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1The Copenhagen Muscle Research Centre and 2Department of Infectious Diseases, Rigshospitalet; and 3August Krogh Institute, University of Copenhagen, Copenhagen, Denmark, DK-2100
Submitted 13 February 2003 ; accepted in final form 16 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODOLOGYRESULTSDISCUSSIONREFERENCES |
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angiogensis; skeletal muscle; peripheral vascular function
VEGF mRNA expression in the muscle is elevated following acute exercise in healthy humans (8, 13), heart failure patients (6) and in renal patients (17). The kinetics of VEGF mRNA expression in response to acute exercise has been examined in rats by Breen et al. (2), who reported that VEGF mRNA expression peaked immediately postexercise and began to decline within 2 h, returning to basal levels within 8 h. In humans, VEGF mRNA expression has been shown to be elevated at 30 min (8) and 1 h (13) postexercise.
Skeletal muscle VEGF protein level has not been measured across the working leg or in muscle tissue in response to acute exercise in humans, although exercise training has been shown to elevate resting skeletal muscle VEGF protein concentration (6, 7). VEGF protein concentration has been shown to both increase (4) and decrease (5) in venous serum samples immediately following a marathon performed in moderate and high altitudes, respectively.
Stimulation of VEGF is of particular interest for patients with impaired peripheral vascular function, and therefore, it is of clinical interest to determine whether exercise modes that are not limited by cardiovascular fitness, such as the leg-kicking model, induce both transcription of the VEGF gene and VEGF protein release. Therefore, one aim of the present study was to determine whether exercise of long duration and low intensity would induce VEGF gene activation. The second aim was to test the hypothesis that VEGF protein would be released from the working skeletal muscle to the circulation.
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METHODOLOGY |
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TOPABSTRACTMETHODOLOGYRESULTSDISCUSSIONREFERENCES |
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Seven physically active males age (means ± SE) 24.14 ± 0.42 yr, body mass 84.29 ± 1.28 kg, height 180.5 ± 0.86 cm, and body mass index (BMI) 25.86 ± 0.34 kg/m2 volunteered to participate in this investigation. Before participation, each subject was medically examined by a physician, and a resting venous blood sample was taken for blood screening. Subjects signed a written consent form after receiving both a verbal and written outline of all procedures and potential risks associated with this study. Ethical approval for this investigation was obtained from the Ethical Committee of Copenhagen and Frederiksberg Communities, Denmark and performed according to the Declaration of Helsinki.
Experimental Protocol
Determination of peak workload. All exercise was performed using the two-legged knee extension "kicking" exercise model. Subjects performed incremental, exhaustive exercise to obtain individual maximal workload (Wmax). After a 5-min warmup at 60 W, subjects continued kicking at 60 extensions/min until volitional exhaustion. Workload was increased by 10 W every 2 min.
Experimental trial. Subjects reported to the (exercise) laboratory at 8 AM in a fasted state having undertaken no vigorous exercise for at least 12 h. Each subject changed into appropriate experimental attire and lay in a supine position for the placement of an indwelling catheter into the femoral artery and femoral vein of one exercising leg under local anesthesia (lidocaine, 20 mg/ml, SAD, Denmark). The femoral artery catheter was inserted 2–5 cm below the inguinal ligament and advanced 5–10 cm proximally. The femoral vein catheter was inserted 
2 cm below the inguinal ligament and advanced 5 cm distally to avoid contamination from blood draining from the lower abdomen and saphenous vein (16). Subjects performed 3 h of two-legged knee extension exercise at 50% of peak workload (W50%). The rate of extension was maintained at 60 extensions/min throughout the exercise period, and the timing was aided by a mechanical metronome and verbal encouragement when necessary. After the completion of exercise, subjects remained in the laboratory for another 3-h recovery period. Water was consumed ad libitum throughout the exercise and recovery period, and no food was permitted until the cessation of the recovery period, at which time lunch was provided. The following morning (9 AM) subjects were asked to return to the laboratory.
Blood samples. Blood samples from the femoral vein and femoral artery were collected before exercise (Pre); after 0.5 h (Ex0.5), 1 h (Ex1), 2 h (Ex2), and 3 h of exercise (Ex3); and 0.5 h (Rec0.5), 1 h (Rec1), and 3 h (Rec3) postexercise.
Muscle biopsies. Muscle biopsies of the vastus lateralis muscle were taken at Pre, Ex0.5, Ex1.5, Ex3, Rec1, Rec3, and Rec20 using the percutaneous needle method (Bergstrom, Sweden) with suction. Local anesthetic (lidocaine, 20 mg/ml; SAD, Denmark) was administered to the site before the biopsies. Immediately after each biopsy, muscle tissue was removed from the needle by using sterile tweezers, and the tissue was immediately frozen in liquid nitrogen.
Skeletal Muscle VEGF mRNA Expression
Total RNA was extracted using the acid-phenol method as described previously (3). Briefly, human skeletal muscle samples (20–30 mg) were homogenized in 500 µl RNAzol B for 15–20 s, and then 50 µl chloroform-isoamyl alcohol were added to homogenized samples, inverted, and placed on ice for 5 min. Samples were centrifuged at 13,000 rpm for 15 min at 4°C, and the upper aqueous layer was carefully pipetted off. An approximately equal volume of isopropanol (ice cold) and 1 µl of 20 mg/ml glycogen were added to the samples, inverted gently, and left for 1 h at –20°C. Samples were centrifuged at 13,000 rpm for 15 min at 4°C, and the top aqueous layer was gently removed. The remaining pellet was washed by adding 500 µl 75% ethanol (diluted in diethylpyrocarbonate water), gently flicking the tube to dislodge the pellet, and centrifuged at 8,000 rpm for 10 min at 4°C. The aqueous supernatant was removed to expose the RNA pellet, which was allowed to dry briefly (10–15 min) and then resuspended in 10–20 µl RNAase free, ice-cold diethylpyrocarbonate-treated water. Finally, the RNA content of each sample was quantified spectrophotometrically using the method of Sambrook et al. (14). Samples (1 µg) were reverse transcribed using Taqman Reverse Transcription Reagents using random hexamer primers according to the manufacturers instructions (Applied Biosystems) and diluted 1:6 in DEPC-treated water.
VEGF mRNA expression was measured by using RT-PCR using real-time PCR (SDS 7700, Applied Biosystems). Primers and probes for amplification of the VEGF gene were constructed from human specific sequence data (NCBI, Bethesda, MD) using DNA analysis software (Primer Express, Applied Biosystems) The sequences used to amplify VEGF were the following: forward primer 5'-CTT GCT GCT CTA CCT CCA CCA T-3', reverse primer 5'-ATG ATT CTG CCC TCC TCC TTC T-3', and TaqMan probe 5'-AAG TGG TCC CAG GCT GCA CCC A-3'. The probe was 5'-FAM and 3'-TAMRA labeled. A blast search of the amplified sequence showed only homology with the target gene. The concentrations of primers and probe were optimized for use in the PCR reaction, the amplification efficiency was determined, and similar amplification efficiency of the target gene and the endogenous control was verified. Samples were analyzed in triplicate using 2.2 µl of the diluted sample in a total volume of 25 µl reaction mixture consisting of Taqman Universal Mastermix (Applied Biosystems) containing AmpliTaq Gold DNA polymerase, AmpErase uracil N-glycosylase, dNTPs with dUTP, carboxy-X-rhodamine as passive reference and buffer components, 0.177 µg each of forward and reverse primers, 0.36-µg probe, and water. The housekeeping gene 18S was measured using a 5'-VIC and 3'-6-carboxy-tetramethyl-rhodamine-labeled predeveloped assay reagent (Applied Biosystems) in all samples in the same PCR run. A two-step PCR run was performed using the general profile: 50°C for 2 min + 95°C for 10 min + [95°C for 15 s and 60°C for 1 min] x 40. A threshold cycle (Ct), where the fluorescence signal from the reporter dye reaches a given level was determined for each sample reflecting the amount of the specific mRNA in the sample. For each sample, the VEGF mRNA content was normalized to the 18S mRNA mRNA (given a 
Ct value). All samples from a given subject were expressed as fold changes relative to the prevalue, which was set to 1, using the Ct method (Applied Biosystems).
Plasma VEGF Concentration and Arteriovenous Difference
Plasma VEGF concentration was measured in femoral arterial and venous blood samples at all sample time points using high-sensitivity ELISA (R&D Systems; Abingdon, UK) according to the manufacturers instructions. The calculation of VEGF arteriovenous balance was calculated by subtracting the femoral vein VEGF concentration from the femoral artery VEGF concentration.
Statistical Analysis
Statistical analysis was performed using SigmaStat for Windows (version 2.03). All data were normally distributed and were therefore expressed as means ± SE. A one-way analysis of variance (ANOVA) for repeated measures was used to determine the effect of acute exercise on VEGF mRNA content and femoral plasma arteriovenous protein balance. The Student-Newman-Keuls test for pairwise multiple comparison procedure was performed to identify the source of any significant differences. At all times, P < 0.05 was used to indicate statistical significance.
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RESULTS |
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All of the subjects completed the 3-h exercise protocol. Heart rate during exercise was 128 ± 2.5 beats/min, and W50% was 70 ± 1.43 W. Predicted 
O2peak was 3.72 ± 0.08 ml·kg·min–1.
Effect of Acute Exercise on Skeletal Muscle VEGF mRNA
Acute exercise increased VEGF mRNA expression (ANOVA, P < 0.001) (Fig. 1). At Ex1.5, VEGF mRNA had increased 4.5-fold from Pre, and remained elevated until Rec3. VEGF mRNA peaked 1 h after exercise (Rec1), increasing over ninefold from Pre levels, and this was significantly higher than all other time points.
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Effect of Acute Exercise on VEGF Arteriovenous Balance
Acute exercise decreased VEGF arteriovenous balance (ANOVA, P = 0.007) (Fig. 2C). From Pre to Ex0.5, arteriovenous balance decreased from 18.06 ± 10.6 to 2.72 ± 3.7 pg/ml. Despite a slight elevation at Ex1 (6.4 1 ± 5.2 pg/ml), VEGF arteriovenous balance continued to gradually decline throughout the exercise period (–3.45 ± 8.3 and –3.11 ± 10.45 pg/ml at Ex2 and Ex3, respectively) and during the first hour of recovery (–14.46 ± 6.7 and –28.8 5 ± 14.4 pg/ml at Rec0.5 and Rec1, respectively). VEGF arteriovenous balance at Rec1 was significantly different from Pre, Ex0.5, Ex1, and Rec3. After 3 h of recovery (Rec3), VEGF had started to return to basal levels (5.26 ± 2.7 pg/ml). The change in VEGF arteriovenous balance over time was predominantly due to an increase in femoral vein VEGF concentration throughout the experimental period (ANOVA, P = 0.019), with no change in femoral artery VEGF concentration (ANOVA, P = 0.397, Fig. 2, B and C, respectively).
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DISCUSSION |
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TOPABSTRACTMETHODOLOGYRESULTSDISCUSSIONREFERENCES |
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To our knowledge, this is the first time that VEGF arteriovenous protein balance has been measured in the working leg in humans. We clearly show that arteriovenous balance decreases during exercise and in recovery, predominantly due to a combination of the maintenance of femoral arterial plasma VEGF concentration and an increase in femoral venous plasma VEGF concentration. The shift from positive to negative arteriovenous balance occurred concurrently to the elevation in VEGF mRNA expression (Ex1.5), and both the greatest decrease in arteriovenous balance and the greatest elevation in VEGF mRNA occurred at Rec1.
An elevation in VEGF mRNA in skeletal muscle may induce a subsequent increase in VEGF protein synthesis, which is released into the extracellular matrix (ECM) and to the circulation. However, it is unlikely in the current study that the decrease in VEGF arteriovenous balance can be entirely accounted for by an increase in protein synthesis. If this were the case, it might be expected that the elevation in VEGF protein release from the working leg might be preceded by an elevation in mRNA content in the muscle tissue rather than the change in both VEGF mRNA content and arteriovenous protein balance occurring at similar time points. This suggests that another mechanism, rather than skeletal muscle protein synthesis, might also contribute to the rapid shift in arteriovenous balance reported here.
An elevation in VEGF protein release from the working muscle tissue may have also contributed to the change in VEGF arteriovenous balance. There is an increase in VEGF protein release into culture media from electrically stimulated skeletal and cardiac myocytes (10, 15). In addition, chronic motor nerve-stimulated rabbit skeletal muscle showed increased VEGF protein predominantly between the muscle fibers using immunohistochemical staining (1), suggesting its release from the myocytes.
Mechanical stimulation is a likely mechanism for VEGF protein release from the myocyte to the ECM. Alginate hydrogel carriers (synthetic polymetric matrices used to simulate the natural ECM) were shown to increase VEGF release when subjected to mechanical compression in vitro (12). In addition, VEGF-loaded hydrogels implanted into murine hindlimb was shown to elevate local vascularization when they were mechanically stimulated for 14 days in vivo (12). This suggests that VEGF protein is released from myocytes when they are subjected to local mechanical stress. In the current study, mechanical stress via contraction of the working muscle is likely to have induced VEGF protein release from myocytes into the ECM, and in addition to exerting local angiogenic effects, would account for the elevation in VEGF protein in the femoral vein.
In conclusion, prolonged, low-intensity exercise induced a rapid decrease in VEGF protein arteriovenous balance across the working leg. This change is likely due to both an increase in VEGF production by, and release from, myocytes to the ECM. This finding demonstrates that exercise modes suitable for humans with low fitness level induce changes in VEGF that is likely to be of clinical significance in angiogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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Significant difference at Rec1 from all other time points (post hoc, P < 0.05).