After that, mononuclear cells (MNCs) were obtained by density gradient centrifugation with Ficoll-Paque Plus (Amersham Biosciences Corp). such as brain derived neurotrophic factor and basic fibroblast growth factor. Astrocyte-EPC signaling required the HMGB1 receptor RAGE since treatment with anti-RAGE antibodies significantly decreased EPC accumulation. Moreover, suppression of HMGB1 with siRNA in vivo significantly decreased EPC numbers in damaged white matter as well as proliferated endothelial cell numbers. Finally, in vitro cell culture systems confirmed that HMGB1 directly affected EPC function such as migration and tube formation. Taken together, our findings suggest that HMGB1 from reactive astrocytes may attract EPCs to promote recovery after white matter injury. 2009). For each independent experiment, spleens from 11C12 weeks old Sprague-Dawley (SD) rats were kept in PBS solution. Under the hood, spleens were mechanically minced, placed at 37C for 15 min and run through a 40-um nylon membrane to obtain cell suspension. After that, mononuclear cells (MNCs) were obtained by density gradient centrifugation with Ficoll-Paque Plus (Amersham Biosciences Corp). Isolated MNCs were Sstr1 shortly washed with red blood cells lysis solution and gently washed twice with complete growth media EGM-2MV (Lonza). MNCs were finally resuspended in EGM-2MV and 3 107 MNCs per well were seeded on collagen I-coated six-well plates (Becton Dickinson Labware) and incubated in a 5% CO2 incubator at 37C. Under daily observation, first media change was performed 3C4 days after plating. Early EPCs (5C7 days after seeding) were used for the migration assay, and late EPCs (1C1.5 months after seeding) were for the tube formation assay. In vitro trans-endothelial migration assay Rat brain endothelial cells (RBE.4) (1105 cells/well) were plated on polycarbonate membrane (3-um pore filters, Corning Costar) coated with collagen I to obtain confluent endothelial monolayer. Ac LDL-labeled EPCs (1105 cells/well) were placed in the upper chamber on top of the RBE.4 monolayer. The chambers were placed in a 24-well culture plate containing HMGB1 (100 ng/ml). After 24 h of incubation at 37C, labeled EPCs migrating into the lower chamber were counted in 4 random microscopic Masitinib mesylate fields. EPCs labeling Cells were incubated with 5 ug/ml 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine (DiI) labeled acetylated low density lipoprotein (ac-LDL; Molecular Probes) at 37C for 120 min in EGM-2MV. In vitro tube formation assay The standard Matrigel assay was used to assess the spontaneous formation of capillary-like structures of the late EPCs. Standard 24-well plates were coated with 150 uL of cold Matrigel and allowed to solidify at 37C for 30 min. Cells (5 104 cells/well) were seeded in that plates, and incubated at 37C for 18 h. Statistical analysis Quantitative data were analyzed by using ANOVA followed by Tukeys honestly significant difference tests. Data are expressed as mean S.E.M. A value of p 0.05 was considered significant. Results As expected, lysophosphatidylcholine (LPC) injections into the corpus callosum induced focal demyelination at 5 days Masitinib mesylate (Figure 1a). HMGB1 expression was increased in the damaged white matter region (Figure 1b), with the majority of signals co-localizing with GFAP-positive reactive astrocytes (Figure 1c). Flow cytometry demonstrated an accumulation of Flk1 and CD34-double positive EPCs in these areas (Figure 2aCb). Further analysis showed that expression levels of brain derived neurotrophic factor and basic fibroblast growth factor were elevated in these EPCs within the damaged white matter regions (Figure 2c). Open in a separate window Figure 1 HMGB1 expression was increased in reactive astrocytes after white matter injury(a) Stereotaxic injection of LPC into the corpus callosum induced myelin damage in white matter tracts (green) on day 5. DAPI (blue) staining showed the cell accumulation inside the injury. N=3. (b) Western blot analysis showed the up-regulation of HMGB1 in an area of ipsilateral corpus callosum compared with contralateral side. C: contralateral side, I: ipsilateral side. N=4. (c) In ipsilateral injured area, reactive astrocytes mostly expressed HMGB1 in cell cytoplasm. N=3. Open in a separate window Figure 2 EPCs accumulation in ipsilateral side after white matter injury(a) Double positive cells for CD34 and Flk1 in ipsilateral side of white matter tract were assessed in order to detect endothelial progenitor cell (EPC) population in flow cytometory. (b) The data analysis showed significant increase of EPCs on day 5 after LPC injection. N=4. **P 0.01. (c) FACS analysis showed that trophic factors (BDNF and FGF-2) were expressed in the Flk1+/CD34+ EPC population. N=3. A key receptor for HMGB1 is the receptor for advanced glycation endproducts (RAGE). Flow cytometry confirmed that EPCs in damaged white matter were Masitinib mesylate positive for RAGE (Figure 3a). These findings therefore suggest that HMGB1 released from reactive astrocytes can bind to RAGE receptors present on EPCs. To assess the functional significance of this cell-cell signaling, mice were treated with vehicle or neutralizing RAGE antibodies. Blockade of RAGE significantly reduced the accumulation of EPCs in LPC-damaged white matter (Figure 3b). Open in a separate window Figure 3 RAGE expression is required.