High-mobility group box 1 from reactive astrocytes enhances the accumulation of endothelial progenitor cells in damaged white matter

Abstract

High-mobility group box 1 (HMGB1) was initially described as a damage-associated-molecular-pattern (DAMP) mediator that worsens acute brain injury after stroke. But, recent findings suggest that HMGB1 can play a surprisingly beneficial role during stroke recovery by promoting endothelial progenitor cell (EPC) function and vascular remodeling in cortical gray matter. Here, we ask whether HMGB1 may also influence EPC responses in white matter injury. The standard lysophosphatidylcholine (LPC) injection model was used to induce focal demyelination in the corpus callosum of mice. Immunostaining showed that within the focal white matter lesions, HMGB1 was up-regulated in GFAP-positive reactive astrocytes, along with the accumulation of Flk1/CD34-double-positive EPCs that expressed pro-recovery mediators such as brain-derived neurotrophic factor and basic fibroblast growth factor. Astrocyte-EPC signaling required the HMGB1 receptor RAGE as treatment with RAGE-neutralizing antibody 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.

Keywords: endothelial progenitor cells (EPCs); high‐mobility group box 1 (HMGB1); neurovascular unit; reactive astrocytes; white matter injury; white matter remodeling.

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.

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.

Figure 3. RAGE expression is required in EPCs accumulation after white matter injury

(a) FACS analysis showed that accumulated Flk1 and CD34 double positive EPC subsets were positive for the HMGB1 receptor RAGE. N=3. (b) Peripheral treatment with neutralizing RAGE antibody significantly reduced EPCs accumulation on day 5 after LPC injection. N=5. *P<0.05.

Figure 4. Astrocytic HMGB1 is required in EPCs accumulation after white matter injury

(a) HMGB1 siRNA was intracerebroventricularly injected on day 2 after LPC injection. Western blot showed HMGB1 expression levels in ipsilateral white matter tract on day 5 after LPC injection. HMGB1 siRNA successfully reduced HMGB1 protein levels in a dose-dependent manner. (b) Immunostaining analysis also showed that HMGB1 siRNA successfully decreased HMGB1 expressed by reactive astrocytes on day 5, without affecting the levels of GFAP-positive cells. (c–d) Flow cytometory showed that treatment with HMGB1 siRNA reduced the EPC accumulation in ipsilateral corpus callosum on day 5. N=5. *P<0.05.

Figure 5. HMGB1-siRNA treatment reduced proliferated endothelial number after white matter injury

(a–b) HMGB1 siRNA was intracerebroventricularly injected on day 2 after LPC injection and brains were taken out three days later. Immunostaining showed that double positive cells with Ki67 and CD31 (i.e. newly emerged endothelial cells, arrows) were observed in the legion area, and the change was attenuated by HMGB1-siRNA treatment. N=5. *P<0.05.

Figure 6. HMGB1 promoted the trans-migration of early EPCs in vitro

(a) Schematics for our in vitro trans-migration assay. Cerebran endothelial RBE.4 cells were plated on the transwell, and once the cells were confluent, DiI-labeled early EPCs were added on the upper side. Twenty-four hours later, labeled EPCs in the lower chamber were counted. (b) HMGB1 (100 ng/mL) significantly promoted the migration of early EPCs. N=4. *P<0.05.

Figure 7. HMGB1 accelerated the tube formation of late EPCs in vitro

(a) Representative images of tube formation in late EPC cultures. HMGB1: 1 ng/mL, anti-RAGE: 5 ug/mL. (b) HMGB1 significantly increased the number of tubes, and the HMGB1-induced tube formation was reduced by co-treatment with anti-RAGE neutralizing antibody. N=5. *P<0.05.