Nitric oxide donor upregulation of stromal cell-derived factor-1/chemokine (CXC motif) receptor 4 enhances bone marrow stromal cell migration into ischemic brain after stroke

Abstract

Stromal cell-derived factor-1 (SDF1) and its chemokine (CXC motif) receptor 4 (CXCR4), along with matrix metalloproteinases (MMPs), regulate bone marrow stromal cell (BMSC) migration. We tested the hypothesis that a nitric oxide donor, DETA-NONOate, increases endogenous ischemic brain SDF1 and BMSC CXCR4 and MMP9 expression, which promotes BMSC migration into ischemic brain and thereby enhances functional outcome after stroke. C57BL/6J mice were subjected to middle cerebral artery occlusion (MCAo), and 24 hours later, the following were intravenously administered (n = 9 mice per group): (a) phosphate-buffered saline; (b) BMSCs (5 x 10(5)); (c) 0.4 mg/kg DETA-NONOate; (d) combination of CXCR4-inhibition BMSCs with DETA-NONOate; and (e) combination of BMSCs with DETA-NONOate. To elucidate the mechanisms underlying combination-enhanced BMSC migration, transwell cocultures of BMSC with mouse brain endothelial cells (MBECs) or astrocytes were performed. Combination treatment significantly improved functional outcome after stroke compared with BMSC monotherapy and MCAo control, and it increased SDF1 expression in the ischemic brain compared with DETA-NONOate monotherapy and MCAo control. The number of BMSCs in the ischemic brain was significantly increased after combination BMSC with DETA-NONOate treatment compared with monotherapy with BMSCs. The number of engrafted BMSCs was significantly correlated with functional outcome after stroke. DETA-NONOate significantly increased BMSC CXCR4 and MMP9 expression and promoted BMSC adhesion and migration to MBECs and astrocytes compared with nontreatment BMSCs. Inhibition of CXCR4 or MMPs in BMSCs significantly decreased DETA-NONOate-induced BMSC adhesion and migration. Our data demonstrate that DETA-NONOate enhanced the therapeutic potency of BMSCs, possibly via upregulation of SDF1/CXCR4 and MMP pathways, and increased BMSC engraftment into the ischemic brain.

Figure 1

Combination DETA-NONOate with BMSC treatment of stroke increases BMSC engraftment into the ischemic brain and improves functional recovery in mice after stroke. (A): mNSS test. (B): Foot-fault test. (C–E): H&E staining showing immunostaining for BrdU-positive BMSCs in BMSC-alone treatment ([C], arrow), combination treatment with chemokine (CXC motif) receptor 4-inhibition BMSCs ([D], arrow), and combination treatment with normal BMSCs ([E], arrow). (F): Quantitative data of BrdU-immunoreactive BMSCs. Scale bar = 50 μm. n = 9 mice per group. Abbreviations: BMSC, bone marrow stromal cell; BrdU, 5-bromo-2′-deoxyuridine; MCAo, middle cerebral artery occlusion; mNSS, modified neurological severity score.

Figure 2

SDF1 expression in the ischemic brain. (A–E): SDF1 immunohistochemical expression in the ischemic boundary zone at 14 days after MCAo. (A): MCAo control; (B): BMSC treatment; (C): DETA-NONOate-alone treatment; (D): combination treatment with chemokine (CXC motif) receptor 4-inhibition BMSCs; (E): combination treatment with normal BMSCs. (F): Quantitative SDF1 data. (G–I): Images of double immunofluorescent staining of SDF1 (G) with GFAP (H). (J): SDF1 gene expression measured by real-time PCR. (K, L): Confocal images of double immunofluorescent staining of SDF1 ([K1–K4]: 0.2 μm/layer; [K]: merged from [K1–K4]) with vWF ([L1–L4]: 0.2 μm/layer; [L]: merged from [L1–L4]). (M): Merged image from (K, L). Scale bars = 100 μm (B), 50 μm (I). n = 9 mice per group. Abbreviations: BMSC, bone marrow stromal cell; GFAP, glial fibrillary acidic protein; MCAo, middle cerebral artery occlusion; SDF1, stromal cell-derived factor-1; vWF, von Willebrand factor.

Figure 3

DETA-NONOate treatment increases BMSC CXCR4 and MMP9 gene and protein expression in vitro. (A, B): CXCR4 immunostaining in nontreatment Con (A) and DETA-NONOate treatment BMSCs (B). (C, D): MMP9 immunostaining in nontreatment Con (C) and DETA-NONOate treatment BMSCs (D). (E, H): Quantitative data of CXCR4-positive (E) and MMP9-positive (H) cell expression. (F, I): CXCR4 (F) and MMP9 (I) gene expression measured by real-time PCR. (G): CXCR4-positive cell expression measured by FACS. (J): MMP9 secretion measured by zymography (n = 3 times per group). Scale bar = 50 μm. Abbreviations: Con, control; CXCR4, chemokine (CXC motif) receptor 4; FACS, fluorescence-activated cell sorting; MMP9, matrix metalloproteinase 9.

Figure 4

Stromal cell-derived factor-1/chemokine (CXC motif) receptor 4 axis and matrix metalloproteinase mediate DETA-NONOate-induced BMSC adhesion and migration in vitro. (A, B): BMSC adhesion assay. Coculture BMSCs with MBECs and astrocytes. BMSC adhesion to MBECs (A) and astrocytes (B) (n = 6 wells per group). (C–E): BMSC migration assay. Culture BMSCs alone or coculture BMSCs with MBECs and astrocytes were used. Shown are BMSC migration alone (C), BMSC migration to MBECs (D), and BMSC migration to astrocytes (E) (n = 4 wells per group). Abbreviations: BMSC, bone marrow stromal cell; MBEC, mouse brain endothelial cell; SDF1, stromal cell-derived factor-1.