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J Appl Physiol 102: 2346-2351, 2007. First published January 25, 2007; doi:10.1152/japplphysiol.00822.2006
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A single bout of exercise activates matrix metalloproteinase in human skeletal muscle

E. Rullman,1,2 H. Rundqvist,2,3 D. Wågsäter,4 H. Fischer,1,2 P. Eriksson,4 C. J. Sundberg,2 E. Jansson,1 and T. Gustafsson1,2

1Department of Laboratory Medicine, Division of Clinical Physiology, and 2Department of Physiology and Pharmacology, 3Department of Cell and Molecular Biology, and 4Department of Medicine, Atherosclerosis Research Unit, King Gustav V Research Institute, Karolinska University Hospital; Karolinska Institutet, Stockholm, Sweden

Submitted 26 July 2006 ; accepted in final form 25 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The aims of this study were 1) to characterize changes in matrix metalloproteinase (MMP), endostatin, and vascular endothelial growth factor (VEGF)-A expression in skeletal muscle in response to a single bout of exercise in humans; and 2) to determine if any exchange of endostatin and VEGF-A between circulation and the exercising leg is associated with a change in the tissue expression or plasma concentration of these factors. Ten healthy males performed 65 min of cycle exercise, and muscle biopsies were obtained from the vastus lateralis muscle at rest and immediately and 120 min after exercise. In the muscle biopsies, measurements of mRNA expression levels of MMP-2, MMP-9, MMP-14, and tissue inhibitor of metalloproteinase; VEGF and endostatin protein levels; and MMP activities were performed. Femoral arterial and venous concentrations of VEGF-A and endostatin were determined before, during, and 120 min after exercise. A single bout of exercise increased MMP-9 mRNA and activated MMP-9 protein in skeletal muscle. No measurable increase of endostatin was observed in the skeletal muscle or in plasma following exercise. A concurrent increase in skeletal muscle VEGF-A mRNA and protein levels was induced by exercise, with no signs of peripheral uptake from the circulation. However, a decrease in plasma VEGF-A concentration occurred following exercise. Thus 1) a single bout of exercise activated the MMP system without any resulting change in tissue endostatin protein levels, and 2) the increased VEGF-A protein levels are due to changes in the skeletal muscle tissue itself. Other mechanisms are responsible for the observed exercise-induced decrease in VEGF-A in plasma.

vascular endothelial growth factor-A; endostatin; gene expression; matrix metalloproteinase-9

IN SKELETAL MUSCLE, angiogenesis occurs as an adaptation to increased work requirements (18, 30). Exercise studies have focused on stimulatory angiogenic factors in skeletal muscle, even though angiogenesis is regulated by both stimulatory (angiogenic) and inhibitory (angiostatic) factors (6, 7, 29). However, not much is known of angiostatic factors in human skeletal muscle and their changes with exercise. Several members of the matrix metalloproteinase (MMP) family have been shown to be associated with extracellular matrix (ECM) remodeling, and these proteases, among others, also mediate the proteolytic release of the angiostatic factor endostatin from collagen XVIII (15). Endostatin is a 20-kDa COOH-terminal fragment of collagen XVIII (36) that was first isolated from a murine hemangioendothelioma. To our knowledge, MMP mRNA levels or MMP activities have not been measured in exercised human skeletal muscle, but it has been reported to be activated in other tissues, such as tendons, in response to exercise training (21, 24). In a recent study, plasma endostatin concentration was shown to increase in response to a single bout of exercise (10). The authors suggested that this was due to release of endostatin from the exercising muscle, which may indicate activation of muscle MMP protease activity. However, as the endostatin release from muscle was not measured, release from other organs could not be excluded. Regardless of the organ, tissue, and/or mechanisms responsible for the changed plasma level of endostatin, their study indicates that exercise regulates circulating levels of angiogenesis regulatory factors that may influence angiogenesis-dependent pathological processes in the vascular system, such as arteriosclerosis.

Further support for exercise-induced changes in circulating angiogenesis-related growth factors toward a lower ratio of stimulatory to inhibitory factors is provided by the fact that the plasma levels of the major angiogenic factor, vascular endothelial growth factor-A (VEGF-A), have been reported to decrease after exercise (4, 11, 12). In skeletal muscle VEGF-A is crucial for exercise-induced angiogenesis (1, 33), and as the skeletal muscle tissue levels of VEGF-A protein increase in response to exercise (12, 31), it is tempting to hypothesize that uptake of VEGF-A from the circulation to skeletal muscle explains the decrease in VEGF-A plasma level. If so, this mechanism would be additive to enhanced gene expression in exercise-induced increases in skeletal muscle tissue levels of VEGF-A protein.

The general aim of this study was to characterize the exercise-induced changes of one major angiogenic and one angiostatic factor in skeletal muscle, and the possible exchange of these factors between the skeletal muscle tissue and circulating blood. More specifically, we hypothesized that a single bout of exercise 1) increases the activity of MMP and thereby the endostatin level in skeletal muscle, leading to a the release of endostatin from the exercising leg to the blood, which in its turn increases the endostatin level in the blood; and 2) leads to an uptake of VEGF-A from the blood to the exercising leg with an increase in VEGF-A protein level in skeletal muscle and a reduction of the VEGF-A protein level in the blood.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects and experimental models.   The study conformed to the standards set by the Declaration of Helsinki and was approved by the Karolinska Institute Ethics Committee (decision 266/01). Written informed consent of all subjects was obtained.

Ten healthy male subjects were included in the study. Their median (range) age, height, and weight were 25 (18–37) yr, 180 (170–190) cm, and 77 (58–82) kg. Median (range) maximal oxygen uptake (VO2 max), determined before the experiments, was 3.7 (3.1–4.3) l/min. The exercise was performed on an electrodynamically loaded cycle ergometer. The subjects performed 65 min of cycle exercise. During the first 20 min, subjects cycled at 60 rpm at a median (range) work rate of 125 (105–148) W, chosen to correspond to 50% of VO2 max, after which the work rate was increased to 165 (138–195) W, corresponding to 65% of VO2 max, for a further 40 min. Finally, the subjects were instructed to cycle at their highest tolerable work rates (median 175 W, range 157–281) that could be sustained for 5 min.

Teflon catheters were inserted into the femoral vein and artery at the level of the inguinal ligament. Blood samples were drawn simultaneously from the femoral artery and the ipsilateral femoral vein at rest before exercise, at 17 and 57 min of exercise, and 120 min after the end of exercise. The time for the sample at 17 minutes was chosen in order to get a sample during steady-state blood flow and oxygen consumption but before any pronounced effects of metabolic perturbation could be expected. The second sample taken during exercise at 57 min was chosen to reflect the exchange over the exercising leg at the end of the exercise bout but without interference from the biopsy sampling at 65 min. The arteriovenous concentration differences were calculated from the measured arterial and venous concentrations of VEGF-A and endostatin. Muscle biopsy samples were obtained using the percutaneous needle biopsy technique (5) from the vastus lateralis muscle at rest, after 65 min of exercise, and 120 min after the end of exercise. All biopsy samples were frozen within 10–15 s in liquid nitrogen and stored at –80°C until analysis. All blood samples were collected in EDTA holders and centrifuged; the plasma was placed on ice within 10 min and stored at –20°C until analysis.

RNA measurements.   Total RNA was prepared by the acid-phenol method (8) and quantified by measuring absorbance at 260 nm on a spectrophotometer. The integrity of the total RNA was determined by 1% agarose-gel electrophoresis. Two micrograms of RNA were reverse transcribed by Superscript reverse transcriptase (Life Technologies, Stockholm, Sweden) using random hexamer primers (Roche Diagnostics, Mannheim, Germany) in a total volume of 20 µl. Detection of mRNA was performed on an ABI-PRISM 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Foster City, CA). VEGF-A oligonucleotide primers and TaqMan probes were designed using Primer Express version 1.0 (Perkin-Elmer Applied Biosystems). The primers for VEGF-A were forward direction, 5'-ACTGCCATCCAATCGAGACC-3'; reverse direction, 5'-GATGGCTTGAAGATGTACTCGATCT-3'; and probe, 5'-TGGTGGACATCTTCCAGGAGTACCCTGA-3'. The 5'-label was FAM, and the 3'-label was TAMRA. The probes were designed to cover exon-exon boundaries to avoid amplification of genomic DNA. MMP-2, MMP-9, MMP-14, and tissue inhibitor of metalloproteinase (TIMP)-1 were ordered as gene assays on demand (Hs00234422_m1, Hs00234579_m1, Hs00237119_m1, Hs00171558_m1, Perkin-Elmer Applied Biosystems). 18S rRNA was selected as an endogenous protein control to correct for potential variations in RNA loading (4310893E, Perkin-Elmer Applied Biosystems). All reactions were performed in 96-well MicroAmp Optical plates, using the ABI-PRISM 7700 Sequence Detector (Perkin-Elmer Applied Biosystems) equipped with a charge-coupled device camera, which detects the signals from the fluorogenic probes during PCR. The amplification mixes contained 5 µl of 1:100 diluted cDNA sample, 2x TaqMan Universal PCR Master mix, 300 nM of each primer, and 200 nM of the probe in a final volume of 25 µl. The mix for 18S rRNA was prepared according to the manufacturer's recommendation and was run in a separate well in a 1:2,000 dilution. Thermal cycling conditions included 2 min at 50°C, 10 min at 95°C, and then 45 cycles each of 15 s at 95°C and 1 min at 65°C. For each gene, all samples were amplified simultaneously in duplicate in one assay run. Dilution series was performed for each mRNA. The threshold cycle was determined for both the experimental gene and the endogenous control gene, and the relative expression was calculated based on the respective standard curve equation. The target gene expression was normalized to 18S rRNA.

Muscle protein measurements.   A portion of the skeletal muscle biopsy was homogenized in ice-cooled buffer (40 µl/mg wet muscle) containing 0.1 M potassium phosphate (pH 7.7), 0.05% BSA, 20 µg/ml leupeptin, 50 µg/ml aprotinin, and 40 µg/ml phenylmethylsulfonyl. The homogenate was rotated for 60 min at 4°C and centrifuged at 15,000 g for 10 min at 4°C. One hundred microliters of diluted supernatant (1:10) was used to quantify VEGF-A and endostatin protein using a sandwich enzyme-linked immunoassay (ELISA) (Quantikine R&D Systems, Minneapolis, MN) according to the manufacturer's directions. The optical density was quantified on a microplate reader (*Quant, Bio-Tek Instruments, Winooski, VT). All samples were assayed in duplicate.

The ELISA-kit used for determination of endostatin has antibodies directed against endostatin, but to our knowledge, the specificity of the kit is not tested for endostatin vs. neostatin-7 and neostatin-14. The kit used cannot therefore be assumed to discriminate between endostatin, neostatin-7, and neostatin-14. The latter most likely make a fraction of the cleavage products generated from collagen XVIII. However, since the biology effects of these products, to our knowledge, are very similar, if not identical to endostatin, this will not interfere with the physiology described and hypothesized in this paper.

Zymography supplies were purchased from Invitrogen (Carlsbad, CA). MMP-2, pro-MMP-9, and active MMP-9 distribution was determined from muscle biopsy homogenate before exercise, immediately after, and 120 min after exercise. Gelatin substrate zymograms were prepared using precast 10% SDS-polyacrylamide gels containing 1 mg/ml of gelatin. Equal volumes of experimental media samples (2.5 µg) were diluted into 2x Tris-glycine SDS sample buffer and electrophoretically separated under nonreducing conditions. Proteins were incubated in renaturing buffer (Invitrogen) for 30 min at room temperature. The gels were incubated overnight at 37°C in developing buffer (Invitrogen). After 1 h staining with Coomassie blue and destaining for 2 days with 10% acetic acid and 40% methanol in water, gelatinase activity was evident by clear bands against a dark blue background (Fig. 1). Quantification of the bands was performed using digital camera Fujifilm LAS-1000 and densiometry software Fujifilm Image gauge version 3.46.


Figure 1
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  		Fig. 1. A representative gelatin zymography of muscle biopsy lysates (2.5 µg) from 3 subjects (1–3) preexercise (B), immediately after (1 h), and 120 min postexercise (3 h). The larger band corresponding to approximately 135 kDa probably reflects a MMP-9 monomer/neutrophil gelatinase-associated lipocalin (NGAL) monomer complex (20). MWM, molecular weight marker.

 
Blood samples.   The plasma concentrations of VEGF-A and endostatin were measured by sandwich ELISA (Quantikine R&D Systems) using similar procedures as described for tissue VEGF-A. All techniques and materials were used according to the manufacturer's instructions. Plasma albumin concentration was measured at the Karolinska University Hospital Laboratory by the Modular P2 equipment.

Statistical analysis.   A one-way ANOVA for repeated measures was used to evaluate the effect of time on mRNA and protein expression. Post hoc analysis used planned comparisons to locate the points of interaction. A two-way ANOVA for repeated measures evaluated the effects of time and localization (artery or vein) on the plasma concentrations of VEGF-A and endostatin, and on the concentration of plasma albumin. MMP-activity was analyzed by a nonparametric method for comparison of multiple dependent samples (Friedman ANOVA and Kendall coefficient of concordance). For all tests, P < 0.05 was considered significant. Before analyzing the changes in the arterial or venous concentrations with time, we corrected the data for hemoconcentration, as indicated by the changes with time in the concentration of plasma albumin. Because the interaction between time and localization was not significant for the plasma albumin concentration and because the arterial and venous concentrations of albumin did not differ significantly, we did not correct the arterial and venous concentrations of VEGF-A or endostatin in the statistical comparison between the arterial and venous concentration. Data presented as means ± SE unless otherwise stated.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Skeletal muscle mRNA levels.   Compared with the preexercise levels, VEGF-A mRNA was significantly increased immediately after exercise (Fig. 2A, P < 0.05). At 120 min after exercise both VEGF-A and MMP-9 mRNA were significantly higher compared with preexercise levels (Fig. 2A and 3A, respectively; P < 0.05). A tendency to increased TIMP mRNA level was observed 120 min after exercise compared to preexercise (P < 0.1) (TIMP: fold change 1.5 ± 0.3 immediately following exercise and 2.6 ± 0.6 120 min after exercise). MMP-2 and MMP-14 mRNA levels were not significantly changed by exercise (MMP-2: fold change 1.2 ± 0.2 immediately following exercise and 1.3 ± 0.3 120 min after exercise; MMP-14: fold change 1.1 ± 0.2 immediately following exercise and 1.2 ± 0.2 120 min after exercise).


Figure 2
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  		Fig. 2. Vascular endothelial growth factor (VEGF)-A mRNA and VEGF-A protein levels in vastus lateralis muscle preexercise (Pre), immediately after (End Ex), and 120 min postexercise (120' post ex). #Significant differences between preexercise and end of exercise (P < 0.05). {dagger}Significant difference between preexercise and 120 min postexercise (P < 0.05). Values are presented as means and SE (n = 10).

 

Figure 3
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  		Fig. 3. Matrix metalloproteinase (MMP)-9 and MMP-9 protein levels in vastus lateralis muscle preexercise, immediately after, and 120 min postexercise. #Significant differences between preexercise and end of exercise (P < 0.05). {dagger}Significant difference between preexercise and 120 min postexercise (P < 0.05). Values are presented as means and SE (n = 10).

 
Skeletal muscle protein levels.   Compared with the preexercise level (29 ± 12 pg/mg wet muscle), VEGF-A protein expression was not significantly different immediately after exercise (33 ± 17 pg/mg wet muscle) but was significantly higher 120 min after exercise (41 ± 15 pg/mg wet muscle, P < 0.05) (Fig. 2B). Endostatin protein expression did not change significantly at either time after exercise (1.2 ± 1.1 ng/mg wet muscle before exercise, 1.8 ± 1.8 ng/mg wet muscle immediately after exercise, and 1.5 ± 1.1 ng/mg wet muscle 120 min after exercise). Immediately and 120 min following exercise, total MMP-2 was not significantly different from the preexercise value. Total MMP-9 (pro- and active MMP-9) was significantly higher immediately following exercise compared with preexercise values (Fig. 3B, P < 0.05) and remained elevated 120 min following exercise.

Plasma albumin concentration.   The arterial albumin concentration did not differ significantly from the venous concentration preexercise or at any measurement time point during (at 17 or 57 min of exercise) or after exercise (120 min postexercise) (Fig. 4). Arterial and venous concentrations of albumin increased at 17 and 57 min of exercise (P < 0.01) (Fig. 4) but did not differ significantly from the preexercise concentration 120 min after exercise (Fig. 4).


Figure 4
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  		Fig. 4. Femoral arterial and venous plasma albumin concentrations pre exercise, during exercise (17 min and 57 min), and 120 min postexercise. #Significant difference compared with arterial preexercise concentration (P < 0.05). {dagger}{dagger}Significant difference compared with venous preexercise concentration (P < 0.05). Values are presented as means and SE (n = 10).

 
Plasma VEGF-A and endostatin concentrations.   The arterial and venous VEGF-A concentrations did not differ significantly before exercise. At 17 min of exercise, the venous concentration of VEGF-A increased significantly above the preexercise value (P < 0.01, Fig. 5), whereas the arterial concentration remained unchanged leading to higher VEGF-A concentration in the venous blood than in the arterial blood (P < 0.05, Fig. 5). This indicates that VEGF-A is released from the muscle into the circulation at 17 min of exercise. At 57 min of exercise, the venous and arterial concentrations of VEGF-A did not differ significantly (Fig. 5). Both the arterial and venous VEGF-A concentrations were significantly lower at 120 min after exercise than pre-exercise (P < 0.01, Fig. 5). The arterial and venous endostatin concentrations did not differ significantly before exercise. The arterial and venous concentrations of endostatin increased above preexercise values at 17 and 57 min of exercise (P < 0.01, Fig. 6). At 17 min of exercise, the venous endostatin concentration was significantly lower than the arterial concentration (P < 0.05, Fig. 6), indicating an uptake from the circulation into the muscle. At 57 min of exercise, venous endostatin concentration was significantly higher than the arterial concentration (P < 0.01, Fig. 6), indicating a release from the muscle into the circulation. At 120 min after exercise, arterial and venous endostatin concentrations did not differ significantly from preexercise values (Fig. 6).


Figure 5
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  		Fig. 5. Femoral arterial and venous plasma VEGF-A concentrations preexercise, during exercise, and 120 min postexercise. {dagger}Significant difference between arterial and venous concentration at the same time point (P < 0.05). #Significant difference compared with arterial preexercise concentration (P < 0.05). {dagger}{dagger}Significant difference compared with venous preexercise concentration (P < 0.05). Values are presented as means and SE (n = 10).

 

Figure 6
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  		Fig. 6. Femoral arterial and venous plasma endostatin concentrations pre exercise, during exercise, and 120 min post-exercise. {dagger}Significant difference between arterial and venous concentration at the same time point (P < 0.05). #Significant difference compared with arterial preexercise concentration (P < 0.05). {dagger}{dagger}Significant difference compared with venous preexercise concentration (P < 0.05). Values are presented as means and SE (n = 10).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of our study were 1) that a single bout of exercise activated the MMP system without any resulting change in tissue endostatin protein levels; 2) that the exercise-induced increase in VEGF-A protein levels are due to changes in the skeletal muscle tissue itself without any influence of a peripheral uptake of VEGF-A from the circulation; and 3) that following exercise, VEGF-A plasma levels are still reduced but without sustained changes in the plasma endostatin levels.

The observations of exercise-induced increases in MMP-9 mRNA levels, increased activity of MMP-9, and a tendency to increased TIMP transcription clearly show that a single bout of exercise activates the MMP system in skeletal muscle. No earlier study has, to our knowledge, explored the exercise-induced expression or activity of MMP in human skeletal muscle. An earlier study on downhill running supports the present finding, as a small but significant increase in serum levels of MMP-9 was found without any measurable change of MMP-2 following exercise (22). In this study, increased MMP-9 activity was evident before any measurable changes in MMP-9 transcription, indicating posttranscriptional mechanisms. In fact, proteolytic cleavage of the MMP-9 propeptide and MMP-9 activation is induced by numerous proteases, including plasmin, and other proteases in the MMP-family. However MMP-2, the only other protease activity measured, did not increase, and thus proteases not measured in this study must be responsible for such actions. In animals, exercise has been reported to increase both MMP mRNA levels and activity (13, 23). Regulation of mRNA is regarded as an important mechanism for changes in MMP-9 activity (34). It has been suggested that such transcriptional activation is induced by muscle damage and inflammatory responses. In the present study, MMP-9 activity and mRNA levels were both increased 120 min after exercise. In exercising human muscle, the number of inflammatory cells is negligible, even after a single bout of hard eccentric exercise (26). Still, the expression of several cytokines, such as IL-1{alpha} and TNF-{alpha}, are known to increase in the skeletal muscle tissue (26–28). Those cytokines have been shown to increase MMP-9 mRNA levels (14, 34) and are therefore possible candidates for regulating the observed increase in MMP-9 transcripts.

The slight release of endostatin from the legs during the later part of exercise could be related to the observed increase in MMP activity. However, it did not cause any measurable increase in tissue levels of endostatin, nor did it influence the overall plasma levels of endostatin following exercise. Therefore, the present data suggest that even if activation of MMP is an early response in exercising skeletal muscle, it does not seem to drive the angiogenic switch toward inhibition through increased production of endostatin. A dual role exists for MMP in ECM remodeling in the angiogenic process. Degradation of the ECM releases numerous growth factors and also allows the endothelial cells to migrate to the site where new capillaries are needed (6, 9). In fact, in the rat, the activation of the MMP is crucial for exercise-induced sprouting angiogenesis (13). However, in that study, only MMP-2 and MMP-14 activities increased, whereas MMP-9 activity did not. An explanation for the absence of increased MMP-9 activity, beside possible species differences, could be that the MMP was measured after two days of stimulation. In other studies, it has been reported that MMP-9 has been activated during microvascular remodeling in the rat (35). Nevertheless, the present observations that a member of the MMP-family is activated in response to exercise, together with an earlier study showing MMP activity to be crucial for exercise-induced angiogenesis in the rat and yet another study showing a role of MMP-9 in microvascular remodeling in the rat, open the possibility that MMP activity might contribute to exercise-induced angiogenesis also in humans. However, further work is required to elucidate the time course of specific MMP changes and the cellular origin of the MMP expression, as well as the stimuli for the changes, i.e., mechanical forces on blood vessels and/or metabolic changes in the skeletal muscle fibers.

VEGF-A protein levels were increased in the skeletal muscle, and this was concurrent with increased mRNA levels. This further supports that VEGF-A is mainly regulated pretranslationally in exercising human muscle. Numerous possible mechanisms for exercise-induced increases in VEGF-A mRNA expression have been shown in both animal and human experimental models (2, 18, 32).

In a study by Hoffner et al. (17) from 2003, the authors estimated the level of VEGF-A protein in the skeletal muscle using the microdialysate technique. Microdialysate samples reflect the interstitium of the tissue, which differs from biopsies since biopsies also reflect intracellular levels. Furthermore, a biopsy represents a time frame of seconds in the tissue, whereas microdialysate sampling represents an average of 15–30 min. Hoffner et. al. (17) found an increase of VEGF-A protein in the muscle during the first 30 min of exercise and speculated that this VEGF-A could be released from the muscle cells by protease activity, since gene expression and exocytosis most likely would take longer than 30 min. In our study, we found a release of VEGF-A to the circulation after 17 min of exercise, which is consistent with the findings of Hoffner et al. (17). Furthermore, as in the study by Hoffner et al., we also found an increase (but insignificant) of VEGF-A protein in the muscle at the end of the exercise bout.

In this study, no uptake of VEGF-A from the circulation to the exercising leg was demonstrated. To our knowledge, only one earlier study has measured arteriovenous differences over the exercising legs in humans. In that study, a release of VEGF-A from the leg was found, but not until 2 h after a 3-h bout of exercise and an increased VEGF-A mRNA expression was observed in skeletal muscle before any VEGF-A was released into the circulation (16). Therefore, the VEGF-A release in that study might have been related to increased VEGF-A protein expression, while in this study, because the release occurred early during exercise, it was probably release of preexisting VEGF-A. These divergent findings might be due to differences in experimental protocols. In the study by Hiscock et al. (16), the duration of exercise was longer, giving both greater exercise stimulation and longer duration for induction of VEGF-A expression. Regardless of this, the exercise-induced decrease in plasma VEGF-A levels is in accordance with previous studies (4, 11, 12). As it was not related to peripheral uptake of VEGF-A to the exercising leg, it must reflect uptake by tissues other than those in the exercising leg. Interestingly, exercise has been shown to increase the plasma levels of endothelial progenitor cells (EPCs) from the bone marrow (25). As VEGF-A is important in the recruitment of EPCs to the bloodstream through high-affinity VEGF receptors (3, 19), it is tempting to speculate that one mechanism behind the decrease in plasma VEGF-A relates to increased binding to EPCs.

In conclusion, the skeletal muscle MMP-9 is activated by a single bout of exercise and seems to be due to a combination of pre- and posttranslational mechanisms. This finding suggests that the MMP system is involved in the early phase of muscle adaptation to exercise. Even though a release of endostatin occurred during the later part of the exercise bout, we could not confirm the earlier report of a sustained exercise-induced increase in the plasma level of endostatin following exercise. No uptake of VEGF-A from the blood to the exercising leg could be demonstrated during a single bout of exercise. Therefore, the exercise-induced increase in VEGF-A protein level in skeletal muscle seems to relate mainly to increased transcription and/or translation in the skeletal muscle itself, without any influence of a peripheral uptake of VEGF-A from the circulation. The lowered plasma VEGF-A level following exercise must therefore reflect mechanisms other than uptake of VEGF-A from the blood to the exercising muscle.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Swedish Heart Lung Foundation, the Swedish Research Council, and the Swedish National Centre for Research in Sports.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Gustafsson, Div. of Clinical Physiology C1-88, Karolinska Univ. Hospital, Huddinge, 141 86 Stockholm, Sweden (e-mail: thomas.gustafsson{at}ki.se)

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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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