Increasing physical education in high school students: effects on concentration of circulating endothelial progenitor cells

Aims: Levels of endothelial progenitor cells (EPCs), that can be increased by regular exercise, correlate with vascular function. In the context of primary prevention, the impact of regular physical activity on the amount and function of EPC has not yet been investigated in school children.

Cited Here...: Four sixth grade classes of high school students (n=92) were randomly assigned to either the intervention group (IG) with daily physical exercise (45 min) at school or to the control group (CG) with conventional physical education (PE) (2 h/week). In addition, one sixth grade class at a high school focused on competitive sports (PE) served as a reference group. After 1 school year, exercise capacity and the amount and function of EPCs were evaluated.

Cited Here...: After 1 year, a significantly higher Vo_2max was evident in the intervention group. Nevertheless, exercise capacity did not reach the level of children from PE. In addition, exercise intervention was successful in increasing the amount of EPCs but failed to increase the migratory capacity of the cells.

Conclusion: The result of this study shows, that intensified, supervised school sports leads to an increase in exercise capacity and EPCs in children. Nevertheless, its effect on primary prevention in cardiovascular disease has to be proven in further longitudinal studies.

Introduction

Childhood obesity is a multifactorial disease that can be characterized by an accelerating increase in bodyweight, type II diabetes, and physical inactivity. Manifestation of cardiovascular disease is likely to follow in the majority of obese children.

Physical exercise has become the mainstay of primary and secondary prevention as its preventive value has been proven [^1,2]. Unfortunately, in the majority of adult patients, persistent motivation and discipline to adhere to a regular training schedule is lacking. Other patients, for example, those suffering from metabolic syndrome, are almost unable to comply to a regular training schedule for various reasons.

Children, however, are still open minded for changes in lifestyle. Therefore, we assumed, that an early adaptation to regular physical activity may result in a long-lasting modification of the personality trait toward an active life-style at least in some of the children.

Circulating endothelial progenitor cells (EPCs) are an important marker associated with cardiovascular risk profile and physical fitness [^3,4]. They originate from bone marrow stem cells and are involved in neo vessel formation and endothelial regeneration [^5-7]. Reduced levels and functional impairment of EPCs has been correlated with the presence of coronary artery disease and has been inversely correlated with the number of cardiovascular risk factors [³]. Although the amount of EPCs decline with increasing age [⁸], exercise training has acute and long-term positive effects [^9-12].

The purpose of this cross-sectional study was to determine whether daily physical exercise during childhood leads to a sufficient elevation and improved function of circulating EPCs.

Methods

Study design

Four sixth grade classes of high school students were recruited. They were offered 2 h of physical education per week, which is standard in Germany. Parents were explained the rationale of the study, the study protocol and potentially untoward side effects. Selection was based on the willingness of all parents without exception to allow their children to participate for at least 1 year.

In addition, one sixth grade class from a specific high school focusing on competitive sports and physical education (PE) was examined as a reference. These pupils received 12 h of high-level exercise session per week and frequently participated in competitive sporting events. Thus, they represent a maximum of physical fitness attainable under reasonable conditions in high school students.

After written consent was obtained classes were randomly assigned to either the intervention group (two classes) or to the control group (two classes). Intervention classes were assigned to one period of physical exercise (45 min) per school day. In addition, all pupils received lessons on healthy life style (smoking, use of illicit drugs, selection of appropriate foods) once monthly. The control classes remained on two exercise lessons per week.

After 1 school year, the measurements including assessment of body composition, blood pressure, heart rate, and treadmill exercise test with spirometry were performed. In addition, the amount and function of EPCs was evaluated.

The investigational protocol was approved by the local ethics committee and the trial was registered (NCT00176371).

Anthropometry and body composition

Measurements of bioelectrical impedance for estimation of body composition were obtained in addition to height and body weight. Bioelectrical impedance was obtained using a STA/BIA device (Akern/RJL, Florence, Italy), single frequency (50 Hz), with the participant lying relaxed on a couch; arms and legs were not in contact with other body parts. Sensing electrodes were placed over the right wrist and ankle; current electrodes were placed over the metacarpals and metatarsals.

Treadmill exercise test with spirometry

All participants underwent a graded treadmill exercise test (Woodway USA, Inc. W229N591 Foster Court, Waukesha, Wisconsin, USA) with spirometry until exhaustion according to a modified Bruce protocol for children starting at 1.7 mph and 0° [¹³]. The workload was increased every 3 min. At stage 9, a speed of 6.0 mph and a grade of 22° was reached. The protocol continues until one of the endpoints (changes in ECG, hypotension or hypertension, fatigue, dyspnea, or arrhythmias) is reached. Recovery period lasted at least 5 min and until heart rate and blood pressure reached normal values. Before testing, children were familiarized with the treadmill exercise test. The exercise test was started when oxygen consumption (Vo₂) at rest reached stable levels. At the end of every exercise step children had to indicate their personal perception of exhaustion according to the Borg Rating. After every stage, blood pressure, heart rate, and Vo_2max were recorded using a portable spirometry system (K4b2, Cosmed Srl, Italy) with special masks for children (Rudolph Inc., USA) and a dead space of 70 ml.

Laboratory tests

Analysis of total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol, and triglycerides were performed using an enzymatic colorimetric test (Roche Diagnostic, F. Hoffmann-La Roche Ltd, Basel, Switzerland). The analysis of the lipid profile was performed on Roche/Hitachi Modular P analyzer. Measurements were made in pairs of samples (baseline and 1 year). Technicians were blinded for group assignments, intraobserver variability was less than 5%.

Measurement of endothelial progenitor cells

EPCs were measured by FACS analysis on 200 μl of peripheral blood after 20-min incubation with different combinations of the following antibodies: PE-conjugated mouse antihuman VEGFR2 also known as KDR (R&D Systems, Wiesbaden, Germany), FITC-conjugated mouse antihuman CD34 (Miltenyi, Bergisch Gladbach, Germany), PerCP-conjugated mouse antihuman CD3, APC-conjugated mouse antihuman CD45 (BD Pharmingen, Heidelberg, Germany), PE-conjugated mouse antihuman CD133 (Miltenyi, Bergisch Gladbach, Germany). After incubation, the erythrocytes were lysed and the remaining cells washed with phosphate buffered saline and fixed in 2% paraformaldehyde before analysis using a FACS-Calibur (Becton, Dickinson, Heidelberg, Germany). To quantify the amount of KDR⁺CD34⁺ double positive cells, the lymphocyte cell fraction was gated and analyzed for the expression of CD34 and CD3. Only the CD34⁺ and CD3^- cells were finally investigated for the content of KDR⁺/CD34⁺ double positive cells. The amount of KDR⁺/CD34⁺ double positive cells is expressed as cells per ml of blood as described recently [^11,12].

Measurement of migratory capacity of endothelial progenitor cells

Mononuclear cells (MNCs) were isolated from venous blood by density gradient centrifugation (Histopaque 1077, Sigma, Deisenhofen, Germany). After washing the cells several times with phosphate buffered saline, 1×10⁷ cells were plated on gelatine-coated cell culture dishes (six well plates, TPP, Berlin, Germany) and cultured in EBM-2 (Cambrex, Verviers, Belgium) as described recently [¹¹]. After 7 days in culture the cells were detached using trypsin/ethylenediaminetetra-acetic acid and the migratory capacity of the cells toward SDF -1 (100 ng/ml) was evaluated using a modified Boyden-chamber as described recently [¹¹].

Statistical analysis

Descriptive statistics were used to compare baseline measurements. Results are given as mean±SEM. Comparisons between the training classes, the control classes and the sports gymnasium were performed with one-way ANOVA test. A probability value of less than 0.05 was considered to indicate statistical significance.

Results

Students characteristics

A total of 108 children (mean age 12.0±0.1 years) took part in the study. Fifty children were in the intervention group and 42 were in the control group. Sixteen children were attending the class of PE. No significant difference was observed among all three groups with respect to age, weight, blood pressure, and lipid profile. A difference in height, fat free mass, and heart rate at rest was evident between the students from PE and the students of the control and intervention groups (Table 1). Furthermore, mean body mass index (BMI) was significantly lower in students of PE compared with students of the two other groups. Regarding BMI distribution in percentiles, five children in the intervention classes (10%) and seven children in the control classes (17%) were overweight (>90. percentile) compared with one child (6%) in PE, respectively.

Table 1

Exercise capacity

Treadmill exercise testing with spirometry was successfully performed in all 108 students after 1 year. No significant difference was observed among the three groups regarding maximal heart rate (199±2/min in the intervention classes vs. 201±3/min in the control classes vs. 200±3 in PE). At this maximal heart rate, students in PE went significantly longer on the treadmill exercise (1215±20 s) as compared with students in the two other groups (1123±10 s in the intervention classes, P<0.01 vs. PE and control; 1066±13 s in the control classes, P<0.01 vs. PE). Analyzing the exercise capacity by spirometry and treadmill exercise, a significant difference between the intervention and control classes was evident (intervention classes: 41.9±0.7 ml/kg×min vs. control classes: 37.6±1.0 ml/kg×min; P<0.01). Nevertheless, the Vo_2max was still significantly lower in the intervention and control classes when compared with PE (49.1±1.6 ml/kg×min, P<0.01) (Fig. 1).

Fig. 1

Endothelial progenitor cells

With respect to the number of circulating CD34^pos cells, no significant difference could be detected between the control and intervention group (control: 2388±155 cells/ml, intervention: 2759±190 cells/ml, P=NS). Comparing the amount of CD34^pos cells of the control and intervention classes with PE, a significant difference was obvious (control: 2388±155 cells/ml, PE: 3458±371 cells/ml; P<0.01). Only a trend was detectable with respect to CD34^pos cells between the intervention class and PE (intervention: 2759±190 cells/ml, PE: 3458±371 cells/ml; P=0.07) (Fig. 2a).

Fig. 2

Analyzing the amount of circulating CD133^pos cells in all three groups, no significant difference was noted between the control (11 104±1498 cells/ml blood) and intervention class (12 347±1074 cells/ml blood), whereas the amount of CD133^pos cells was significantly higher in the children from PE (17 110±4032 cells/ml blood; P<0.01 vs. control and intervention class) compared with the other ones (Fig. 2b).

The evaluation of more differentiated EPCs revealed a different picture among all three groups: CD34⁺/KDR⁺ was 466±28 (control) versus 600±34 (intervention) cells/ml blood; P<0.01 (Fig. 2c) and CD133⁺/KDR⁺ double positive cells were 189±10 (control) versus 301±27 (intervention) cells/ml blood; P<0.05 (Fig. 2d). Beside this higher level of EPCs in the intervention class, a significant difference compared with PE was still evident (CD34⁺/KDR⁺ 768±42 cells/ml blood, P<0.05 vs. intervention class, P<0.001 vs. control class; CD133+/KDR+: 529±83 cells/ml blood, P<0.05 vs. intervention class, P<0.001 vs. control class).

No difference was observed with respect to CD45^pos cells among all groups (control: 2.01×10⁶±88 663; intervention: 2.05×10⁶±10 6001; PE: 2.05×10⁶±11 1514 cells/ml blood; P=NS).

Migratory capacity of endothelial progenitor cells

As depicted in Fig. 3, no significant difference in the migratory capacity of EPCs could be documented between the intervention and the control group (intervention: 116±37 vs. control: 98±19 cells/1000 seeded cells; P=NS). Analyzing the migratory capacity in the children from PE, a significantly elevated value was obtained when compared with the other two groups (PE: 289±43 cells/1000 seeded cells; P<0.001 vs. control, P<0.01 vs. intervention).

Fig. 3

Correlation analysis between endothelial progenitor cell amount and exercise capacity

A weak, but significant correlation between the amount of EPCs and exercise capacity of the children was detected (r=0.33, P<0.001; Fig. 4).

Fig. 4

Discussion

Childhood obesity has increased by approximately three-fold in most industrialized countries over the past 20 years, mostly owing to a decline in physical activity [^14-16]. Endothelial dysfunction is often observed in obese children [¹⁷] and is closely linked to the amount and function of EPCs [¹⁸], which have the potential to participate in neovascularization and repair of damaged endothelium. Over the last years, it has become evident that exercise training has the potential to increase the amount of EPCs [^9,11], however, the impact of a structured exercise intervention program in school children was unknown.

Four important messages emerge from this study. First, additional exercise, resulting in five instead of two exercise lessons per week, results in a higher exercise capacity. Second, students attending sports lessons every day have significantly higher levels of EPCs when compared with students from the control group. Third, despite an increase in EPCs owing to 1 year of additional exercise, these students still have less EPCs as compared with students performing routine exercise on a high level every day (students attending PE). Fourth, the amount of exercise performed during the intervention seems not to be enough to alter the migratory capacity of EPCs.

Taken together, these results imply that structured exercise intervention in school seems to have a positive impact on exercise capacity and the amount of EPCs. This may be an important factor for primary prevention of cardiovascular diseases for the future.

Prevention and intervention programs in children and adolescents

Several prevention programs for children have been started so far [¹⁹]. Despite all efforts, the results published are disappointing so far [²⁰]. In this cross-sectional randomized study, in contrast to other studies, we applied intensified school sport (5 days/week, 45 min/day) with a special focus on endurance training. It is worth pointing out, that this form of exercise intervention can be integrated into everyday life at school, and that it showed a positive effect on exercise capacity and the amount of circulating EPCs.

Endothelial progenitor cells

This study is the first to demonstrate the effect of a structured exercise program on EPCs in school children. Thus, it can be concluded that regular exercise training is effective in children also. When comparing the concentration of EPCs from the investigated children with data we published for patients with either coronary artery disease or peripheral artery occlusive disease [^11,12], it became evident that elderly individuals (around the age of 60 years) with cardiovascular diseases have less EPCs (around 100 CD34/KDR double positive cells/ml blood) than healthy children (around 500 CD34/KDR double positive cells/ml blood). This decline in EPCs with age and disease is in line with findings of other groups [^3,8]. In addition, in an earlier study of our group, we concluded that transient ischemia in exercise program seems to be necessary for the increase in number of EPCs [¹¹]. What is the difference to the present study, were an increase in EPC could be documented without ischemia? Possible explanations are different disease severity (patients with CHF/PAOD vs. healthy school children), age (older individuals vs. school children), and training duration (4 weeks vs. 1 year in this study).

Analyzing the less differentiated CD34^pos hematopoietic stem cells, no difference was observed between the control and intervention class. This finding is in contrast to a recently published study by Zaldivar and colleagues [²¹], who showed a significant increase in CD34^pos cells in healthy children and adolescents (age-range: 8-17 years). One reason for the difference may be, that the authors used a single intermittent high-intensity exercise test, and not an exercise-training program over a longer period of time as described in our study.

Using the training strategy as described here, no alteration in migratory capacity was evident between the control and the intervention group. Is there room for improvement in migratory capacity also in healthy children? This question can be clearly answered with yes, because the EPCs isolated from children performing exercise training on a high level exhibit a three-fold increase in migratory capacity. On the basis of these data, we have to conclude, that the exercise intensity of one additional hour sports lesson per day is sufficient to increase the amount of EPCs, but not their function.

What is the physiological impact of EPC increase in the intervention group? The answer to this question can only be speculative at present. Several animal and human studies provided evidence, that EPCs enhance angiogenesis, promote vascular repair, and improve endothelial function, [^22,23]. Therefore, one may speculate, that the elevated level of EPCs in the intervention group may represent one component mediating the beneficial effect of exercise training even in children. This notion is finally supported by the significant, but weak correlation between EPCs and exercise capacity.

A limiting factor of this study is its cross-sectional design. As yet there is no information on the longitudinal change of EPCs in the intervention classes.

In summary, this is the first study proving the important relationship between regular exercise training implemented into daily life at school and an improved exercise capacity as well as EPC amount. Nevertheless, a longitudinal and long-term study is warranted to confirm that increased exercise during childhood will lead to an effective primary prevention of cardiovascular diseases.

Acknowledgements

The authors thank Claudia Weiss, Angela Kricke, and Nicolle Urban for their excellent technical assistance. The study was supported by a grant from Novartis and the Deutsche Forschungsgemeinschaft (KO 3512/1-1).

Conflicts of interest: none.