Contribution of endogenous endothelial progenitor cells to neovascularization and astrogliosis in spinal cord injury +1Kamei, N; 2Ishikawa, M; 3Asahara, T; 1Ochi, M

+1Hiroshima University, Hiroshima, Japan, 2Case Western Reserve University, Cleveland, OH, 3Institute of Biomedical Research and Innovation/ RIKEN Center for Developmental Biology, Kobe, Japan

nahkamei@hiroshima-u.ac.jp

INTRODUCTION: Spinal cord injury causes initial mechanical damage, followed by ischemia-induced, secondary degeneration deteriorating the tissue damage. While endothelial progenitor cells (EPCs) have been reported to play an important role for pathophysiological neovascularization in various ischemic tissues, the EPC kinetics following spinal cord injury has never been elucidated. In this study, we therefore assessed the in vivo kinetics of bone marrow-derived EPCs by EPC colony forming assay and Bone marrow transplantation (BMT) from Tie2/lacZ transgenic mice into wild type mice with spinal cord injury. METHODS Bone Marrow Transplantation (BMT) model with Tie2/lacZ transgenic mice: Bone marrow mononuclear cells (MNCs) were collected from Tie2/lacZ transgenic mice, which constitutively express β-galactosidase (β-gal) encoded by lacZ under the transcriptional regulation of an endothelium-specific promoter, Tie2. Wild mice (C57BL/6, 6 weeks old) were lethally irradiated with 12 Gy and received intravenous infusion of 2×106 BM-MNCs from Tie2/lacZ transgenic mice. At 4 weeks post-BMT, by which time the bone marrow of the recipient mice was reconstituted, surgical operation was performed to induce spinal cord injury. Mouse Spinal Cord Injury Models: After laminectomy at the 10th thoracic spinal vertebrae, spinal cord crush injury was performed by compressing the cord laterally from both sides with number 5 Dumont forceps for 10 seconds. EPC colony forming assay using peripheral blood MNCs: To assess the endothelial commitment and differentiation capacity, an EPC colony forming assay (EPC-CFA) established in our laboratory was performed using circulating MNCs. Peripheral blood (0.6~1.0 ml/ mouse) was collected from non-injured mice (pre SCI) and injured ones at days 0 (immediately after SCI), 3, 7 and 14 after spinal cord injury. The EPC-CFA was performed by culturing 1×105 PB-MNCs in methyl cellulose-containing medium with several growth factors. Ten days later, two kinds of EPC colonies can be morphologically identified as follows; primitive EPC colony forming units (EPC-CFUs) consisting of small, round-shaped cells and definitive EPC-CFUs consisting of large, spindle-shaped cells (Figure 1A). The numbers of primitive, definitive and total EPC-CFUs formed from 1×105 PB-MNCs were serially examined. Transplantation of cultured EPCs into mice with SCI: BM-MNCs obtained from C57BL/6 mice were cultured in endothelial cell basal medium-2 supplemented with 5% FBS, VEGF, bFGF, IGF. The floating cells were removed at day 4 and the adherent cells were cultured for 3 more days. Then, the adherent cells defined as cultured EPCs were labeled by DiI-Ac-LDL, and intravenously administered to mice immediately after SCI (1×106 EPCs/mouse). Immunohistochemistry: Spinal cord sections at days 3, 7, 14 or 28 after injury were stained with the following primary antibodies: nestin, β-gal, glial fibrillary acidic protein (GFAP) and CD31. Statistical analysis: All measured values were expressed as mean ± standard deviation (SD). Statistical analysis was performed using StatView software. Student’s unpaired t test was used for the comparison between 2 groups. One-way ANOVA followed by Scheffe’s post hoc test was performed for multiple comparisons. RESULTS Serial change in EPC colony formation from PB-MNCs PB-MNCs slightly decreased immediately after SCI, however gradually increased at day 3 or later. The number of PB-MNCs was significantly greater at days 7 and 14 post injury than pre injury. The vasculogenic potential of PB-MNCs was serially assessed by EPC-CFA. The number of primitive, definitive and total EPC-CFUs peaked at day 3 after SCI but gradually decreased at days 7 and 14. Notably, definitive EPC-CFUs were hardly observed either pre or immediately post SCI and obviously fewer than primitive EPC-CFUs at days 7 and 14 (Figure 1). These results suggest that spinal cord injury may augment mobilization and differentiation capacity of BM-derived and circulating EPCs, possibly for the spinal cord tissue repair.

Figure 1: The number of EPC-CFUs from PB-MNCs after spinal cord injury. *, p<0.05 vs pre injury; **, p<0.05 vs day 0; †, p<0.05 vs day 7; ‡, p<0.05 vs day 14. Recruitment of BM-derived EPCs into the damaged spinal cord The spinal cord sections were immunostained for β-gal. The β-gal positive cells, which were considered to be BM-derived EPCs recruited into sites of neovascularization, were abundantly observed around the injury site at days 3, 7, 14 and 28 after SCI (Figure 2A-D). The number of β-gal positive cells markedly increased at day 7 after SCI, and then gradually decreased at days 14 and 28 (Figure 6E). The β-gal positive cells are also labeled with CD31 (Figure 6G). Although the β-gal positive cells were scattered around the injury site at day 3 after SCI, they were aligned along vessels at day 7 or later after SCI (Figure 6F-I).

E Figure 2: Immunohistochemistry for β-gal (red) at days 3 (A), 7 (B), 14 (C) and 28 (D) after injury. Nuclei were counterstained with DAPI (blue). E: The number of β-gal positive cells in the injured tissue peaked at day 7 after injury and gradually decreased at days 14 and 28. F-I: Immunohistochemical staining for CD31 (green) and β-gal (red) at days 3 (F), 7 (G), 14 (H) and 28 (I) after injury. *, p<0.05 vs day 3; **, p<0.05 vs day 14; †, p<0.05 vs day 28. Administration of exogenous EPCs for repairing injured spinal cord To clarify the contribution of EPC recruitment to histological changes in the injured spinal cord, cultured EPCs labeled with DiI-acLDL (EPC group) or PBS (PBS group) were administered systemically just after spinal cord injury. DiI positive cells indicating administered EPCs were observed around the injury site at day 3. These DiI positive cells were labeled with CD31. Immunohistochemistry revealed that immature reactive astrocytes co-labeled with GFAP and nestin were more frequently observed in EPC group compared with PBS group. Immunostaining for CD31 disclosed that enlarged blood vessels were more abundantly observed in EPC group compared to PBS group. DISCUSSION Our findings in this study suggest that bone marrow-derived EPCs may contribute to the tissue repair by augmenting neovascularization and astrogliosis following spinal cord injury.

Poster No. 0590 • ORS 2012 Annual Meeting