| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1Copenhagen Muscle Research Centre, 2Rigshospitalet section 7652, Copenhagen, 3Department of Sport Science, Århus, Denmark, 4Department for Exercise and Sport Science, University of Copenhagen; and 5Centre of Inflammation and Metabolism, Department of Molecular Biology, University of Copenhagen, Denmark
Submitted 13 November 2007 ; accepted in final form 23 January 2008
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
angiogenesis; hypertrophy; vascular endothelial growth factor; rHuEpo; cancer; tumor
Interestingly, cell culture studies indicate that Epo-R is present in cardiomyocytes (35), suggesting that the Epo-R may also be present in skeletal muscle. Therefore, provided that Epo-R is present in skeletal muscle tissue, Epo may also be of importance for the regulation of angiogenesis and, hence, prolonged Epo treatment could result in an increased muscle capillarization.
Epo has been described as exerting effects similar to VEGF on the angiogenic process, and one of the mechanisms by which Epo appears to promote angiogenesis is by enhancing the level of VEGF in tissue. A close association between VEGF and Epo in angiogenesis has been proposed (1, 4) and Epo treatment has been found to enhance the release of VEGF from marrow stromal cells (37) and to increase levels of VEGF in brain (14, 32). Considering the importance of VEGF in skeletal muscle capillary growth (22), it is therefore plausible that one of the angiogenic effects of Epo is mediated by promoting VEGF levels in the muscle.
Another interesting potential physiological role of Epo in skeletal muscle is in muscle fiber growth. Epo-R activation stimulates the signal transducer and activator of transcription (STAT)5, which is known to modulate cell proliferation and differentiation (7). STAT5 also activates the PI3 kinase-Akt signaling pathway (24, 31), which is believed to result in activation of AKT and p70s6K, which in turn plays a role in transcription and cell-cycle progression. This pathway has been suggested to be critical in regulation of skeletal muscle hypertrophy (5, 29). On the basis of the above findings it appears plausible that Epo-R activation may contribute in regulating skeletal muscle fiber growth.
In addition, it may be speculated that Epo is regulating the expression of genes involved in iron homeostasis as recent findings demonstrate that 8 days of altitude exposure alters the level of several ion-regulating proteins including myoglobin (28).
In the present study we examined the presence and distribution of Epo-R in human skeletal muscle and we tested the hypothesis that prolonged treatment with rHuEpo in humans causes angiogenesis and hypertrophy in skeletal muscle. For this purpose eight subjects received rHuEpo for 14 wk where muscle biopsies were obtained before and after the treatment period. In addition, in another group of 11 subjects, the effect of acute injections of 15,000 IU rHuEpo on mRNA expression of genes related to angiogenesis and selected muscle characteristics in human skeletal muscle was investigated.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
Study A: Eight healthy male volunteers (age: 27 ± 7 yr; height: 180 ± 4 cm; weight: 83 ± 7 kg) participated in the study. The study was approved by the local ethical committee of the communities of Copenhagen and Frederiksberg (KF 01–269-637) and conformed to the Declaration of Helsinki. All subjects gave written informed consent to participate.
Study B: Eleven healthy male subjects [age: 25.5 ± 3.9 yr, height: 183.4 ± 4.8 cm, weight: 78.0 ± 11.7 (mean ± SD)] participated in the study. All subjects were informed about possible risks and discomfort involved before giving their written consent to participate. This study was carried out according to the Declaration of Helsinki and was approved by the ethical committee for the communities of Copenhagen and Frederiksberg (KF-01–291917).
Experimental Design
Study A: Long-term Epo administration.
EPO ADMINISTRATION:
Every injection contained a dose of 5,000 IU (
60 IU/kg body mass) of rHuEpo (NeoRecormon, Roche, Mannheim, Germany) in 0.3 ml saline. For the first 2 wk one injection was administered every second day; during the third week three injections were given on three consecutive days, and during weeks 4 to 15 one injection was given every week. From 2 wk prior to rHuEpo treatment, all subjects received 100 mg iron/day orally, and this was maintained throughout the entire study period. Blood samples were obtained throughout the study period to ensure that hematocrit was kept close to 50%. These data are presented in Table 1. Please see our companion paper (18) for full details on blood parameters.
|
Study B: Acute Epo administration.
The study was performed in a double-blind, placebo, crossover, experimental design, and nine of the subjects reported to the laboratory on two occasions, separated by at least 14 days, whereas two subjects only completed the Epo trial. From 2200 the day before the experiments, all subjects were asked to refrain from food and liquids (except water). They thus arrived overnight fasted at the laboratory the next morning at 0700, where they were served a light standardized breakfast adjusted for body weight and activity level (34). After consumption, the subjects were put in a hospital bed in the supine position where they remained until the last muscle biopsy had been taken. A 20-gauge venflon (BD Venflon, Sweden) was inserted in an antecubital vein for injection of either rHuEpo or saline as well as blood sampling. After 2 h of supine resting, blood was drawn and a muscle biopsy (Pre) was obtained using the Bergström technique with suction. The biopsy specimen was immediately transferred to liquid nitrogen, and thereafter kept in a –80°C freezer until time of analysis. After the Pre samples were obtained subjects received either 15,000 IU rHuEpo (200 IU/kg body mass; NeoRecormon, Roche) in 0.9 ml saline (Epo) or 0.9 ml iv saline (Con). Hereafter muscle biopsies were taken 2, 4, 6, and 10 h after the injection. After the 6 h biopsy, the subjects were served a sandwich (same on both study days). Venous blood samples were obtained at the same time points as the biopsies were taken.
Analyses
Immunohistochemical localization of Epo-R.
The cellular localization of Epo-R was determined on 8 µm transverese sections of frozen skeletal muscle samples. The sections were fixed in 2% formaldehyde for 2 min at room temperature and –20°C acetone for 30 s. The sections were rinsed with PBS containing 1% BSA and thereafter blocked with 1% PBS containing BSA. The sections were then incubated for 1 h at room temperature with a monoclonal antibody, mouse 
-Epo-R (25 µg/ml; MAB 307, R&D Systems, Abingdon, UK) for the detection of Epo-R. Primary monoclonal antibody, mouse -CD31 (50 µg/ml; Clone JC70A, DAKO A/S, Glostrup, Denmark) or Ulex Europeaus Agglutinin-1 (UEA-1; Sigma Aldrich) was used for detection of endothelial cells. All sections, with the exception of those incubated with UEA-1, were rinsed and thereafter incubated with either a biotin coupled goat -rabbit (E0432, DAKO A/S) or a biotin-coupled rabbit -mouse antibody (E0354; DAKO A/S). UEA-1 was directly coupled to TRITC. Antibody binding was visualized with either streptavidin coupled to FITC (F0422, DAKO A/S) or ABC complex with alkaline phosphatase (ABC/Complex/AP, DAKO A/S). Negative controls were achieved with staining without the primary antibody. Immunoreactive cells were examined and photographed in a Zeiss Axioplan Microscope.
Immunohistochemical analysis of MHC I, MHC IIa, and capillaries. The distribution of muscle fibers containing MHC I or MHC IIa was determined by immunohistochemistry with the use of the antibodies and anti-human MHC I (M8421, Sigma, MO) or MHC IIa (N2.261, Developmental Studies Hybridoma Bank, University of Iowa). Capillaries were identified with the use of CD-31 (50 µg/ml; Clone JC70A, DAKO A/S) and proliferating endothelial cells were visualized using an antibody to human Ki67 (10 ug/ml; BD Pharmingen, Cat no. 5560003). Positive binding was visualized as described for immunohistochemical localization of Epo-R. The number of MHC I, MHC IIa-positive fibers, and their respective fiber area, and the number of capillaries were determined using the software program Tema version 1.04 by CheckVision. From the pre rHuEpo treatment biopsy a total of 122 (range: 70–165) fibers were included for the analysis and 120 (range: 89–149) from the post rHuEpo specimen.
RNA isolation and reverse transcription. Total RNA was isolated from 20–25 mg of tissue. RNA was resuspended in 2 µl/mg original tissue in diethyl pyrocarbonate (DEPC)-treated H2O containing 0.1 mM EDTA. Reverse transcription (RT) of 2.9 µg total RNA of each sample was performed using the Superscript II RNase H– system with Oligo dT (Invitrogen, Carlsbad, CA). RT products were diluted in nuclease-free H2O to a total volume of 170 µl. For details see Ref. 21.
PCR. The mRNA content of selected genes was determined by fluorescence-based real-time PCR (ABI PRISM 7900 Sequence Detection System, Applied Biosystems). Forward (FP) and reverse (RP) primers and TaqMan probes were designed from human specific sequence data (Entrez-NIH and Ensembl, Sanger Institute) using computer software (Primer Express, Applied Biosystems). The oligo sequences and accession numbers are given in Table 1. The probes were 5',6-carboxyfluorescein (FAM) and 3',6-carboxy-N,N,N',N'-tetramethylrhodamine (TAMRA) labeled. Prior optimization was conducted determining optimal primer concentrations, probe concentration, and verifying the efficiency of the amplification. PCR amplification was performed (in triplicates) in a total reaction volume of 10 µl with 17 ng cDNA as previously described (17).
Determination of single-stranded cDNA content. The amount of single-stranded DNA (ssDNA) was determined in the RT samples using the OliGreen reagent (Molecular Probes). Samples were analyzed in a 96-well white microplate (Thermo Labsystems) with a total reaction volume of 200 µl in each well. Each sample was run in triplicate with 5 µl RNAse-treated cDNA sample, 95 µl TE, and 100 µl of OliGreen reagent in each well. For further analytical details please see Ref. 16.
Determination of plasma hormones. Venous blood was centrifuged at 2,465 g for 15 min and stored at –40°C until analyzed. The plasma concentration of insulin, testosterone, growth hormone, and cortisol were determined in duplicates by ELISA (Electra-Box Diagnostica, Tyresö, Sweden; IBL-Hamburg, Germany; BioSource, Nivelles, Belgium).
Statistical Analysis
In Study A, statistical differences were assessed by paired t-test, and in Study B a two-way ANOVA for repeated measures with Student Newman-Keuls post hoc test to locate differences was applied. Statistical difference was set to P < 0.05. All values reported are means ± SE if not stated otherwise.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
Staining for Epo-R was conducted with specific antibody that showed the receptor to be primarily localized in the region of capillaries, small arterioles, and venules as verified by colocalization of the Epo-R staining with CD-31 staining in serial sections and by double stain with UEA-1 on the same section as Epo-R (Fig. 1). Epo-R staining was also present in regions of the sarcolemma adjacent to endothelium as well as in some regions of the sarcolemma where no endothelial cells were located (Fig. 1). However, there was no continuous staining of the sarcolemma. Positive staining was also observed in the smooth muscle cells. Incubation of the rabbit Epo antibody with the matching peptide abolished the staining pattern and staining without primary antibody resulted in no visible staining.
|
Blood parameters. Hemoglobin concentration was 14.2 ± 0.6 before treatment and reached a peak of 17.1 ± 0.5 g/dl after 12 wk of rHuEpo administration (P < 0.05 from weeks 7 to 15). The hematocrit values followed a similar pattern, being increased from 42 ± 3 to 49 ± 3% at week 12 (P < 0.05 from weeks 6 to 15; Table 2).
|
|
Study B
Hormones. The single Epo injection had no effect on the plasma levels of either insulin, testosterone, cortisol, or growth hormone within 10 h after injection.
Content of mRNA. The Epo injection reduced the mRNA content of myoglobin to 68% of the prelevel 10 h after injection, the transferrin receptor mRNA content increased twofold 2 h after Epo injection, but had returned to pre levels already at the 4 h biopsy. In addition, Epo-R levels tended to change over time in the Epo group, and MRF4 mRNA levels were significantly elevated 6 h after the injection (Fig. 2). VEGF, HIF-1a, IGF-IEa, ferroportin, MyoD, and myogen were all unaffected at the mRNA level at the investigated time points following Epo injection.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
mRNA levels in skeletal muscle, and even 14 wk of rHuEpo treatment did not increase angiogenic activity or muscle fiber hypertrophy. Combined these findings suggest that Epo administration may have effects within the human skeletal muscle, but that Epo alone cannot initiate angiogenesis or cause muscle fiber hypertrophy in the muscle tissue during basal conditions. Until recently, the role for Epo was thought to be strictly related to erythropoiesis. However, within the last few years, new and very exciting functions of Epo have emerged (6). The current demonstration that the Epo-R is present in the sarcolemma as well as in endothelial and smooth muscle cells in human skeletal muscle tissue supports the notion that Epo may also be involved in physiological processes in skeletal muscle. Within the skeletal muscle, three effects of Epo-R activation could be of interest: angiogenesis, hypertrophy, and iron homeostasis in human skeletal muscle.
In a previous study, we showed that altitude-induced erythropoiesis is associated with decreases in skeletal muscle transferrin receptor, ferritin, and myoglobin after 7–9 days of exposure to 4,559 m (27). On the basis of the decreased mRNA levels of myoglobin after 10 h of elevated rHuEpo in the present study, it may be speculated that the altitude-associated effects can be attributed directly to circulating Epo, whereas the transient increase in transferrin-receptor mRNA in the current study does not directly support that Epo mediates the altitude-induced decrease in this protein. In agreement with an effect on iron homeostasis-regulating proteins is a recent observation that prolonged rHuEpo treatment decreases muscle L-ferritin in healthy humans (G. Cairo, unpublished data). This combined would suggest that there is a functional role of Epo-R within the skeletal muscle.
The scientific evidence supporting a promoting role for Epo in angiogenesis in tissue undergoing growth or repair is quite clear (6). The Epo-R has been localized on endothelial cells in vivo and in vitro (2), and the activation of Epo-R on these cells has shown a response including mitogenesis, chemotaxis, and endothelin-1 release. Among the first investigations regarding the proliferation of vessels by Epo stimulation was the study by Anagnostou et al. (2). These investigators demonstrated a dose-dependent increase in proliferation of umbilical vein endothelial cells with rHuEpo added to the medium. In line with this, it was recently made clear that rHuEpo stimulates both the early invasive phase of the angiogenic process that leads to endothelial sprouting, as well as the late differentiation, and that VEGF and Epo amplify each others' activities (25). The present observation that 14 wk of rHuEpo infusion did not induce capillarization is therefore not in agreement with the many previous observations on a role for Epo in angiogenesis. Nevertheless, the present observation does not exclude a role for Epo in promoting capillary growth in skeletal muscle, considering the fact that angiogenesis is a complex process involving many compounds. Thus it may be suggested that Epo cannot induce capillarization on its own when the tissue is in a normal maintenance state, as other angiogenic compounds need to be present in sufficient amounts concomitantly with Epo. Previous studies demonstrating a role of Epo have all used either cell culture or models where the tissue is undergoing growth or repair and in which other angiogenic compounds necessary for capillary growth are likely to be present in an enhanced concentration relative to during maintenance. Thus it could be speculated that rHuEpo injections would be more effective in promoting angiogenesis if infused in combination with a growth factor such as VEGF, considering the proposed close association between these two factors (1, 4, 9).
On the basis of the fact that several previous studies have shown that Epo treatment can induce an enhanced expression of VEGF in different tissues (14, 32, 37), we hypothesized that Epo would promote the potential for angiogenesis in skeletal muscle tissue by increasing the content of VEGF mRNA. However, this single Epo injection did not affect VEGF or HIF-1 mRNA levels in muscle biopsies obtained from 2 to 10 h after injection, thus the proposition that Epo would enhance VEGF gene expression in skeletal muscle was not supported. Previous experiments from our laboratory have shown that VEGF and HIF-1 are rather easily induced genes that are increased between 1 and 6 h after a single endurance exercise bout (13, 15). Thus the timing of the obtained biopsies in the present study corresponds well with these previously observed induction patterns of both VEGF and HIF-1 mRNA and suggest that the lack of VEGF and HIF-1 mRNA responses was not a result of inadequate sampling. It rather appears that Epo may not be a stimulator of VEGF or HIF-1 gene expression in basal skeletal muscle tissue, unlike in other tissues (14).
It should be noted that the skeletal muscle is know as a tissue with a high level of plasticity. With endurance type of training for instance, it is well documented that the fiber composition is altered from IIb to IIa, and with strength training hypertrophy is universally observed, whereas inactivity studies, such as confinement to bed rest for weeks, clearly show a decrease in the cross-sectional area of the muscle fibers (30). The rHuEpo treatment regimen had the expected outcome on blood variables and was effective in increasing hematocrit from 42 to 49% (Table 1), which was the greatest increase possible considering the cardiovascular risks involved at high hematocrit levels. On the basis of the effects at the red blood cell level, it appears likely that the Epo dose given should have been sufficient to induce physiological effects. Nevertheless, as no effects were observed on capillarization or the muscle ultrastructure it cannot be excluded that the lack of effect on the muscle tissue was a consequence of a lower receptor density or a lower receptor sensitivity on the muscle cells compared with on erythrocyte progenitor cells, therefore requiring greater concentrations of Epo. In support of this possibility is that a single injection of Epo resulting in very high plasma Epo concentrations did increase MRF4 mRNA content in the hours after injection, suggesting a role in muscle fiber differentiation or growth as resistance exercise has been shown to increase MRF4 mRNA in human skeletal muscle as well (23).
In conclusion, the Epo-R is located in the sarcolemma and the vascular cells in muscle tissue, suggesting a physiological role of Epo in skeletal muscle myocytes as well as in the blood vessels. A single Epo injection regulates the mRNA expression of myoglobin, transferrin receptor, Epo-R, and MRF4, but prolonged Epo administration does not promote angiogenesis or stimulate muscle fiber growth in nongrowing skeletal muscle tissue. This indicates that Epo may contribute directly in regulating iron homeostasis, whereas an effect of Epo on capillary growth and ultrastructure may require concomitant stimuli from other angiogenic factors or tissue already undergoing angiogenesis.
|
GRANTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
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.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  H. Rundqvist, E. Rullman, C. J. Sundberg, H. Fischer, K. Eisleitner, M. Stahlberg, P. Sundblad, E. Jansson, and T. Gustafsson Activation of the erythropoietin receptor in human skeletal muscle Eur. J. Endocrinol., September 1, 2009; 161(3): 427 - 434. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Robach, S. Recalcati, D. Girelli, C. Gelfi, N. J. Aachmann-Andersen, J. J. Thomsen, A. M. Norgaard, A. Alberghini, N. Campostrini, A. Castagna, et al. Alterations of systemic and muscle iron metabolism in human subjects treated with low-dose recombinant erythropoietin Blood, June 25, 2009; 113(26): 6707 - 6715. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Lundby Reply to Boning, Maassen, and Pries J Appl Physiol, December 1, 2008; 105(6): 1989 - 1989. [Full Text] [PDF]
|
|
|
|
|
|
C. Lundby, P. Robach, R. Boushel, J. J. Thomsen, P. Rasmussen, M. Koskolou, and J. A. L. Calbet Does recombinant human Epo increase exercise capacity by means other than augmenting oxygen transport? J Appl Physiol, August 1, 2008; 105(2): 581 - 587. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Lundby, N. J. Achman-Andersen, J. J. Thomsen, A. M. Norgaard, and P. Robach Testing for recombinant human erythropoietin in urine: problems associated with current anti-doping testing J Appl Physiol, August 1, 2008; 105(2): 417 - 419. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
