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European Journal of Cell Biology 90 (2011) 642–648

Contents lists available at ScienceDirect

European Journal of Cell Biology

journa l homepage: www.e lsev ier .de /e jcb

verexpression of osteopontin induces angiogenesis of endothelial progenitorells via the av�3/PI3K/AKT/eNOS/NO signaling pathway in glioma cells

ingyi Wanga,1, Wei Yana,1, Xiaoming Lua,1, Chunfa Qianb,1, Junxia Zhanga, Ping Lia, Lei Shic,eng Zhaoa, Zhen Fua, Peiyu Pud, Chunshen Kangd, Tao Jiange, Ning Liua,∗, Yongping Youa,∗

Department of Neurosurgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR ChinaDepartment of Neurosurgery, The Affiliated Brain Hospital of Nanjing Medical University, Nanjing 210029, PR ChinaDepartment of Neurosurgery, The First People’s Hospital of Kunshan Affiliated with Jiangsu University, Suzhou 215300, PR ChinaDepartment of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052, PR ChinaGlioma Therapy Center, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, PR China

r t i c l e i n f o

rticle history:eceived 17 July 2010eceived in revised form 21 March 2011ccepted 21 March 2011

eywords:lioma

a b s t r a c t

Angiogenesis, a hallmark of tumor growth, is regulated by various angiogenic factors. Recent studieshave shown that osteopontin (OPN) is a secreted, integrin-binding protein that contributes to gliomaprogression. However, its effect on the angiogenesis of gliomas is not fully understood. To elucidatethe role of OPN in the process of glioma angiogenesis, endothelial progenitor cells (EPCs) were treatedwith conditioned media of human glioma SHG44 cells overexpressing OPN. Here, we identified that OPNsecreted by glioma cells accelerated EPCs angiogenesis in vitro, including proliferation, migration, and

steopontinngiogenesis

tube formation. OPN also induced the activation of AKT and endothelial nitric oxide synthase (eNOS) andincreased NO production without affecting the expression of VEGF, VEGFR-1, or VEGFR-2. Moreover, theav�3 antibody, the PI3-K inhibitor LY294002 and the eNOS inhibitor NMA suppressed the OPN-mediatedincrease in NO production and angiogenesis in EPCs. Taken together, these results demonstrate that OPNdirectly stimulates angiogenesis via the av�3/PI3-K/AKT/eNOS/NO signaling pathway and may play animportant role in tumorigenesis by enhancing angiogenesis in gliomas.

ntroduction

Angiogenesis is one of the hallmarks of cancer, as it enablesells to evolve from benign to malignant tumors. Previous studiesave demonstrated that the angiogenic process is regulated by var-

ous pro-angiogenic factors that are secreted by tumor cells (Dunnt al., 2000; Lamszus et al., 2004). Endothelial progenitor cellsEPCs) have the capacity to proliferate and differentiate into maturendothelial cells, which facilitate angiogenesis. Recent studies haverovided ample evidence that EPCs contribute to the formationf new blood vessels in tumors (Ahn et al., 2010). Moreover, the

evelopment of a new vascular network that occurs primarily byngiogenesis also plays an important role in glioma progressionJouanneau, 2008). The cooperation of angiogenic cytokines and

Abbreviations: OPN, osteopontin; CM, conditioned media; EPCs, endothelialrogenitor cells; RT-PCR, reverse-transcription polymerase chain reaction; VEGF,ascular endothelial growth factor; VEGFR-1, vascular endothelial growth factoreceptor 1; VEGFR-2, vascular endothelial growth factor receptor 2.∗ Corresponding authors.

E-mail addresses: liuning0853@126.com (N. Liu), YYPL9@njmu.edu.cn (Y. You).1 These authors contributed equally to this article.

171-9335/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.ejcb.2011.03.005

© 2011 Elsevier GmbH. All rights reserved.

EPCs may play an important role in the angiogenic process ingliomas.

Osteopontin (OPN) is a secreted, integrin-binding protein that isassociated with tumor progression in several cancer types, includ-ing hepatocellular, breast, prostate, and colon carcinomas. ElevatedOPN levels in tumors of various tissue types were associated withpoor prognosis (Wai and Kuo, 2004). And OPN plays an importantrole in various aspects of malignancy, particularly in tumor angio-genesis (Chakraborty et al., 2006). OPN secreted by cancer cellsacts as a potent angiogenic factor contributing to tumor growth inseveral cancer types (Chakraborty et al., 2008; Tang et al., 2007;Takahashi et al., 2002; Colla et al., 2005). Further, it has beenreported that the expression level of OPN correlates with malig-nancy in human glioma cells (Saitoh et al., 1995) and that thenumber of newly formed blood vessels is higher in tumors show-ing high interstitial OPN expression (Matusan-Ilijas et al., 2008).However, the effect of OPN on angiogenesis and the correspond-ing mechanism have not yet been extensively studied in humangliomas.

In the present study, we analyzed the role of OPN secretedby glioma cells on the angiogenesis of EPCs. Overproductionof OPN by glioma cells induced angiogenesis of EPCs via theav�3/PI3K/AKT/eNOS/NO signaling pathway. To the best of our

Y. Wang et al. / European Journal of Cell Biology 90 (2011) 642–648 643

Fig. 1. Expression of OPN mRNA in glioma tissues and cell lines. (A) OPN expressionlevels in human normal adult brains and glioma tissues as determined by Real-timequp

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Fig. 2. The level of OPN expression and secretion in SHG44 cells overexpressingOPN. (A) SHG44 cells were infected by OPN-carrying lentivirus with GFP reporter,and the efficiency of infection was assessed by the fluorescence microscope (mag-nification, 100×). (B) SHG44 cells were infected by OPN-carrying lentivirus, OPN

RT-PCR. (B) Expression of OPN mRNA in different glioma cell lines. �-Actin wassed as an internal control in the lower panel. (C) Real-time qRT-PCR analysis waserformed to validate the expression levels in glioma cell lines.

nowledge, it is the first time to report that OPN plays an importantole in EPCs angiogenesis in glioma.

aterials and methods

ell culture

EPCs were isolated as described in our previous study (Zhangt al., 2008). Briefly, mononuclear cells were isolated from humanord blood by density gradient centrifugation. CD133+ cells puri-ed by magnetic-activated cell sorting columns (MACS; Miltenyiiotech) were then suspended in EGM-2 MV medium (Cambrex)ontaining endothelial basal medium (EBM), 5% fetal bovine serum,EGF, VEGF, hFGF-B, IGF-1, ascorbic acid, and heparin, and seeded

n fibronectin-coated (Chemicon) culture dishes. After 72 h, nonad-erent cells were removed.

Human glioma cell lines SHG44, U251, and U87 were purchased

rom the Chinese Academy of Sciences Cell Bank. U251 and U87ells were cultured in DMEM culture medium supplemented with0% fetal bovine serum (FBS); SHG44 cells were cultured in 1640ulture medium supplemented with 10% FBS. Conditioned medium

mRNA and protein expression was measured by RT-PCR and Western blot analysis.(C) Conditioned medium (CM) was collected and subjected to ELISA. Data shown inthe graphs are the means ± SE of at least three individual experiments.

(CM) was prepared as follows. Each group of SHG44 cells wasgrown to 80% confluence and the culture medium was replacedwith serum-free medium for 24 h. The culture media were thenharvested, centrifuged to remove any particulates and cell debris,and stored at −70 ◦C until they were applied to the EPCs. Experi-ments were divided into three groups including the blank controlgroup (SHG44), the negative control (SHG44/NC) group, and theOPN group (SHG44/OPN). Anti-av�3 antibody and the chemicalinhibitors of PI3-K (LY294002) and eNOS (NMA) (Sigma) were usedfor further experiments.

Lentiviral vector construction and infection

A lentivirus-based vector for human OPN expression was

constructed with technical support from Shanghai GeneChem.The full-length OPN cDNA was annealed and inserted into thepGC-LV expression vector (Invitrogen) containing the humancytomegalovirus promoter and herpes simplex virus thymidine

644 Y. Wang et al. / European Journal of Cell Biology 90 (2011) 642–648

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ig. 3. OPN promotes EPC proliferation, migration, and tube formation. EPCs wereormation were determined by cell cycle assay (A), Transwell assay (B) and in vitro fif at least three individual experiments. *P < 0.05 versus negative control.

inase polyadenylation signal. The OPN expression vector (pGC-LV)nd packaging vectors (pHelper 1.0 and pHelper 2.0) (Invitrogen)ere cotransfected into 293FT cells with Lipofectamine 2000 (Invit-

ogen). The culture supernatants were collected, concentrated, andtored in a −70 ◦C freezer. Lentivirus-mediated infection of SHG44ells with OPN resulted in the expression of stable OPN protein.nhanced green fluorescent protein (eGFP) was expressed in aentiviral vector to titer and measure the infection efficiency innfected cells. SHG44 cells were infected with lentiviral vectors atmultiplicity of infection (MOI) of 10 in the presence of polybrene

10 �g/mL).

T-PCR and real-time qRT-PCR

Glioma tissues were obtained from the Department of Neuro-urgery of the First Affiliated Hospital of Nanjing Medical University

fter informed consent was obtained from adult patients diagnosedith glioma. Total RNAs were isolated from these glioma tissues,

ell lines, and EPCs using TRIzol reagent. The visualized bands werehown via RT-PCR. Real-time qRT-PCR was performed using the

ulated with CM from SHG44/OPN cells, and cell proliferation, migration and tubel angiogenesis assay (C), respectively. Data shown in the graphs are the means ± SE

SYBR green Real-time qRT-PCR system and �-actin was used asan internal control. The following primers were used: OPN, 5′-TGATGA ATC TGA TGA ACT GGT C-3′ and 5′-GGT GAT GTC CTC GTC TGTAG-3′; �-actin, 5′-AAG ACC TGT ACG CCA ACA CAG T-3′ and 5′-AGAAGC ATT TGC GGT GGA CGA T-3′; VEGF, 5′-CAT GAA CTT TCT GCTGTC TTG G-3′ and 5′-TCA CCG CCT CGG CTT GTC ACAT-3′; VEGFR-1, 5′-TCA TGA ATG TTT CCC TGC AA-3′ and 5′-GGA GGT ATG GTGCTT CCT GA-3′; VEGFR-2, 5′-GTG ACC AAC ATG GAG TCG TG-3′ and5′-CCA GAG ATT CCA TGC CAC TT-3′.

ELISA assay

To analyze the secretion of OPN protein from the SHG44/OPNinfectants, OPN in the culture supernatant was measured with theR&D ELISA kit according to the manufacturer’s instructions. Briefly,SHG44, SHG44/NC, and SHG44/OPN cells were plated at a den-

sity of 5 × 105 cells per well in six-well culture plates in 2 mL ofmedium containing 10% FCS. After 72 h, culture supernatants werecollected and subjected to ELISA analysis. Absorbance at 450 nmwas measured with a microplate reader (Bio-Rad).

al of Cell Biology 90 (2011) 642–648 645

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Fig. 4. OPN does not alter the expression of VEGF, VEGFR-1, and VEGFR-2 in EPCs.

Y. Wang et al. / European Journ

ell cycle assay

EPCs treated with CM from each group for 24 h were collected,ashed in phosphate-buffered saline (PBS), and fixed with 70% cold

thanol for at least 1 h. After extensive washing, the cells were sus-ended in HBSS containing propidium iodide (69 �M; Sigma) andNase A (0.1 mg/mL; Sigma), incubated for 1 h at room temperature,nd analyzed by FACScan (Becton Dickinson).

igration assay

Transwell membranes coated with 10 �g/mL of gelatin (Sigma)n the lower surface were used to assay the migration of EPCs initro. EPCs were plated at a density of 5 × 104 cells per well in thepper chamber in serum-free medium. The CM of SHG44/OPN wasdded to the lower chamber. After 24 h of incubation, nonmigrat-ng cells were removed from the top well with a cotton swab, andhe bottom cells were fixed with 75% alcohol and stained with 0.1%rystal violet. Images were captured with a video graphic systemsing three independent 10× fields for each well. The migrationbility of EPCs was quantified by counting the cells that migrated tohe lower side of the filter in low-power (100×) fields. Three inde-endent experiments were performed and used to calculate theold migration relative to the negative control; error was calculateds the SE.

ube formation assay

To evaluate tube formation in EPCs treated with OPN, an in vitrobro gel angiogenesis kit (CHEMICON) was used. EPCs were seeded

n 96-well culture plates that were coated with fibro gel for 30 mint 37 ◦C according to the manufacturer’s instructions. EPCs weretimulated with CM from the respective groups for 24 h. Tube for-ation in the EPCs was observed under an inverted phase contrasticroscope and photographed at 100× magnification.

estern blotting assay

To determine the levels of protein expression, total proteinas isolated in lysis buffer (137 mM NaCl, 15 mM EGTA, 15 mMgCl2, 0.1 mM sodium orthovanadate, 0.1% Triton X-100, 25 mMOPS, 100 �M phenylmethylsulfonyl fluoride, and 20 �M leu-

eptin, adjusted to pH 7.2). Equal amounts of protein (30 �g) wereoaded into the sample wells and separated on a 12% sodiumodecyl sulfate-polyacrylamide gel. The electrophoresed proteinsere transferred to PVDF membranes (Millipore) and incubatedith primary antibodies against av�3, AKT, phospho-AKT, eNOS,hospho-eNOS, VEGF, VEGFR-1 and VEGFR-2 (Santa Cruz). GAPDHas reblotted to check for equal loading of the gel.

easurement of NO

The intracellular NO levels in EPCs were measured in situ usingAF-FM diacetate (NO Molecular Probes, Beyotime). After CM

reatment for 24 h, EPCs were washed three times with PBS andncubated for 1 h at 37 ◦C with 5 �M DAF-FM diacetate. After thexcess probe was removed and cells were washed three times withBS, EPCs were incubated for an additional 20 min. The fluorescenceas captured with a confocal laser microscope from at least 10 ran-omly selected cells for each well. The relative levels of intracellularO were determined from the fluorescence intensity of DAF-FMiacetate.

tatistical analysis

All tests were repeated three times and analyzed with the SPSSraduate Pack 11.0 statistical software. All data are presented as

EPCs were treated with CM from SHG44/OPN cells, and OPN mRNA and proteinexpression was measured by RT-PCR (A) and Western blot analysis (B). �-Actin andGAPDH were regarded as the endogenous normalizer.

the means ± SE. One-way ANOVA was used to determine significantdifferences. A value of P < 0.05 was considered to indicate statisticalsignificance.

Results

Expression of OPN mRNA in glioma tissues and cell lines

To examine the expression of OPN mRNA in glioma tissues andcell lines, PCR was carried out using specific primers for OPN. Thelevels of OPN mRNA in WHO III and IV grade glioma tissues weresignificantly higher than those in WHO II grade glioma and normalbrain tissues (Fig. 1A). The high expression of OPN mRNA was con-firmed in U251 and U87 cells, but only faint bands were observedin SHG44 cells (Fig. 1B). To validate the high expression levels ofOPN in human glioma cell lines, real-time qRT-PCR was performed.Based on the ��Ct relative to SHG44 cells, OPN was expressed ata significantly higher level (1000 fold) in U251 and U87 cells thanthat in SHG44 cells (Fig. 1C). Therefore, we used SHG44 cells as aglioma cell model for OPN overexpression in the present study.

OPN secreted by glioma cells is critical for the angiogenesis of EPCs

To obtain CM with a high level of OPN produced by gliomacells, SHG44 cells were treated by OPN-carrying lentivirus at a highefficiency of infection (Fig. 2A). And Fig. 2B represented that a sig-nificant induction of OPN mRNA was detected in SHG44/OPN cellscompared to the control groups, with a similar trend in OPN pro-tein expression. To further verify the secretion of OPN protein inSHG44/OPN cells, ELISA assay was employed. As shown in Fig. 2C,

SHG44/OPN cells secreted a notable level of OPN protein into theculture medium.

In vitro angiogenesis is a complex biological process thatrequires the precise coordination of cell proliferation, migration,

646 Y. Wang et al. / European Journal of Cell Biology 90 (2011) 642–648

Fig. 5. OPN activates the av�3/PI3-K/AKT/eNOS/NO signaling pathway in EPCs. EPCs were pre-treated with an anti-av�3 antibody, a PI3-K inhibitor (100 nM, LY294002) ora cells.b onfocac

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DAF-FM, respectively. An obvious activation of eNOS and pro-

n eNOS inhibitor (1 mM, NMA) for 30 min before exposure to CM from SHG44/OPNlot analysis (A). The relative levels of intracellular NO were determined with a control; **P < 0.05 versus the group only treated with CM from SHG44/OPN cells.

nd tube formation. To determine whether CM from SHG44/OPNells affects the cell cycle of EPCs, a flow cytometric analysis toetermine the cellular DNA content was performed after treatmentith CM for 24 h. As shown in Fig. 3A, the S phase percentages

n EPCs treated with CM from SHG44/OPN cells were significantlyigher than in the negative and blank controls. Next, the Tran-well assay showed that CM from SHG44/OPN cells significantlyncreased the migration proportion of EPCs (Fig. 3B). Further-

ore, we found that the tube formation capacity of EPCs in theHG44/OPN group was significantly increased compared with theegative and blank controls, respectively (Fig. 3C). These findings

ndicate that OPN secreted by glioma cells is critical for the angio-enesis of EPCs.

PN induces angiogenesis without affecting VEGF, VEGFR-1, andEGFR-2

To explore the molecular mechanism of OPN involved in angio-enesis, VEGF, VEGFR-1 and VEGFR-2 expression of EPCs wasested. RT-PCR analysis showed that CM from SHG44/OPN cellsid not affect VEGF, VEGFR-1 or VEGFR-2 mRNA levels in EPCs

The levels of phosphorylated and total AKT and eNOS were determined by Westernl laser microscope (B). Data shown are the means ± SD. *P < 0.05 versus negative

(Fig. 4). More, we also measured the protein levels of VEGF,VEGFR-1 and VEGFR-2. Consistently, there was no detectableexpression of these proteins, suggesting that OPN-induced gliomaangiogenesis is not mediated by VEGF, VEGFR-1 and VEGFR-2signaling.

OPN activates AKT and eNOS and elevates NO production in EPCs

It has been shown that AKT and eNOS phosphorylation and NOproduction are important cellular signaling events for endothe-lial cell angiogenesis. First, we determined the effects of OPNon the phosphorylation of AKT. As shown in Fig. 5, the CMof SHG44/OPN triggered an increase of AKT phosphorylation.Then, eNOS phosphorylation and NO production in EPCs weredetected by the Western blot assay and the NO-specific probe

duction of NO was observed after treatment with the CM ofSHG44/OPN. These results suggested that OPN secreted by gliomacells promotes the angiogenesis of EPCs by activation of AKT andeNOS.

Y. Wang et al. / European Journal of Cell Biology 90 (2011) 642–648 647

Fig. 6. OPN promotes angiogenesis via the av�3/PI3-K/AKT/eNOS/NO-dependent signaling pathway. EPCs were pre-treated with an anti-av�3 antibody, a PI3-K inhibitor(100 nM, LY294002) or an eNOS inhibitor (1 mM, NMA) for 30 min before exposure to CM from SHG44/NC and SHG44/OPN cells. Cell proliferation, migration and tubef o fibrom P < 0.0

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ormation were determined by cell cycle assay (A), Transwell assay (B) and in vitreans ± SD of three independent experiments. *P < 0.05 versus negative control; **

PN promotes angiogenesis via thevˇ3/PI3-K/AKT/eNOS-dependent signaling pathway

Osteopontin (OPN) is a phosphorylated acidic RGD contain-ng glycoprotein that binds certain CD44 variants and integrineceptors, including av�3. To further examine the role of av�3,I-3K, AKT, and eNOS in OPN-induced angiogenesis, the anti-v�3 antibody and the chemical inhibitors of PI3-K (LY294002)nd eNOS (NMA) were employed. The phosphorylation of AKTnduced by CM from SHG44/OPN cells was inhibited by thenti-av�3 antibody and LY294002, but not by NMA. However,NOS activation was suppressed by all these inhibitors (Fig. 5A).dditionally, NO expression was significantly downregulated by

he anti-av�3 antibody, LY294002 and NMA (Fig. 5B). Theseesults indicate that OPN stimulates the PI3K/AKT signaling viainding to av�3, and subsequently activates the eNOS/NO path-ay. We next investigated the effects of these inhibitors onPN-mediated angiogenesis. Treatment with the anti-av�3 anti-ody, LY294002, and NMA resulted in significant decreases ofhe S phase of the cell cycle (Fig. 6A), cell migration (Fig. 6B)

nd tube formation (Fig. 6C) of EPCs incubated with CM fromHG44/OPN cells. These results suggest that OPN induces angio-enesis by activating the PI3K/AKT/eNOS/NO pathway via bindingo av�3.

gel angiogenesis assay (C), respectively. Data shown in the graphs (D–F) are the5 versus the group only treated with CM from SHG44/OPN cells.

Discussion

OPN has been shown to promote vascular formation in murineneuroblastoma (Takahashi et al., 2002) and inhibition of OPN levelsinduces a suppression of angiogenesis in gastric cancer (Tang et al.,2007). However, the effect of OPN on angiogenesis in glioma andthe corresponding mechanism remain unclear. In addition, EPCscontribute to the formation of new blood vessels in tumors andmight be a better model for investigating human tumor endothelialcells than either human micro-vascular endothelial cells (HMVEC)or human umbilical vein endothelial cells (HUVEC) (Bagley et al.,2003). Our previous study has also demonstrated that glioma cellsenhance the angiogenesis of EPCs isolated from human cord blood(Zhang et al., 2008). Thus, EPCs were selected as a cell model forangiogenesis in this study. Our data found that OPN secreted byglioma cells accelerated EPCs angiogenesis, including proliferation,migration, and tube formation, via the av�3/PI3-K/AKT/eNOS/NOsignaling pathway but produced no change in the level of VEGF,VEGFR-1, or VEGFR-2.

PI3-K/AKT activation induced by pro-angiogenic factors has

been evidenced to participate in the proliferation, migration, andmorphogenesis of endothelial cells (Abid et al., 2004). Thus, the sup-pression of PI3-K/AKT activation inhibits angiogenesis in vitro andin vivo (Chen and Meyrick, 2004). Our results showed that OPN

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48 Y. Wang et al. / European Journ

roduced by glioma cells increased PI3-K/AKT activation and thengiogenesis of EPCs and the PI3-K inhibitor (LY294002) effectivelynhibited AKT activation and angiogenesis in vitro. These findingsndicate that OPN increases EPCs angiogenesis through the PI3-/AKT pathway, in line with the previous study (Dai et al., 2009).

The eNOS/NO signaling pathways are recognized as importantediators of angiogenic processes, knockdown of the eNOS gene

r downregulation of endogenous NO reduced new blood vesselormation in an animal model (Smith et al., 2002; Murohara et al.,998). Recently, eNOS/NO signaling has been shown to play a criti-al role in angiogenesis in gliomas (Bulnes et al., 2010; Zheng et al.,007).

Furthermore, phosphorylation of eNOS and NO production cane upregulated by the PI3-K/AKT pathway in the angiogenesis ofndothelial cells (Tanimoto et al., 2002). In the present study, weound that OPN increases AKT and eNOS phosphorylation and NOroduction thereby stimulates angiogenesis. The inhibition of AKThosphorylation by treatment with the PI3-K inhibitor LY294002educed eNOS phosphorylation and NO production, leading to theuppression of angiogenesis in vitro. However, the eNOS inhibitorMA decreased OPN-induced NO production and its angiogenicctivity, accompanying no AKT activation. These results demon-trate that the av�3/PI3-K/AKT/eNOS/NO pathway is required forPCs angiogenesis induced by OPN in glioma.

Some angiogenic factors have been shown to induce angio-enesis by activating angiogenic signals via upregulation ofro-angiogenic genes, including VEGF (Huang and Bao, 2004). Rasncogenes upregulate the expression of VEGF in various tumor cellypes, which is in part responsible for their role in the angiogenicrocess (Rak et al., 1995). However, in our study, treatment withM from SHG44/OPN cells did not alter VEGF, VEGFR-1, or VEGFR-2xpression, suggesting that the mechanism of the angiogenic effectf OPN is not likely to be associated with VEGF, VEGFR-1, or VEGFR-expression.

In conclusion, the present study demonstrates, for the firstime, that the overexpression of OPN in glioma cells stimu-ates the in vitro angiogenesis of EPCs via activation of thev�3/PI3-K/AKT/eNOS/NO-dependent signaling pathway, but notpregulation of VEGF-related signaling molecules. Therefore, theesults of the present study suggest that the modulation of theechanism responsible for EPC-mediated glioma angiogenesisay be used as a therapeutic strategy to affect pathological angio-

enesis and thus warrants further investigation.

cknowledgements

This work was supported by the China National Naturalcientific Fund (81072078, 81000963 and 30872657), Jiangsurovince’s Medical Major Talent Program (RC2007061), Jiangsurovince’s Natural Science Foundation (BK2008475, 2009444 and010580),the Program for Development of Innovative Researcheam in the First Affiliated Hospital of NJMU, and Priority Academicrogram Development of Jiangsu Higher Education Institutions.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.ejcb.2011.03.005.

ell Biology 90 (2011) 642–648

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