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Basic science: Experiment research
e0193 A critical role of ckit in CXCR4-mediated progenitor cell niche maintenance and mobilisation
  1. Cheng Min,
  2. Zeng Qiutang,
  3. Losordo Douglas
  1. Wuhan Union Hospital

Abstract

Therapeutic mobilisation of bone marrow progenitor cells (BM PCs) is a novel strategy for cardiovascular repair. Both CXCR4 antagonism and c-kit blockade can rapidly and potently mobilise BM PCs; however, the functional interaction between CXCR4 and c-kit remains unclear. We treated c-kit-deficient (c-kit W/W-V) and wild-type (WT) mice with CXCR4 antagonist AMD3100 and evaluated PCs in the peripheral blood (PB) with a colony-forming assay. AMD3100 treatment for 2 h dramatically increased the number of PCs in the PB in WT mice but not in c-kit W/W-V mice (c-kitW/W-V vs WT: 30 vs 194 colonies/ml blood, p<0.01). To confirm that c-kit deficiency impairs BM PC mobilisation by AMD3100, we developed an in vivo BM niche clearance/occupation assay. The c-kit W/W-V and WT mice were firstly treated with AMD3100 to remove PCs from the niche, and 2 h later, transplanted with eGFP transgenic BM cells to competitively occupy the niche. After 3 h, BM cells were isolated and analysed by FACS. Despite the total donor-derived (eGFP+) cells were similar between WT and c-kit W/W-V recipients, the donor-derived CXCR4-expressing PCs, including eGFP+CXCR4+Lin-, eGFP+CXCR4+Lin-Sca1+, and eGFP+CXCR4+Lin-ckit+ cells, were much fewer in the c-kit W/W-V mice (c-kitW/W-V vs WT: 19.7% vs 30.6%, p<0.01; 20.3% vs 29.1%, p<0.05; and 6.7% vs 17.9%, p<0.001, respectively), indicating that c-kit deficiency specifically reduced the capacity of AMD3100 to clear the CXCR4+ PC niche. To better understand the mechanisms, we designed an ex vivo adhesion assay. Mouse BM mononuclear cells were isolated and applied onto plates pre-coated with BM stromal protein VCAM-1, followed by addition of AMD3100. The adhesion of BM cells to VCAM-1 resulted in marked c-kit phosphorylation. Interestingly, AMD3100 significantly attenuated the c-kit phosphorylation. In vivo, AMD3100 treatment for 15 min significantly reduced the level of phospho-c-kit in the BM as assessed by Western blotting of the BM lysates. Consistently, immunofluorescence staining of BM niche demonstrated a significantly lower ratio of phospho-ckit+/total ckit+ PCs in AMD3100-treated mice as compared to PBS-treated mice. We conclude that c-kit plays a critical role in CXCR4-mediated BM PC niche maintenance and mobilisation.

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