INTRODUCTION

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ONE OF THE MANY skeletal muscle adaptations to exercise is the increased capillary network that develops to satisfy elevated tissue needs (5, 11, 30). In terms of O₂ requirements, we have previously demonstrated that in trained humans and other species, O₂ transport conductance (diffusion of O₂ from capillary to myocyte) interacts with convective O₂ delivery to determine maximum exercise capacity (14, 21). Because O₂ transport conductance is positively related to the number of capillaries per muscle fiber, it is apparent that angiogenesis plays an important role in both skeletal muscle function and dysfunction (2, 7). Vascular endothelial growth factor (VEGF) is undoubtedly involved in the regulation of this angiogenic process, but the mechanisms behind this response to exercise are not yet well understood.

Recent investigations demonstrated that VEGF, in addition to increasing vascular permeability (27), increases endothelial cell proliferation in vitro (17) and angiogenesis in vivo (29). Furthermore, Breen et al. (4) recently documented an elevation in skeletal muscle VEGF mRNA after a single exercise bout in intact rats, and Hang et al. (11) supported this finding in electrically stimulated rat skeletal muscle. In a recent attempt to uncover the mechanisms behind this response, Roca et al. (24) demonstrated that passive hyperperfusion did not elevate VEGF mRNA levels in dog skeletal muscle, whereas electrically stimulated exercise with similar blood flows resulted in a threefold increase in VEGF mRNA abundance. This suggests that the exercise-induced elevation in muscle blood flow alone does not stimulate increased VEGF mRNA levels. Breen et al. (4) also reported a greater increase in VEGF mRNA levels when rats were exercised in hypoxia, which is in accordance with the observation that VEGF is induced by hypoxia (18, 24). Because intracellular PO₂ falls with exercise, and falls to a greater extent in hypoxic exercise (22), it has been suggested that intracellular PO₂ may play role in the skeletal muscle angiogenic response to exercise (4).

Similar, but lessened, effects of normoxic exercise were reported for basic fibroblast growth factor (bFGF) (4), which is also recognized as a direct angiogenic factor (9). However, because VEGF contains a signal sequence peptide and can be secreted from cells, it is more likely to be directly related to angiogenic control (15, 17). To our knowledge there are no published data elucidating the human skeletal muscle VEGF or bFGF mRNA responses to acute exercise and their relationship to cellular O₂ levels.

Prompted by the physiological significance of angiogenesis in health and disease, the purpose of this study was to test the hypotheses that in the skeletal muscle of sedentary humans 1) VEGF mRNA abundance will be increased after a single exercise stimulus; 2) breathing hypoxic gas during exercise will further increase the abundance of VEGF mRNA; 3) bFGF mRNA, although a direct angiogenic factor, will be less obviously upregulated in response to exercise in normoxia or hypoxia than VEGF mRNA; and 4) there will be an inverse relationship between skeletal muscle intracellular PO₂ during exercise and VEGF mRNA abundance after exercise.

	METHODS

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Subjects. Six sedentary males, weighing 79.8 ± 2.9 kg (mean ± SD), ages 26.7 ± 1.9 yr, and with heights of 175.3 ± 3.2 cm, volunteered to participate in this study after health histories and physical examinations were completed and informed written consent was obtained according to the University of California, San Diego, Human Subjects Committee requirements. None of the subjects had performed endurance exercise on a regular basis before the study and, as indicated by the mean maximum O₂ consumption (O_{2 max}) measured during conventional cycle ergometer exercise (O_{2 max} = 33.9 ± 1.8 ml · kg¹ · min¹), they were appropriately classified as sedentary.

Exercise apparatus. The knee-extensor ergometer used to produce an acute exercise stimulus was designed to limit exercise to the quadriceps muscles of the left leg (21). Briefly, subjects were semirecumbent in an adjustable chair with a special ankle boot placed on the left leg that was connected by a bar to the ergometer (Fig. 1 in Ref. 22). Contractions of the quadriceps muscles caused the lower part of the leg to extend from an angle of 90° to 170°. Therefore, the lower leg traveled with an arc-shaped trajectory of ~80°. The momentum of the ergometer passively returned the relaxed leg to the start position, and, as a result, the quadriceps muscle was functionally isolated (20). During exercise, the contraction rate was maintained at 60 min¹.

View larger version (46K): [in this window] [in a new window]   Fig. 1.   Northern blots showing sedentary human mRNA signals for vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Blots were reprobed and normalized to 18S ribosomal RNA (18S). Muscle biopsies were taken from muscle at rest (lane 1) and at 1 h after acute knee-extensor exercise in normoxia (lane 2) or hypoxia (lane 3). Initials (right) identify data from individual subjects.

Acute exercise stimulus. One week after a brief familiarization period, subjects underwent an acute knee-extensor exercise bout with their left leg, consisting of a 5-min unloaded warm-up followed by a graded maximal exercise test and then by 30 min of knee-extensor exercise at 50% of the maximum work rate achieved. The graded exercise test required subjects to maintain each work rate for 60 s, after which the work rate was incremented by 5 W. The subjects continued until they were unable to maintain a cadence of 60 rpm for the entire minute.

Muscle biopsies. Approximately 1 h (50-70 min) after knee-extensor exercise was completed, a muscle biopsy was taken from the exercised leg (left) and the rested leg (right). Several weeks later a muscle biopsy was taken from the right leg after acute knee-extensor exercise in hypoxia [fractional inspired O₂ (FI_O2) 0.12]. Because peak work rate was not affected by hypoxia, the absolute and relative work rates were identical in hypoxia and normoxia. Thus the exercise stimulus before the biopsy procedure was identical in either condition. The use of a single resting biopsy for either condition is supported by our observation that resting levels of human VEGF mRNA are consistently low with varying environmental/physiological changes preceding the sample, even with 8 wk of training (unpublished observation). All biopsies were taken from the vastus lateralis ~3.5 cm deep, 15 cm proximal to the knee, and slightly distal to the ventral midline of the muscle. The 5-mm diameter biopsy needle was attached to sterile tubing and a syringe to apply a negative pressure to assist in the muscle sample collection (12). Lidocaine (2%) was used as a local anesthetic and was infiltrated beyond the depth of the biopsy. The muscle samples from each biopsy were immediately frozen in liquid nitrogen and stored at 80°C. In summary, muscle biopsies were collected from muscles in three different states: 1) rested, 2) acutely exercised, and 3) acutely exercised in hypoxia.

RNA isolation and Northern analysis. Total cellular RNA was isolated from each muscle sample (muscle sample mass range 65-110 mg) by the method of Chomczynski and Sacchi (6). RNA preparations were quantitated by absorbance at 260 nm, and intactness was assessed by ethidium bromide staining after separation by electrophoresis in 6.6% formaldehyde-1% agarose gel. Fractionated RNA was transferred by Northern blot to Zeta probe membrane (Bio-Rad, Hercules, CA). RNA was cross-linked to the membrane by ultraviolet irradiation for 1 min and stored at 4°C. The blots were then probed with oligo-labeled [-³²P]deoxycytidine triphosphate cDNA probes, which had a specific activity of 1 × 10⁹ dpm/µg DNA (8). The human VEGF is a 0.93-kb cDNA fragment isolated from the EcoR I site of pUC-derived plasmid (17). The bFGF is a 1-kb Xho I fragment of human bFGF cDNA (16). 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 pyrrolidine), 50 mM sodium phosphate (pH 6.5), 1% SDS, and 250 µg/ml salmon sperm DNA at 37 or 42°C. Blots were washed with 2× SSC and 0.1% SDS at 50°C for the VEGF mRNA. Blots were exposed to XAR-5 X-ray film (Eastman Kodak, New Haven, CT) with the use of a Cronex Lightning Plus screen at 70°C. Autoradiographs were quantitated by densitometry (Media Cybernetics, Silver Spring, MD). Each blot was subsequently reprobed (after prior complexes were stripped) with a cDNA specific for 18S ribosomal RNA, and this signal was used to normalize the mRNA signal for minor variations in lane loading. All samples from a single subject were run on the same gel, producing optimum conditions for quantitative analyses across conditions.

Determination of intracellular PO₂. The same exercise paradigm was performed in a 2.0-Tesla Oxford imaging magnet (Fig. 1 in Ref. 22). Spectra were collected from the muscle region below the 7-cm-diameter surface coil that was double-tuned to proton (85.45 MHz) and phosphorus (34.59 MHz) and placed over the rectus femoris portion of the quadriceps group (26) ~20-25 cm proximal to the knee. For these studies, this "sensitive region" was <100 cm³ of muscle, which isolated signal detection predominantly to the rectus femoris (1). Details of the theory behind O₂-sensitive myoglobin signals have been published previously (3, 22). Briefly, the heme iron exhibits O₂-dependent spin states that, in turn, influence nearby protons. The N- proton on proximal histidine F8, one of the ligands coordinated to the iron, is particularly sensitive to these changes. When O₂ is bound to the active site, the resonance of this proton is hidden beneath the dominant water signal. However, when myoglobin becomes deoxygenated, changes in the iron spin state shift this peak to a temperature-dependent position that is clearly distinct from all other resonances. Fractional deoxymyoglobin (^fdeoxy-Mb) was determined by normalizing the signal areas to the average signal obtained during the 9th and 10th minutes of cuff ischemia at suprasystolic pressure (270 mmHg). Intramuscular O₂ depletes within 6-8 min of occlusion (28). Therefore, the plateaued signals obtained during the last 2 min of cuff occlusion represent complete deoxygenation of myoglobin and are used to estimate total myoglobin content within the muscle. Conversion to PO₂ values was then calculated from the O₂ binding curve for myoglobin as
where ^fMbO₂ is the fraction of myoglobin that is oxygenated and MbP₅₀ is the O₂ pressure at which 50% of the myoglobin binding sites are bound with O₂. The temperature-dependent myoglobin half-saturation (P₅₀) of 3.2 mmHg was used (25). On the basis of the previously published observation that intracellular PO₂ does not vary from submaximal efforts (50% of work rate maximum) (22), the data collected in several of the current subjects progressing toward their maximum work rate, and the inclusion of maximal exercise in the original exercise paradigm, intracellular PO₂ values in the magnet were measured during maximal knee-extensor exercise. Intracellular PO₂ data were only successfully collected on five of the six subjects because of a signal-to-noise problem.

Statistical analysis. The acute effect on resting muscle of either normoxic or hypoxic exercise was qualitatively assessed from the densitometry values for the VEGF and bFGF mRNA levels (normalized by the 18S values) using a repeated-measures ANOVA. Differences between groups were then identified using a Newman-Keuls post hoc analysis. Intracellular PO₂ and functional measurements of skeletal muscle work rate in hypoxia and normoxia were compared using paired t-tests. The relationship between intracellular PO₂ and VEGF mRNA was assessed with a regression analysis. Statistical significance was accepted if P < 0.05.

	RESULTS

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Functional response to acute normoxic and hypoxic exercise. The average maximal knee-extensor work rate was not statistically different between exercise in normoxia (62 ± 5 W) and hypoxia (58 ± 6 W). Thus for these sedentary subjects both the absolute and relative intensities for the 30-min exercise stimulus in these two conditions were also not statistically different between normoxia and hypoxia.

VEGF response to acute normoxic and hypoxic exercise. As shown in Fig. 1, it is clear that at rest the VEGF mRNA levels of these sedentary subjects were very low. It is also evident that VEGF mRNA abundance in human skeletal muscle increased significantly 1 h after an acute small muscle mass exercise bout (Figs. 1 and 2). This VEGF mRNA increase is apparent whether the exercise was performed in normoxic or hypoxic conditions. In fact, Fig. 2 shows the observation that the VEGF mRNA responses to exercise in normoxia or hypoxia were significantly increased compared with resting control values, but the exercise values were not statistically different from each other. Figure 3 shows that no significant relationship exists between intracellular PO₂ during exercise and VEGF mRNA abundance after exercise.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Quantitative densitometry of VEGF mRNA levels at rest and after acute knee-extensor exercise in hypoxia and normoxia. Lane loading was normalized for by densitometry on 18S ribosomal RNA. * P < 0.05; # P < 0.05 vs. resting condition.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Individual intracellular PO2 values measured during hypoxic and normoxic exercise and values for VEGF mRNA abundance 1 h after exercise in either condition. Repeated measurements are linked by solid lines.

bFGF response to acute normoxic and hypoxic exercise. Significant levels of bFGF mRNA were seen in the control condition (rested skeletal = 0.9 ± 0.07 bFGF/18S arbitrary units; Fig. 1.). Acute exercise in normoxia or hypoxia did not statistically alter the baseline values recorded in this skeletal muscle (0.8 ± 0.7 and 1.2 ± 0.2 bFGF/18S arbitrary units, respectively; Fig. 1). Thus, in contrast to the VEGF mRNA response, the bFGF mRNA response to exercise was not measurably different from control values (Fig. 1).

Intracellular PO₂ response to acute normoxic and hypoxic exercise. The use of myoglobin as an endogenous probe for intracellular PO₂ is limited by the fact that when myoglobin is bound to O₂, it is not visible by magnetic resonance spectroscopy. However, on the basis of our previous observations, a P₅₀ of 3.2 mmHg, and the signal-to-noise ratio in the present study, a myoglobin signal of ~20% of the maximal cuff signal should be detectable. Because there was no such signal at rest in these subjects, we can conclude that the intracellular PO₂ at rest was >13 mmHg. Thus intracellular PO₂ was reduced from resting levels during exercise in either normoxia or hypoxia. Intracellular PO₂ during exercise in these subjects was significantly lower during exercise in hypoxia (3.8 ± 0.3 mmHg) than during exercise in normoxia (7.2 ± 0.6 mmHg). The relationship between individual subject VEGF mRNA response to exercise and the intracellular PO₂ recorded at maximal exercise is shown in Fig. 3 (note that intracellular PO₂ was measured in only 5 subjects). There was no relationship between intracellular PO₂ measured during exercise and the level of VEGF mRNA after exercise (Fig. 3).

	DISCUSSION

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The principal finding of this study is that VEGF mRNA in untrained human skeletal muscle was substantially increased 1 h after a single acute exercise bout (Fig. 1). Because VEGF functions as a direct angiogenic factor with a high specificity for vascular endothelial cells (17), these findings are in line with the theory that VEGF is involved in the well-documented formation of new blood vessels within human skeletal muscles in response to exercise (5, 11, 30). This is an essential adaptive response in skeletal muscle to repeated exercise (i.e., training), resulting in an increase in the number of capillaries per muscle fiber that enhance O₂ transport conductance between the microcirculation and mitochondria (2). Interestingly, the present data reveal a fall in intracellular PO₂ from rest (>13 mmHg) to exercise in normoxia (7.2 ± 0.6 mmHg), but an even greater fall in intracellular PO₂ in hypoxic exercise (3.8 ± 0.3 mmHg) did not demonstrate an additive effect on VEGF mRNA abundance. Perhaps this observation can be reconciled with the concept that hypoxia is a stimulus for angiogenesis by the existence of an intracellular PO₂ "threshold" beyond which no greater angiogenic stimulus is produced. Exercise in normoxia appears to achieve this threshold.

VEGF response to hypoxic exercise. As recognized above, VEGF mRNA increased significantly after exercise in either hypoxia or normoxia, but the response was not significantly different or correlated with intracellular PO₂ (Figs. 1-3). This disproves our hypothesis of an inverse relationship between intracellular PO₂ and VEGF mRNA level and disagrees with the findings of Breen et al. (4), who recorded a doubling of the VEGF message in rats exercised in hypoxia compared with that in rats exercised in normoxia.

bFGF response to acute exercise. In human skeletal muscle at rest there were significant levels of bFGF mRNA, contrasting with the low levels of VEGF mRNA in the same condition. Additionally, exercise in either normoxia or hypoxia did not lead to increased bFGF mRNA levels. Thus, in agreement with the findings and inferences of other work, we support the concept that bFGF may play a less significant role in the control of the angiogenic process than VEGF (4, 11, 15, 17). However, it should be recognized that this study is limited by the single muscle sample taken 1 h after exercise and that bFGF may increase beyond the measured time frame, although this was not evident in the previous exercise study in rats, which employed several sampling times after exercise (4).

Limitations to present study. There are several unavoidable limitations to our experimental design, including the fact that measurements of intracellular PO₂ (dorsal area of quadriceps) were not recorded in the same anatomic site as the biopsies (vastus lateralis of the quadriceps). However, it has been documented that all quadriceps muscles are equally recruited in this exercise model (20). The PO₂ measurements/biopsies were not taken at the same time, but the logistical complexity of this study precluded this. A single muscle sample limits conclusions, because the time course for VEGF mRNA may be affected by hypoxic exercise. Either a shortening or lengthening of the response time may lead to an erroneous conclusion, based on one sample. In the future, multiple biopsy samples need to be taken that will reveal both time-course issues and information beyond transcription to translation of angiogenic growth factors and receptors. Additionally, the unexpected trend toward a reduced VEGF mRNA response with decreasing PO₂ (Fig. 3) may have achieved significance with a greater number of subjects in this study. However, such an observation is contrary to our final hypothesis and to the commonly accepted dogma that VEGF expression appears to be inversely related to O₂ availability.