Ras Induction of Superoxide Activates ERK-dependent Angiogenic Transcription Factor HIF-1α and VEGF-A Expression in Shock Wave-stimulated Osteoblasts
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m308013200
ISSN1083-351X
AutoresFeng‐Sheng Wang, Ching‐Jen Wang, Yeung-Jen Chen, Per-Rong Chang, Yu‐Ting Huang, Yi‐Chih Sun, Hueng-Chen Huang, Ya‐Ju Yang, Kuender D. Yang,
Tópico(s)Heat shock proteins research
ResumoVascular endothelial growth factor (VEGF) released by osteoblasts plays an important role in angiogenesis and endochondral ossification during bone formation. In animal studies, we have reported that shock waves (SW) can promote osteogenic differentiation of mesenchymal stem cells through superoxide-mediated signal transduction (Wang, F. S., Wang, C. J., Sheen-Chen, S. M., Kuo, Y. R., Chen, R. F., and Yang, K. D. (2002) J. Biol. Chem. 277, 10931–10937) and vascularization of the bone-tendon junction. Here, we found that SW elevation of VEGF-A expression in human osteoblasts to be mediated by Ras-induced superoxide and ERK-dependent HIF-1α activation. SW treatment (0.16 mJ/mm2, 1 Hz, 500 impulses) rapidly activated Ras protein (15 min) and Rac1 protein (30 min) and increased superoxide production in 30 min and VEGF mRNA expression in 6 h. Early scavenging of superoxide, but not nitric oxide, peroxide hydrogen, or prostaglandin E2, reduced SW-augmented VEGF-A levels. Inhibition of superoxide production by diphenyliodonium, an NADPH oxidase inhibitor, was found to suppress VEGF-A expression. Transfection of osteoblasts with a dominant negative (S17N) Ras mutant abrogated the SW enhancement of Rac1 activation, superoxide synthesis, and VEGF expression. Further studies demonstrated that SW significantly promoted ERK activation in 1 h and HIF-1α phosphorylation and HIF-1α binding to VEGF promoter in 3 h. In support of the observation that superoxide mediated the SW-induced ERK activation and HIF-1α transactivation, we further demonstrated that scavenging of superoxide by superoxide dismutase and inhibition of ERK activity by PD98059 decreased HIF-1α activation and VEGF-A levels. Moreover, culture medium harvested from SW-treated osteoblasts increased vessel number of chick chorioallantoic membrane. Superoxide dismutase pretreatment and anti-VEGF-A antibody neutralization reduced the promoting effect of conditioned medium on angiogenesis. Thus, modulation of redox reaction by SW may have some positive effect on angiogenesis during bone regeneration. Vascular endothelial growth factor (VEGF) released by osteoblasts plays an important role in angiogenesis and endochondral ossification during bone formation. In animal studies, we have reported that shock waves (SW) can promote osteogenic differentiation of mesenchymal stem cells through superoxide-mediated signal transduction (Wang, F. S., Wang, C. J., Sheen-Chen, S. M., Kuo, Y. R., Chen, R. F., and Yang, K. D. (2002) J. Biol. Chem. 277, 10931–10937) and vascularization of the bone-tendon junction. Here, we found that SW elevation of VEGF-A expression in human osteoblasts to be mediated by Ras-induced superoxide and ERK-dependent HIF-1α activation. SW treatment (0.16 mJ/mm2, 1 Hz, 500 impulses) rapidly activated Ras protein (15 min) and Rac1 protein (30 min) and increased superoxide production in 30 min and VEGF mRNA expression in 6 h. Early scavenging of superoxide, but not nitric oxide, peroxide hydrogen, or prostaglandin E2, reduced SW-augmented VEGF-A levels. Inhibition of superoxide production by diphenyliodonium, an NADPH oxidase inhibitor, was found to suppress VEGF-A expression. Transfection of osteoblasts with a dominant negative (S17N) Ras mutant abrogated the SW enhancement of Rac1 activation, superoxide synthesis, and VEGF expression. Further studies demonstrated that SW significantly promoted ERK activation in 1 h and HIF-1α phosphorylation and HIF-1α binding to VEGF promoter in 3 h. In support of the observation that superoxide mediated the SW-induced ERK activation and HIF-1α transactivation, we further demonstrated that scavenging of superoxide by superoxide dismutase and inhibition of ERK activity by PD98059 decreased HIF-1α activation and VEGF-A levels. Moreover, culture medium harvested from SW-treated osteoblasts increased vessel number of chick chorioallantoic membrane. Superoxide dismutase pretreatment and anti-VEGF-A antibody neutralization reduced the promoting effect of conditioned medium on angiogenesis. Thus, modulation of redox reaction by SW may have some positive effect on angiogenesis during bone regeneration. Angiogenesis is an essential component of skeletal development, and vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial growth factor; SW, shock wave; ERK, extracellular signal-regulated kinase; HIF-1α, hypoxia inducible factor-1α; CAM, chick chorioallantoic membrane; SOD, superoxide dismutase; l-NAME, N-nitro-l-arginine methyl ester; DPI, diphenyliodonium; RT-PCR, reverse transcription PCR; Ras, Rous sarcoma kinase; Raf-1, Rous sarcoma-associated factor-1; PEG, polyethylene glycol; ELISA, enzyme-linked immunosorbent assay; BMP, bone morphogenetic protein; TGF, transforming growth factor; IGF, insulin-like growth factor. signaling plays an important role in this process (1Deckers M.M. Karperien M. van der Bent C. Yamshita T. Papapoulos S.E. Lowik C.W.G.M. Endocrinology. 2000; 141: 1667-1674Crossref PubMed Scopus (396) Google Scholar). VEGF is secreted in four biologically active isoforms that arise from alternative splicing of the VEGF primary transcription. VEGF-A is the most abundant of the four isoforms and is commonly used in studies investigating the biological effects of VEGF (2Ferrara N.K. Houck L. Jakeman L. Leung D.W. Endocr. Rev. 1992; 13: 18-32Crossref PubMed Scopus (1554) Google Scholar, 3Thomas K.A. J. Biol. Chem. 1996; 271: 603-606Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar). Exogenous VEGF was found to enhance blood vessel formation, ossification, and new bone (callus) maturation in mouse femur fractures (4Street J. Bao M. deGuzman L. Bunting S. Peale Jr., F.V. Ferrara N. Steinmetz H. Hoeffel J. Cleland J.L. Daugherty A. van Bruggen N. Redmond H.P. Carano R.A. Filvaroff E.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9656-9661Crossref PubMed Scopus (1165) Google Scholar). Targeted deletion of VEGF gene caused skeletal defect in mice by impairing angiogenesis and endochondral bone formation (5Maes C. Carmeliet P. Moermans K. Stockmans I. Smets N. Collen D. Bouillon R. Carmeliet G. Mech. Dev. 2002; 111: 61-73Crossref PubMed Scopus (358) Google Scholar). During bone formation, there is a cross-talk between endothelial cells and osteoblasts. VEGF was reported to induce endothelial cell migration, proliferation, and capillary permeability of vascularization process in endochondral bone (6Ferguson C. Alpern E. Miclau T. Helms J.A. Mech. Dev. 1999; 87: 57-66Crossref PubMed Scopus (444) Google Scholar). These findings indicate that osteoblast-derived VEGF in the bone microenvironment has an important role in angiogenic activities during bone formation. Bone regeneration and blood vessel formation of fractured callus can be promoted by physical modalities (7Aroson J. Shen X.C. Skinner R.A. Hogue W.R. Badger T.M. Lumpkin C.K. J. Orthop. Res. 1997; 15: 221-226Crossref PubMed Scopus (77) Google Scholar, 8Azuma Y. Ito M. Harada Y. Takagi H. Ohta T. Jingushi S. J. Bone Miner. Res. 2001; 16: 671-680Crossref PubMed Scopus (296) Google Scholar). Acoustic energy and pressure released by shock waves (SW) have been shown to have a positive effect on fracture healing and tendon repair (9Romp J.D. Rosendahl T. Schollner C. Theis C. Clin. Orthop. 2001; 387: 102-111Crossref PubMed Scopus (136) Google Scholar, 10Wang C.J. Chen H.S. Chen C.E. Yang K.D. Clin. Orthop. 2001; 387: 95-101Crossref PubMed Scopus (195) Google Scholar, 11Loew M. Daecke W. Kusnierczak D. Rahmanzadeh M. Ewerbeck V. J. Bone Jt. Surg. Br. 1999; 81: 863-867Crossref PubMed Scopus (245) Google Scholar), and mechanical stimulation has been found to raise adaptive modeling response of bone microenvironment via induction of anabolic molecules (12Burger E.H. Klein-Nulend J. FASEB J. 1999; 13: S102-S112Google Scholar, 13Salter D.M. Wallace W.H.B. Robb J.E. Caldwell H. Wright M.O J. Bone Miner. Res. 2001; 15: 1746-1755Crossref Scopus (46) Google Scholar). Previous studies have demonstrated that SW can stimulate bone marrow mesenchymal stem cell differentiation into osteoprogenitors, which has been associated with increases in osteogenic factor expression (15Rivilis I. Milkiewicz M. Boyd P. Goldstein J. Brown M.D. Egginton S. Hansen F.M. Hudlicka O. Haas T.L. Am. J. Physiol. 2002; 283: H1430-H1438Crossref PubMed Scopus (150) Google Scholar). Moreover, several cell types have been reported to respond to mechanical stimulation by elevating VEGF mediation of angiogenic responses (15Rivilis I. Milkiewicz M. Boyd P. Goldstein J. Brown M.D. Egginton S. Hansen F.M. Hudlicka O. Haas T.L. Am. J. Physiol. 2002; 283: H1430-H1438Crossref PubMed Scopus (150) Google Scholar, 16Shay-Salit A. Shushy M. Wolfovitz E. Yahav H. Breviario F. Dejana E. Resnick N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9462-9467Crossref PubMed Scopus (267) Google Scholar). In animal studies, we have also demonstrated that SW treatment can induce neovascularization of tendon-bone junction and may be associated with increases in VEGF-A expression (17Wang C.J. Wang F.S. Yang K.D. Huang C.S. Hsu C.C. Yang L.C. J. Orthop. Res. 2003; 21: 984-989Crossref PubMed Scopus (497) Google Scholar). However, the exact molecular mechanism by which SW promotes angiogenesis has remained undetermined. Hypoxia-inducible factor-1 (HIF-1), a heterodimeric basic helix-loop-helix transcription factor, acts as a critical regulator of VEGF expression. Active HIF-1 has been found to accumulate in the cell nucleus, bind to the target DNA sequence, and enhance hypoxia-inducible gene transcription (18Guillemin K. Krasnow M.A. Cell. 1997; 89: 9-12Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Evidence has suggested that reactive oxygen radicals mediate hypoxia, arsenite, and vanadate activation of HIF-1-dependent VEGF expression (19Duyndam M.C. Hulscher T.M. Fontijn D. Pinedo H.M. Boven E. J. Biol. Chem. 2001; 276: 48066-48076Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 20Gao N. Ding M. Zheng J.Z. Zhang Z. Leonard S.S. Liu K.J. Shi X. Jiang B.H. J. Biol. Chem. 2002; 277: 31963-31971Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). One of our recent studies has shown that superoxide mediated SW-promoted extracellular signal-regulated kinase (ERK) activation and mesenchymal stem cell differentiation into osteogenic lineage (21Wang F.S. Wang C.J. Sheen-Chen S.M. Kuo Y.R. Chen R.F. Yang K.D. J. Biol. Chem. 2002; 277: 10931-10937Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). This finding implies that induction of reactive oxygen molecules by SW potentially may provoke intracellular signaling transduction, which in turn may activate angiogenic activity of bone cells. We hypothesize that SW induce the reactive oxygen radical as a mediator in the activation of intracellular signal transduction and angiogenic transcription. The purposes of this study were to examine the effect of SW on the angiogenic response of human osteoblasts and to investigate whether SW promotion of angiogenic activity can be linked to the induction of reactive oxygen radicals, the activation of HIF-1α, and the promotion of VEGF-A production. Cell Culture—Human fetal preosteoblastic cells (CRL-11372, American Type Culture Center, Manassas, VA) were maintained in a mixture of phenol red-free Ham's F12 medium and Dulbecco's modified Eagle's medium (1:1) containing 10% fetal bovine serum and 2.5 mml-glutamine (Invitrogen) in a 5% CO2, 34 °C incubator for 6 days. Cells were harvested by trypsinization and resuspended in medium for further studies. Cell viability was determined using trypan blue exclusion. Shock Wave Treatment—Cells (1 × 106 cells/dish, 35-mm Petri dishes) were cultured for 48 h and subjected to SW treatment using OssaTron® SW equipment (HMT High Medical Technologies, Kreuzlingen, Switzerland) as described previously (14Wang F.S. Wang C.J. Huang H.C. Chung H. Chen R.F. Yang K.D. Biochem. Biophys. Res. Commun. 2001; 287: 648-655Crossref PubMed Scopus (159) Google Scholar). Briefly, culture dishes were floated in a thermostastically controlled water bath. A sterile SW probe was immersed vertically into each culture dish and placed to just touch the surface of the medium. The distance between the probe and the cells was ∼5–6 mm. Cells were exposed to a single SW treatment at 0.16 mJ/mm2 energy flux density, 1 Hz, 500 impulse. This SW energy enhances osteogenic differentiation of mesenchymal stem cells (14Wang F.S. Wang C.J. Huang H.C. Chung H. Chen R.F. Yang K.D. Biochem. Biophys. Res. Commun. 2001; 287: 648-655Crossref PubMed Scopus (159) Google Scholar). Control samples were prepared in the same manner except that they were not exposed to SW. Cells were harvested at 15 min, 30 min and 1, 3, 6, 12, and 24 h after SW treatment for RT-PCT and immunoblotting. To verify whether SW-augmented VEGF expression was regulated by osteogenic factors, SW-treated osteoblasts were co-cultured with or without 10 ng/ml monoclonal antibodies against BMP-2, TGF-β1, and IGF-I (R&D Systems) for 24 h, respectively. To investigate the role of bioactive radical in SW promotion of VEGF expression, subconfluent cell cultures were pretreated with or without 500 units/ml polyethylene glycol (PEG)-coupled bovine erythrocyte superoxide dismutase (SOD) and PEG-catalase (Sigma) to scavenge superoxide and hydrogen peroxide. To determine whether prostaglandin E2 was involved in the SW-enhanced VEGF expression, cells were pretreated with or without 10 μm indomethacin to suppress PGE2 production by inhibiting cyclooxygenase-2 activity. To elucidate the role of nitric oxide in the SW-promoted VEGF expression, osteoblasts were pretreated with or without 100 μm N-nitro-l-arginine methyl ester (l-NAME, Sigma) to inhibit nitric oxide production. To differentiate which oxidase was responsible for SW-induced superoxide production, osteoblasts were pretreated with 30 μm DPI (an NADPH oxidase inhibitor), 50 μm allopurinol (a xanthine oxidase inhibitor) or 50 μm rotenone (a mitochondrial oxidase inhibitor; Sigma). In some experiments, subconfluent cell cultures were pretreated with 20 μm PD98059 (Calbiochem) to inhibit ERK activity for 4 h before SW treatment. RT-PCR—Total RNA was extracted and purified from 106 cells with and without SW treatment using Tri-reagent (Sigma). One microgram of total RNA was reverse transcribed into cDNA followed by PCR amplification using human gene-specific primers: VEGF-A, forward, 5′-TTA TAC CGG GAT TTC TTG CG-3′), and reverse, 5′-CCC ACT GAG GAG TCC AAC AT-3′ (209 base pair expected); β-actin, forward, 5′-CGC CAA CCG CGA GAA GAT-3′, β-actin reverse, 5′-CGT CAC CGG AGT CCA TCA-3′) (168 base pair expected). The parameters for RT-PCR cycling were set as described previously (14Wang F.S. Wang C.J. Huang H.C. Chung H. Chen R.F. Yang K.D. Biochem. Biophys. Res. Commun. 2001; 287: 648-655Crossref PubMed Scopus (159) Google Scholar). All signals were quantified by scan densitometry, and the final value was obtained by calculating the VEGF-A/β-actin ration value. The -fold of promotion was calculated as the increase over the value of its corresponding control sample. Determination of Superoxide Production—Superoxide production by cell cultures with or without SW was determined using a horse heart cytochrome c reduction assay in the absence and presence of SOD and calculated from the molar extinction coefficient of 0.0282 μm–1 cm–1 as described previously (21Wang F.S. Wang C.J. Sheen-Chen S.M. Kuo Y.R. Chen R.F. Yang K.D. J. Biol. Chem. 2002; 277: 10931-10937Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Measurement of VEGF-A Production in the Culture Supernatants— The VEGF-A levels of culture supernatant were determined using ELISA kits (Quantikine®, R&D Systems) according to manufacturer's instructions. Results were calculated using an interpolation determined from a standard curve made by a series of VEGF-A concentrations. Transient Transfection of a Dominant Negative Ras Plasmid— cDNAs encoding wild type and mutant (S17N) H-Ras proteins (DN-Ras) were ligated and cloned into pUSE vectors (Upstate Biotechnology, Lake Placid, NY), respectively. Stable transfection and selection were accomplished according to previously described methods (22Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar). Briefly, osteoblasts (5 × 105 cells/well, 6-well plate) were plated to reach 60–80% confluence. Cells were transfected with 5 μg of wild type Ras and DN-Ras mutant cDNA plasmid (14Wang F.S. Wang C.J. Huang H.C. Chung H. Chen R.F. Yang K.D. Biochem. Biophys. Res. Commun. 2001; 287: 648-655Crossref PubMed Scopus (159) Google Scholar) using FuGENE™ 6 transfection reagent (Roche Diagnostics) according to manufacturer's instructions. Cells stably transfected with the plasmid were selected in medium containing 600 μg/ml G418 (Invitrogen). Cytosolic and Nuclear Extracts—Cytosolic and nuclear extracts of cell cultures were prepared as described previously (21Wang F.S. Wang C.J. Sheen-Chen S.M. Kuo Y.R. Chen R.F. Yang K.D. J. Biol. Chem. 2002; 277: 10931-10937Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Briefly, cytosolic extracts were harvested by lysing cells with buffer containing 10 mm Tris-HCl (pH 7.9), 10 mm KCl, 2 mm MgCl2, 0.1 mm EDTA, and 0.7% Nonidet P-40 for 10 min at 4 °C and centrifuged at 500 × g for 5 min, and then supernatants were harvested. Pellets were further lysed with buffer containing 40 mm Tris-HCl (pH 7.9), 350 mm NaCl, 2 mm MgCl2, 1 mm EDTA, 0.2 mm EGTA, 20% glycerol, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 2 μm dithiothreitol, 2 μg/ml leupeptin, and 1 μg/ml aprotinin for 20 min at 4 °C, and supernatants were harvested after centrifugation at 12,000 × g at 4 °C for 10 min. Protein concentrations in cytosolic and nuclear extracts were determined using a Bio-Rad assay kit. Measurement of Ras and Rac1 Activation by Raf-1 and PAK-1 Agarose Conjugate—Ras and Rac1 activation were determined using Ras and Rac1 activation assay kits (Upstate Biotechnology). Briefly, cell lysates were precleared with glutathione-agarose followed by incubation with specific Raf-1 Ras binding domain and PAK-1 p21 binding domain agarose conjugates, respectively. The immunoprecipitates were reacted with Laemmli buffer containing 200 mm Tris, pH 6.8, 10% glycerol, 4% SDS, 50 μm dithiothreitol, and 0.05% bromphenol blue for 5 min at 95 °C. The mixtures were subject to Western blot assay. Activated Ras and Rac1 proteins on the blot were recognized by a mouse anti-Ras and anti-Rac1 antibodies (Upstate Biotechnology) followed by goat anti-mouse horseradish peroxidase-conjugated IgG as the second antibody. The activated Ras protein and Rac1 levels were visualized using chemiluminescence agents (SuperSignal®, Pierce) (14Wang F.S. Wang C.J. Huang H.C. Chung H. Chen R.F. Yang K.D. Biochem. Biophys. Res. Commun. 2001; 287: 648-655Crossref PubMed Scopus (159) Google Scholar). Determination of ERK, p38, and HIF-1α Phosphorylation—Cytosolic and nuclear extracts (500 μg) were reacted with anti-ERK, anti-p38 antibodies (Upstate Biotechnology), and HIF-1α antibodies (Santa Cruz Biotechnology) and were precipitated with protein A (Sigma), respectively. Immunoprecipitates (20 μg) were subjected to Western blot assay (21Wang F.S. Wang C.J. Sheen-Chen S.M. Kuo Y.R. Chen R.F. Yang K.D. J. Biol. Chem. 2002; 277: 10931-10937Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Total ERK, p38, and HIF-1α on the blots were recognized by anti-ERK, anti-p38, and HIF-1α antibodies followed by horseradish peroxidase-conjugated IgG as the second antibody and were visualized with chemiluminescence agents. The phosphorylated ERK, p38, and HIF-1α were recognized by stripping the membrane in a buffer containing 62.5 mm Tris-HCl (pH 6.7), 2% SDS, and 100 mm mercaptoethanol for 30 min at 50 °C and then reprobed with mouse anti-phospho-ERK, anti-phospho-p38, and anti-phosphotyrosine antibodies (Upstate Biotechnology), respectively, using a similar procedure. Electrophoretic Mobility Shift Assay—A HIF-1 consensus VEGF-A promoter (underlined) oligonucleotides probe (5′-CCA CAG CAT ACG TGG GCT CCA ACA-3′, 3′-GGT GTC GTA TGC ACC CGA GGT TGT-5′) was 5′ end-labeled with γ-32P using T4 polynucleotide kinase (New England Biolabs Inc.) and [γ-32P]ATP (3000 Ci/mmol at 10 mCi/ml; Amersham Biosciences). Nuclear extracts (10 μg) were incubated with a binding buffer containing 10 mm HEPES (pH 7.9) 1 mm dithiothreitol, 1 mm EDTA, 80 mm KCl, 20% glycerol, and 0.25 mg/ml poly(dI-dC) (Amersham Biosciences) and 1.75 pmol of γ-32P-labeled oligonucleotide probe (30000–40000 cpm; 2 μl). To specify protein/DNA binding reactions, 1 μl of anti-HIF-1α antibodies was added to binding buffer, incubated for 30 min at 4 °C, and mixed with γ-32P-labeled oligonucleotide probe. Samples were electrophoresed through 6% polyacrylamide gel in 0.5% TBE (45 mm Tris, 45 mm boric acid, 10 mm EDTA, pH 8.3). The gel was dried and the radioactive band visualized using Kodak Bio-Max film with an intensifying screen at –70 °C. In Vivo Angiogenesis Assay—Concentrated culture supernatants were harvested by lyophilizing 1 ml of culture supernatants of osteoblasts with and without SW treatment in the presence or absence of 500 units/ml PEG-SOD and then resuspending them in 20 μl of phosphate-buffered saline. VEGF-A concentrations in the mixtures were determined using a VEGF-A ELISA kit. To confirm whether VEGF-A was involved in angiogenesis, culture supernatants were neutralized with 50 ng/ml monoclonal anti-VEGF-A antibodies (R&D Systems). Concentrated culture supernatants were subjected to assessment of angiogenesis using a chick chorioallantoic membrane (CAM) assay as described previously (23Bellahcene A. Bonjean K. Fohr B. Fedarko N.S. Robey F.A. Young M.F. Fisher L.W. Castronovo V. Circ. Res. 2000; 86: 885-891Crossref PubMed Scopus (100) Google Scholar). Briefly, fertile White Leghorn chicken eggs were incubated at 37 °C and 70% relative humidity. On the third day of incubation, the eggs were windowed by gentle sanding to expose an opening on the CAM. The openings were sealed with UV-sterilized adhesive tape, and the eggs were further incubated until day 5. On day 5, silastic rings were placed on the CAM surface. Concentrated culture supernatants were applied inside the rings. The eggs were resealed and incubated for 5 days. CAMs were examined and photographed with a Nikon color camera under microsurgery microscope. A vascular index was determined by counting all discernible vessels traveling the ring and was expressed as the relative increase of the number of vessels under different conditions in comparison with the control (23Bellahcene A. Bonjean K. Fohr B. Fedarko N.S. Robey F.A. Young M.F. Fisher L.W. Castronovo V. Circ. Res. 2000; 86: 885-891Crossref PubMed Scopus (100) Google Scholar). Statistical Analysis—All values were expressed as mean ± S.E. Student's paired t test was used to evaluate the difference between the sample of interest and its respective control. For analysis of the time course, a multiple range of analysis of variance was used. A p value of <0.05 was considered significant. SW Treatment Raised VEGF-A Expression—We first determined whether SW augmented VEGF-A gene expression in osteoblasts. Cell cultures were treated with SW at 0.16 mJ/mm2 energy flux density, 1 Hz, 500 impulses. There was no significant difference in cell viability between SW and control groups (data not shown). RT-PCR results indicated that VEGF-A mRNA expression significantly increased in 6 h, peaking at 12 h (Fig. 1A). ELISA results also showed that osteoblasts subjected to SW treatment significantly increased VEGF-A production in 12 h (Fig. 1B). SW Promotion of VEGF-A Expression Mediated by Superoxide but Not by Osteogenic Factors or Nitric Oxide, Hydrogen Peroxide, or Prostaglandin E2—Previous studies have demonstrated that osteogenic factors can regulate VEGF expression of osteoblasts (24Goad D.L. Rubin J. Wang H. Tashjian Jr., A.H. Patterson C. Endocrinology. 1996; 137: 2262-2268Crossref PubMed Scopus (205) Google Scholar, 25Deckers M.M.L. van Bezooijen R.L. van der Horst G. Hoogendam J. van der Bent C. Papapoulos S.E. Lowik C.W.G.M. Endocrinology. 2002; 143: 1545-1553Crossref PubMed Scopus (457) Google Scholar, 26Chua C.C. Hamdy R.C. Chua B.H. Biochim. Biophys. Acta. 2000; 1497: 69-76Crossref PubMed Scopus (58) Google Scholar) We sought to elucidate whether osteogenic factors were involved in SW-augmented VEGF expression, SW-treated osteoblasts were co-cultured with BMP-2, TGF-β1, and IGF-I monoclonal antibodies for 24 h, respectively. BMP-2, TGF-β1, IGF-I neutralization did not significantly alter SW-enhanced VEGF-A mRNA expression (Fig. 2A) or VEGF-A production (Fig. 2B). The accumulated evidence suggests that cells responded to SW by altering biological activities through influx of bioactive molecules such as oxygen radicals or PGE2 (21Wang F.S. Wang C.J. Sheen-Chen S.M. Kuo Y.R. Chen R.F. Yang K.D. J. Biol. Chem. 2002; 277: 10931-10937Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 27Maier M. Averbeck B. Milz S. Refior H.J. Schmitz C. Clin. Orthop. 2003; 406: 237-245Crossref PubMed Scopus (121) Google Scholar). We investigated which reactive radical might be responsible for mediating SW increase in VEGF-A expression. Scavenging of hydrogen peroxide by 500 units/ml PEG catalase did not influence SW-promoted VEGF-A expression. Inhibition of cyclooxygenase-2 activity by 10 μm indomethacin and nitric oxide synthase activity by 100 μml-NAME did not affect SW-promoted VEGF-A mRNA expression (Fig. 2C) or VEGF-A production (Fig. 2D). Nevertheless, PEG-SOD pretreatment (500 units/ml) significantly reduced SW enhancement of VEGF-A expression (Fig. 2C) and VEGF production (Fig. 2D). These findings suggest that superoxide, but not nitric oxide, hydrogen peroxide, or prostaglandin E2, was involved in the SW promotion of VEGF-A expression. DPI Pretreatment Reduced SW-induced Superoxide and VEGF-A Expression—Osteoblasts with SW treatment significantly increased superoxide production in 30 min. This higher production of superoxide persisted for 24 h (Fig. 3A). We determined whether SW promotion of superoxide production was linked to mitochondrial oxidase, xanthine oxidase, or NADPH oxidase. Pretreatment with DPI (an NADPH oxidase inhibitor), but not with other oxidase inhibitors, significantly reduced SW-promoted superoxide production (Fig. 3B), VEGF-A mRNA expression (Fig. 3C), and VEGF-A production (Fig 3D). This suggests that NADPH oxidase was responsible for SW-augmented superoxide production. Ras Regulated SW-promoted Rac1 Activation, Superoxide Production, and VEGF-A Expression—There has been some evidence that Ras and Rac1 proteins are involved in NADPH oxidase-derived superoxide synthesis (28Sundaresan M. Yu Z.X. Ferrans V.J. Sulciner D.J. Gutkind J.S. Irani K. Goldschmidt-Clermont P.J. Finkel T. Biochem. J. 1996; 318: 379-382Crossref PubMed Scopus (440) Google Scholar, 29Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.J. Feron E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1436) Google Scholar). We sought to examine whether SW-induced superoxide production could be associated with Ras or Rac1 activation. SW rapidly activated Ras protein in 15 min and Rac1 protein in 30 min (Fig. 4A), respectively. To verify whether SW promotion of superoxide production and VEGF-A expression was regulated by Ras protein, we subjected wild type Ras- and dominant negative Ras-transfected osteoblasts to SW treatment. Transfection of the mutant-Ras completely reduced SW-induced Ras and Rac1 activation (Fig. 4B). Superoxide synthesis (Fig. 4C) and VEGF-A mRNA expression (Fig. 4D) were also significantly suppressed in the mutant Ras-transfected cells. SW promoted Phosphorylation of ERK and HIF-1α—Experiments were done to elucidate whether SW-increased VEGF-A expression was linked to mitogen-activated protein kinase and angiogenic transcription. Immunoblotting indicated that SW increased ERK activation in 1 h as demonstrated by phosphorylated ERK expression (Fig. 5A). SW did not affect p38 phosphorylation throughout the study period (Fig. 5B). Furthermore, SW increased nuclear HIF-1α phosphorylation, as demonstrated by phosphotyrosine expression of HIF-1α, in 3 h (Fig. 6A) and promoted HIF-1 binding to VEGF-A promoter, as determined by electrophoretic gel shift (Fig. 6B). We employed monoclonal antibodies against HIF-1α to confirm the DNA-protein binding activity. An electrophoretic mobility shift assay radiograph showed that nuclear extract harvested from SW-treated osteoblasts was indeed super-shifted by anti-HIF-1α antibodies (Fig. 6C). These findings indicate that SW activates HIF-1α binding to the VEGF promoter.Fig. 6SW-induced HIF-1α activation. A, SW activated nuclear HIF-1α phosphorylation in 3 h. Nuclear HIF-1α immunoprecipitates isolated from nuclear extracts of osteoblasts with and without SW treatment were subjected to immunoblotting. Phosphorylated HIF-1α was probed using anti-phosphotyrosine antibodies. B, SW promoted binding activity of HIF-1α with VEGF-A promoter in 3 h as determined by electrophoretic mobility shift assay. C, supershift of HIF-1α. Nuclear extracts of SW-treated osteoblasts were incubated with HIF-1 probe in the presence or absence of anti-HIF-1α antibodies. FP, free probe, SS, supershift.View Large Image Figure ViewerDownload Hi-res image Download (PPT) SOD and PD98059 Pretreatments Reduced SW-augmented ERK and HIF-1α Activation—Scavenging of superoxide by PEG-SOD (500 units/ml) a
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