We investigated whether acute systemic exercise increases vascular endothelial growth factor (VEGF), VEGF receptor (KDR and Flt-1) mRNA, and VEGF protein in sedentary humans. Twelve sedentary subjects were recruited and performed 1 h of acute, cycle ergometer exercise at 50% of maximal oxygen consumption. Muscle biopsies were obtained from the vastus lateralis before exercise and at 0, 2, and 4 h postexercise. Acute exercise significantly increased VEGF mRNA at 2 and 4 h and increased KDR and Flt-1 mRNA at 4 h postexercise. The sustained increase in VEGF mRNA through 4 h and the increases in KDR and Flt-1 at 4 h are different from their respective time course responses in rats. In contrast to the increase in VEGF mRNA postexercise, VEGF protein levels were decreased at 0 h postexercise. These results provide evidence in humans that 1) VEGF, KDR, and Flt-1 mRNA are increased by acute systemic exercise; 2) the time course of the VEGF, KDR, and Flt-1 mRNA responses are different from those previously reported in rats (Gavin TP and Wagner PD. Acta Physiol Scand 175: 201–209, 2002); and 3) VEGF protein is decreased immediately after exercise.
- skeletal muscle capillary supply
- time course
- vascular endothelial growth factor
it is well known that endurance exercise training promotes many adaptations in skeletal muscle, including increases in oxidative enzymes and in the number of capillaries surrounding the muscle fibers, the latter being known as angiogenesis (2, 17, 21, 28). Acute exercise increases the expression of vascular endothelial growth factor (VEGF) mRNA 3- to 5-fold in response to systemic exercise in rats (4, 10–12, 22) and >10-fold in response to knee extensor (KE) exercise in humans (15, 24, 25). VEGF is important for the maintenance of the skeletal muscle capillary supply, as well as the expansion of the capillary supply in response to exercise training (1, 30).
Exercise training has been shown to increase skeletal muscle VEGF protein in both human and animal models (1, 14). In human skeletal muscle, VEGF protein was approximately twofold greater 24 h after the last exercise bout in response to 10 days of KE exercise training (14). In rat skeletal muscle, VEGF protein was approximately twofold greater 2 h after the last exercise bout in response to 3 days of exercise training (1), suggesting that VEGF protein is increased during the initial phases of an exercise training program; however, the response to an initial acute exercise bout is unknown.
The biological activity of VEGF is produced through ligand binding to the VEGF receptors KDR and Flt-1. These high-affinity VEGF receptors are localized predominantly to the vascular endothelium, both on proliferating endothelial cells as well as on quiescent cells (8). It is currently hypothesized that it is KDR that is essential to induce the full spectrum of the VEGF biological response and that Flt-1 functions in angiogenesis as a ligand-binding molecule, rather than as a signal-transducing receptor (9). Gavin and Wagner (12) have demonstrated that acute systemic exercise increases both Flk-1 (the murine analog of KDR) and Flt-1 mRNA in rats, suggesting that in rats at least there is an increase in both VEGF and VEGF receptor expression in response to acute exercise.
Therefore, the purposes of this study were to investigate in sedentary humans whether acute systemic exercise increases VEGF, KDR and Flt-1 mRNA, and VEGF protein. We hypothesized that, in response to acute systemic exercise, VEGF, KDR, and Flt-1 mRNA and VEGF protein would be increased in human skeletal muscle. We demonstrate here that in humans 1) VEGF, KDR, and Flt-1 mRNA are increased by acute systemic exercise; 2) the time course of the VEGF, KDR, and Flt-1 mRNA responses are different from those previously reported in rats; and 3) VEGF protein is decreased immediately after exercise.
Subjects. Twelve sedentary men volunteered to participate in the study after written and verbal explanations of the content and intent of the study in accordance with the University and Medical Center Institutional Review Board. All subjects were healthy, nonsmokers, with no history of cardiopulmonary disease, and they completed a Physical Activity Readiness Questionnaire before the commencement of the study. The physical characteristics of the subjects are listed in Table 1.
Maximal oxygen consumption. Maximal oxygen consumption (V̇o2 max) was measured on a Monark 886 cycle ergometer. Minute ventilation, oxygen uptake (V̇o2), and carbon dioxide production were continuously monitored via open-circuit spirometry (True Max 2400, Parvo Medics, Salt Lake City, UT). Heart rate was measured continuously (model Accurex Plus, Polar Electro, Woodbury, NY). The test began with a 5-min warm-up at 70 W. After the warm-up, the workload was increased 35 W every 2 min until volitional fatigue. Subjects were verbally encouraged to continue for as long as possible. The criterion used to assess V̇o2 max included 1) a heart rate in excess of 90% of age predicted maximum (220 - age); 2) a respiratory exchange ratio ≥1.10; and 3) identification of a plateau (≤150 ml increase) in V̇o2 despite a further increase in workload. In all tests, at least two of three criteria were met.
Body composition. Body density (Db) was determined via hydrostatic weighing. Residual volume was measured by oxygen dilution (33). Body fat percentage was determined from Db on the basis of the two-compartment model of Siri et al. (29).
Submaximal exercise and muscle biopsies. At least 1 wk after the V̇o2 max test, subjects performed a 1-h exercise bout at 50% of V̇o2 max. This intensity of exercise has been shown to promote exercise-induced angiogenesis in humans (28). Before the commencement of exercise, and at 0, 2, and 4 h postexercise, a muscle biopsy was obtained from the vastus lateralis for the measurement of mRNA and protein. Biopsies were alternated between legs and sites were separated by at least 3 cm (7). The leg for the resting biopsy samples was alternated between subjects. Muscle biopsy sampling at sites separated by 3 cm and 2 h does not increase VEGF mRNA expression in resting biopsy samples (unpublished observation). Samples were stored at -80°C until analysis.
For muscle morphometry, a section of the resting muscle sample was used for the measurement of the skeletal muscle capillary supply. Muscle tissue was oriented in an OCT-tragacanth mixture, frozen in liquid nitrogen cooled isopentane, and stored at -80°C until processing.
RNA isolation and Northern blot analysis. Total cellular RNA was isolated from each sample by the method of Chomczynski and Sacchi (6). These RNA preparations were quantitated by absorbance at 260 nm, and RNA intactness and integrity were assessed by ethidium bromide staining after separation by electrophoresis in a 6.6% form-aldehyde-1% agarose gel. Fractionated RNA was transferred by Northern blot to Zeta-probe membrane (Bio-Rad, Hercules, CA). After transfer, RNA was cross-linked to the membrane by ultraviolet irradiation for 1 min and stored at 4°C. The blots were then probed with oligolabeled [α-32P]deoxycytidine triphosphate cDNA probes. The human VEGF probe was a 675-bp EcoRI fragment (kind gift of N. Ferrara, Genentech); the human KDR probe was a 709-bp EcoRI fragment (kind gift of L. Aiello, Joslin Diabetes Center); and the human Flt-1 probe was a 123-bp EcoRI fragment (kind gift of M. Shibuya, University of Tokyo, Tokyo, Japan). Prehybridization and hybridization were performed in 50% formamide, 5× SSC (20× SSC is 0.3 M sodium chloride, 0.3 M sodium citrate), 10× Denhardt's solution (100× Denhardt's solution is 2% Ficoll, 2% polyvinyl pyrrolidone, 2% bovine serum albumin factor V), 50 mM sodium phosphate (pH 7.0), 1% SDS, and 250 μg/ml salmon sperm DNA at 42°C. Blots were washed with 2× SSC and 0.1% SDS at room temperature and 0.1× SSC and 0.1% SDS at 45°C (Flt-1), 55°C (KDR), or 65°C (VEGF). Blots were exposed to XAR-5 X-ray film (Eastman Kodak, New Haven, CT) by use of a Cronex Lightning Plus screen at -80°C. Autoradiographs were quantitated by densitometry within the linear range of signals and normalized to β-actin mRNA levels.
Protein isolation and analysis. A portion of the muscle biopsy sample was homogenized in RIPA (1× PBS, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS with protease inhibitors). Total protein was measured by the bicinchoninic acid method (BCA protein assay kit, Bio-Rad Laboratories, Hercules, CA). For each sample, VEGF protein was measured from 25 μg of total protein and was analyzed in duplicate. A commercial VEGF ELISA kit was used according to the manufacturer's instructions (model DVE00, R&D Systems, Minneapolis, MN). According to the manufacturer, the VEGF ELISA kit recognizes both the 121-amino acid and 165-amino acid isoforms of VEGF and VEGF, and within the physiological range it recognizes both free and receptor-bound VEGF protein. VEGF levels were obtained by use of a microplate reader at 450 nm and corrected by readings at 540 nm (Titertek Multiscan MCC-340: Eflab, Turrku, Finland).
Morphometric and morphologic analysis. Muscle tissue mounted in OCT and tragacanth was sectioned to a thickness of 10 μm on a cryostat, mounted on slides, and kept at -20°C until fixation. Sections were stained for capillaries using a modified ATPase method (Rosenblatt), which simultaneously provides fiber typing (type I and II) and capillary visualization (5, 26). There is no difference in the number of capillaries visualized with frozen biopsy samples using the Rosenblatt technique and the number visualized with muscle perfusion fixation (18).
Muscle sections were viewed under a light microscope (Nikon 400) and a digital image taken of the section (Nikon Coolpix 990). Capillaries were quantified manually from the digital image on individual fibers. The following indexes as outlined by Hepple et al. (17) were measured: 1) the number of capillaries around a fiber (capillary contacts), 2) the capillary-to-fiber ratio on an individual-fiber basis, and 3) the number of fibers sharing each capillary [sharing factor (SF)]. Quantitation of the capillary supply was performed on at least 50 fibers by randomly selecting a fiber in an artifact-free region. Fiber area and perimeter were measured with the image-analysis system and commercial software (SigmaScan, Jandel Scientific), calibrated to transform the number of pixels (viewed on a computer monitor) into micrometers. Capillary density and capillary-to-fiber perimeter exchange (CFPE) index were measured according to Hepple et al. (17). CFPE is a relative measure of the capillary supply accounting for differences in fiber size, and in particular it estimates the capillary-to-fiber surface area, which represents the greatest resistance to oxygen flux (17). The morphological characteristics of the subjects are listed in Table 2.
Statistical treatment. For both mRNA and protein, a repeated-measures one-way analysis of variance (time postexercise) was used. After a significant F ratio, a Dunnett's post hoc analysis was used to determine significance between rest and postexercise time points. Paired Student's t-tests were used to compare differences in skeletal muscle morphometry between type I and II fibers. The following mRNA samples were lost during isolation: three samples at rest, two samples at 0 h, one sample at 2 h, and one sample at 4 h. The following protein samples were lost during isolation: three samples at rest and one sample at the 0 h. Significance was established at P ≤ 0.05 for all statistical sets, and data are reported are means ±SE.
The mRNA responses to acute systemic exercise between 0 and 4 h are shown for VEGF in Fig. 1, KDR in Fig. 2, and Flt-1 in Fig. 3 with representative Northern blots in Figs. 1A, 2A, and 3A and quantitative densitometry normalized to β-actin mRNA in Figs. 1B, 2B, and 3B, respectively. Gene expression was analyzed at rest and 0, 2, and 4 h after a single, 1 h submaximal exercise bout in the vastus lateralis. Systemic exercise significantly increased VEGF (∼ 4.5-fold) at 2 and 4 h and increased KDR (∼5-fold) and Flt-1 (∼2.5-fold) mRNA at 4 h postexercise.
The VEGF protein response to acute systemic exercise is shown in Fig. 4. In contrast to the increase in VEGF mRNA observed after acute exercise, VEGF protein declined at 0 h postexercise.
The principal findings of the present study are 1) VEGF, KDR, and Flt-1 mRNA are increased by acute systemic exercise; 2) the time course of the VEGF, KDR, and Flt-1 mRNA responses are different than those previously reported in rats (12); and 3) VEGF protein is decreased immediately after exercise. In support of our hypothesis, acute systemic exercise increases the gene expression of the major components of the VEGF pathway in humans, including the VEGF receptors (KDR and Flt-1), suggesting a role for increased receptor expression in skeletal muscle angiogenesis. In contrast to our hypothesis, VEGF protein is not increased by an initial acute exercise bout but is in fact decreased immediately postexercise.
VEGF mRNA expression. It is well known that acute systemic exercise increases VEGF mRNA in rats (4, 10–12, 22). In addition, there are several reports that acute KE exercise increases the expression of VEGF in healthy humans (15, 24, 25) and patients with chronic renal failure (32). In the present report, we have demonstrated for the first time that acute systemic exercise increases VEGF mRNA in humans.
There are several differences between KE and traditional systemic exercise. Muscle mass-specific blood flow (ml·min-1· 100 g-1) and mitochondrial oxidative rates (ml O2·min-1·cm-3) are approximately twofold higher during KE (23), suggesting that KE may provide a greater stimulus than more traditional exercise modalities. Thus the larger increases observed in VEGF mRNA after KE (∼6- to 18-fold compared with ∼4.5-fold in the present study) are consistent with these differences (24, 32). However, most individuals performing exercise training do not use exercise modalities such as KE but rather more traditional systemic exercise such as bicycle ergometry, thus our investigation using systemic exercise is consistent with the exercise-induced angiogenesis observed with traditional exercise in humans (2, 17, 28).
In previous reports, the time course of the VEGF mRNA response to exercise was documented in rats (4, 11). During the preparation of this manuscript, Hiscock et al. (19) reported VEGF mRNA expression during (at 0.5, 1.5, and 3 h) and after (at 1, 3, and 20 h postexercise) a 3-h two-legged KE exercise bout. The greatest VEGF mRNA response was observed 1 h postexercise, which is 4 h after the commencement of exercise. In the current report, we have demonstrated a nonsignificant 2-fold increase in VEGF mRNA at 0 h postexercise and a 4.5-fold increase at 2 and 4 h postexercise (Fig. 1), which are 3 and 5 h after the commencement of exercise and are thus in close agreement with Hickson et al. (19). Interestingly, there are differences in the time course of the VEGF mRNA response to acute exercise between rats and humans. In rats, VEGF mRNA is significantly increased immediately postexercise, with the greatest increase observed at 1 h postexercise, and with a return to baseline by 4 h postexercise (11). Whether the maintained increase in VEGF mRNA through 4 h in humans is a function of an increase in mRNA stability or an increase in transcription remains to be determined.
VEGF protein expression. Of particular interest in the present report is the VEGF protein response to acute exercise. We observed a decrease in VEGF protein at 0 h (Fig. 4). To our knowledge, this is the first report investigating the VEGF protein response in muscle to an acute exercise bout.
The reduction in VEGF protein at 0 h is in contrast to our hypothesis that the VEGF protein would increase in response to an acute exercise bout. The decrease in VEGF protein as measured from muscle biopsies may reflect the secretion of VEGF from the skeletal muscle into the circulation during and after exercise. Recently, Höffner et al. (20) have demonstrated that VEGF protein increases in the skeletal muscle interstitial space during the first 30 min of KE exercise, suggesting that VEGF is secreted by the skeletal muscle into the interstitial space where it can then enter into the vasculature. Consistent with this, VEGF levels in the femoral vein tend to increase during 3 h of KE exercise and reach statistical significance 1 h postexercise (19). Given the small nonsignificant increase in VEGF mRNA immediately after the completion of exercise (Fig. 1), it would be difficult to suggest that the increase observed in interstitial VEGF during the first 30 min of exercise is the result of new protein synthesis and thus probably reflects the release of stored VEGF from the skeletal muscle. Our present results demonstrating that VEGF protein is lower at 0 h postexercise (Fig. 4) are consistent with VEGF leaving the skeletal muscle and entering into the circulation during and after exercise (19, 20).
It is also possible that increased VEGF in the interstitial space during exercise and reduced VEGF in skeletal muscle immediately after exercise may result from proteolytic cleavage of VEGF from extracellular binding sites. VEGF is known to exist in several different isoforms, including 121, 165, 189, and 209 amino acids. The 121 and 165 isoforms are secretable, whereas the 189 and 209 isoforms are membrane bound. Proteolytic cleavage of membrane-bound VEGF may occur through VEGF-induced production of matrix metalloproteinases (MMP) from endothelial cells (31). The increased production of MMPs may then lead to cleavage of membrane-bound isoforms of VEGF (9). In response to electrical stimulation, MMP production is increased in skeletal muscle (16), consistent with the suggestion that membrane-bound VEGF may be released during exercise.
The return of VEGF protein to baseline levels may reflect translation of the increased VEGF mRNA observed during (19) and immediately postexercise (Ref. 19 and Fig. 1), suggesting that whereas the increased VEGF mRNA observed after a single acute exercise bout in otherwise untrained humans does not increase the skeletal muscle content of VEGF, it is required to return VEGF to baseline levels. A second possibility is that the return of VEGF protein occurs because of an uptake of VEGF from the circulation. At rest, skeletal muscle takes up VEGF from the circulation (13, 19). One hour postexercise, skeletal muscle is secreting VEGF into the circulation, but by 3 h VEGF secretion has ceased and muscle returns toward net VEGF uptake (19). A third possible mechanism is that the changes in VEGF protein observed here merely reflect changes in protein half-life. However, given the large increases in VEGF mRNA observed, this explanation does not appear likely.
VEGF-receptor mRNA expression. The biological activity of VEGF is produced through ligand binding to the two predominant VEGF receptors, KDR and Flt-1. Gavin and Wagner (12) have demonstrated that acute systemic exercise increases both Flk-1 and Flt-1 mRNA in rats. To our knowledge, the present report is the first in humans demonstrating that acute exercise increases the expression of both KDR and Flt-1 mRNA. An increase in VEGF, KDR, and Flt-1 gene expression with exercise is consistent with a coordinated VEGF specific angiogenic response. A coordinated VEGF response is a prerequisite for tumor angiogenesis as tumors express VEGF, KDR/Flk-1, and Flt-1 (3). Thus the increase in VEGF, KDR, and Flt-1 mRNA with exercise is consistent with a coordinated increase in the major components of the VEGF system in human skeletal muscle.
As observed with VEGF, there is a different time course of the VEGF receptor mRNA response to acute exercise between rats and humans. Although exercise was shown to increase Flk-1 mRNA in rats, post hoc analysis was unable to reveal the time point at which Flk-1 was elevated (12). In the present report, we demonstrate in humans that KDR mRNA is significantly increased at 4 h postexercise (Fig. 2). In rats, Flt-1 mRNA is increased at 1 h postexercise and returns to baseline by 2 h, with an additional increase at 24 h postexercise (11). In contrast, there is an increase in Flt-1 mRNA at 4 h postexercise in humans (Fig. 3). The exact nature of these differences remains to be evaluated; however, the major finding that both KDR mRNA and Flt-1 mRNA are significantly increased by acute exercise in humans and rats demonstrates a consistent upregulation of the components of the VEGF system across species.
It might be hypothesized that the secretion of VEGF from the muscle may be responsible for the increase in VEGF-receptor mRNA expression. VEGF can regulate both KDR and Flt-1 mRNA expression in endothelial cells (3, 27). Thus, if VEGF is secreted from skeletal muscle as suggested by the present results and as observed by Höffner et al. (20), this might result in an increase in both KDR and Flt-1 gene expression, consistent with a coordinated increase in the VEGF pathway by exercise.
In summary, we have demonstrated in humans that 1) VEGF, KDR, and Flt-1 mRNA are increased by acute systemic exercise; 2) the time course of the VEGF, KDR, and Flt-1 mRNA responses are different than those previously reported in rats (12); and 3) VEGF protein is decreased immediately after exercise. Our data suggest, that in humans, there is a coordinated increase in VEGF and VEGF receptor mRNA expression in response to a single acute exercise. In contrast, VEGF protein content is reduced immediately postexercise, consistent with the secretion of VEGF from the skeletal muscle.
The authors thank Brian A. Annex, MD, G. Lynis Dohm, PhD, Russell T. Hepple, PhD, Mike Thompson, Ellis Jensen, and David Lang for assistance.
This study was supported in part by State of California Tobacco Related Disease Research Program Grant 8KT-0081, American Heart Association Grant 9960238U, and National Institute on Aging Grants AG-021891, AG-18407, and AG-19209.
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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