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J Appl Physiol 98: 2137-2146, 2005. First published January 20, 2005; doi:10.1152/japplphysiol.01402.2004
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VEGF-A splice variants and related receptor expression in human skeletal muscle following submaximal exercise

T. Gustafsson,1,2 H. Ameln,1,2 H. Fischer,1,2 C. J. Sundberg,2 J. A. Timmons,2,3 and E. Jansson1

1Department of Physiology and Pharmacology, and 2Division of Clinical Physiology, Department of Laboratory Medicine, Karolinska University Hospital, and 3Center for Genomics and Bioinformatics, Karolinska Institutet, Stockholm, Sweden

Submitted 21 December 2004 ; accepted in final form 15 January 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
VEGF-A contributes to muscle tissue angiogenesis following aerobic exercise training. The temporal response of the VEGF-A isoforms and their target receptors has not been comprehensively profiled in human skeletal muscle. We combined submaximal exercise with and without reduced leg blood flow to establish whether ischemia-induced metabolic stress was an important physiological stimuli responsible for regulating the VEGF-A system in humans. Nine healthy men performed two 45-min bouts of one-leg knee-extension exercise, with and without blood flow restriction. Muscle biopsies were obtained at rest and 2 and 6 h after exercise. Expression (mRNA) of the VEGF-A splice variants and related receptors [VEGF receptor (VEGFR)-1, VEGFR-2, and neuropilin-1] was determined by using qPCR. VEGF-Atotal expression increased more robustly after exercise with reduced blood flow, and initially this principally reflected an increase in VEGF-A165. Six hours after exercise, there was a relatively greater increase in VEGF-A189, and this response was not influenced by blood flow conditions. VEGFR-1 mRNA expression increased 2 h after exercise, and neuropilin-1 expression was transiently reduced, while all three receptors increased by 6 h. There was no evidence for the expression of the inhibitory VEGF-A165B variant in human skeletal muscle. Our study, reflecting both VEGF-A ligand and receptors, implicates metabolic perturbation as a regulator of human muscle angiogenesis and demonstrates that VEGF-A splice variants are distinctly regulated. Our findings also indicate that all three receptor genes exhibit different pretranslational regulation, in response to exercise in humans.

vascular endothelial growth factor; angiogenesis; ischemia

INCREASED MUSCLE CAPILLARY density is a well-established and functionally important adaptation, following a period of endurance training (26, 40, 46, 53). Many in vitro and in vivo studies have shown that, during angiogenesis, VEGF-A expression is associated both spatially and temporally with de novo capillarization (6, 31, 35), indicating that VEGF-A may be an important mediator of the aforementioned endurance training response. The single VEGF-A gene gives rise, by differential splicing, to at least six different mRNA species. In humans, these are named VEGF-A121, VEGF-A145, VEGF-A165, VEGF-A165B, VEGF-A189, and VEGF-A206 (3, 23, 31). Each mRNA codes for a distinct protein, and each protein induces vessel growth with different structural characteristics (6, 44), reflecting their distinct tissue localization and receptor-binding characteristics (6, 23, 31, 57). More recently, an inhibitor splice variant of VEGF-A165, named VEGF-A165b, was described (3, 8), increasing the complexity of VEGF-A signaling. VEGF-A165b inhibits VEGF-A165-mediated endothelial cell proliferation and migration (3, 8) and may represent a counterregulatory mechanism, limiting the rate of de novo muscle capillarization.

We were the first group to demonstrate that VEGF-A gene expression is increased within human skeletal muscle tissue in response to acute submaximal exercise (19) and that this was related to the degree of increase in hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) gene expression. This observation has been confirmed (15, 19, 21, 25, 26, 37), while it has been demonstrated that 8 wk of training attenuates the acute VEGF-A response to exercise in humans (38), and short-term exercise training increases baseline expression (18, 30). More recently, Gavin et al. (15) demonstrated that muscle VEGF121/VEGF165 protein expression is reduced immediately postexercise, while there is a simultaneous increase in VEGF-Atotal mRNA. Furthermore, VEGF121/VEGF165 protein increases in parallel with VEGF-Atotal mRNA over 10 days of training (18). However, none of these studies addressed the impact of exercise on VEGF-A gene splicing. We hypothesized that differential splicing may explain why the angiogenic response during muscle contraction is enhanced by a modest reduced oxygen delivery, as there appears to be no greater increase in total VEGF-A mRNA expression (19, 37). During the completion of the present study, Jensen et al. (26) published a study characterizing some of the VEGF-A splicing variants in human skeletal muscle, following 3 h of aerobic exercise. They did not, however, use specific primers for the individual splice variants, nor did they directly quantify VEGF-A121, despite arguing that VEGF-A121 was one of the most effective proangiogenic isoforms (26). They were able to demonstrate a statistically significant increase in VEGF-Atotal postexercise, somewhat reminiscent of Hiscock et al. (21), while changes in VEGF-A165 underscored the majority of the statistical significance.

The primary aim of our study was to establish a precise picture of VEGF-A gene splicing in human skeletal muscle, and thus we utilized an analysis strategy that quantified all of the detectable VEGF-A splice variants directly. Furthermore, given that VEGF-A121 gives rise to a highly diffusible form of VEGF-A, important for the recruitment of endothelial precursor cells (6, 8), we felt that it was critical to directly measure this variant. Restriction of blood flow to the exercising muscle augments the increase in exercise-induced angiogenesis (46, 53). This indicates that a reduction in oxygen tension and/or increased metabolic disturbance (e.g., altered ATP/ADP ratio or lower pH) provides a greater stimulus to exercise-induced angiogenesis (1, 46). Although complex and poorly understood, it is known that gene splicing can be influenced by metabolic stress and ischemia (45), and thus differential VEGF-A splicing may be the principal physiological mechanism for enhanced angiogenesis during exercise with low blood flow (46, 53). Given the somewhat heterogeneous nature of human skeletal muscle, we also examined, for the first time, the VEGF-A splice variant expression in pooled "type I" (slow and oxidative) and "type IIa + IIb" (mixture of fast oxidative and glycolytic) dissected human muscle fibers, as the metabolic responses in these fibers can differ during muscle contraction (11, 12).

Finally, three receptors have been described to bind VEGF-A, two of which, VEGF receptor (VEGFR)-1 [fms-like tyrosine kinase 1 (Flt-1)] and VEGFR-2 [kinase insert domain-containing receptor (KDR)], exhibit tyrosine kinase activity (31, 42). A third receptor, neuropilin-1 (NP-1), also binds VEGF-A (43), modulating the binding of VEGF-A165 to VEGFR-2, promoting enhanced VEGFR-2-induced angiogenic activity (43). A single bout of exercise in humans increases the expression of VEGFR-1 and VEGFR-2 in a manner distinct from that observed in rodents (15). As an alternative hypothesis, it is plausible that exercise with low blood flow differentially impacts on VEGFR expression, and this underpins the greater muscle angiogenesis. We, therefore, assessed, for the first time, the response of all three VEGFR genes following exercise with and without partially reduced blood flow (9, 46). The results of our study indicate that VEGFRs and VEGF-A splice variants respond in a manner sensitive to the degree of metabolic perturbation during exercise and that VEGF-A121, VEGF-A165, and VEGF-A189 demonstrate a distinct expression pattern with time, whereas VEGF-A145, VEGF-A165B, and VEGF-A206 are not present. Unlike rodents, VEGF-A mRNA responses do not appear to be fiber-type dependent, while the NP-1 mRNA expression response is sensitive to alterations in blood flow during exercise.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Subjects.   Nine healthy and physically active men participated in the study. Their average (range) age, height, and weight were 22 (18–26) yr, 180 (170–188) cm, and 74 (65–84) kg, respectively. Before the study, the experimental protocol was explained to all subjects, and informed consent was obtained. The Ethics Committee of the Karolinska Institutet approved the study.

Experimental protocol.   A method first described by Eiken and Bjurstedt (9) was used to restrict (R) blood flow during exercise. This model reduces leg blood flow during one-legged cycle exercise by 15–20% and augments the metabolic perturbation more than observed during exercise with nonrestricted (NR) blood flow, for any given absolute submaximal workload (46).

At least 1 wk before the first experiment, each subject was familiarized with the experimental procedures on two separate occasions. During familiarization, maximal one-legged knee-extension performance capacity was determined under normal NR blood flow conditions. The test began at a workload of 5 W, and workload increased by 5 W each minute until fatigue. Five subjects were selected at random to perform the first exercise bout under NR conditions, while the remaining four subjects performed the first exercise period under R blood flow conditions. Each leg was assigned randomly to either NR or R conditions. At least 10 days separated these two exercise sessions. On the day of the experiment, dynamic constant-load, knee-extension exercise (45 min, 60 rpm) was performed, as described previously (19). The relative workload used in all subsequent experiments was 26 ± 4% of the one-legged peak load in the NR exercise condition (10 ± 1 W). To establish differences between the two exercise conditions, heart rate was continuously recorded from the electrocardiogram by means of a linear beat-to-beat meter. Systolic arterial blood pressure (SAP) was determined by the cuff method while in the supine position, and local rating of perceived exertion (L-RPE) was obtained by using the Borg 6–20 RPE scale. Venous blood samples were obtained from a superficial vein before exercise and 15, 30, and 45 min after exercise for determination of lactate concentration by a fluorometric enzymatic procedure, as described earlier (19), and confirmed a comparable metabolic response to previous observations.

Muscle biopsies and RNA extraction.   Muscle biopsy samples were obtained from the vastus lateralis muscle before each of the two exercise bouts (with and without R blood flow) and 2 and 6 h after each exercise bout, using a percutaneous needle biopsy. Biopsy samples were frozen in liquid nitrogen within 10–15 s of sampling and stored at –80°C until analysis. Using biopsies obtained before and 2 h after exercise in the R condition (n = 6), ~400 single-fiber fragments were dissected from each freeze-dried sample (12). These were classified using histochemistry (ATPase staining) as either "type I" or "type II" fibers and pooled (i.e., ~200 type I and type II fibers) (12). Total RNA was prepared from frozen biopsy samples (n = 9 subjects, 3 biopsies in each leg, 2 legs/subject) and from freeze-dried fiber pools (n = 6 subjects, 2 biopsies/individual) by the acid phenol method (7). Total RNA concentration was measured using the optical density at 260 nm and quality controlled by visual inspection of the denaturing gels. Two micrograms of total RNA from each biopsy, or the entire RNA yield from each pool of dissected fibers, were reverse transcribed by Superscript RNase H reverse transcriptase (GIBCO, BRL) using random hexamer primers, according to the manufacturer’s specifications.

mRNA measurements.   To explore which major splice variants exist in human skeletal muscle tissue, PCR was run with primers located as shown in Table 1 and displayed in Fig. 1. Bands were cut out and sequenced by KIseq (Karolinska Institutet, Stockholm, Sweden) to confirm identify (Fig. 2). To establish if VEGF165b existed in human skeletal muscle tissue, we used the primers designed by Bates et al. (3) to detect exon 9-containing isoforms (now called exon 8b). For nested RT-PCR, VEGF-A forward (Table 1) and 3'-UTR primer (3) was used, followed by PCR with the exon 7a forward primer and exon 9 reverse primer. Two different Taq polymerases were used, Platinum Taq polymerase (Invitrogen) and Accuprime Taq DNA Polymerase High Fidelity (Invitrogen), that amplify nucleic acid templates using antibody-mediated hot-start, which is a blend of Taq DNA polymerase and a proofreading enzyme. The same PCR conditions were used as previously described (3). PCR products were run on 3% agarose gels (0.5 µg/ml ethidium bromide) and visualized in a UV transilluminator.


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  		Table 1. Oligonucleotide sequences for primer and probe pairs used in TaqMan analysis

 


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  		Fig. 1. The localization of primers and probes used for the measurements of total VEGF and for the specific isoforms VEGF-A121 (121), VEGF-A165 (165), and VEGF-A189 (189). For specific primer and probe sequences, see Table 1.

 


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  		Fig. 2. Top left: the PCR product from skeletal muscle biopsy obtained before (pre) and 2 h (2h) after 45-min exercise with restricted blood flow. The primers used were located in exons 4 and 8 to capture all splice variants. The band observed corresponded to the calculated size of VEGF-A121, VEGF-A165, and VEGF-A189. The bands were sequenced to further establish the origin of the different bands. The light band below the VEGF-A165 was sequenced on 4 occasions without any formal identification of a VEGF-A species. Top right: the PCR product (Pr) from skeletal muscle biopsies obtained with the designed specific primers for each splice variant. The calculated size of the product ranged between 75 and 125 bp. Each band was sequenced to ensure the specificity of the designed primers. Bottom left: no PCR product was obtained either before or 2 h after exercise with restricted blood flow when using the primers designed by Bates et al. (2) to detect exon 9-containing isoforms (now exon 8b). C, control.

 
Real-time qPCR was used to quantify mRNA (ABI-PRISMA 7700 Sequence Detector, Perkin-Elmer Applied Biosystems). Oligonucleotide primers and TaqMan probes were designed by using Primer Express version 1.5 (Perkin-Elmer Applied Biosystems). {beta}-Actin was used as an endogenous control to correct for potential variation in RNA loading or efficiency of the amplification procedure. Both the target and control gene were run at the same concentration, in the same well, thereby helping to control for dilution errors. To exclude potential changes in {beta}-actin expression, its expression was controlled using 18S, and it was found not to vary in this particular experiment. {beta}-Actin primers and probe were supplied as a TaqMan Reagents kit from Applied Biosystems with either a TAMRA quencher or Dark Quencher [part no. 4310881E (TAMRA), 4326315E (DQ)]. The primer pairs and probes used for total VEGF-A, VEGF-A121, VEGF-A165, VEGF-A189, VEGFR-1 (Flt-1), VEGFR-2 (KDR), and NP-1 are shown in Table 1. A schematic map for the primer and probe pairs used is shown in Fig. 1 and clarifies why our analysis ensures that we obtained isoform-specific measurements and how it differs from Jensen et al. (26) in that our probes spanned exon-exon boundaries to avoid genomic DNA contamination.

All reactions were performed in 96-well MicroAmp Optical plates. Amplification mixes (25 µl) contained the sample cDNA diluted twofold with TaqMan Universal PCR Mastermix and the primers and probe (Table 1). Before the PCR cycles, samples were incubated for 2 min at 50°C and for 10 min at 95°C. Thermal cycling consisted of 50 cycles at 95°C (15 s) and 65°C for 1 min. Control experiments revealed approximately equal efficiencies over different starting template concentrations for target genes and {beta}-actin (Fig. 3). For each subject, all samples were analyzed in one assay run. Relative quantification of the samples was carried out using dilution curves for each target gene analogous to a standard curve. The relative distribution of VEGF-A121, VEGF-A165, and VEGF-A189 was calculated for each individual. A threshold cycle ({Delta}Ct) value was obtained by subtracting {beta}-actin Ct values from respective target gene Ct values. VEGF121 was used as a reference and was subtracted from the VEGF165 and VEGF189 to derive a {Delta}-{Delta}Ct value. The relative expression of each isoform was then calculated by 2–{Delta}-{Delta}Ct (52).



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  		Fig. 3. TaqMan efficiency curves. Efficiency and dilution curves for each gene were analyzed in multiplex reactions together with {beta}-actin, VEGF-A, VEGF receptor (VEGFR)-1, VEGFR-2, neuropilin (NP)-1, VEGF-A121, VEGF-A165, and VEGF-A189. The x-axis represents the log cDNA concentration (which should reflect RNA concentration), and the y-axis represents threshold cycle (Ct) values.

 
Statistical analysis.   All values are expressed as means (SD), unless stated otherwise. Hemodynamic parameters, venous lactate, L-RPE, and mRNA levels were analyzed with a two-way ANOVA (time and exercise condition as the dependent factors). Fiber-type-specific expression of VEGF-A, VEGF-A121, VEGF165, and VEGF189 was analyzed using a Wilcoxon matched-pairs test. Because of a correlation for within-subject changes, the relative distribution of specific VEGF isoforms was evaluated using a one-way ANOVA (time). For post hoc analysis, planned comparisons were used to locate the points of interaction. For all tests, P < 0.05 was considered as significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Hemodynamic parameters, locally perceived exertion, and venous plasma lactate levels.   Exercise induced a similar increase in heart rate during both exercise conditions (NR: before exercise, 62 ± 8 beats/min; after 15-min exercise, 84 ± 15 beats/min; R: before exercise, 63 ± 6 beats/min; after 15-min exercise, 89 ± 13 beats/min). Exercise induced an increase in SAP and mean L-RPE, and both were significantly greater during the R exercise condition (SAP NR: before exercise, 127 ± 13 mmHg; after 15-min exercise, 140 ± 14 mmHg; R: before exercise, 126 ± 8 mmHg; after 15-min exercise, 156 ± 11 mmHg, P < 0.05; L-RPE NR: 15-min exercise, 10 ± 2; 30-min exercise, 12 ± 2; 45-min exercise, 12 ± 2; R: 15-min exercise, 16 ± 1; 30-min exercise, 16 ± 2; 45-min exercise, 17 ± 1; P < 0.05). The resting venous plasma lactate concentration did not differ at rest and increased at the onset of exercise only during exercise with R blood flow (Fig. 4).



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  		Fig. 4. Venous plasma lactate levels during exercise with nonrestricted and restricted blood flow. R-leg, exercise with restricted blood flow; NR-leg, exercise with nonrestricted blood flow; 15', 30', and 45': 15, 30, and 40 min. Values are presented as means (n = 9). {dagger}Significant differences between the two exercise conditions (P < 0.05).

 
VEGF-Atotal and VEGF-A isoform expression.   The splice variants VEGF-A121, VEGF-A165, and VEGF-A189 were routinely observed, whereas VEGF-A145 and VEGF-A206 were not detected. Moreover, we did not observe the expression of the VEGF-A165B splice variant in any human muscle sample (Fig. 2). Before exercise, there were no differences in VEGF-Atotal or the specific isoform distribution between legs (data not shown). At rest, the abundance of VEGF-A165 was significantly greater than VEGF-A121 and VEGF-A189. The percentage distribution of isoforms was 29 ± 9% for VEGF-A121, 41 ± 5% for VEGF-A165, and 30 ± 8% for VEGF-A189 (n = 9 for each). Following exercise with R blood flow, VEGF-Atotal and expression of the three isoforms increased 2 h after exercise and to a greater extent than with NR blood flow (Fig. 5). This particular data point is also reported in a concurrent analysis (2), where the biology of HIF-1{alpha} activation is being studied and where VEGF-A represents a HIF-1{alpha} target gene. An increase in the relative proportion of VEGF-A165 was observed 2 h after exercise, followed by an increase in the relative proportion of VEGF-A189 6 h after exercise (Fig. 6). The pattern of change was similar following exercise with or without R blood flow (Fig. 6).



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  		Fig. 5. mRNA levels of VEGFtotal, VEGF121, VEGF165, and VEGF189 after exercise with NR and R blood flow. mRNA quantitative analysis was performed by real-time PCR in human vastus lateralis muscle at rest and postexercise at 2 and 6 h. Values are presented as medians (n = 9). {dagger}Significant differences in total VEGF-A between the two exercise conditions (P < 0.05). {ddagger}{ddagger}Significant difference within the NR exercise condition, between 2 and 6 h postexercise (P < 0.05).

 


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  		Fig. 6. The percentage distribution of VEGF121, VEGF165, and VEGF189 isoforms, calculated using the 2–{Delta}-{Delta}Ct values (52), where VEGF121 was the reference from which the percentage distribution was calculated for each isoform. Measurements were made at rest and postexercise (2 and 6 h) with NR and R blood flow. Values are presented as medians (n = 9 data samples per data point). {dagger}Significant change preexercise vs. 2 h postexercise for VEGF165 (P < 0.05). {ddagger}Significantly increase from 2 to 6 h (P < 0.05) for VEGF189.

 
VEGF splice variants and muscle fiber type.   At rest, there were no differences in VEGF-Atotal or in the individual VEGF-A isoforms expressed in pooled dissected type I and II human muscle fibers (Fig. 7). With R blood flow, VEGF-Atotal and VEGF-A121, VEGF-A165, and VEGF-A189 increased to a similar extent in type I and type II fiber pool, 2 h postexercise (Fig. 8). The relative distribution of the VEGF isoform in type I or II fibers was not altered 2 h postexercise, compared with resting muscle tissue (data not shown).



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  		Fig. 7. The relative expression (distribution) of the detectable isoforms VEGF121, VEGF165, and VEGF189 in type I and II fibers dissected from preexercise human skeletal muscle biopsies. The relative expression was determined from the 2–{Delta}-{Delta}Ct data (52), where VEGF121 was the reference (type I and II fibers) from which the percentage distribution was calculated for each isoform. Values are presented as medians (n = 5).

 


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  		Fig. 8. Exercise-induced changes in type I and II fibers of the detectable VEGF isoforms VEGF121, VEGF165, and VEGF189 preexercise and at 2 h postexercise in the R blood flow condition only. Values are presented as medians (n = 5). {dagger}Significant change preexercise vs. 2 h postexercise (P < 0.05).

 
VEGF-R1, VEGF-R2, and NP-1 mRNA expression.   There was no difference in VEGF-R1, VEGF-R2, and NP-1 mRNA expression between legs at rest. VEGF-R1 mRNA increased 2 h after exercise with R blood flow (Fig. 9). VEGF-R1 and -R2 expression increased in response to exercise with NR blood flow only at the 6-h postexercise time point (Fig. 9). Both VEGFR-1 and VEGFR-2 increased to a greater extent 6 h after exercise with R than with NR blood flow (Fig. 9). The NP-1 receptor mRNA expression significantly decreased 2 h after exercise with NR condition, but had increased by 6 h compared with levels before exercise (Fig. 9). We specifically rechecked the housekeeping gene expression data for this observation, and NP-1 mRNA was reduced at 2 h, regardless of normalization. In contrast, there was no change in NP-1 receptor mRNA at 2 h following exercise with R condition. The increase in NP-1 mRNA expression 6 h after exercise was greater with R than with NR blood flow (Fig. 9).



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  		Fig. 9. mRNA levels of VEGFR-1, VEGFR-2, and NP-1 after exercise with NR and R blood flow. mRNA quantitative analysis was performed by real-time PCR in human vastus lateralis muscle at rest and postexercise at 2 and 6 h. Values are presented as medians (n = 9). {dagger}Significant differences between the two exercise conditions (P < 0.05) between preexercise and 2 h postexercise. {dagger}{dagger}Significant differences between the two exercise conditions (P < 0.05) between 2 and 6 h postexercise. {ddagger}Significant differences from preexercise (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In the present study, we demonstrate a number of novel observations regarding the interaction between human muscle contraction, blood flow, and VEGF-A-related gene expression. We also demonstrate that basal human VEGF-A mRNA expression is not fiber-type dependent, consistent with a previous study in rats (4). We demonstrate that VEGF-A splice variant abundance demonstrates a specific temporal pattern, following exercise, in humans. Most importantly, we conclude that, when one considers all available human data, VEGF-A gene expression is enhanced during submaximal exercise when combined with reduced oxygen delivery. Furthermore, regulation of VEGFR expression may also contribute to the greater skeletal muscle angiogenesis following endurance training with reduced oxygen delivery (34, 46, 53), as both NP-1 and VEGFR-1 demonstrated a distinct expression response during exercise with reduced blood flow.

VEGF-A splice variants in human skeletal muscle.   In resting human skeletal muscle, the VEGF-A165 isoform was most abundantly expressed (~40%), followed by VEGF-A121 (~25%) and VEGF-A189 (~30%), and this is similar to adult murine skeletal muscle (32). Furthermore, isoform abundance measured in mixed muscle corresponded to the isoform distribution measured in the dissected single fibers (Fig. 7). Our analysis indicates that VEGF-A splice variant expression is not influenced by human muscle phenotype, as defined by ATPase staining. We also demonstrated that VEGF-A165 represents the majority of the increased expression of VEGF-Atotal 2 h after submaximal exercise, while the variant coding for the less diffusible VEGF-A189 protein contributes more to the 6-h postexercise change. It may be hypothesized that this pattern of expression reflects an early endothelial activation followed by a later chemoattractant and differentiation role for VEGF-A. For example, isoforms containing the domain encoded by exon 6, such as VEGF-A189, are bound tightly to cell surface heparin-sulfate proteoglycans, whereas isoforms lacking this domain, such as VEGF-A121 and VEGF-A165, are more diffusible (23, 31). Although all isoforms contribute to the angiogenic process, VEGF-A165 has been regarded as the major variant promoting vascular growth (6, 44). Nevertheless, when we specifically measured changes in VEGF-A121 mRNA, we were able to demonstrate a significant increase following exercise, and thus changes in the lower abundance VEGF-A121 species may also be of functional importance.

The present results contrast somewhat with a recent report by Jensen et al. (26). In that study, VEGF165 splice variant appeared to explain all of the statistical increase in VEGF-A mRNA 2 and 6 h following endurance exercise. There are, however, some problems with the interpretation of this data (26). First, because of the design of the qPCR primers, the VEGF-A165 variant contributed to the amplicon signal in three out of four "splice variant" groups. This creates a statistical problem related to additional repeated measures, but also an analytical hurdle, in that changes in the higher abundance VEGF-A165 could have overshadowed alterations in the lower abundance VEGF-A121 and VEGF-A189 species. The authors argue that relative fold changes from the analysis "groups" could be used to deduce net changes in VEGF-A121. There is no evidence, given the relative proportion of the three detectable VEGF-A mRNA variants (Fig. 7), that this is a sensitive or validated method for detecting changes in VEGF-A121. Thus it may be argued that Jensen et al. (26) robustly confirmed what was known (19, 21, 37) and raised the possibility that such changes reflect mainly the VEGF-A165 splice variant. Therefore, our data represent the first detailed study of VEGF-A gene splicing in human skeletal muscle and indicate that all three detectable VEGF-A mRNA species contribute to the postexercise increase in VEGF-Atotal. Finally, it has been hypothesized that changes in an inhibitory splice variant may regulate tumor angiogenesis (3, 8). We were unable to find any evidence for the expression of the VEGF-A165b splice variant in human skeletal muscle, suggesting that VEGF-A165b is not a physiological regulator of muscle angiogenesis.

The postexercise changes in the relative distribution of the various VEGF-A isoforms (Fig. 6) did not differ between the two exercise conditions, implying that VEGF-A transcript splicing is not influenced by the degree of metabolic perturbation induced during contraction. The increase in VEGF-A mRNA expression following exercise with R blood flow was similar in type I and type II pools of dissected human muscle fibers. This is consistent with the described pattern of angiogenesis around type I and type II human muscle fibers following high-intensity endurance training (25). Our observation may indicate that the exercise-induced pretranslational regulation of VEGF-A expression is not influenced by the myocyte response to exercise (11). Alternatively, reduced blood flow can have a greater impact on type I fiber metabolism compared with type II fibers (17). This would ensure that the metabolic disturbance observed in both "fiber-type" populations was actually more similar, when blood flow was reduced. Thus, if metabolic factors influence VEGF-A gene expression and reduced blood flow "normalized" the metabolic responses between type I and type II, then it is not surprising that a similar increase in VEGF-A mRNA expression was observed. It can be concluded, therefore, that VEGF-A splice variants are equally abundant in human muscle fibers of varying phenotype (in resting tissue), but fiber-type-dependent responses, following submaximal exercise, require further investigation.

The stimulus for increased VEGF-A gene expression.   In previous human studies (19, 37), increased expression of VEGF-Atotal was similar during submaximal single-leg exercise with or without a modest reduction in normal oxygen delivery. We, therefore, hypothesized that regulation of VEGF-A gene splicing may be important for the greater muscle angiogenesis observed under such circumstances (34, 46). In the present study, and in contrast with previous work by Richardson et al. (37) and our own laboratory (19), increased VEGF-Atotal expression was more robust during exercise with reduced blood flow (Fig. 5). This new observation is more consistent with some (5) but not all rodent (49) studies from the Wagner laboratory. However, it is also clear that a great deal of intersubject variation (5, 19, 37) exists, and failure to detect an additive effect of reduced oxygen delivery with submaximal muscle contraction may have reflected this heterogeneity. It is worth emphasizing that a variety of detection methods and normalization procedures have produced both positive and neutral data in support of an additive effect of reduced oxygen delivery on contraction-induced increases in VEGF-A mRNA expression (5, 19, 37, 49). Thus methodological considerations are unlikely to explain these divergent observations. Because reduced flow per se did not alter VEGF-A mRNA splice variant expression, but did appear to result in a more robust VEGF-A mRNA response, we must reject one of our initial hypothesis.

The present data support a role for a link between reduced oxygen delivery and VEGF-A gene activation in exercising human skeletal muscle. In fact, HIF-1{alpha} protein, the major hypoxic transcriptional activator of VEGF-A (13), is increased concurrently with an increase in VEGF-A mRNA concentration, following acute and chronic skeletal muscle ischemia (39). Furthermore, we have recently established that HIF-1{alpha} protein expression and nuclear localization are enhanced during submaximal exercise (2) in the subjects from the present study. HIF-1{alpha} induces an increase in expression of the VEGF-A isoforms (10), supporting a role for HIF-1{alpha} in human skeletal muscle angiogenesis. However, we did not observe a greater HIF-1{alpha} response during submaximal exercise when combined with reduced blood flow (2), suggesting that HIF-1{alpha} activation does not explain the greater VEGF-A response.

A recent report in rat skeletal muscle (49) also provides support for this conclusion. In the study by Tang et al. (49), a combination of hypoxia and muscle electrical stimulation was used to examine the synergy between the aforementioned stimuli on VEGF-A and HIF-1{alpha} levels in rodent muscle. When all muscle fibers in the preparation were activated, no further increase in HIF-1{alpha} stabilization was noted with hypoxia. Unfortunately, the design of the study does not convincingly address the interaction between oxygen availability and HIF-1{alpha} stabilization during submaximal contraction, as fiber recruitment (by altering the voltage applied) was varied, rather than modifying the duty cycle to increase the work done within each fiber (See Ref. 50 for more discussion on this topic). Thus changes in muscle tissue gene expression reflected fiber recruitment (hence a "mixture" of active and inactive fibers) rather than the impact of oxygen tension on a contracting muscle fiber per se. Intracellular mechanisms associated with an increased stabilization of VEGF mRNA have also been reported in skeletal muscle tissue. For example, Hu protein R increases in ischemic rat skeletal muscle in parallel with an increase in VEGF-A mRNA levels (47). Hu protein R stabilizes VEGF-A mRNA in vitro by interacting with the VEGF-A-3' untranslated region (28). Clearly, the impact of reduced blood flow during exercise can influence VEGF-A mRNA expression by a variety of mechanisms.

VEGF-A-related receptor mRNA is regulated by exercise.   The angiogenic properties of VEGF-A are conveyed via activation of a promiscuous class of receptors. Three of these receptors have been described to bind VEGF-A, two of which, VEGFR-1 and VEGFR-2, exhibit tyrosine kinase activity (31, 42). A third receptor, NP-1, also binds VEGF-A (43), modulating the binding of VEGF-A165 to VEGFR-2, which then enhances receptor-induced angiogenic activity (43). Activation of VEGFR-1 and VEGFR-2 induces different biological effects (31, 42, 44). For example, homozygous knockout of any of the three receptor genes is embryonically lethal, yet each model demonstrates a distinct vascular pathology (42). Tang et al. (48) have recently demonstrated that, by targeted knock-down of VEGF-A expression, adult muscle capillarization is disrupted. This indicates that VEGF-A signaling, via VEGFR-1, VEGFR-2/NP-1 is important for baseline muscle angiogenesis in adult skeletal muscle.

The importance of increased VEGFR-1 and VEGFR-2 expression for exercise-induced angiogenesis has recently been explored in rat skeletal muscle (29). The study by Lloyd et al. (29) utilized the tyrosine kinase inhibitor ZD4190 to block VEGFR-2 activity in vivo during a period of vessel remodeling precipitated by exercise combined with arterial ligation. Unfortunately, the relevance of the data to the specific role of VEGF in skeletal muscle arteriogenesis and angiogenesis can be challenged, as ZD4190 is equipotent (20) at blocking endothelial cell proliferation induced by either VEGF or epidermal growth factor. Careful examination of the selectivity profile of ZD4190 (20) also highlights that true binding affinities are not determined, while the selectivity data reflect human rather than rat receptor proteins. Overall, it is difficult to conclude that the effects observed reflect only the VEGFR system.

Coexpression of the VEGF-A, NP-1, and VEGFR-2 receptors is important for an effective increase in tissue angiogenesis (24). In the present study, submaximal exercise induced an increase in all three receptors responsible for VEGF-A signaling (VEGFR-1, VEGFR-2, and NP-1) at the mRNA level. Furthermore, we demonstrate for the first time that the pattern of expression changes during submaximal exercise is influenced by reduced blood flow (Fig. 9). In rats, Gavin and Wagner (14) reported a bimodal increase in VEGFR-1 mRNA levels postexercise: the first peak occurred 1 h after, and a second peak 24 h, after a single bout of treadmill running. Gavin et al. (15) also demonstrated that VEGFR-1 and VEGFR-2 increased 4 h after a single bout of exercise in humans. The present data support their observations; however, our data also indicate that different stimuli regulate VEGFR-1 (Flt-1) and VEGFR-2 (KDR) in response to exercise. During exercise, with R blood flow, there was an increase in VEGFR-1 mRNA expression at 2 h, whereas there was no change in muscle VEGFR-2 or NP-1 mRNA content. Furthermore, during submaximal exercise with intact blood flow, NP-1 mRNA expression declined and then demonstrated a modest increase.

It is important to discuss why such a pattern of expression was observed. VEGFR-1 has, in contrast to VEGFR-2 and NP-1, a hypoxic response element in its promoter region (16, 42). The decrease in muscle oxygen tension during exercise is within the range known to activate such elements (27, 36). We can speculate that, in the present study, reduced oxygen tension in skeletal muscle tissue may have contributed to the more immediate increase in VEGFR-1 mRNA expression compared with VEGFR-2. Furthermore, although hypoxia induces VEGFR-2 expression in cell culture, this response reflects feedback regulation from increased levels of VEGF-A (41, 42). Similarly, VEGF-A-induced NP-1 mRNA expression has also been reported (33). In the present study, a significant increase in VEGFR-2 and NP-1 mRNA was only apparent 6 h after exercise, and this was preceded by the significant increase in VEGF-A mRNA 2 h postexercise. However, Gavin et al. (15) recently demonstrated that, following submaximal cycle exercise, there is a temporary decline in VEGF-A121/VEGF-A165 protein. This suggests that factors other than VEGF-A protein signaling may be responsible for the changes in VEGF-A-activated receptor mRNA observed (see Ref. 51). Alternatively, Höffner et al. (22) demonstrated that VEGF-A121/VEGF-A165 protein increases in skeletal muscle dialysate during one-legged knee-extension exercise, similar in nature to our exercise protocol. It is plausible to interpret their observation as evidence for a net loss of VEGF protein from the muscle (15), and this would be consistent with Hiscock et al. (21). Neither of these observations rules out the possibility that VEGF-A secretion into the interstitial fluid (22, 25) results in a paracrine-mediated promotion of VEFGR-2 and NP-1 gene expression. Although we are profiling regulation of these receptors at the mRNA level, it can be argued that the distinct temporal pattern of expression may influence the magnitude of the angiogenesis response, and hence this observation supports one of our main hypotheses.

In conclusion, a single bout of exercise in humans enhances the VEGF system at both the ligand and receptor mRNA level in skeletal muscle, whereas ischemia-induced metabolic perturbation enhances or alters this process. The relative distribution of VEGF-A splice variant expression is not influenced by metabolic perturbation over and above that observed with submaximal exercise alone, whereas there is a distinctive temporal pattern of splice variant expression following exercise that involves changes in VEGF-A165, VEGF-A121, and VEGF-A189 expression. Importantly, our findings suggest that VEGFR-1 exhibits different pretranslational regulation, in response to exercise in humans, compared with VEGFR-2 or NP-1.


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This work was supported by the Swedish Heart-Lung Association, the Swedish Medical Research Council (Vetenskapsrådet), and the Swedish National Center for Research in Sports (Centrum für Idrotts Forskning).


   ACKNOWLEDGMENTS
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 ABSTRACT
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We thank Laura Svensson and Kristina Miras de Gea for providing technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Timmons, Karolinska Institute, Berzelius Väg 35, Stockholm 171 77, Sweden (E-mail: Jamie.Timmons{at}cgb.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.


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  1. Adair TH, Gay WJ, and Montani JP. Growth regulation of the vascular system: evidence for a metabolic hypothesis. Am J Physiol Regul Integr Comp Physiol 259: R393–R404, 1990.[Abstract/Free Full Text]
  2. Ameln H, Gustafsson T, Sundberg CJ, Poellinger L, Jansson E, Okamoto K, and Makino Y. Physiological activation of hypoxia inducible factor-1 in human skeletal muscle. FASEB J In press.
  3. Bates DO, Cui TG, Doughty JM, Winkler M, Sugiono M, Shields JD, Peat D, Gillatt D, and Harper SJ. VEGF165b, an inhibitory splice variant of vascular endothelial growth factor, is down-regulated in renal cell carcinoma. Cancer Res 62: 4123–4131, 2002.[Abstract/Free Full Text]
  4. Birot OJ, Koulmann N, Peinnequin A, and Bigard XA. Exercise-induced expression of vascular endothelial growth factor mRNA in rat skeletal muscle is dependent on fibre type. J Physiol 552: 213–221, 2003.[Abstract/Free Full Text]
  5. Breen EC, Johnson EC, Wagner H, Tseng HM, Sung LA, and Wagner PD. Angiogenic growth factor mRNA responses in muscle to a single bout of exercise. J Appl Physiol 81: 355–361, 1996.[Abstract/Free Full Text]
  6. Carmeliet P, Ng YS, Nuyens D, Theilmeier G, Brusselmans K, Cornelissen I, Ehler E, Kakkar VV, Stalmans I, Mattot V, Perriard JC, Dewerchin M, Flameng W, Nagy A, Lupu F, Moons L, Collen D, D’Amore PA, and Shima DT. Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Nat Med 5: 495–502, 1999.[CrossRef][ISI][Medline]
  7. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[ISI][Medline]
  8. Cui TG, Foster RR, Saleem M, Mathieson PW, Gillatt DA, Bates DO, and Harper SJ. Differentiated human podocytes endogenously express an inhibitory isoform of vascular endothelial growth factor (VEGF165b) mRNA and protein. Am J Physiol Renal Physiol 286: F767–F773, 2004.[Abstract/Free Full Text]
  9. Eiken O and Bjurstedt H. Dynamic exercise in man as influenced by experimental restriction of blood flow in the working muscles. Acta Physiol Scand 131: 339–345, 1987.[ISI][Medline]
  10. Elson DA, Thurston G, Huang LE, Ginzinger DG, McDonald DM, Johnson RS, and Arbeit JM. Induction of hypervascularity without leakage or inflammation in transgenic mice overexpressing hypoxia-inducible factor 1 alpha. Genes Dev 15: 2520–2532, 2001.[Abstract/Free Full Text]
  11. Essen B and Haggmark T. Lactate concentration in type I and II muscle fibers during muscular contraction in man. Acta Physiol Scand 95: 344–346, 1975.[ISI][Medline]
  12. Essen B, Jansson E, Henriksson J, Taylor AW, and Saltin B. Metabolic characteristics of fiber types in human skeletal muscle. Acta Physiol Scand 95: 153–165, 1975.[ISI][Medline]
  13. Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, Koos RD, and Semenza GL. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 16: 4604–4613, 1996.[Abstract]
  14. Gavin TP and Wagner PD. Attenuation of the exercise-induced increase in skeletal muscle Flt-1 mRNA by nitric oxide synthase inhibition. Acta Physiol Scand 175: 201–209, 2002.[CrossRef][ISI][Medline]
  15. Gavin TP, Robinson CB, Yeager RC, England JA, Nifong LW, and Hickner RC. Angiogenic growth factor response to acute systemic exercise in human skeletal muscle. J Appl Physiol 96: 19–24, 2004.[Abstract/Free Full Text]
  16. Gerber HP, Condorelli F, Park J, and Ferrara N. Differential transcriptional regulation of the two vascular endothelial growth factor receptor genes. Flt-1, but not Flk-1/KDR, is up-regulated by hypoxia. J Biol Chem 272: 23659–23667, 1997.[Abstract/Free Full Text]
  17. Greenhaff PL, Soderlund K, Ren JM, and Hultman E. Energy metabolism in single human muscle fibers during intermittent contraction with occluded circulation. J Physiol 460: 443–453, 1993.[Abstract/Free Full Text]
  18. Gustafsson T, Knutsson A, Puntschart A, Kaijser L, Nordqvist AC, Sundberg CJ, and Jansson E. Increased expression of vascular endothelial growth factor in human skeletal muscle in response to short-term one-legged exercise training. Pflügers Arch 444: 752–759, 2002.[CrossRef][ISI][Medline]
  19. Gustafsson T, Puntschart A, Kaijser L, Jansson E, and Sundberg CJ. Exercise-induced expression of angiogenesis-related transcription and growth factors in human skeletal muscle. Am J Physiol Heart Circ Physiol 276: H679–H685, 1999.[Abstract/Free Full Text]
  20. Hennequin LF, Stokes ES, Thomas AP, Johnstone C, Ple PA, Ogilvie DJ, Dukes M, Wedge SR, Kendrew J, and Curwen JO. Novel 4-anilinoquinazolines with C-7 basic side chains: design and structure activity relationship of a series of potent, orally active, VEGF receptor tyrosine kinase inhibitors. J Med Chem 45: 1300–1312, 2002.[CrossRef][ISI][Medline]
  21. Hiscock N, Fischer CP, Pilegaard H, and Pedersen BK. Vascular endothelial growth factor mRNA expression and arteriovenous balance in response to prolonged, submaximal exercise in humans. Am J Physiol Heart Circ Physiol 285: H1759–H1763, 2003.[Abstract/Free Full Text]
  22. Höffner L, Nielsen JJ, Langberg H, and Hellsten Y. Exercise but not prostanoids enhance levels of vascular endothelial growth factor and other proliferative agents in human skeletal muscle interstitium. J Physiol 550: 217–225, 2003.[Abstract/Free Full Text]
  23. Houck KA, Ferrara N, Winer J, Cachianes G, Li B, and Leung DW. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol 5: 1806–1814, 1991.[Abstract]
  24. Ishida S, Shinoda K, Kawashima S, Oguchi Y, Okada Y, and Ikeda E. Coexpression of VEGF receptors VEGF-R2 and neuropilin-1 in proliferative diabetic retinopathy. Invest Ophthalmol Vis Sci 41: 1649–1656, 2000.[Abstract/Free Full Text]
  25. Jensen L, Bangsbo J, and Hellsten Y. Effect of high intensity training on capillarization and presence of angiogenic factors in human skeletal muscle. J Physiol 557: 571–582, 2004.[Abstract/Free Full Text]
  26. Jensen L, Pilegaard H, Neufer PD, and Hellsten Y. Effect of acute exercise and exercise training on VEGF splice variants in human skeletal muscle. Am J Physiol Regul Integr Comp Physiol 287: R397–R402, 2004.[Abstract/Free Full Text]
  27. Jiang BH, Semenza GL, Bauer C, and Marti HH. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. Am J Physiol Cell Physiol 271: C1172–C1180, 1996.[Abstract/Free Full Text]
  28. Levy NS, Chung S, Furneaux H, and Levy AP. Hypoxic stabilization of vascular endothelial growth factor mRNA by the RNA-binding protein HuR. J Biol Chem 273: 6417–6423, 1998.[Abstract/Free Full Text]
  29. Lloyd PG, Prior BM, Li H, Yang HT, and Terjung RL. VEGF receptor antagonism blocks arteriogenesis, but only partially inhibits angiogenesis, in skeletal muscle of exercise trained rats. Am J Physiol Heart Circ Physiol 288: H759–H768, 2005.[Abstract/Free Full Text]
  30. Lloyd PG, Prior BM, Yang HT, and Terjung RL. Angiogenic growth factor expression in rat skeletal muscle in response to exercise training. Am J Physiol Heart Circ Physiol 284: H1668–H1678, 2003.[Abstract/Free Full Text]
  31. Neufeld G, Cohen T, Gengrinovitch S, and Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 13: 9–22, 1999.[Abstract/Free Full Text]
  32. Ng YS, Rohan R, Sunday ME, Demello DE, and D’Amore PA. Differential expression of VEGF isoforms in mouse during development and in the adult. Dev Dyn 220: 112–121, 2001.[CrossRef][ISI][Medline]
  33. Oh H, Takagi H, Otani A, Koyama S, Kemmochi S, Uemura A, and Honda Y. Selective induction of neuropilin-1 by vascular endothelial growth factor (VEGF): a mechanism contributing to VEGF-induced angiogenesis. Proc Natl Acad Sci USA 99: 383–388, 2002.[Abstract/Free Full Text]
  34. Olfert IM, Breen EC, Mathieu-Costello O, and Wagner PD. Skeletal muscle capillarity and angiogenic mRNA levels after exercise training in normoxia and chronic hypoxia. J Appl Physiol 91: 1176–1184, 2001.[Abstract/Free Full Text]
  35. Park JE, Keller GA, and Ferrara N. The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol Biol Cell 4: 1317–1326, 1993.[Abstract]
  36. Richardson RS, Noyszewski EA, Kendrick KF, Leigh JS, and Wagner PD. Myoglobin O2 desaturation during exercise. Evidence of limited O2 transport. J Clin Invest 96: 1916–1926, 1995.[ISI][Medline]
  37. Richardson RS, Wagner H, Mudaliar SR, Henry R, Noyszewski EA, and Wagner PD. Human VEGF gene expression in skeletal muscle: effect of acute normoxic and hypoxic exercise. Am J Physiol Heart Circ Physiol 277: H2247–H2252, 1999.[Abstract/Free Full Text]
  38. Richardson RS, Wagner H, Mudaliar SR, Saucedo E, Henry R, and Wagner PD. Exercise adaptation attenuates VEGF gene expression in human skeletal muscle. Am J Physiol Heart Circ Physiol 279: H772–H778, 2000.[Abstract/Free Full Text]
  39. Rissanen TT, Vajanto I, Hiltunen MO, Rutanen J, Kettunen MI, Niemi M, Leppanen P, Turunen MP, Markkanen JE, Arve K, Alhava E, Kauppinen RA, and Yla-Herttuala S. Expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in ischemic skeletal muscle and its regeneration. Am J Pathol 160: 1393–1403, 2002.[Abstract/Free Full Text]
  40. Saltin B and Gollnick PD. Skeletal muscle adaptability: significance for metabolism and performance. In: Handbook of Physiology. Skeletal Muscle. Bethesda, MD: Am. Physiol. Soc., 1983, sect. 10, chapt. 19, p. 539–554.
  41. Shen BQ, Lee DY, Gerber HP, Keyt BA, Ferrara N, and Zioncheck TF. Homologous up-regulation of KDR/Flk-1 receptor expression by vascular endothelial growth factor in vitro. J Biol Chem 273: 29979–29985, 1998.[Abstract/Free Full Text]
  42. Shibuya M, Nobuyuki L, and Claesson-Welsh L. Structure and function of VEGF receptor-1 and 2. In: Vascular Growth Factors and Angiogenesis, edited by Claesson-Welsh L. Berlin: Springer-Verlag, 1999.
  43. Soker S, Takashima S, Miao HQ, Neufeld G, and Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 9: 735–745, 1998.
  44. Stalmans I, Ng YS, Rohan R, Fruttiger M, Bouche A, Yuce A, Fujisawa H, Hermans B, Shani M, Jansen S, Hicklin D, Anderson DJ, Gardiner T, Hammes HP, Moons L, Dewerchin M, Collen D, Carmeliet P, and D’Amore PA. Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms. J Clin Invest 109: 327–336, 2002.[CrossRef][ISI][Medline]
  45. Stamm S. Signals and their transduction pathways regulating alternative splicing: a new dimension of the human genome. Hum Mol Genet 11: 2409–2416, 2002.[Abstract/Free Full Text]
  46. Sundberg CJ. Exercise and training during graded leg ischaemia in healthy man with special reference to effects on skeletal muscle. Acta Physiol Scand 615, Suppl: 1–50, 1994.
  47. Tang K, Breen EC, and Wagner PD. Hu protein R-mediated posttranscriptional regulation of VEGF expression in rat gastrocnemius muscle. Am J Physiol Heart Circ Physiol 283: H1497–H1504, 2002.[Abstract/Free Full Text]
  48. Tang K, Breen EC, Gerber HP, Ferrara NM, and Wagner PD. Capillary regression in vascular endothelial growth factor-deficient skeletal muscle. Physiol Genomics 18: 63–69, 2004.[Abstract/Free Full Text]
  49. Tang K, Breen EC, Wagner H, Brutsaert TD, Gassmann M, and Wagner PD. HIF and VEGF relationships in response to hypoxia and sciatic nerve stimulation in rat gastrocnemius. Respir Physiol Neurobiol 144: 71–80, 2004.[CrossRef][ISI][Medline]
  50. Timmons JA, Constantin-Teodosiu D, Poucher SM, and Greenhaff PL. Acetyl group availability influences phosphocreatine degradation even during intense muscle contraction. J Physiol 561: 851–859, 2004.[Abstract/Free Full Text]
  51. Veikkola T, Karkkainen M, Claesson-Welsh L, and Alitalo K. Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res 60: 203–212, 2000.[Free Full Text]
  52. Winer J, Jung CK, Shackel I, and Williams PM. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 270: 41–49, 1999.[CrossRef][ISI][Medline]
  53. Yang HT, Ogilvie RW, and Terjung RL. Low-intensity training produces muscle adaptations in rats with femoral artery stenosis. J Appl Physiol 71: 1822–1829, 1991.[Abstract/Free Full Text]



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