In the present study, we demonstrated that (1) AMD3100-treatment could mobilize endogenous EPCs to peripheral blood (PB) of rats after pMCAO, and continuous AMD3100-treatment mobilized more endogenous EPCs to PB than single AMD3100-treatment. (2) Single AMD3100 treatment in an early phase rather than continuous AMD3100 treatment could recruit endogenous EPCs in IBZ after pMCAO. (3) Single AMD3100 administration in an early phase attenuated infarct volume and improved neurological outcome. (4) a single AMD3100 administration in later stage (on day 14 ) could still mobilize a little more endogenous EPCs to the PB compared with saline-treatment, but had no effects on neurological outcome and could not recruit endogenous EPCs to IBZ after pMCAO. (5) the SDF-1/CXCR4 axis could be modulated differently in single and continuous AMD3100-treatment groups. Together, these observations highlighted the importance of endogenous EPCs mobilization and recruitment in IBZ after pMCAO and suggested that enhanced recruitment of endogenous EPCs via a single AMD3100-treatment in an early phase has potential to be an alternative for the treatment of stroke.
Formation of new blood vessels, either angiogenesis or vasculogenesis, plays an important part in neovascularization and regeneration after stroke(24). Bone marrow-derived endothelial progenitor cell (EPC) is angioblast that is believed to take part in the formation of the new blood vessels in cardiovascular diseases(25). Stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, play important roles in stem cell homing, chemotaxis, modulating the expression of adhesion molecules, engraftment, proliferation, and cell survival(26). Intravenously transplanted hEPC was capable of homing into ischemic areas of brain, promoting angiogenesis, and improving neurobehavioral outcome in MCAO mice(27). Moreover, studies have shown that SDF-1 is expressed along the ischemic boundary zone of the brain and facilitates the migration of transplanted cells into the ischemic zone(28).
Previous studies have documented that most of EPCs expressed CXCR4 receptors(27) ,increased SDF-1 could, by combining with CXCR4 receptors, mobilize EPC from BM to PB and circulating EPC homed into ischemic area(27, 29). AMD3100 is a highly selective CXCR4 receptor antagonist and can rapidly mobilize stem cells from the BM to PM by reversibly blocking the interactions between SDF-1 and CXCR4(30). Moreover, AMD3100 can also suppress the recruitment of stem cells to the area expressing SDF-1 by blocking CXCR4(29). The plasma half-life of AMD3100 is 2–3 hours(29). Also AMD3100 is known to mobilize bone marrow derived stem cells (BMCs) in high concentrations, some low concentrations can block CXCR4 without stimulating mobilization(21).
In the current study, we determined how AMD3100, a CXCR4 antagonist could intervene endogenous EPCs in rats after pMCAO and whether AMD3100-treatment could improve prognosis of rats after pMCAO or not. The results presented here indicate that a single AMD3100-treatment in an early phase could mobilize endogenous EPCs to peripheral blood (PB) and recruit them in IBZ of brain after pMCAO, attenuating infarct volume and improving prognosis of rats after pMCAO. In addition, continuous AMD3100-treatment could mobilize more endogenous EPCs to the PB, but could not recruit endogenous EPCs to IBZ after pMCAO and had worse prognosis than a single AMD3100-treatment.
The findings presented here raise questions about the mechanism responsible for the determinant of endogenous EPCs recruitment in IBZ in rat brain after pMCAO. We hypothesized that the SDF-1/CXCR4 axis was modulated differently in single and continuous AMD3100-treatment groups. Then, we detected SDF-1 expression in IBZ in ischemic rats dynamically via real-time PCR. The data demonstrated that SDF-1 relative expression in IBZ peaked from day1 to day 3, then decreased on day 7, and had a further decrease on day 14, which were all significantly greater than in sham-operate group. Previous studies observed that on day 7, after MCAO, SDF-1 protein expression was significantly upregulated in the injured hemisphere of PBS-treated rats via Immunofluorescence staining, especially in the penumbral regions and in addition, Western blot analysis of the SDF-1 protein further confirmed this result(30).
The underlying mechanism might be that, after pMCAO, a single AMD3100 administration in an early phase first mobilized endogenous EPCs from BM to PB, then AMD3100 was degraded, and mobilized circulating EPCs were recruited to the IBZ by chemotaxis of SDF-1, which presented in a high level within 7 days. However, when AMD3100 was continuously injected, endogenous EPCs mobilized to the PB were inhibited by combining with injected AMD3100 through CXCR4 receptors of themselves before recruitment in IBZ.
The benefits associated with a single injection of AMD3100 in an early phase after pMCAO were not observed with AMD3100 treatment in later phase. Instead, the number of circulating EPCs in AMD3100 treatment group was just about 0.05% higher in AMD3100 treatment group on day 21 after pMCAO. What’s more, recruited EPCs in IBZ in AMD3100 treatment group did not increase, and the prognosis of rats did not improve. There were several possible reasons. Firstly, though circulating EPCs were mobilized in AMD3100 administration group in later phase, the number of EPCs (0.11 ± 0.03%) was too few to be recruited in IBZ. Secondly, the expression of SDF-1 in ischemic area had decreased in later phase so that the level of SDF-1 at day 21 may be not enough to function with EPCs. Thirdly, in later phase, blood brain barrier (BBB) around IBZ was almost repaired, and it was difficult for endogenous EPCs to be recruited in IBZ from the PB. Thus, in later phase after pMCAO, mobilized EPCs could not be recruited in IBZ and could not improve prognosis of rats after pMCAO.
Yang et al demonstrated that AMD3100 significantly attenuated leukocyte accumulation and infiltration into the infarct perifocal region, and effectively reduced the level of proinflammatory cytokines in the ischemic brain tissue. The dose of AMD3100 in his study was 1 mg/kg/day, which is sufficient for blocking CXCR4 without causing stem cell mobilization. Because AMD3100 is known to mobilize bone marrow derived stem cells (BMCs) in high concentrations(21). In the present study, the dose of AMD3100 we chose was 3 mg/kg/day, which could also cause stem cell mobilization. We attributed the benefits from AMD3100 treatment to the increase of endogenous EPCs mobilization and recruitment in IBZ after pMCAO, but whether inhibition inflammation effects caused by AMD3100 participated partially in the improvement after pMCAO needs further research. In addition, whether there are other mechanisms playing a role in these associated benefits, like increasing NPCs(31), caused by AMD3100 are still not known.