Four-week-old male Wistar rats (80–100 g) were used in our study. The rats were purchased from the Laboratory Animal Center of China Medical University. All experiments using animals were approved by the Animal Care and Use Committee of China Medical University and in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. BMSCs were prepared as previously described with some modifications, according to their adherence to plastic (16,17). In brief, the rats were sacrificed by cervical dislocation after they were anesthetized with 10% chloral hydrate (0.3 ml/100 g) by intraperitoneal injection. Bilateral tibias and femurs were dissected aseptically, and bone marrow was collected and suspended in low glucose Dulbecco’s modified Eagle’s medium (L-DMEM; Gibco, Invitrogen Corp., Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco). The cell suspension was then transferred into 25-cm² culture flasks. After 48 h of cultivation, the culture medium was replaced to remove the non-adherent hemato poietic lineage cells, and adherent cells were further cultured and expanded. BMSCs at passage 3 were used for the studies.

Rat C6 glioma cells (American Type Culture Collection, Rockville, MD) were maintained in L-DMEM supplemented with 10% FBS.

In our experiments, 24-well Transwell inserts with an 8-μm pore size (Corning Costar) were used to evaluate the migration of BMSCs under different conditions. C6 glioma cells and BMSCs were trypsinized and resuspended in serum-free L-DMEM at 5x10⁵/ml and 1x10⁶/ml, respectively. To explore the migration of BMSCs toward C6 glioma cells, 1 ml of C6 glioma cell suspension was added into the lower chambers and 6 h later, a 200-μl BMSC suspension was added into the upper chambers. Moreover, to ascertain whether VEGF promotes the migration of BMSCs towards C6 glioma, we examined the migration of BMSCs in response to a suspension of C6 glioma cells supplemented with or without a VEGF neutralizing antibody (1 μg/ml, Abcam, Cambridge, UK), which were placed in the lower chambers, respectively. In addition, we applied VEGF₁₆₄, one major isoform of rat VEGF, to test the chemotactic effect of VEGF on BMSCs. Serum-free L-DMEM with recombinant rat VEGF₁₆₄ (R&D Systems, Minneapolis, MN, USA) at concentrations of 20 ng/ml was added into the lower wells. VCAM-1 blocking antibody (Covance, Princeton, NJ, USA) was added to the suspension of BMSCs at 10 μg/ml to evaluate the role of VCAM-1 in the VEGF₁₆₄-induced migration of BMSCs. Furthermore, we used the PI3K selective inhibitor, LY294002, to assess the role of PI3K in the VEGF-induced migration of BMSCs. BMSCs were treated with LY294002 (20 µM; Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min prior to, and for the duration of, the stimulation of 20 ng/ml VEGF₁₆₄. The contents of the upper and lower wells were separated by a polycarbonate membrane (8-μm pore size). Cell migration was allowed for 36 h at 37°C. Following incubation, the media were aspirated, and the cells remaining on the upper surface of the polycarbonate membrane were removed with a cotton swab. The cells migrating to the lower surface were stained with Giemsa stain for 15 min. Cell counting was performed under an inverted microscope by two researchers separately. The average numbers of migrated cells were determined by counting the cells in 6 random high-power fields (x400). Serum-free L-DMEM alone served as negative controls.

To investigate the change in VEGF-induced VCAM-1 expression of BMSCs and the relevance of PI3K to this process, the cells were incubated with L-DMEM containing 10% FBS in the presence of VEGF₁₆₄ (20 ng/ml) with or without LY294002 (20 μmol/l) for 12 h. The expression of VCAM-1 mRNA was evaluated by reverse transcriptase-polymerase chain reaction analysis (RT-PCR). The cells incubated with L-DMEM containing 10% FBS served as a negative control. Total RNA was extracted from the cultured cells using TRIzol reagent (Invitrogen) and cDNA was generated from 1 μg of total RNA from each sample. The primers used were as follows: VCAM-1, 5′-ACACCTCCCCCAAGAATACAG-3′ (forward) and 5′-GCT CATCCTCAACACCCACAG-3′ (reverse) (18); β-actin, 5′-TCA GGTCATCACTATCGGCAAT-3′ (forward) and 5′-AAAGA AAGGGTGTAAAACGCA-3′ (reverse). PCR conditions for both were as follows: 32 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 2 min. The PCR products were subjected to electrophoresis on 1.5% agarose gels. All of the cDNA bands were scanned using Chemi Imager 5500 V2.03 software, and the integrated density values (IDV) were calculated by computerized image analysis system (Fluor Chen 2.0) and normalized with that of β-actin.

For immunofluorescence assays, BMSCs were cultured on glass coverslips coated with 0.1% gelatin. After incubation was performed as mentioned above, the cells were fixed with acetone and immunostained with mouse monoclonal anti-VCAM-1 antibody (diluted 1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4°C overnight. Subsequent visualization was performed with anti-mouse IgG conjugated with rhodamine (TRITC) (diluted 1:200; Santa Cruz Biotechnology, Inc.) for 1 h at 37°C in darkness. Coverslips were mounted in mounting media, and images were captured with an Olympus BX60 upright fluorescence microscope with appropriate filters and objectives, using identical acquisition parameters per experiment. L-DMEM containing 10% FBS served as a control.

All results are described as mean ± SD for each group. A Student’s t-test was used to assess a significant difference between two groups. One-way analysis of variance (ANOVA) was performed to determine significant differences between multiple groups. P<0.05 was considered to indicate a statistically significant result.

We used an in vitro migration asssay to evaluate the migratory ability of BMSCs towards C6 glioma cells. Consistent with previous studies, in our experiment, we found that BMSCs migrated directionally towards glioma (2). As shown in Fig. 1, the average number of migrating cells towards C6 glioma cells was significantly higher than that in the control.

To determine whether VEGF has a role in the BMSC migration toward gliomas, a VEGF neutralizing antibody was added into the lower chamber together with the C6 glioma cells. We found that the neutralization of VEGF significantly reduced the migration-enhancing effect of C6 glioma cells and the number of migrating cells decreased compared with the control (Fig. 1). These results suggest that VEGF participates in mediating the migration of BMSCs toward C6 glioma in vitro.

To evaluate the chemotactic effect of VEGF on rat BMSCs, we analyzed the effect of recombinant rat VEGF₁₆₄ on the migration of BMSCs. Our data showed that the addition of recombinant rat VEGF₁₆₄ caused chemotaxis activity of BMSCs and induced them to migrate towards the lower chambers. When compared to the control, VEGF₁₆₄, at a concentration of 20 ng/ml, led to a significant increase in the number of migrating BMSCs (Fig. 2).

Our data of the in vitro migration assays demonstrated that the addition of a VCAM-1 neutralizing antibody significiantly decreased the number of migrating BMSCs towards VEGF₁₆₄ (Fig. 3). These results imply that VCAM-1 is a key adhesion molecule mediating the migration of BMSCs induced by VEGF₁₆₄.

The results of RT-PCR and immunofluorescence assays revealed that the BMSCs expressed a low level of VCAM-1 in the culture without VEGF₁₆₄, while the cells treated with 20 ng/ml VEGF₁₆₄ for 12 h exhibited a higher VCAM-1 expression than the cells without the stimulation of VEGF₁₆₄ (Figs. 4 and 5).

The results of the in vitro migration assays showed that the migration of BMSCs toward recombinant rat VEGF₁₆₄ was found to be partially blocked and the number of migrating BMSCs significantly decreased with the addition of LY294002. This indicates that VEGF₁₆₄-mediated PI3K activation may correlate with the migration of BMSCs induced by VEGF₁₆₄ (Fig. 2). We also found that the upregulation of VCAM-1 mRNA and protein expression decreased following treatment with LY294002, which suggests that PI3K activation may play an active role in VCAM-1 upregulation of BMSCs stimulated by VEGF₁₆₄ (Figs. 4 and 5).