Wagner Nana-Maria, Bierhansl Laura, Butschkau Antje, Noeldge-Schomburg Gabriele, Roesner Jan Patrick, Vollmar Brigitte
Clinic for Anesthesiology and Critical Care Medicine, University Hospital Rostock Rostock, Germany.
Int J Clin Exp Pathol. 2013 Nov 15;6(12):2813-23. eCollection 2013.
Toll-like receptor 2 (TLR2)-deficiency is associated with the preservation of vascular function and TLR2-deficient (TLR2(-/-)) mice exhibit increased neovascularization following induction of hindlimb ischemia. Hematopoietic stem cells play an important role in ischemia-induced angiogenesis and we now investigated whether the effects observed in TLR2(-/-) mice may be attributed to TLR2 deficiency on bone marrow-derived stem cells.
cKit-positive (cKit(+)) bone marrow cells (BMC) were isolated from wild type (WT) and TLR2(-/-) mice employing MACS-bead technology. Co-incubation of TLR2(-/-)cKit(+) BMC with mature endothelial cells (ECs) resulted in increased tube formation of ECs on matrigel, augmented sprouting in a 3D-collagen matrix and increased migratory capacity compared to co-incubation with WT cKit(+) BMC. In an in vivo matrigel plug assay, TLR2(-/-)cKit(+) BMC exhibited enhanced formation of capillary-like networks. In a murine model of hindlimb ischemia, administration of TLR2(-/-) cKit(+) BMC to WT mice augmented capillary density and reperfusion of ischemic M. gastrocnemius muscle tissue to the level of TLR2(-/-) mice. Western Blot analysis revealed comparable expression of CXCR4 on TLR2(-/-)cKit(+) BMC but increased activation of the PI3K downstream signaling molecule protein kinase B (PKB/AKT) compared to WT cKit(+) cells.
The absence of TLR2 on cKit(+) BMC is associated with augmented potency to support angiogenic processes in vitro and in vivo. Functional inhibition of TLR2 may therefore provide a novel tool to enhance stem cell function for the treatment of vascular diseases.
Toll样受体2（TLR2）缺陷与血管功能的保留相关，且TLR2缺陷（TLR2(-/-)）小鼠在诱导后肢缺血后表现出新生血管形成增加。造血干细胞在缺血诱导的血管生成中起重要作用，我们现在研究在TLR2(-/-)小鼠中观察到的效应是否可归因于骨髓来源干细胞上的TLR2缺陷。
采用MACS磁珠技术从野生型（WT）和TLR2(-/-)小鼠中分离cKit阳性（cKit(+)）骨髓细胞（BMC）。与成熟内皮细胞（ECs）共孵育时，与WT cKit(+) BMC共孵育相比，TLR2(-/-)cKit(+) BMC导致ECs在基质胶上的管形成增加、在三维胶原基质中的芽生增强以及迁移能力增加。在体内基质胶栓试验中，TLR2(-/-)cKit(+) BMC表现出毛细血管样网络形成增强。在小鼠后肢缺血模型中，向WT小鼠施用TLR2(-/-) cKit(+) BMC可使缺血的腓肠肌组织的毛细血管密度和再灌注增加至TLR2(-/-)小鼠的水平。蛋白质印迹分析显示，与WT cKit(+)细胞相比，TLR2(-/-)cKit(+) BMC上CXCR4的表达相当，但PI3K下游信号分子蛋白激酶B（PKB/AKT）的激活增加。
cKit(+) BMC上TLR2的缺失与在体外和体内支持血管生成过程的能力增强相关。因此，TLR2的功能抑制可能为增强干细胞功能以治疗血管疾病提供一种新工具。
