Stromal Derived Factor-1/CXCR4 Axis Involved in Bone Marrow Mesenchymal Stem Cells Recruitment to Injured Liver

Abstract

The molecular mechanism of bone marrow mesenchymal stromal stem cells (BMSCs) mobilization and migration to the liver was poorly understood. Stromal cell-derived factor-1 (SDF-1) participates in BMSCs homing and migration into injury organs. We try to investigate the role of SDF-1 signaling in BMSCs migration towards injured liver. The expression of CXCR4 in BMSCs at mRNA level and protein level was confirmed by RT-PCR, flow cytometry, and immunocytochemistry. The SDF-1 or liver lysates induced BMSCs migration was detected by transwell inserts. CXCR4 antagonist, AMD3100, and anti-CXCR4 antibody were used to inhibit the migration. The Sprague-Dawley rat liver injury model was established by intraperitoneal injection of thioacetamide. The concentration of SDF-1 increased as modeling time extended, which was determined by ELISA method. The Dir-labeled BMSCs were injected into the liver of the rats through portal vein. The cell migration in the liver was tracked by in vivo imaging system and the fluorescent intensity was measured. In vivo, BMSCs migrated into injured liver which was partially blocked by AMD3100 or anti-CXCR4 antibody. Taken together, the results demonstrated that the migration of BMSCs was regulated by SDF-1/CXCR4 signaling which involved in BMSCs recruitment to injured liver.

Figure 1

Expression of CXCR4 in BMSCs. (a) RT-PCR results of CXCR4 expression in BMSCs from passages 1 to 8; CXCR4 was stably expressed; 293T cells as negative control; (b) immunocytochemistry results of CXCR4 expression, which indicated both membrane and intracellular CXCR4 expression; (c) the result of flow cytometry indicated only 4.2% of cells were positive for membrane CXCR4.

Figure 2

SDF-1 induces BMSCs migration in vitro. (a) SDF-1 at different concentration; the migrated cells were observed under fluorescence microscope and the graph indicated the number of migrated cells; (b) SDF-1 at 100 ng/mL; AMD3100 was added at different concentration, the migrated cells were observed and counted under fluorescence microscope and the graph indicated the number of migrated cells; (c) SDF-1 at 100 ng/mL; anti-CXCR4 antibody was added at different concentration, the migrated cells were observed and counted.

Figure 3

Injured liver lysates induce BMSCs migration in vitro. (a) Changes of serum ALT and AST level after TAA injection; (b) HE stained liver tissue sections after TAA injection; (c) the concentrations of SDF-1 of liver lysates detected by ELISA at different time point of TAA induced liver injury; (d) the migrated cells induced by normal liver lysates and TAA injured liver lysates. More cell migration was induced by TAA injured liver lysates which could be partially blocked by AMD3100 (24 μg/mL) or anti-CXCR4 antibody (20 μg/mL). The migrated cells were observed under fluorescence microscope and the graph indicated the number of migrated cells.

Figure 4

Difference between portal vein injection and tail vein injection. (a) The fluorescent intensity of whole rats; the BMSCs were injected through tail vein; (b) the fluorescent intensity of dissected livers; the BMSCs were injected through tail vein; (c) the fluorescent intensity of whole rats; the BMSCs were injected through portal vein; (d) the fluorescent intensity of dissected livers; the BMSCs were injected through portal vein; (e) Dir-labeled BMSCs before injection were as positive control; (f) rats without cell injection were as negative control; (g) the fluorescent intensity value and fold changes of the dissected livers of two groups; the difference was statistically significant.

Figure 5

BMSCs migration in injured liver inhibited by anti-CXCR4 antibody and AMD3100. (a) The fluorescent intensity of normal rats after Dir-labeled BMSCs injection; (b) the fluorescent intensity of dissected normal livers after Dir-labeled BMSCs injection; (c) the fluorescent intensity of rats with TAA induced liver injury after Dir-labeled BMSCs injection; (d) the fluorescent intensity of dissected TAA injured livers after Dir-labeled BMSCs injection; (e) the fluorescent intensity of rats with TAA induced liver injury after Dir-labeled BMSCs and AMD3100 injection at the same time; (f) the fluorescent intensity of dissected injured livers after Dir-labeled BMSCs and AMD3100 injection at the same time; (g) the fluorescent intensity of rats with TAA induced liver injury after anti-CXCR4 antibody pretreated and Dir-labeled BMSCs injection; (h) the fluorescent intensity of dissected injured livers after anti-CXCR4 antibody pretreated and Dir-labeled BMSCs injection; (i) Dir-labeled BMSCs before injection were as positive control; (j) rats without cell injection were as negative control; (k) the fluorescent intensity value and fold changes of the dissected livers of each group; the differences were statistically significant.