George P. Livanos Laboratory, Critical Care Department and Pulmonary Services, Evangelismos Hospital, University of Athens, Athens 10675, Greece
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ABSTRACT |
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Vascular endothelial growth factor (VEGF) is a potent
angiogenic stimulus, the expression of which increases in skeletal
muscle after exercise. Because exercise is also accompanied by
increased intramuscular reactive oxygen species (ROS) generation, we
tested the hypothesis that ROS stimulate VEGF production from skeletal myotubes. Differentiated C2C12 skeletal
myotubes exposed to ROS-producing agents exhibited a
concentration-dependent increase in VEGF production, whereas
undifferentiated myoblasts did not respond to oxidants. Moreover,
conditioned medium from ROS-treated myotubes increased the bovine lung
microvascular cell proliferation rate. To study the mechanism(s)
involved in the stimulation of VEGF production by ROS, myotubes were
pretreated with a selective phosphatidylinositol 3-kinase (PI3K)
inhibitor, LY-294002, before being exposed to hydrogen peroxide or
pyrogallol. LY-294002 attenuated both Akt phosphorylation and VEGF
production. In addition, oxidants increased nuclear
factor-B-dependent promoter activity in transiently transfected myotubes; however, pretreatment with the pharmacological inhibitor of
nuclear factor-
B, diethyldithiocarbamate, did not affect the oxidant-stimulated VEGF release. We conclude that ROS induce VEGF release from myotubes via a PI3K/Akt-dependent pathway.
exercise; vascular endothelial growth factor; phosphatidylinositol 3-kinase; protein kinase B
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INTRODUCTION |
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SKELETAL MUSCLE
RESPONDS to intense exercise by altering both its structural and
biochemical properties (27). It has been observed that
exercise training leads to increased capillary density and
capillary-to-fiber ratio (6). The increase in the
vascularity of exercised skeletal muscle is thought to be an adaptive
response that allows for greater blood flow, O2 transport,
and metabolic clearance to support higher workloads. In line with these
observations, the mRNA levels of angiogenic growth factors such as
vascular endothelial growth factor (VEGF) and transforming growth
factor-1 are significantly higher in skeletal muscle homogenates
after intense exercise (5, 16, 31) and might contribute to
the angiogenic response of skeletal muscle tissue to exercise.
VEGF is a potent endothelial cell mitogen that is known to play a key role in both vasculogenesis and angiogenesis (12). Targeted disruption of either VEGF or VEGF receptor loci leads to a lethal phenotype associated with a lack of endothelial cells or an inability of the endothelium to form vascular structures during development (8, 13, 36). Adenovirus-mediated gene transfer of VEGF or administration of the recombinant protein stimulates neovascularization in vivo (24, 29). In addition, neutralizing antibodies and soluble receptors that neutralize the action of VEGF inhibit tumor growth (20, 21). Various stimuli including decreased oxygen tension and increased nitric oxide and prostaglandin production, all of which are encountered during exercise, have been implicated in the increased VEGF mRNA expression (3, 12). In vitro studies (9, 22) have demonstrated increased VEGF secretion from endothelial and epithelial cells on oxidative stress; however, the mechanism underlying this response remains largely unknown.
The lipid kinase phosphatidylinositol 3-kinase (PI3K), as well as its downstream target Akt, has been implicated in a number of cellular responses linked to angiogenesis, including endothelial cell migration and survival (19, 30, 39). Overexpression of constitutively active PI3K or Akt promotes angiogenesis in vivo and increases VEGF expression in avian endothelial cells (19). Recently, Ushio-Fukai et al. (38) showed that hydrogen peroxide (H2O2) activates the PI3K/Akt pathway in vascular smooth muscle cells. In the present study, we utilized an in vitro system of C2C12 skeletal myotubes to examine the ability of reactive oxygen species (ROS) to stimulate VEGF production. We show that ROS stimulate VEGF release from skeletal myotubes and that activation of the PI3K/Akt signaling pathway is necessary for the ROS-induced VEGF release.
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MATERIALS AND METHODS |
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Reagents and cell culture.
Tissue culture plates were obtained from Nalgen Nunc International.
Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS),
antibiotics, and LipofectAMINE were obtained from GIBCO BRL (Life
Technologies, Paisley, UK). The ELISA kit for murine VEGF
(VEGF164 and VEGF120) was purchased from R&D
Systems (Minneapolis, MN). The luciferase reporter gene assay was
purchased from Boehringer Mannheim (Mannheim, Germany); pNF-B
and pTAL were obtained from Clontech Laboratories. Bradford dye and
nitrocellulose membranes were obtained from Bio-Rad (Hercules, CA); the
enhanced chemiluminescence Western blotting analysis system was
purchased from Amersham Life Science. The antibodies for Akt were
obtained from New England Biolabs (Beverly, MA). The Myo D antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other
reagents including horse serum, xanthine/xanthine oxidase (X/XO),
pyrogallol (Pyr), H2O2, actinomycin D,
cycloheximide, LY-294002, and diethyldithiocarbamate (DETC) were
obtained from Sigma (St. Louis, MO).
VEGF measurements.
C2C12 cells were cultured for 2 days in
24-multiwell clusters until they reached confluence and were then
allowed to differentiate into myotubes for 48 h. Myotubes were
incubated with Pyr, X/XO, or H2O2 with and
without pretreatment with either LY-294002 (a PI3K inhibitor), DETC [a
pharmacological inhibitor of nuclear factor (NF)-B], actinomycin D
(an inhibitor of transcription), or cycloheximide (an inhibitor of
protein synthesis). After 24 h, the supernatants were collected
and centrifuged for 5 min at 6,000 rpm in a tabletop microcentrifuge to
remove floating cells. After centrifugation, the pellets were
discarded, and the supernatants were used for ELISA in accordance with
the manufacturer's instructions. C2C12 cell
monolayers in the multiwell plates were lysed with 1 N NaOH. Protein
amounts per well were determined by the Bradford method and used to
normalize the values obtained for cytokine release.
Bovine lung microvascular endothelial cell proliferation. C2C12 myotubes were exposed to H2O2 (300 µM) or Pyr (500 µM) and incubated for 24 h. Bovine lung microvascular endothelial cells (BLMVECs) were seeded in six-well plates (15 × 104 cells/well) and allowed to attach overnight. BLMVECs were then serum starved for 18 h and exposed for 48 h to conditioned medium (2 ml/well) from ROS-treated myotubes. Cells were then trypsinized, centrifuged at 1,500 rpm for 5 min, and counted with a hemacytometer.
Transfections.
C2C12 myoblasts were plated in six-well plates
at a density of 2 × 104/cm2 and allowed
to reach 40-60% confluence. Cells were transfected with vector
alone (pTAL) or a plasmid containing the luciferase coding sequence
under the control of six B sites (pNF-
B). Transfections were
performed with LipofectAMINE in a DNA-to-lipid ratio of 2.5 µg
plasmid DNA/9 µl lipid. When the transfected cells reached confluence, the growth medium was replaced by differentiation medium
for 48 h. The myotubes were then exposed to ROS and after 24 h were lysed and assayed for luciferase activity. To normalize for
transfection efficiency, we cotransfected a plasmid coding for lacZ
(under the control of an SV40 promoter) with either pTAL or pNF-
B.
-Galactosidase activity was measured with standard methods.
Western blotting. C2C12 cells were cultured in six-well plates, pretreated, and lysed in lysis buffer (1% Nonidet P-40, 50 mM NaCl, 0.1% SDS, 50 mM NaF, 1 mM Na3VO4, 50 mM Tris · HCl, 0.1 mM EGTA, 0.5% deoxycholic acid, 1 mM EDTA, 10 µg/ml of aprotinin, 10 µg/ml of leupeptin, and 1 mM phenylmethylsulfonyl fluoride). The cell lysates were rocked for 30 min at 4°C followed by a brief centrifugation at 14,000 rpm. Sample aliquots (50 µg/lane) were electrophoresed on 7.5% (or 6% for Myo D experiments) SDS-polyacrylamide gels and transferred to a nitrocellulose membrane at 20 V overnight at 4°C in a buffer containing 25 mM Tris and 700 mM glycine. The membranes were subsequently incubated for 2 h at room temperature with 5% dry milk in buffer containing 0.1% (vol/vol) Tween 20 in Tris-buffered saline (TBS-T). The following day, the membranes were incubated with the primary antibody in TBS-T containing 1% milk for 2 h at room temperature and then washed three times with TBS-T for 20 min each time. Finally, the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody and washed again two times with TBS-T and once with TBS. Immunoreactive protein bands were visualized with the enhanced chemiluminescence system.
Data analysis and statistics. Data are presented as means ± SE of the indicated number of observations. VEGF values are expressed as picomoles per milligram of protein or as a percentage of the control value. Statistical comparisons between groups were performed with one-way ANOVA followed by a post hoc test or a Student's t-test as appropriate. Differences among means were considered significant when P < 0.05.
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RESULTS |
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Effects of ROS-producing agents on VEGF production by myoblasts and
myotubes.
To test whether ROS-producing agents can stimulate VEGF release from
skeletal myoblasts and myotubes, C2C12 cells
were exposed to either X/XO or Pyr. Differentiation of myoblasts into
myotubes was confirmed both by light microscopy (appearance of
elongated, multinucleated cells that fuse) and by the increased
expression of the myogenic basic helix-loop-helix
transcription factor Myo D (Fig.
1A). Skeletal
myoblasts exhibited higher basal release of VEGF compared with myotubes
(0.38 ± 0.1 vs. 0.15 ± 0.02 pg/mg protein) but did not
respond by increasing VEGF release when exposed to Pyr or X/XO (Fig.
1B). On the other hand, exposure of myotubes to Pyr or X/XO
led to a 2.7- or 2-fold increase, respectively, in VEGF release. The
effects of Pyr, X/XO, and H2O2 on VEGF
production were concentration dependent, showing a maximal response at
500 µM, 500 µM/50 mU, and 300 µM, respectively (Fig.
2). When C2C12 cells were incubated with 300 µM Pyr or 500 µM
H2O2, we noticed a reduction in cell number
(52.8 and 39.8% with Pyr and H2O2, respectively). Moreover, cultures exposed to 250 µM Pyr, 165 µM/25 mU of X/XO, or 500 µM H2O2 for 24 h had
41.5, 31.2, and 12.2% less protein, respectively, than control cells.
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Effect of conditioned medium from ROS-stimulated myotubes on
endothelial cell proliferation.
To investigate whether treatment of C2C12
myotubes with ROS results in the production of factors that promote
endothelial cell proliferation, myotubes were treated with vehicle,
H2O2, or Pyr for 24 h, and conditioned
medium was collected. As shown in Fig. 3,
conditioned medium from H2O2- or
Pyr-treated myotubes stimulated BLMVEC growth over a 48-h period,
suggesting that myotube-conditioned medium contained biologically
active growth factors in addition to the immunologically
active VEGF. It should be noted that incubation of BLMVECs with 300 µM H2O2 or 500 µM Pyr reduced the
proliferation of serum-starved cultures stimulated by 10%
FCS-containing medium (data not shown).
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Mechanism(s) of ROS-induced VEGF production.
To investigate whether the ROS-induced increase in VEGF release is the
result of increased transcription or translation, skeletal myotubes
were pretreated for 2 h with either actinomycin D or cycloheximide
and then incubated with H2O2 or X/XO.
Pretreatment with actinomycin D or cycloheximide completely abolished
the H2O2- and X/XO-induced VEGF increase (Fig.
4), suggesting that ROS stimulate VEGF
production via a transcription-dependent mechanism.
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DISCUSSION |
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The aim of this study was to investigate whether ROS are able to stimulate VEGF production from skeletal myocytes and to dissect the mechanisms involved in this response. Our major findings are that 1) ROS increase VEGF production from skeletal myotubes but not from myoblasts in a concentration-dependent manner, 2) conditioned medium from ROS-treated myotubes contains biologically active growth factors that stimulate the growth of endothelial cells, 3) ROS-stimulated VEGF production is transcription dependent, 4) ROS activate the PI3K/Akt signaling pathway in myotubes, and 5) pharmacological inhibition of PI3K inhibits the ROS-induced VEGF release.
Strenuous exercise is accompanied by increased generation of ROS
intramuscularly. Several mechanisms have been proposed to contribute to
this increase, including disruption of the mitochondrial respiratory
chain, increased levels of enzymes such as XO and NADPH oxidase,
leukocyte infiltration, and an insufficiency of antioxidant defense
mechanisms (10, 17, 32). In addition, exercise or chronic
motor nerve stimulation of skeletal muscle is associated with increased
gene expression of VEGF and transforming growth factor-1, suggesting
that release of these factors might contribute to the increase in
capillary density associated with exercise training (1, 5, 16,
31). Recent evidence (15) shows that inhibition of
nitric oxide release during exercise attenuates the increase in
steady-state VEGF mRNA by 50%. However, the source of VEGF in the
exercised muscle remains to be elucidated. In situ hybridization data
(5) revealed the presence of increased mRNA for VEGF in
skeletal muscle fibers after exercise, suggesting that myocytes might
be the source of this growth factor. In the present study, we show that
murine C2C12 myoblasts and myotubes produced
VEGF under basal conditions, with myoblasts producing greater amounts
of this growth factor. However, unlike myoblasts, differentiated
skeletal myotubes exhibited an increase in the release of VEGF when
exposed to ROS-producing agents. This observation is in agreement with
the finding that VEGF expression is increased in vascular endothelial
cells, retinal pigment epithelial cells, and keratinocytes when exposed
to H2O2 and that VEGF expression in
atherosclerotic lesions correlates with the generation of ROS (4,
9, 22, 34). In addition, VEGF is elevated in the bronchoalveolar
lavage fluid of cancer patients receiving radiotherapy and tumor
chemotherapy, both of which promote oxidative stress (2).
Consistent with the role of ROS in promoting angiogenesis is the
observation that ROS scavengers inhibit differentiation of endothelial
cells in vitro and that antioxidants such as dimethylthiourea, catalase, and allopurinol attenuate reperfusion-induced VEGF expression in vivo (22, 23). The differential effect of
ROS-generating agents on VEGF release from myoblasts versus myotubes
might reflect differences in redox status, suggesting that increased
antioxidant mechanisms exist in myoblasts. Alternatively,
differentiation of myoblasts into myotubes might induce the expression
of critical signaling components required for ROS-stimulated VEGF expression.
Most growth factors that promote angiogenesis, including VEGF, are endothelial cell mitogens. To assess whether ROS-treated C2C12 cells secrete biologically active growth factors in addition to immunoreactive VEGF, conditioned medium was collected, and its ability to stimulate endothelial cell growth was determined. Indeed, BLMVECs exposed to conditioned medium from ROS-stimulated C2C12 cells exhibited an increased proliferation rate; however, the identity of the growth-promoting factor remains to be elucidated.
Free radicals in excess amounts are damaging to cells, leading to lipid peroxidation, DNA damage, protein dysfunction, and apoptosis (18). ROS can also act as signaling molecules, mediating cellular responses to vasoactive peptides, growth factors, and inflammatory stimuli, leading to phosphorylation events and activation of transcription factors, thus controlling gene expression (35). VEGF expression has been shown to be regulated both at the level of transcription and through changes in mRNA stability. For example, exposure of human epithelial cells to X/XO prolonged the VEGF mRNA half-life without affecting the rate of transcription (22). Exposure of C2C12 myotubes to actinomycin D suppressed the oxidant-stimulated VEGF production, suggesting that ROS-induced VEGF release from myotubes is associated with increased transcriptional activity.
To identify the signaling pathways mediating the ROS-induced VEGF release, we tested the ability of ROS to activate the PI3K/Akt pathway. Activation of this pathway has been documented to play a role in the increased VEGF promoter activity induced by hypoxia (28). In addition, expression of constitutively active PI3K or Akt is associated with an increased steady state of VEGF mRNA levels in endothelial cells and fibroblasts (19). Incubation of C2C12 myotubes with H2O2 or Pyr promoted Akt phosphorylation on Ser473 in an LY-294002-sensitive manner, suggesting that these ROS-producing agents activate PI3K. This is in agreement with the observation that H2O2 stimulates the PI3K/Akt pathway in fibroblasts and epithelial cells (40). It should be noted that Akt phosphorylation in response to ROS-producing agents was also observed in myoblasts (data not shown). Because PI3K and Akt are expressed in both myoblasts and myotubes, with the latter in greater amounts in myotubes (14), the signaling molecule responsible for the differential effects of ROS on VEGF release in myotubes versus myoblasts must be downstream from Akt. More importantly, PI3K inhibition also blocked H2O2- and Pyr-induced VEGF production, suggesting that this pathway plays an important role in the response of C2C12 cells to ROS. The difference in the efficacy of LY-294002 in inhibiting the Pyr- and H2O2-induced VEGF release might be attributed to the different ROS generated from the two agents in terms of both species and amount. Chua et al. (9) have provided a link between PKC activation and increased steady-state VEGF mRNA levels in endothelial cells. Because cross talk between PKC and PI3K pathways has been described, the possibility exists that the PI3K pathway is also involved in the increased VEGF expression by endothelial cells in response to H2O2.
Several transcription factors have been identified as downstream
targets of Akt. These include the forkhead family member Daf-16, E2F,
cAMP response element binding protein, and NF-B (7, 11,
33). Akt was recently shown to activate I
B kinase, thus
increasing I
B phosphorylation and degradation and upregulating NF-
B-mediated gene expression (33). Moreover, putative
B sites are located 90 and 185 bp upstream from the initiation site
of the mouse promoter (37). Treatment of
C2C12 cells with ROS increased NF-
B-dependent promoter activity in skeletal myotubes transiently transfected with plasmids coding for the luciferase reporter gene; this
effect was almost completely inhibited by DETC, a pharmacological inhibitor of NF-
B. This finding is in line with observations that
ROS-producing agents cause activation and nuclear translocation of
NF-
B in several cell types, including skeletal myocytes (25, 26). In contrast, DETC failed to inhibit the ROS-stimulated VEGF
release from skeletal myotubes; however, we cannot exclude the
possibility that the small degree of NF-
B activation in the presence
of DETC is sufficient to drive VEGF production. Alternatively, other
transcription factors such as activator protein (AP)-1 and AP-2 might
play a role in the ROS-induced VEGF release. Several consensus
sequences for transactivating AP-1 and AP-2 complexes are present in
the murine VEGF promoter, and various stimuli known to activate these
transcription factors also increase VEGF gene expression
(37). In our experiments, AP-1-dependent promoter activity
was increased in response to Pyr in myotubes (data not shown);
moreover, oxidative stress has been shown to upregulate c-fos and c-jun expression (35).
In conclusion, we have shown that the predominant cell in skeletal muscle tissue, the myocyte, can be a cellular source of angiogenic growth factors and that oxidative stress is a potential stimulus for the angiogenic response of skeletal muscle. In addition, we have provided a link between activation of the PI3K/Akt pathway and VEGF production in response to ROS. These results may provide an insight into the regulation of growth factor expression in skeletal muscle tissue in conditions such as exercise and ischemia-reperfusion that are associated with increased free radical production.
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ACKNOWLEDGEMENTS |
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We acknowledge the expert technical assistance of Athanasia Hatzianastasiou.
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FOOTNOTES |
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This study was supported by a grant from the Greek Secretariat of Research and Technology and by the Thorax Foundation.
Address for reprint requests and other correspondence: A. Papapetropoulos, George P. Livanos Laboratory, Ploutarchou 3, 5th floor, Athens 10675, Greece (E-mail: andreaspap{at}altavista.net).
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.
Received 28 July 2000; accepted in final form 30 October 2000.
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