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J Appl Physiol 103: 474-483, 2007. First published April 19, 2007; doi:10.1152/japplphysiol.01160.2006
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Effects of short-term vibration and hypoxia during high-intensity cycling exercise on circulating levels of angiogenic regulators in humans

Frank Suhr,1,4,* Klara Brixius,2,4,* Markus de Marées,1,4 Birgit Bölck,3 Heinz Kleinöder,1,4 Silvia Achtzehn,1,4 Wilhelm Bloch,2,4 and Joachim Mester1,4

1Institute of Training Science and Sport Informatics and 2Department of Molecular and Cellular Sport Medicine, German Sport University Cologne; 3Laboratory of Muscle Research and Molecular Cardiology, Department of Internal Medicine III, University of Cologne; and 4The German Research Center of Elite Sport, German Sport University Cologne, Cologne, Germany

Submitted 13 October 2006 ; accepted in final form 18 April 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
This study aimed to investigate the biological response to hypoxia as a stimulus, as well as exercise- and vibration-induced shear stress, which is known to induce angiogenesis. Twelve male cyclists (27.8 ± 5.4 yr) participated in this study. Each subject completed four cycle training sessions under normal conditions (NC) without vibration, NC with vibration, normobaric hypoxic conditions (HC) without vibration, and HC with vibration. Each session lasted 90 min, and sessions were held at weekly intervals in a randomized order. Five blood samples (pretraining and 0 h post-, 0.5 h post-, 1 h post-, and 4 h posttraining) were taken from each subject at each training session. Hypoxia was induced by a normobaric hypoxic chamber with an altitude of 2,500 m. The mechanical forces (cycling with or without vibration) were induced by a cycling ergometer. The parameters VEGF, endostatin, and matrix metalloproteinases (MMPs) were analyzed using the ELISA method. VEGF showed a significant increase immediately after the exercise only with exogenously induced vibrations, as calculated with separate ANOVA analysis. Endostatin increased after training under all conditions. Western blot analysis was performed for the determination of endostatin corresponding to the 22-kDa cleavage product of collagen XVIII. This demonstrated elevated protein content for endostatin at 0 h postexercise. MMP-2 increased in three of the four training conditions. The exception was NC with vibration. MMP-9 reached its maximum level at 4 h postexercise. In conclusion, the results support the contention that mechanical stimuli differentially influence factors involved in the induction of angiogenesis. These findings may contribute to a broader understanding of angiogenesis.

mechanical stimuli; angiogenesis

ANGIOGENESIS is the outgrowth of new capillaries from preexisting vessels (12, 19, 47). There is evidence that physical exercise induces angiogenesis (30), but the molecular mechanisms that regulate this reaction are poorly understood (35). The formation of new capillaries is a complex and multistep phenomenon that includes the migration, differentiation, and proliferation of endothelial cells (ECs), as well as microtube formation and degradation of the extracellular matrix (ECM). The process is highly controlled and coordinated and seems to depend on the local balance between pro- and anti-angiogenic regulators (26, 42), as well as different proteinases in the affected tissue (5, 8, 38).

In some sports, such as cycling and alpine skiing, the human body is exposed to vibrations. Furthermore, vibration has been used as a specific training method for strength training for many years. Different authors have demonstrated significant increases in maximal muscle strength due to vibration training (7, 14). Measurements of cardiovascular parameters have demonstrated that the effects of whole body vibration on the total peripheral resistance to the blood flow are not a simple matter. On the one hand, vibration tends to increase the resistance through the increase of viscosity, while on the other hand the small vessels might be dilated under certain conditions during the body vibration. This would greatly reduce the total peripheral resistance (37, 59, 61). Hydrodynamic analysis indicates that the shear stress at the wall of vessels is considerably increased under certain conditions during body vibration (37, 59, 60). The release of angiogenic agents can therefore be hypothesized.

An appropriate supply of oxygen is essential for almost all biological tissues. To protect the organism, hypoxic situations (reduced oxygen supply) trigger a number of physiological and pathophysiological responses and adaptations such as vasculogenesis, angiogenesis, or erythropoiesis (56). During normal exercise, local hypoxic conditions in the stressed skeletal muscle can occur. These can be elevated by inducing environmental hypoxic circumstances.

In our study, vibrations and normobaric hypoxia were applied to the subjects during cycling performance, and the effects on the following angiogenic agents were investigated.

VEGF.   VEGF is a potent mitogen in endothelial cells (18, 53). Therefore, it plays a critical role in the induction and regulation of angiogenesis in physiological processes such as embryonic development and the menstrual cycle as well as in pathological conditions (e.g., tumor growth and atherosclerosis) (17, 21, 45).

Endostatin.   Endostatin corresponding to a 22-kDa fragment derived from the carboxyl-terminal noncollagenous NC1 domain of collagen XVIII (43, 49) was investigated. This molecule is highly expressed in the basement membrane zones around blood vessels (40) and has been shown to be an inhibitor of VEGF-induced angiogenesis by preventing proliferation and migration of endothelial cells (28, 38, 54). In contrast to the running opinion of endostatin function and signaling, different authors have recently demonstrated that endostatin seems to operate as an angiogenic modulator rather than an anti-angiogenic agent (50, 51, 57). Up until now, there is incomplete understanding of endostatin signaling pathways and functions.

Matrix metalloproteinases.   These represent a family of about 20 proteolytic enzymes that comprise a zinc ion at the active side of catalysis (36). Because of their proteolytic and processing properties, matrix metalloproteinases (MMPs), and especially MMP-2 and MMP-9, play a crucial role in the development of new capillaries (2, 20).

VEGF, endostatin, MMP-2, and MMP-9 have been shown to play a central role as mediators/inductors of exercise-induced angiogenesis. These growth factors and cytokines have been shown to be regulated by different stimuli, such as hypoxia and mechanical loadings. We therefore investigated the influence of hypoxia and mechanical loadings, separately and in combination, in the angiogenic process. The secretion patterns of proangiogenic growth factors (VEGF), extracellular matrix (ECM) cleavage fragments (endostatin), and proteolytic enzymes (MMP-2 and MMP-9) were analyzed during high-intensity cycling exercise training with normobaric hypoxic and vibration stimuli.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Selection of subjects.   Twelve healthy male subjects participated in this study. Characteristics of basic anthropometric and physical parameters of the subjects are summarized in Table 1. The subjects represented cyclists with different performance backgrounds, including national class athletes and recreational cyclists. In addition, all subjects abstained from alcohol consumption for 24 h before and during the training intervention and were nonsmokers. All subjects gave written informed consent to contribute to the study. The protocols used in the study have been granted a license from the institutional office to conduct the human research described in the present study and are in line with the Declaration of Helsinki.


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  		Table 1. Anthropometric data of subjects

 
Determination of training intensity.   In advance of the study, we carried out tests to determine the correct training intensity for each subject for the experiments. Two incremental cycling step tests until exhaustion were carried out under normoxic and normobaric hypoxic conditions for every subject at intervals of 3 days and at least 1 wk before the start of the investigation. The subjects had to perform at high intensities without aborting the training session. The intensity of the test started at 100 W and was increased every 5 min by 40 W. Heart rate (HR) (Polar S810i, Polar Electro, Ort, Germany) and maximal oxygen uptake (VO2max) (Zan 600, Zan Messgeräte, Oberthulba, Germany) were measured. For lactate determination, 20 µl blood was withdrawn from the earlobe. Capillary blood lactate concentrations were measured with EBIO plus (EKF Diagnostic Sales, Magdeburg, Germany). A Cyclus 2 (RBM Elektronik, Automation, Leipzig, Germany) cycling ergometer was used to generate the intervals during the incremental cycling tests under both normobaric hypoxic and normoxic conditions.

Exercise study protocol.   For the study itself, an exercise protocol was developed for both normoxic conditions and normobaric hypoxic conditions. Exercise was carried out on either a conventional cycling ergometer (Cyclus 2, as above) or a cycling ergometer fixed on a vibration platform with a peak-to-peak amplitude of 4 mm and a frequency of 30 Hz that enhanced the mechanical forces within the skeletal muscle tissue by inducing vibrations.

The combination of two physiological conditions with or without artificial mechanical stimuli resulted in four different training conditions: normoxic conditions (NC) without vibration, NC with vibration, normobaric hypoxic conditions (HC) without vibration, and normobaric HC with vibration. The hypoxic conditions were induced by using a normobaric hypoxic-chamber (Hypoxic Training Systems, Hypoxico, New York) with an average altitude of ~2,500 m (O2 15.5–15.3%). The O2 and CO2 concentrations were measured during the whole performance period with a Dräger Multiwarn O2 and CO2 Gas Analyser (Dräger, Lübeck, Germany). To keep the CO2 concentration within a physiologically tolerable range (0.03–0.3%), a CS 2210 CO2 absorber (SK Engineering, Kiel, Germany) was used. Each subject completed the four training sessions at weekly intervals in a random order. Two incremental cycling tests until exhaustion for every subject were carried out under both conditions to calculate the correct training intensity (see above). Each training session lasted 90 min and consisted of a 10-min warmup at 50% of VO2max followed by 10 intervals of 3-min high load at 80–85% of VO2max and 5 min recovery at 55–60% of VO2max (Fig. 1).


Figure 1
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  		Fig. 1. The exercise protocol lasted a total of 90 min and consisted of a 10-min warmup at 50% of maximal O2 uptake (VO2max) followed by a high-intensity phase (10 x 3 min at 80–85% VO2max). A recovery phase (5 min at 55–60% VO2max) followed each high-intensity interval. Blood samples were taken before the training and at 0 h post-, 0.5 h post-, 1 h post-, and 4 h postcompletion.

 
Measurement of VEGF, endostatin, MMP-2, and MMP-9 serum levels.   One venous blood sample was taken before exercise, and four samples were taken at 0 h, 0.5 h, 1 h, and 4 h postexercise. Nine and a half milliliters of blood was collected by the Vacutainer blood withdrawal system (Becton Dickinson). After storage at 7°C for ~30 min for deactivation of coagulation factors, the blood samples were centrifuged for 10 min at 1,861 g and 4°C (Rotixa 50, Hettich Zentrifugen, Mühlheim, Germany). The serum was stored at –80°C. Serum levels of VEGF (polyclonal antibody) (pg/ml), endostatin (ng/ml), MMP-2 (ng/ml), and MMP-9 (ng/ml) were determined by using human ELISA kits (R & D Systems, Wiesbaden, Germany).

Immunoblot analysis.   Fifty micrograms of proteins was thawed on ice and suspended in buffer (0.5 mM Tris·HCl, 10% glycerol, 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 0.05% bromphenol blue). Proteins were separated using discontinuous PAGE with 4% and 17% acrylamide and transferred to polyvinylidene difluoride (PVDF) membranes (Roche, Mannheim, Germany) by wet blotting. Transfer efficiency was verified by total protein staining of the gels with Coomassie brilliant blue and by staining of the protein bands on PVDF membranes (Western blots) with Ponceau S solution (Sigma Chemical, St. Louis, MO; ready-to-use, 5 min). The blots were blocked in 5% low-fat milk and washed with Tris-buffered saline and Tris-buffered saline with Tween [150 mmol/l NaCl, 10 mmol/l Tris·HCl, 0.05% Tween 20 (pH 7.5)]. Membranes were incubated with rabbit anti-endostatin antibody (1:100) (Chemicon International, Temecula, CA). Proteins were detected by an enhanced chemiluminescence assay (ECL Kit, Amersham-Life Science, Buckinghamshire, UK) exposed to X-ray film (Kodak X-OMAT Engineering, Eastman Kodak, Rochester, NY).

Statistical analysis.   Statistical analyses of the data were performed by using a statistics software package (Statistica for Windows, 7.0, Statsoft, Tulsa, OK). Descriptive statistics of the data are presented as means ± SE unless described otherwise. For the comparison of different terms (preexercise, 0 h postexercise, 0.5 h postexercise, 1 h postexercise, and 4 h postexercise), repeated-measures ANOVA with Bonferroni post hoc test was used. For comparison between the groups of more highly trained subjects vs. less-trained subjects, an unpaired Student's t-test was used. For VEGF, the individual responses under all training conditions for every subject are presented by using the relative difference ({Delta}rel) between VEGF concentration at 0 h postexercise ([VEGF]0h post) and the VEGF concentration before training ([VEGF]pre). {Delta}rel was calculated using the following formula: ([VEGF]0h post – [VEGF]pre)/[VEGF]pre. The two incremental cycling tests under normoxic and normobaric hypoxic conditions were compared by using a paired Student's t-test. Statistical differences were considered to be significant for values of (P < .05 or P < .01).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
VO2max.   Every subject completed two incremental cycling tests until individual exhaustion. There was a significant difference (P < 0.01) between normoxic conditions (VO2max: 54.7 ± 2.6 ml·kg–1·min–1) and normobaric hypoxia (VO2max: 50.9 ± 2.4 ml·kg–1·min–1, fraction of inspired O2 {approx} 15.3%).

ELISA analyses.   The circulated serum VEGF levels in response to acute exercise for trained subjects are shown in Fig. 2. Separately calculated ANOVA with repeated measurements only showed a significant (P < 0.05) increase in the VEGF levels at 0 h postexercise when artificial vibrations were an additional input during the training sessions (NC with vibration: preexercise 248.9 ± 46.0 pg/ml, 0 h postexercise 332.9 ± 75.3 pg/ml; HC with vibration: preexercise 288.8 ± 56.3 pg/ml, 0 h postexercise 360.9 ± 77.2 pg/ml). The training interventions without vibration did not show a significant increase at any time after the performance compared with the vibration treatments when calculated separately with repeated measures ANOVA (NC without vibration: preexercise 282.6 ± 56.9 pg/ml, 0 h postexercise 318.2 ± 69.1 pg/ml; HC without vibration: preexercise 270.0 ± 54.4 pg/ml, 0 h postexercise 326.3 ± 75.9 pg/ml). However, there was a tendency toward increased values of VEGF for HC without vibration (P = 0.07) when calculated by a separate ANOVA analysis. After 4 h postexercise, VEGF tended toward the resting levels (Fig. 2).


Figure 2
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  		Fig. 2. Changes in circulating VEGF protein content at different time points in 12 healthy athletes preexercise and at 0 h post-, 0.5 h post-, 1 h post-, and 4 h postexercise training. VEGF protein levels were measured using the ELISA method. Significant increases (*P < 0.05) were observed only in vibration situations. Values are presented as means ± SE. NC, normoxic conditions; HC, hypoxic conditions.

 
During the analysis we observed highly interindividual variations in the VEGF response (Fig. 3). Therefore the subjects were divided into two groups based on their anaerobic thresholds (data not shown): highly trained (HT) subjects (S2, S4, S5, S7, S8, S10, S11; n = 7) and less trained (LT) subjects (S1, S3, S6, S9, S12; n = 5). We observed the highest variations in the individual VEGF response (compared with pretraining levels) at 0 h postexercise. Therefore we compared the relative difference between these time points in the two groups by using an unpaired Student's t-test. The HT subjects showed a significant increase in VEGF at 0 h postexercise under NC without vibration (P = 0.01) and under HC with vibration (P = 0.01; Fig. 4, A and D, respectively). Under NC with vibration, the increase tended to be significant (P = 0.055; Fig. 4B). The treatment under hypoxic conditions did not show a significantly elevated VEGF level (P = 0.1; Fig. 4C). Figure 5 gives an overview of the relative difference between the two time points for all four treatments for every subject. As shown in Fig. 5, the VEGF response is highly individual. In particular, the HT subjects showed a very strong increase in VEGF content under situations when vibrations were applied (e.g., S4, S7, and S8), whereas the LT subjects tended to reduce the VEGF level (e.g., S1, S3, and S6).


Figure 3
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  		Fig. 3. Individual VEGF response of the subjects. A: NC without vibration. B: NC with vibration. C: HC without vibration. D: HC with vibration. Black bars represent the preexercise value; gray bars represent the 0-h postexercise value.

 

Figure 4
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  		Fig. 4. Individual relative difference of VEGF between preexercise and 0-h postexercise for the lesser-trained group (LT) and the highly trained group (HT). A: NC without vibration. B: NC with vibration. C: HC without vibration. D: HC with vibration. Values are presented as means ± SE. **Significantly increased relative VEGF values for the HT group at NC without vibration and HC with vibration (P < 0.01).

 

Figure 5
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  		Fig. 5. Relative difference in VEGF between the preexercise value and the 0-h postexercise value under the 4 different treatments.

 
Endostatin showed a significant increase at 0 h postexercise compared with pretraining values for all treatment types (NC without vibration: preexercise 87.3 ± 5.7 ng/ml, 0 h postexercise 108.4 ± 6.0 ng/ml; NC with vibration: preexercise 86.3 ± 4.7 ng/ml, 0 h postexercise 107.7 ± 4.5 ng/ml; HC without vibration: preexercise 86.1 ± 5.1 ng/ml, 0 h postexercise 100.5 ± 7.2 ng/ml, P < 0.01, respectively; HC with vibration: preexercise 89.9 ± 6.5 ng/ml, 0 h postexercise 99.2 ± 7.9 ng/ml, P < 0.05). After training in normoxia without vibration, the endostatin levels were still significantly elevated at 0.5 h (P < 0.05) and 1 h (P < 0.01) postexercise (preexercise 87.3 ± 5.7 ng/ml, 0.5 h postexercise 98.9 ± 5.5 ng/ml, 1 h postexercise 100.9 ± 6.6 ng/ml). By 4 h postexercise, there were no significant differences detectable as endostatin levels had nearly returned to the pretraining values. The results for endostatin are presented in Fig. 6A.


Figure 6
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  		Fig. 6. A: changes in circulating endostatin protein content at different time points in 12 healthy athletes: preexercise and at 0 h post-, 0.5 h post-, 1 h post-, and 4 h postexercise training. Endostatin protein levels were measured using the ELISA method. At 0-h postexercise, endostatin was significantly increased in all training interventions (*P < 0.05, **P < 0.01). NC without vibration showed significant increases at 0.5 h postexercise (*P < 0.05) and 1 h postexercise training (**P < 0.01). Values are presented as means ± SE. B: by using Western Blot analysis, we observed an increase in endostatin fragment at 0-h postexercise compared with the pretraining situation. B shows the protein content for NC without vibration.

 
The MMP-2 values were significantly (P < 0.01) increased after all training sessions at 0 h postexercise with the exception of NC with vibration where no correlation could be found (NC without vibration: preexercise 223.5 ± 11.1 ng/ml, 0 h postexercise 244.8 ± 11.7 ng/ml; HC without vibration: preexercise 223.5 ± 10.9 ng/ml, 0 h postexercise 243.0 ± 10.7 ng/ml; HC with vibration: preexercise 237.7 ± 10.8 ng/ml, 0 h postexercise 254.3 ± 11.3 ng/ml). MMP-2 level at 0 h postexercise for NC with vibration increased (preexercise 235.9 ± 9.8 ng/ml, 0 h postexercise 244.8 ± 10.5 ng/ml), but the change was not significant (P > 0.09). Similar to the behaviors of VEGF and endostatin, MMP-2 also declined to pretraining values (Fig. 7) at 4 h post exercise.


Figure 7
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  		Fig. 7. Changes in circulating matrix metalloproteinase (MMP)-2 protein content at different time points in 12 healthy athletes: preexercise and at 0 h post-, 0.5 h post-, 1 h post-, and 4 h postexercise training. MMP-2 protein levels were measured using the ELISA method. At 0 h postexercise, MMP-2 was significantly increased in all training interventions (**P < 0.01) except NC with vibration (P > 0.05). NC without vibration and HC with vibration showed significantly elevated MMP-2 levels at 0.5-h postexercise (**P < 0.01, *P < 0.05, respectively). Values are presented as means ± SE.

 
In contrast, MMP-9 showed a completely different pattern for all training interventions. In all cases, the MMP-9 levels were approximately twofold greater and significantly increased (P < 0.05 for NC without vibration, P < 0.01 for the other 3 combinations) at 4 h after the training sessions compared with the resting levels (NC without vibration: preexercise 428.6 ± 77.5 ng/ml, 4 h postexercise 843.8 ± 162.8 ng/ml; NC with vibration: preexercise 446.5 ± 69.5 ng/ml, 4 h postexercise 866.8 ± 163.6 ng/ml; HC without vibration: preexercise 468.1 ± 56.4 ng/ml, 4 h postexercise 930.3 ± 135.2 ng/ml; HC with vibration: preexercise 488.1 ± 85.6 ng/ml, 4 h postexercise 919.7 ± 159.3 ng/ml). The results for MMP-9 are shown in Fig. 8. Because of the different temporal release pattern of MMP-9 compared with MMP-2, it appears that there is a different mechanism of activation for these molecules.


Figure 8
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  		Fig. 8. Changes in circulating MMP-9 protein content at different time points in 12 healthy athletes: preexercise and at 0 h post-, 0.5 h post-, 1 h post-, and 4 h postexercise training. MMP-9 protein levels were measured using the ELISA method. At 4-h postexercise, MMP-9 was significantly increased in all training interventions (*P < 0.05, **P < 0.01). NC without vibration, NC with vibration, and HC without vibration showed significantly elevated MMP-9 levels at 1-h postexercise (*P < 0.05, **P < 0.01, *P < 0.05, respectively). Values are presented as means ± SE.

 
Immunoblot analysis.   Because the ELISA technique observes all proteolytic fragments of collagen XVIII, Western Blot analysis was performed to detect the collagen XVIII cleavage fragment and biologically active form endostatin. The original blot is shown in Fig. 6B. In line with the results obtained from ELISA experiments, an increase in endostatin protein content was observed.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGMENTS
 REFERENCES
 
This study investigated the influence of physical exercise with physiological (hypoxia) and mechanical (vibration) interventions on different growth factors and mechanisms that had been shown to be involved in angiogenesis. It was shown that VEGF is only increased in situations of exogenous-induced mechanical loading but not by exercise itself, as reported in RESULTS. The VEGF release seems to be dependent on the subject's training status because HT individuals showed a stronger VEGF release than LT subjects. In addition, we observed an exercise-induced increase in endostatin that was unaffected by additional hypoxic conditions or vibration. This increase in endostatin was paralleled by an increase in MMP-2. Under all experimental conditions, we observed a delayed but prolonged increase in MMP-9 following physical exercise.

VEGF is a key regulator of physiological angiogenesis (11, 18, 47). However, there is an ongoing discussion about the underlying mechanisms. VEGF was shown to be increased in situations of physical exercise performance (23). On the one hand, it has been shown that there is an insufficient O2 supply in the skeletal muscle tissue during exercise, and this leads to the elevation of VEGF mRNA and/or protein levels within the stressed skeletal muscle tissue (3, 24, 29, 30, 35, 54). These increased VEGF mRNA and/or protein levels could lead to formation of new capillaries along the chemical gradient of VEGF (1). Compared with endothelial cells, parenchymal cells in the vasculature are a rich source of VEGF and therefore play a critical role in the formation of the important VEGF gradient (1). On the other hand, increased physical exercise elevates the total skeletal muscle blood flow, and the resulting mechanical stress may play a crucial role in angiogenesis by increasing VEGF production (8, 32). VEGF is not increased under normoxic conditions without vibration but tended to increase (P = 0.07) under normobaric hypoxic conditions without vibration. This may indicate that the exercise-induced alterations in PO2 in the circulating blood and skeletal muscle fibers, as probably the greatest source of VEGF, do not affect VEGF release under NC without vibration, but that the VEGF release tended to increase under HC without vibration. In our experiments, the VEGF serum concentration only increased in situations of increased mechanical vibration. Our findings are in line with the findings of Kim et al. (32), who showed that a single 90-min bout of in vivo interval cycling exercise increased the VEGF protein content only if the training is combined with additional exogenous-induced mechanical forces. Our results are comparable to the observations of Petersen et al. (46) and Pufe et al. (48), who demonstrated in in vitro experiments that the VEGF level in tendon fibroblasts and cartilage disks is elevated after inducing mechanical overloading. Yue and Mester (59), Mester et al. (37), and Yue and Mester (60) demonstrated the effects of increased maximal shear stress at the vessel wall as a result of mechanical vibrations. Therefore, shear stress could be a factor under NC for the exercise-induced release of proangiogenic VEGF. Under HC without vibration, a tendency for an increase in VEGF level at 0 h postexercise was observed (P = 0.07). Exercise under hypoxic conditions combined with exogenous-induced vibration led to an increase in VEGF (P = 0.03). In our study, we observed high interindividual variations in VEGF content (Fig. 3). These data are in line with the observations made by Gunga et al. (24) and Heits et al. (27), who demonstrated big fluctuations in the VEGF release for trained subjects. Interestingly, the subjects with a high relative difference ({Delta}rel) between the 0 h postexercise value and the preexercise value were highly trained (Fig. 5). These subjects seem to get a beneficial release in their VEGF content when vibrations are applied to the training session (Fig. 3). By performing an unpaired t-test to compare the {Delta}rel between the LT group and the HT group, we observed a significant increase in the HT group for {Delta}rel compared with the LT group. Therefore the induced vibration stimulus seems to be a benefit for the HT subjects as it increases their VEGF concentration directly after performance (Fig. 4). Exercise leads to an insufficient O2 supply to the stressed skeletal muscle tissue, which induces a local hypoxic condition. Highly trained athletes are exposed to these local hypoxic conditions regularly because of their training schedule. These data could lead to the assumption that the regular training of highly trained athletes may induce a higher VEGF content that is stored in endothelial cells, parenchymal cells, or even in the ECM by heparan sulfate proteoglycans in the stressed tissue of athletes. By completing a high-intensity training session as in our study, these VEGF pools could be released into the circulation. A further speculation could be that the increased VEGF release in highly trained athletes may result from the larger cross-sectional area of capillaries in their skeletal muscle tissue (47). This leads to a higher number of VEGF-producing cell types (ECs, parenchymal cells), which can release VEGF molecules very quickly. Our data support these conjectures, but they will have to be investigated and proved in further studies.

Endostatin has been suggested to act as an anti-angiogenic factor by inhibiting VEGF-induced endothelial cell migration and proliferation (28, 38, 52, 55, 58) and by the induction of endothelial cell apoptosis (4, 31, 52). However, Schmidt and co-workers (50, 51) demonstrated both pro-angiogenic and anti-angiogenic effects of endostatin in a dose-dependent manner. Our data reveal evidence of a significant increase in the endostatin plasma concentration following physical exercise that is independent of exogenously induced stimuli such as normobaric hypoxia and/or mechanical loading. Interestingly, it was shown in a recent study that endostatin evokes vascular relaxation by increasing the cytosolic NO production in vitro (57). Therefore it can be speculated that endostatin is one of the mechanisms that regulate the local blood supply during high intensity physical activity.

The results of our study are also in line with the data of Gu et al. (22), who observed elevated endostatin levels after performing a treadmill run. Previous studies demonstrated that an upregulation of endostatin is triggered by hypoxic conditions (13, 16, 44). At first glance these findings seem to be in contrast with our results since immediately after training sessions we observed a significant increase in endostatin levels that was not caused by exogenously induced normobaric hypoxia. However, the increase may have been due to local hypoxic conditions in the peripheral skeletal muscle tissue. Our data indicate an increased physiological collagen XVIII turnover induced by physical exercise, which results in an elevation of the endostatin serum concentration.

In the angiogenic process, MMPs play a critical role. These enzymes can regulate endothelial cell adhesion, proliferation, and migration and can therefore affect neovascularization (28, 39). MMPs seem to have bilateral functions in angiogenesis. On the one side, in their active form these enzymes facilitate the degradation of ECM compounds and neovascularization. On the other, indirectly, they are able to inhibit the angiogenic process of endothelial cell growth by generating anti-angiogenic growth factors and cytokines, such as endostatin (15, 28, 41). MMPs have been shown to be upregulated after physical activity (9, 10, 33, 34).

Our data indicate evident kinetic differences in the release of the MMP-2 and MMP-9 isoforms (see Figs. 7 and 8, respectively). It has been suggested by different authors that MMP-2 and MMP-9 play different roles in the regulation of angiogenic processes. MMP-2 may possess the ability to stabilize and contribute to the maturation of new capillaries (10), whereas MMP-9 has been shown to interfere in the primary activation and stimulation of endothelial cells and thus contribute to the initial steps of forming new blood vessels (10). These observations from different authors seem to be in contrast to our data because we observed a time-delayed maximum in MMP-9 concentration at 4 h postexercise. Normally, plastic angiogenic regulation and processes persist for a longer period, e.g., several days to weeks (30). It must be noted that the results of our study characterize short-term responses in the capillary bed. Therefore, the increased levels in MMP-2 and MMP-9 at 0 h postexercise and 4 h postphysical performance, respectively, seem to express functional rather than structural modulations and adaptations of the cardiovascular system in the peripheral skeletal muscle tissue. The current knowledge about the functions of MMP-2 and MMP-9 and the period of time needed for plastic and structural angiogenic modulations is limited. Therefore, a direct transfer of the MMP-2 and MMP-9 results to angiogenic regulation seems not to be accomplishable.

Our findings provide evidence of a similar increase in the pro-angiogenic growth factor VEGF and the proteolytic enzyme MMP-2 (Figs. 2 and 7). This relation may be explained by the ability of MMP-2 to digest several extracellular matrix compounds, e.g., proteoglycans. Proteoglycans have been shown to bind to different growth factors (such as VEGF), and therefore they can contribute to the storage property of the extracellular matrix. VEGF has been demonstrated to possess the ability to be attached to heparan sulfate proteoglycans, indicating the ability of proteoglycans to store different growth factors (e.g., VEGF) (8, 53, 62). The similar increases in VEGF and MMP-2 suggest a close interrelation of these parameters and may contribute to the hypothesis that pro-angiogenic growth factors are rapidly released from the extracellular matrix into the interstitial space in addition to their release from endothelial cells. This is because MMP-2 possesses the ability to cleave growth factors (such as VEGF) from heparan sulfate proteoglycans (53, 62). This hypothesis must be mentioned, although we observed no clear statistical evidence to support it, because after the NC without vibration and HC without vibration interventions, MMP-2 was significantly increased and VEGF showed a tendency to increase, although the results were not statistically significant. The biological evidence that growth factors like VEGF can be cleaved from heparan sulfate proteoglycans by MMP-2, however, may indicate a relation between growth factors and their ability to bind to heparan sulfate proteoglycans.

In conclusion, VEGF release seems to depend on exogenously induced mechanical loadings as calculated by separate repeated-measures ANOVA. Our results show that all conditions with the exception of NC without vibration seem to be amenable for a possible elevated VEGF release.

The time course of the endostatin increase may provide evidence of its possible vasoregulatory effect (57), i.e., that endostatin could regulate blood supply during physical exercise in addition to its angiogenic effect (50, 51). The endopeptidases MMP-2 and MMP-9 are elevated after physical performance as well. In summary, intensified research on angiogenic regulators with respect to special physiological and mechanical stimuli can lead to better understanding of vascular signaling and to new aspects in the support of high-performance endurance athletes and in preventing cardiovascular diseases.

Limitations of the study.   A limitation of the study could be the heterogeneous group of subjects, consisting of subjects who show a high "recreational" level and subjects reflecting a "national class" level.

A further limitation of this study may be that we only measured the VEGF level in the venous blood. We did not carry out measurements determining the VEGF content within the stressed tissue.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Katja Rössler from the Laboratory of Muscle Research and Molecular Cardiology, Department of Internal Medicine III, University of Cologne for expert technical assistance and support during the Western blot analyses.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Suhr, Institute of Training Science and Sport Informatics, German Sport Univ. Cologne, Carl-Diem-Weg 6, 50933 Köln, Germany (e-mail: Suhr{at}dshs-koeln.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* F. Suhr and K. Brixius contributed equally to this study. Back


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