Role of CXCR4-FAK signaling pathway in migration and adhesion of hypoxia-preconditioned bone marrow mesenchymal stem cells towards damaged tissues resulting from spinal cord ischemia-reperfusion injury in rats

Abstract

Objective To investigate the role of CXC chemokine receptor 4-focal adhesion kinase (CXCR4-FAK) signaling pathway in migration and adhesion of hypoxia-preconditioned bone marrow mesenchymal stem cells (BMSCs) towards damaged tissues resulting from spinal cord ischemia-reperfusion (I/R) injury in rats.Methods Part I Rat BMSCs transfected with recombinant adenovirus-mediated green fluorescent protein gene 3 were seeded in 24-well plates and randomly divided into 5 groups (n =18 wells each):control group (group C),normoxia-incubated group (group N),HP group (group H),HP + CXCR4 antagonist AMD3100 group (group HA) and HP + FAK inhibitor FAK-related nonkinase group (group HF).In group C,BMSCs were incubated in DMEM culture medium.In group N,BMSCs were exposed to 21％ O2-74％ N2-5％ CO2 for 36 h.In group H,BMSCs were exposed to 0.5％O2-94.5％ N2-5.0％CO2 for 24 h followed by 12 h exposure to normoxia.In groups HA and HF,5 μg/ml AMD3100 and 10 μg/ml FAK-related nonkinase were added to the culture medium before HP,respectively.The expression of stromal derived factor-1α (SDF-1α),CXCR4 and phosphorylated FAK (p-FAK) in BMSCs was determined by Western blot.The migratory capability and adhesive ability of BMSCs were measured by Transwell invasive assay and fibronectin adhesive assay,respectively.Part Ⅱ Two hundred and sixteen male Sprague-Dawley rats weighing 300-350 g were used and 210 out of the 216 rats underwent spinal cord ischemia by occlusion of the thoracic aorta combined with controlled hypotension.Thirty-six rats were chosen and sacrificed before spinal cord I/R and at 12 h and 1,3,5 and 7 days of reperfusion (T0-5) and the lumbar segment of spinal cord was removed for detection of the content of SDF-1α.The left 180 rats with spinal cord I/R were randomly divided into 5 groups (n =36 each).IT DMEM medium 300 μl was injected in group C and BMSC suspension 300 μl (1 × 106/ml) was injected in groups N,H,HA and HF immediately after onset of reperfusion.Neurological function was scored at T0-5.The animals were then sacrificed and the lumbar segment of spinal cord was removed for detection of the degree of BMSC aggregation.Results There was no significant difference in the expression of SDF-1α,CXCR4 and p-FAK,migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation between groups C and N (P ＞ 0.05).Compared with group N,the expression of SDF-1α,CXCR4 and p-FAK was significantly up-regulated,and migratory capability and adhesive ability of BMSCs,neurological function scores and the degree of BMSC aggregation were increased in group H,while no significant change was found in the expression of p-FAK,migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation in HA and HF groups (P ＞ 0.05).Compared with group H,the expression of p-FAK was down-regulated and the migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation were decreased in groups HA and HF (P ＜0.05).The content of SDF-1α was significantly higher at T2,3 than at T0.Conclusion HP can promote migration and adhesion of BMSCs towards damaged tissues resulting from spinal cord I/R injury through CXCR4-FAK signaling pathway in rats; thus CXCR4-FAK signal pathway provides the protective effect on spinal cord. Key words: Receptors, CXCR4; Focal adhesion protein-tyrosine kinases; Mesenchymal stem cell transplantation ; Cell Hypoxia ; Ischemic preconditioning ; Cell movement; Cell adhesion; Spinal cord ; Reperfusion injury