INTRODUCTION

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THE ADAPTATION OF SKELETAL muscle in humans to endurance-type training (low-resistance, high-repetition contractile activity) is well characterized and includes increases in the number and size of mitochondria as well as the activity of enzymes controlling oxidative metabolism. Furthermore, numerous cross-sectional and longitudinal studies in humans have demonstrated increased capillary density and/or increased capillary-to-fiber ratio in response to endurance exercise training (for references, see Ref. 35).

During the last decade, several angiogenic factors have been characterized (15), and much attention has been focused on vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). They both stimulate angiogenesis in vitro and in animal species in vivo as well as in humans (11, 15, 27). Furthermore, because VEGF and FGF are upregulated after electrical stimulation and after acute exercise in the rat, these two factors have been proposed to be possible mediators in the exercise-induced capillary growth in skeletal muscle (4, 8, 20, 31, 37).

VEGF mRNA is induced by hypoxia in cultured cells (36) and in rat skeletal muscle in vivo (8, 30). Hypoxia-inducible factor 1 (HIF-1), a transcription factor composed of the two subunits HIF-1 and HIF-1, seems to be necessary for the hypoxia-induced VEGF gene expression (16). Jiang et al. (25) have shown in vitro that HIF-1 operates within a range of oxygen tensions that is physiologically relevant during exercise in humans. FGF-2 has not been shown to be induced by hypoxia in vitro (9) or in vivo (8), but increased levels have been found in serum in patients with ischemic heart disease (21) and in ischemic animal skeletal muscle (10).

During exercise, local muscle oxygen tension falls considerably (34), and exercise under conditions of restricted blood flow reduces the oxygen tension even more (34, 39). We have earlier reported a higher capillary-to-fiber ratio after training with restricted blood flow than after training at the same power output but with normal blood flow in the leg (14). Those findings indicate that lowered oxygen tension may play a role in exercise-induced angiogenesis. One possible mediating mechanism could be angiogenic factors induced by muscle hypoxia during exercise. Support for this hypothesis comes from Breen et al. (8) and Asano et al. (5). In the former study, one single bout of exercise in the rat increased the expression of VEGF mRNA, an increase that was further augmented when the exercise was performed under hypoxemia. In the same study, FGF-2 mRNA was increased by exercise but was not further increased during exercise under hypoxemia. In the study by Asano et al. (5), increased serum levels of VEGF were observed during high-altitude swimming training in humans.

To our knowledge, there are no published studies on the effects of exercise on the mRNA expression in human skeletal muscle of angiogenesis-related growth factors VEGF and FGF-2 and the transcription factor subunits HIF-1 and HIF-1. Therefore, we wanted to test the hypothesis that there is an increased mRNA expression of these factors in response to a single bout of endurance exercise. Furthermore, we wanted to investigate whether there was a graded response to the oxygen delivery and thus to the metabolic stress. To do this, we applied a human experimental model in which blood flow in the legs was reduced in a controlled fashion during exercise.

	METHODS

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Subjects. Fifteen healthy males participated in the main study. Their average age, height, and weight were 24 (range: 20-32) yr, 184 (range: 175-194) cm, and 79 (range: 70-94) kg. The leisure-time physical activity amounted to 3.5 (range: 1.5-6) h of moderate intensity physical activity per week.

Before the study, the experimental protocol was explained to all subjects and informed consent was obtained. The study was approved by the Ethics Committee of the Karolinska Institute.

Experimental protocol. A method first described by Eiken and Bjurstedt (13) was employed for induction of restricted blood flow during exercise. Local application of external pressure over the working leg was used to reduce blood flow in a controlled fashion. The subject was positioned supine in the opening of a large pressure chamber with both legs inside the chamber and with a pad strapped to the calf of one leg. The pad was connected, via a metallic bar, to a crank-arm of an electrically braked cycle ergometer with locked flywheel, the center of rotation being at the level of the heart. The chamber opening was sealed off at the level of the crotch by a rubber diaphragm with holes and self-sealing sleeves for the legs. Shoulder supports were used to prevent craniad displacement of the body as the chamber pressure was increased. For exercise under restricted blood flow, the chamber pressure acting on the exercising leg was elevated to 50 mmHg above atmospheric pressure. This has been shown to reduce leg blood flow during one-legged cycle exercise by 15-20% (39). Exercise under nonrestricted blood flow was performed using the same experimental arrangements but at normal atmospheric pressure.

Familiarization. At least 1 wk before the first experiment each subject was familiarized twice with the experimental model and procedures. During familiarization, the maximal one-legged performance capacity was determined under normal, nonrestricted blood flow conditions in a test where the workload, beginning at 5 W, was increased by 5 W at 1-min intervals to fatigue.

Experiments. In the main study, seven of the 15 subjects were randomly selected to perform exercise under nonrestricted blood flow conditions (NR) and the remaining eight subjects to perform exercise under restricted blood flow conditions (R). In each group, four of the subjects used their right leg. One hour before exercise, Teflon catheters were inserted into the brachial artery and the femoral vein. The dynamic constant-load knee-extension exercise (45 min, 60 rpm) was performed in a similar fashion to that described by Andersen and Saltin (3). Each voluntary contraction extended the leg from 70 to 150 degrees knee angle. Flexion was performed passively using the ergometer flywheel momentum to reposition the leg for the next extension. One-legged peak workload was assessed in the NR condition. The relative workload subsequently used in the experiments was 24 ± 3% (mean ± SD) of the one-legged peak load; in absolute values it was 9 ± 1 and 10 ± 1 W in the R and NR condition, respectively.

In an additional study, the oxygen saturation, PO₂, and pH during exercise under restricted and nonrestricted blood flow were measured in the effluent femoral venous blood. Six of the subjects performed two 5-min exercise bouts at the same workload as in the main study. A Teflon catheter was inserted into the femoral vein, and blood samples were obtained before and after 5 min of exercise. The blood samples were immediately put on ice and analyzed within 2 h.

Muscle biopsies and blood samples. Muscle biopsies were obtained, 1 wk before the experimental day and 30 min after the exercise bout, from the vastus lateralis muscle using the percutaneous needle biopsy technique (6). The time period between the first and second biopsy was chosen to minimize a possible traumatic effect of the first biopsy on the mRNA measurements in the second biopsy. The choice of the 30-min time period between end of exercise and postexercise biopsy was based on the results of Breen et al. (8), who showed a maximal increase of VEGF and FGF-2 mRNA between 0 and 30 min after 1 h of exercise in Wistar rats. All biopsy samples were frozen within 10-15 s in liquid nitrogen and stored at 80°C until subsequent analysis. In the main study, arterial and venous blood samples for lactate determination were obtained before and after 15, 30, and 45 min of exercise.

Systolic blood pressure, heart rate, and local rate of perceived exertion. Before exercise, systolic blood pressure and heart rate (HR) were determined by the cuff method in the supine position. During exercise, HR was recorded continuously from the electrocardiogram by means of a linear beat-to-beat meter, and systolic pressure was obtained sphygmomanometrically from the brachial artery in the noncatheterized arm by an automatic blood pressure recorder (Criticon Exercise Monitor 1165). Local rates (leg) of perceived exertion (L-RPE) were assessed using Borg's 6-20 RPE scale (7).

Plasma analysis. Plasma lactate concentration was analyzed by a fluorometric enzymatic procedure as modified from Hohorst (22). Venous oxygen saturation, PO₂, and pH were determined with an automatic spectrophotometric technique (ABL 520, Copenhagen, Denmark).

Muscle fiber type and citrate synthase activity. Serial cross sections (10 µm) were cut at 20°C and stained for myofibrillar ATPase after preincubation at pH 10.3 and 4.3. The fibers were classified as type I and II; ~300 fibers were counted in each biopsy. Citrate synthase was analyzed by a fluorometric method according to the principles of Lowry and Passonneau (29) as described by Lin et al. (28).

RNA extraction and reverse transcription. Total RNA was prepared by the acid phenol method (12) as described previously (33) and was reverse transcribed by Superscript RNase H-reverse transcriptase (GIBCO BRL) using random hexamer priming according to the manufacturer's specifications. After 1 h at 37°C, the enzyme was inactivated by incubation at 95°C for 10 min. Thereafter, the solution was diluted to 200 µl with Tris-EDTA buffer yielding the reverse transcription (RT) mix used in the polymerase chain reaction (PCR).

The following primer locations were chosen: VEGF (human heparin-binding VEGF, GenEMBL AC M32977), 5' primer: bases 140-166, 3' primer: bases 296-270, expected PCR fragment: 157 bp; 5' Biotin-probe: bases 215-234. Four isoforms of VEGF mRNA are known to be produced via alternative splicing (23). The PCR fragment used for the present study includes all four isoforms: 1) FGF-2 (human basic FGF, GenEMBL AC M27968), 5' primer: bases 1,358-1,385, 3' primer: bases 1,593-1,566, expected PCR fragment: 236 bp; 5' Biotin-probe: bases 1,390-1,409; 2) 28S (human 28S ribosomal RNA gene, GenEMBL AC M11167), 5' primer: bases 4,535-4,564, 3' primer: bases 4,667-4,638, expected PCR fragment: 133 bp; 5' Biotin-probe: bases 4,283-5,006; 3) HIF-1 (human HIF-1, GenEMBL AC U22431), 5' primer: bases 2,308-2,332, 3' primer: bases 2,552-2,533, expected PCR fragment: 245 bp; 5' Biotin-probe: bases 2,372-2,391; and 4) HIF-1 (human aryl hydrocarbon receptor nuclear translocator, GenEMBL AC M69238), 5' primer: bases 1,207-1,234; 3' primer: bases 1,382-1,355, expected PCR fragment: 176 bp; 5' Biotin-probe: bases 1,271-1,290. The specificities of the primers were tested by diagnostic restriction cuts of the PCR products.

PCR. Quantification of specific RNAs was performed using a statistical PCR approach (33). For every PCR run, a master mix was prepared on ice using the 10× buffer supplied by the manufacturer, 0.2 µM of each primer, 40 µM dATP, dCTP, dGTP, 38 µM dTTP, and 2 µM digoxigenin-dUTP (PCR DIG labeling mix, Boehringer Mannheim), and 1.6 U/100 µl DynaZyme DNA polymerase (Finnzymes Oy). Two microliters of RT mix (further diluted 1:10 for all 28S rRNA measurements) were pipetted to 0.2-ml, 96-tube plates (Thermo-Fast, Biolabo) on ice, and 38 µl of the master mix were added to each tube. In each PCR run, a duplicate reference sample (33) was amplified in parallel. A control without template was also run each time. The tube plates were transferred into the preheated (95°C) thermocycler with heated lid (Biometra UNO), and DNA was denatured for 2 min. PCR steps were, for 28S and HIF-1: denaturation at 95°C for 10 s, annealing at the appropriate temperature (60°C) for 90 s, and extension at 72°C for 10 s; for HIF-1: denaturation at 95°C for 15 s, annealing at the appropriate temperature (60°C) for 60 s, and extension at 72°C for 10 s. A two-step PCR (95°C for 10 s, 70°C for 90 s) was performed in the case of VEGF and FGF-2. Cycle numbers were as follows: VEGF, 29×; FGF-2, 32×; 28S, 16×; HIF-1, 33×; and HIF-1 34×. After the amplification, tube plates were stored at 4°C until further processing.

ELISA quantification of PCR products. The amounts of PCR products were quantitated by hybridization of specific biotinylated probes (see above) and detection by ELISA. Ten microliters of PCR product was denatured by adding 40 µl of Bio-probe [0.1 µM biotinylated probe (see PCR) in 0.1 M NaOH] for 10 min at room temperature in a 96-well microplate. Next, 50 µl of hybridization solution were added (6× SSC, 10 µM Tris · HCl pH 7.4, 10 mM EDTA, 0.2 M HCl), and samples were incubated at room temperature for 1 h. Fifty microliters of the hybrids were then transferred to a streptavidin-coated microplate [coating: 100 µl of 10 µg/ml streptavidin (Boehringer Mannheim) in PBS at 4°C overnight or longer], which had been washed 4 times with 100 µl TBS-T (20 mM Tris pH 7.6, 192 mM glycine, 0.9% Tween-20). Hybrids were bound to streptavidin by incubation for 30 min at room temperature. Plates were again washed four times with 100 µl TBS-T and then incubated for 30 min at room temperature with 50 µl of an alkaline phosphatase conjugated anti-digoxigenin antibody (Fab fragments, Boehringer Mannheim), diluted 1:1,000 in TBS-T. Plates were washed four times with 100 µl TBS-T, and 50 µl of freshly prepared substrate was added: 10% (vol/vol) diethanolamine, 0.5 mM MgCl₂, 0.08 M HCl, 4 mg/ml 4-nitrophenyl phosphate (Boehringer Mannheim). After incubation for 10-90 min, absorbance at 405 nm was determined in an ELISA reader (Bio-Rad). The control without template served as background, which was subtracted to yield the muscle sample absorbances. These were related to the mean of the duplicate reference sample. The amounts of each PCR product were corrected for the different RNA lengths.

Statistical analysis. All values are expressed as means ± SD. Hemodynamic parameters, arterial and femoral venous lactate, L-RPE, and mRNA were statistically analyzed with a two-way ANOVA (condition as independent factor and time as dependent factor), which tested for differences over time between the R and NR condition. Planned comparison was used (i.e., post hoc test) to locate differences corresponding to the significant interaction in the ANOVA model and also to locate differences between rest and exercise within the two conditions (arterial and femoral venous lactate). A multiple-regression analysis was carried out to estimate the influence of lactate on the mRNA changes and to test differences between condition in mRNA exercise-induced increase, adjusted for the mRNA preexercise values. A least-squares regression line was performed between exercise-induced changes in VEGF and FGF-2 mRNA to HIF-1 and HIF-1. Student's t-test was used to test difference in fiber types and citrate synthase between the two conditions.

	RESULTS

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Venous oxygen saturation, PO₂, and pH. The venous oxygen saturation, PO₂, and pH were reduced after 5 min of exercise during both R and NR conditions (P < 0.001, Table 1) and to a greater extent during the R condition (P < 0.01, Table 1).

VEGF, FGF-2 mRNA, HIF-1, and HIF-1

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Exercise-induced percentage changes in mRNA in nonrestricted (NR) and restricted (R) blood flow conditions. VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor; HIF, hypoxia-inducible factor.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Relationship between individual exercise-induced changes (postexercise  preexercise) and preexercise value in mRNA for VEGF (A), HIF-1 (B), and HIF-1 (C) in NR and R conditions.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Relationship between individual exercise-induced changes (postexercise  preexercise) in different mRNAs: VEGF and HIF-1 (A) and VEGF and HIF-1 (B) in NR and R conditions.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Relationship between individual exercise-induced changes (postexercise  preexercise) in VEGF mRNA and exercise-induced changes in lactate concentration. Graph shows residual exercise-induced changes in VEGF mRNA against exercise-induced changes in lactate concentration plus the contribution of VEGF mRNA preexercise value in the regression model. This plot is discussed in detail by Larsen and McCleary (26).

	DISCUSSION

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The main findings in the present study were that a single 45-min bout of one-legged dynamic knee-extension exercise increased VEGF mRNA in human skeletal muscle by almost threefold and that this increase was correlated to the changes in HIF-1 and HIF-1 mRNA as well as to the exercise-induced increase in femoral venous lactate concentration.

Endurance training increases the capillarization of skeletal muscle (2, 24). What underlies the capillary growth process in skeletal muscle is not known exactly, but reduced oxygen tension and/or related metabolic consequences have been suggested as possible stimuli (1). Furthermore, growth factors, e.g., VEGF and FGF-2, have been proposed to be of importance in the angiogenesis process in skeletal muscle (4, 8, 20, 31, 37). Our study is the first to demonstrate that VEGF at the mRNA level increases in human skeletal muscle after an exercise bout.

It is not known in which cell type or types the mRNAs were expressed in the current study. The skeletal muscle fiber is one possible site for VEGF mRNA expression as shown in the rat (8). However, other cells represented in the skeletal muscle biopsy, e.g., endothelial and smooth muscle cells, have also been shown to increase mRNA and protein VEGF under specific conditions such as, e.g., hypoxia (18, 32). Finally, the increase in VEGF mRNA could be due to enhanced mRNA stability and/or increased transcription because both mechanisms have been shown to operate in vitro (38).

Hypoxia seems to be a potent stimulus for VEGF gene expression in intact animals (30) and in vitro (36). In the present experimental setup, the femoral venous oxygen saturation and oxygen tension decreased significantly during exercise in both conditions, indicating a lower local oxygen tension in the working muscle. Richardson et al. (34) have recently shown that the mean oxygen tension in the muscle tissue itself, as calculated from myoglobin O₂ saturation curves, decreases to ~2-3 mmHg during exercise performed at an intensity above 50% of maximal O₂. Therefore, one possible stimulus for the increase of VEGF mRNA in our study could be the exercise-induced reduction in the oxygen tension in the working muscle, even if we could not exclude involvement by other exercise-related factors.

On the basis of the enhanced capillary growth response after training under restricted blood flow (14) and the further exercise-induced increase of VEGF mRNA under hypoxemia in the rat (8), we hypothesized that a VEGF mRNA increase would be greater after exercise with restricted blood flow. Even if there was no significant difference (P = 0.1) in exercise-induced VEGF mRNA expression between the two exercise conditions, a correlation was found between the increase in VEGF mRNA and femoral venous lactate, indicating that VEGF expression to some extent was related to "metabolic stress." The lack of a further increase in VEGF mRNA when flow restriction was "added" may be explained by the smaller further reduction in oxygen saturation and oxygen tension compared with the rest-to-exercise transition, even if this difference was large enough to induce a greater lactate concentration increase in the R condition. Another explanation could be that, even if there was no significant VEGF mRNA difference before exercise between the NR and R conditions, a strong influence of the preexercise value of VEGF mRNA on the exercise-induced increase makes a difference between the two conditions more difficult to detect.

In contrast to the clear increase in VEGF mRNA with exercise, no effect was observed on FGF-2 mRNA. Our results and those found in the rat by Breen et al. (8) resemble each other in that exercise induced a clear increase in VEGF mRNA expression and a lower response in FGF-2 mRNA expression. Furthermore, the FGF-2 mRNA response was not altered by reduced oxygen delivery either in their or in our study.

Because, in the present study, a single bout of exercise did not induce significantly higher VEGF and FGF-2 mRNA expression under restricted than under nonrestricted blood flow, an importance of these factors for the augmented capillary neoformation after training with restricted blood flow may presume repetitive bouts of exercise.

There are numerous in vitro studies that show activation of HIF-1 by hypoxia and several that show that HIF-1 is necessary for the hypoxia-induced increase of VEGF (16, 17, 25). If exercise increases VEGF protein concentration and if hypoxia is one underlying mechanism behind this increase, a correlation between exercise-induced changes in VEGF and HIF-1 proteins would be expected. The observations in this study that there was a significantly increased skeletal muscle expression of HIF-1 mRNA after exercise and that there was a correlation between the exercise-induced changes in VEGF mRNA and the changes in both HIF-1 and HIF-1 mRNA are, to our knowledge, novel findings in vivo. We have earlier found correlations between the basal values of VEGF mRNA to both HIF-1 and HIF-1 mRNA and between HIF-1 and HIF-1 mRNA (19). We do not know if these correlations reflect cause-effect relationships. If not, one possibility is that the same or similar stimuli or mechanisms influence transcription and/or stabilization of the mRNAs for VEGF and both subunits of HIF-1. However, from in vitro studies under hypoxia the main regulation of HIF-1 and thereby HIF-1-regulated gene expression seems to be at the translational and posttranslational levels rather than at the transcriptional level (17).

In contrast to VEGF mRNA, no correlation between the changes in FGF-2 mRNA and HIF-1 or HIF-1 was observed in the present study. To our knowledge, there are no publications from either in vitro or in vivo conditions where the influence of HIF-1 on the FGF-2 gene expression has been studied.

It is concluded that 1) VEGF but not FGF-2 gene expression is upregulated in human skeletal muscle by a single bout of dynamic exercise and that there is a graded response in VEGF mRNA expression to the metabolic stress and 2) the increase in VEGF mRNA expression correlates to the changes in both HIF-1 and HIF-1 mRNA. This last finding may indicate that HIF-1 influences the exercise-induced VEGF gene expression or alternatively that VEGF and HIF expression are coregulated at the transcriptional level in human skeletal muscle.