1 hour at room temperature with horseradish peroxidise-conjugated streptavidin (1:400; Dako, Milano, Italy), which gives a brown reaction product in the presence of hydrogen peroxide and diaminobenzidine. Morphometric analysis was performed by a Leitz Diaplan microscope provided with a Leica DC 200 digital camera. Images of interest were processed by Image-Pro Plus software (Media Cybernetics, Silver Spring, MD) to count the number of capillary blood vessels per examined area. Capillary diameters 5–15 µm thick were considered in this study. Five tissue sections per animal per each experimental group were examined. The number of capillaries per 20 fields was measured on each section by two independent operators, blind to treatment (V.C., G.G.A.). Then, mean values of the measurements from five sections per animal per experimental group were calculated and plotted. The final values were expressed as capillaries-to-fiber ratio and as mean capillary number per unit area equivalent to 1000 µm2.

Western Blot Analysis of VEGF and HIF-1{alpha} Protein Expression
VEGF determination was performed on whole-cell protein extracts obtained after the homogenization of ischemic and contralateral gastrocnemius muscles of both young and old animals harvested 7 days after limb ischemia. Total (50 µg) and nuclear (20 µg) proteins were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted on nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in 0.1% Tween–Tris-buffered saline (TBS-T) and then probed with antihuman VEGF antibody (1:1000, goat polyclonal, sc-1836; Santa Cruz Biotechnology, Santa Cruz, CA) and with HIF-1{alpha} antibody (1:500, goat polyclonal, sc-8711; Santa Cruz Biotechnology) overnight at 4°C. Afterwards, blots were washed three times in TBS-T and then incubated for 1 hour with antigoat horseradish peroxidise-conjugated immunoglobulin G (IgG) (1:2000; Santa Cruz Biotechnology). Blots were washed in TBS-T, and antigen–antibody complexes were visualized after incubation for 1 minute with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL), followed by exposure to the ChemiDoc System (Bio-Rad Laboratories, Milano, Italy).

Systemic VEGF Determination
Serum levels of VEGF were measured by highly sensitive enzyme-linked immunosorbent assays (ELISAs; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Results were compared with standard curves, and the lower detection limits was 9 pg VEGF/mL. The intraassay and interassay variabilities were both < 10%. Measurements were performed in duplicate.

Statistical Analysis
All values are expressed as mean ± standard error of the mean. Comparison between groups was made by a two-tailed Student t test or analysis of variance (ANOVA) with the Bonferroni post hoc test for multiple comparisons. A two-factor (age, training) ANOVA was performed on data presented in Table 1, Figure 1C, Figure 2B and C, and Figure 3. A value of p <.05 was considered to denote statistical significance.


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  		Table 1. Hemodynamic Data and Physical Characteristics of Young and Old Animals.

 

Figure 01
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  		Figure 1. A and B, Maximal exercise capacity evaluated in both age groups at baseline, at the end of 12 weeks sedentary or training protocol, and at 3 weeks after limb ischemia. At baseline: *p <.001 for old trained and untrained animals versus young ones. At 12 weeks from starting protocol: *p <.0001 for trained versus untrained animals in both age groups. At 3 weeks from ischemia: *p <.0001 for untrained ischemic versus untrained at baseline in both age groups, {dagger}p <.0001 for old untrained animals versus young untrained ones, {ddagger}p <.0001 for trained versus untrained animals in both age groups. C, Percent loss of exercise capacity induced by ischemia in untrained and trained rats of both age groups. *p <.001 for young untrained and trained versus old ones, p <.001 for Age x Training interaction (two-factor analysis of variance). Values are expressed as mean ± standard error of the mean (n = 15 for each group)

 

Figure 02
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  		Figure 2. A, Digital angiography obtained after maximal vasodilation (nitroglycerin, 20 µg i.a.) from old untrained and trained animals at 21 days from femoral artery resection (black arrow). Collateral circulation and limb blood flow distal to the occlusion were detectable in old trained (white arrow) but not in old untrained rats. B, Thrombolysis in myocardial infarction (TIMI) frame count: *p <.0001 for ischemic versus nonischemic, {dagger}p <.0001 for old ischemic untrained and trained animals versus young ones, {ddagger}p <.0001 for ischemic trained versus untrained ones in both age groups. p <.001 for Age x Training interaction (two-factor analysis of variance [ANOVA]). C, Dyed bead assay expressed by ischemic-to-nonischemic ratio of dyed bead content per milligram of hind-limb muscle tissue. *p <.001 for old untrained versus young untrained animals, {dagger}p <.0001 for trained versus untrained ones in both age groups. p =.004 for Age x Training interaction (two-factor ANOVA). Values are expressed as mean ± standard error of the mean (n = 10 for each group)

 

Figure 03
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  		Figure 3. A, Lectin Bandeiraea simplicifolia I (BS-I) staining of capillaries in the ischemic hind limb from both age untrained and trained rats. Magnification x40; bar = 50 µm. B, Capillary-to-fiber ratio and capillaries per area (mm2) in ischemic muscles of both age groups. *p <.005 for old trained versus untrained animals, {dagger}p <.001 for young trained versus untrained animals. p =.002 and p <.001 for Age x Training interaction on capillary-to-fiber and capillaries/mm2 (two-factor analysis of variance). Values are expressed as mean ± standard error of the mean (n = 10 for each group)

 

   RESULTS
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 Materials and Methods
 Results
Discussion
 References
 
Effects of Age and Exercise on Hemodynamic Data and Physical Characteristics
Table 1 shows hemodynamic data and physical characteristics of the animal population before surgery and on day 21 after limb ischemia. Heart rate and systolic blood pressure were significantly affected by age. Training significantly reduced systolic blood pressure in both age groups. Aging and training were significantly associated with greater body and left ventricular weights. Furthermore, a significant Age x Training interaction on the reduction of left ventricle to body weight ratio was found. Limb ischemia did not affect hemodynamic and physical data in young and old animal groups.

Exercise Improves Ischemia-Related Alterations of Hind-Limb Trophism in Old Rats
No animals among the young trained, untrained, and old trained groups developed trophism alterations or limb necrosis at surgical follow-up performed on day 21 after surgery. In contrast, only 6 of 15 old untrained animals showed ungual atrophy, and one had severe necrosis and autoamputation of the distal hind limb (data not shown).

Exercise Attenuates Ischemia-Induced Decrease in Maximal Exercise Capacity in Old Rats
Before starting training or sedentary protocols, and after randomization, no intragroup difference in exercise capacity was found. In contrast, exercise levels were higher in young than in old rats, as expected. Training induced a significant increase in maximal exercise capacity in both young and old animals. Hind-limb ischemia was associated with a dramatic decrease in exercise capacity in untrained young and even more in untrained old rats. Training attenuated the negative effect of ischemia in both age groups (Figure 1A and B). Nevertheless, the effect of training on reducing the percent loss of function was visible in old but not in young animals. In this regard, a significant Age x Training interaction contributed to the overall effect on the recovery of physical performance (Figure 1C).

Exercise Improves Blood Flow and Native Angiogenesis in Aged Ischemic Muscles
Digital angiography was performed in all animals at day 21 after surgery (Figure 2A). In both age groups, ischemia induced a significant reduction of limb perfusion as expressed by the higher TIMI score in ischemic versus nonischemic limbs. The negative effect of ischemia on limb perfusion was more evident in old rats. TIMI score was comparable in the nonischemic limbs of all groups (Figure 2B). Analogous results were obtained with the dyed bead assay. Blood flow in ischemic hind limbs was lower in untrained old animals compared to untrained young ones. Prior training was associated with a significant blood flow increase in ischemic limbs of both age groups. A significant Age x Training interaction was found on both TIMI score and dyed bead assay (Figure 2C). Angiographic and blood flow data were paralleled by histologic results. Limb ischemia induced a muscle capillaries rarefaction in both untrained young and old rats. Training increased capillary number in ischemic muscles of both age groups. In this case also, age and training significantly increased muscle capillarization after ischemia (Figure 3A and B).

Exercise Increases Muscle HIF-1{alpha} and VEGF Production After Hind-Limb Ischemia and Reduces Systemic VEGF Levels
In trained young rats, HIF-1{alpha} expression increased in ischemic versus nonischemic muscles. This increase was evident also in untrained young animals, although to a lower extent. In untrained old animals, muscle protein levels remained unchanged after ischemia, whereas they significantly increased in trained old ones at levels comparable to those observed in the young animals (Figure 4). In untrained young rats, VEGF protein expression significantly increased in ischemic versus nonischemic muscles, whereas no difference was found between ischemic and nonischemic limbs of untrained old animals. VEGF levels increased in both nonischemic and ischemic muscles of trained young animals, but more in the latter. Interestingly, exercise restored ischemia-related increase in VEGF production in trained aged muscles (Figure 5). At day 21 after hind-limb ischemia, serum VEGF was higher in untrained old animals compared to other groups. In trained old rats, protein levels decreased at values comparable to those observed in the young rats (Figure 6).


Figure 04
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  		Figure 4. Western blot analysis of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) protein expression in ischemic and nonischemic muscles harvested 7 days after limb ischemia in young and old trained and untrained rats. *p <.001 and {dagger}p <.0001 for young ischemic versus nonischemic animals, {ddagger}p <.001 for old ischemic versus nonischemic ones). Actin protein level is used as a loading control. Values are expressed as mean ± standard error of the mean (n = 10 for each group)

 

Figure 05
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  		Figure 5. Western blot analysis of vascular endothelial growth factor (VEGF) protein expression in ischemic and nonischemic muscles harvested 7 days after limb ischemia in young and old trained and untrained rats. *p <.001 for ischemic versus nonischemic in both young untrained and trained rats, {dagger}p <.001 for trained versus untrained ones, {ddagger}p <.001 for old ischemic versus nonischemic ones. Actin protein level is used as a loading control. Values are expressed as mean ± standard error of the mean (n = 10 for each group)

 

Figure 06
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  		Figure 6. Enzyme-linked immunosorbent assay (ELISA) quantitative analysis of serum vascular endothelial growth factor (VEGF) in old trained and untrained animals at 21 days after surgery. *p <.001 for old trained versus old untrained animals, {dagger}p <.001 for old untrained versus all animals. Values are expressed as mean ± standard error of the mean (n = 10 for each group)

 

   DISCUSSION
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This study focuses on the positive role of exercise on age-dependent impairment of angiogenesis in response to tissue ischemia. In old rats, a training program performed before the induction of hind-limb ischemia promotes vascular growth in the ischemic limb, improves blood perfusion, and prevents the dramatic decrease of exercise performance induced by the ischemic insult. The increase of HIF-1{alpha} and VEGF protein expression in aged ischemic muscles mediates the favorable effects of exercise on neoangiogenesis.

Effects of Exercise on Age-Related Impairment of Angiogenesis and Exercise Performance After Hind-Limb Ischemia
Age-dependent impairment of angiogenesis has been demonstrated in peripheral (1,24) and cardiac (25–29) vascular beds and may represent one of the reasons for the increased severity of cardiovascular diseases in the geriatric population. In old mice and rabbits with critical limb ischemia, limb perfusion and muscle capillarization are significantly reduced compared with younger animals (1). Although the present study confirms these previous observations, it shows that age-related attenuation of the protective mechanism of neoangiogenesis represents, at least in part, a reversible phenomenon. In fact, as shown here, prior exercise is able to improve new vessel development, blood perfusion, and trophism of ischemic limbs of old animals. These findings are associated with a significant improvement of maximal exercise capacity, thus indicating the favorable effects of enhanced angiogenesis on functional parameters. In this study, the significant Age x Training impact on exercise performance, ischemic limb perfusion, and capillarization could be due to at least two main reasons: (i) Training may have a greater effect on improving exercise capacity in those animals showing the higher levels of physical deconditioning, i.e., the older ones; and (ii) the positive effect of training on tissue responses to ischemia could be more evident when molecular mechanisms regulating angiogenesis are impaired rather than preserved, i.e., the younger ones.

It is noteworthy that our study looking at the effects of training performed before the induction of limb ischemia is the first one to demonstrate that prior exercise has a preventive role in the loss of tissue neoangiogenesis properties and exercise performance occurring with age.

Effects of Exercise on Muscle HIF-1 Activity and VEGF Expression
Previous studies have shown that age-dependent impairment of angiogenesis is primarily due to a defect in the transcriptional regulation of VEGF (1). T cells are important sources of VEGF, and their reduction in aged ischemic muscles may be responsible, at least in part, for lower VEGF levels (1). More recently, it has been demonstrated that age-dependent alterations of transcriptional and posttranscriptional regulation of HIF-1 are key determinants of VEGF dysregulation occurring with age (8). HIF-1 is shown to be specifically involved in the regulation of muscle adaptations after hypoxia. In human and animal studies, high-intensity exercise physiologically increases HIF-1 (17,18) and muscle VEGF expression (14,19,30,31). Furthermore, recent data indicate that exercise training improves aging-induced downregulation of VEGF angiogenic signaling cascade in the heart (32). Our data agree with these results and demonstrate for the first time that prior exercise is able to restore HIF-1-dependent VEGF upregulation in aged ischemic muscles. The ability of exercise to increase the recruitment of T lymphocytes (33), which are an important source of VEGF protein, and the mobilization and functional activation of endothelial circulating progenitor cells (20) could represent further mechanisms accounting for enhanced angiogenesis after exercise. It is likely that in older animals the overall improvement of age-impaired endothelial function induced by exercise (34) may improve restoration of VEGF production in response to ischemia. Accordingly, previous data from our group demonstrated the ability of physical activity to counteract age-dependent biochemical abnormalities related to endothelial dysfunction and to enhance vascular reactivity (35).

Chronic Exercise Affects Systemic VEGF Levels in Old Animals With Critical Limb Ischemia
Exercise training increases plasma VEGF levels in a murine model of carotid injury and in humans with coronary artery disease (14). Patients with peripheral artery disease show increased VEGF serum levels after training (20). This increase reflects, at the systemic level, the local increase of protein production induced by activation of angiogenesis responses to repetitive episodes of ischemia occurring during endurance training. These data are only in apparent contrast with those obtained in the present study showing a significant reduction of serum VEGF in old trained rats at day 21 after ischemia. In fact, we hypothesize that overall improvement in local ischemia evoked by prior exercise may progressively lead to a reduction of local and systemic VEGF. Another potential mechanism to explain the reduction of serum VEGF might be the attenuation of local inflammation with a lower recruitment of T lymphocytes, which significantly contribute to tissue VEGF expression (1). These results confirm recent data from our group on the relationship between improved angiogenesis, enhanced limb perfusion, and reduction of systemic VEGF levels in spontaneously hypertensive rats (23). Furthermore, it has been demonstrated that increased systemic VEGF levels induced by chronic ischemia return to baseline values when the ischemia is eliminated (36,37).

Clinical Implications and Conclusions
The prevalence of peripheral atherosclerotic disease dramatically increases with aging. This syndrome represents a major cause of mortality and morbidity in the elderly population (38,39). Our data strengthen the role of exercise training as a valid strategy to counteract anatomical impairment and functional limitations associated with this syndrome. These experimental observations may be translated into the clinical setting and explain the results of previous studies indicating the ability of exercise in reducing disability and improving the quality of life of older persons affected by peripheral atherosclerotic disease (40,41). In addition, this study adds information on the molecular mechanisms by which physical activity exerts its therapeutic role and emphasizes the role of an active lifestyle in the prevention, treatment, and prognosis of cardiovascular diseases in elderly persons.


   Footnotes
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Decision Editor: Huber R. Warner, PhD

Received July 30, 2006

Accepted December 12, 2006


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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D. Leosco, G. Rengo, G. Iaccarino, L. Golino, M. Marchese, F. Fortunato, C. Zincarelli, E. Sanzari, M. Ciccarelli, G. Galasso, et al.
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