CXCR4-dependent tissue remodeling after experimental stroke

CXCR4趋化因子受体在造血和神经干细胞中表达。受体引导来自这些干细胞的白细胞和前体细胞的迁移。同样，一种CXCR4拮抗剂（plerixafor, AMD3100）最近被批准用于造血干细胞动员。CXCR4在肿瘤、炎症和再生过程中的功能被深入研究。在这种情况下，有报道称cxcr4介导的趋化因子CXCL12的信号引导干细胞和炎症细胞向受损组织移动。因此，CXCR4被认为参与组织重塑、新生血管形成和肿瘤形成。然而，要对这些过程进行适当的研究，需要对表达cxcr4的干细胞发育的组织进行明确的鉴定。此外，需要对CXCR4进行可控的失活。由于缺乏CXCL12和cxcr4的小鼠会在围产期死亡，因此有条件的方法是必要的。因此，申请人产生了含有cxcr4敲除/ creer敲除等位基因（Cxcr4CreER）和已建立的cre报告基因的小鼠。在这些小鼠中，他莫昔芬诱导荧光蛋白TdTomato在具有活性Cxcr4启动子的细胞中永久表达。当Cxcr4CreER等位基因与Cxcr4LoxP等位基因结合时，Cxcr4- creer允许Cxcr4消融。对Cxcr4CreER/WT和Cxcr4CreER/LoxP小鼠的全面表征表明，对tdtomo阳性细胞的分析允许在Cxcr4CreER/WT存在和Cxcr4CreER/LoxP缺失的情况下，追踪表达CXCR4的干细胞的谱系。申请人计划使用该模型评估CXCR4在实验性脑卒中后重塑过程中的功能（单核细胞、中性粒细胞和未成熟神经元的募集、受损区域的胶质反应和新生血管形成）。在第二个重点中，干细胞将在体内用Cxcr4-CreER标记，在体外繁殖和再植入过程中分离和表征。该项目将阐明CXCR4是否是卒中中合适的药物靶点的问题，并严格验证CXCR4在缺血性病变重塑过程中指导干细胞的概念。

CXCR4 chemokine receptors are expressed in hematopoietic and neural stem cells. The receptor guides migration of leukocytes and precursor cells that are derived from these stem cells. Consistently, a CXCR4 antagonist (plerixafor, AMD3100) was recently approved for hematopoietic stem cell mobilization. Functions of CXCR4 in neoplastic, inflammatory and regenerative processes are intensively investigated. In this context, it was reported that the CXCR4-mediated signal of the chemokine CXCL12 directs stem cells and inflammatory cells towards lesioned tissues. Accordingly, CXCR4 is thought to be involved in tissue remodeling, neovascularization and tumor formation. However, proper investigation of these processes requires non-ambiguous identification of tissues that develop from Cxcr4-expressing stem cells. In addition, controllable inactivation of CXCR4 is desirable. Since CXCL12- and CXCR4-deficient mice die perinatally, a conditional approach is necessary. The applicant thus generated mice harboring a Cxcr4-knockout/CreER-knockin allele (Cxcr4CreER) and an established Cre-reporter. In these mice, tamoxifen induces permanent expression of the fluorescent protein TdTomato in cells with an active Cxcr4 promoter. When the Cxcr4CreER allele is combined with a Cxcr4LoxP allele, Cxcr4-CreER permits Cxcr4 ablation. Thorough characterization of Cxcr4CreER/WT and Cxcr4CreER/LoxP mice showed that analysis of TdTomato-positive cells permits to trace the lineage of Cxcr4-expressing stem cells in the presence (Cxcr4CreER/WT) and in the absence (Cxcr4CreER/LoxP) of functional CXCR4. The applicant plans to use this model to assess the function of CXCR4 in remodeling processes after experimental stroke (recruitment of monocytes, neutrophils and immature neurons, glial reaction and neovascularization in the lesioned area). In the second focus, stem cells will be in vivo-labeled with Cxcr4-CreER, isolated and characterized during in vitro propagation and reimplantation. The project will shed light on the question if CXCR4 is a suitable drug target in stroke and rigorously test the concept that CXCR4 directs stem cells during remodeling of ischemic lesions.