Butschkau Antje, Wagner Nana-Maria, Genz Berit, Vollmar Brigitte
Institute for Experimental Surgery, University Hospital Rostock, Rostock, Germany.
Clinic for Anesthesiology and Critical Care Medicine, University Hospital Rostock, Rostock, Germany.
PLoS One. 2014 Dec 4;9(12):e113554. doi: 10.1371/journal.pone.0113554. eCollection 2014.
Protein Z (PZ) is a vitamin K-dependent coagulation factor without catalytic activity. Evidence points towards PZ as an independent risk factor for the occurrence of human peripheral arterial disease. However, the role of PZ in ischemia-driven angiogenesis and vascular healing processes has not been elucidated so far.
Angiogenic potency of PZ was assessed in established in vitro assays using endothelial cells. PZ-deficient (PZ(-/-)) mice and their wild-type littermates (PZ(+/+)) were subjected to hindlimb ischemia. Furthermore, PZ(-/-) mice were exposed to PZ expressing adenovirus (AdV-PZ) or control adenovirus (AdV-GFP). In an additional set of animals, PZ(-/-) mice were exposed to AdV-PZ and AdV-GFP, each in combination with the CXCR4 antagonist AMD3100.
In vitro, PZ stimulated migratory activity and capillary-like tube formation of endothelial cells comparable to SDF-1. PZ(-/-) mice exhibited diminished hypoxia-driven neovascularization and reperfusion in post-ischemic hindlimbs, which was restored by adenoviral gene transfer up to levels seen in PZ(+/+) mice. The stimulatory impact of PZ on endothelial cells in vitro was abolished by siRNA targeting against PZ and PZ was not able to restore reduced migration after knock-down of CXCR4. The increased surface expression of CXCR4 on PZ-stimulated endothelial cells and the abrogated restoration of PZ(-/-) mice via AdV-PZ after concomitant treatment with the CXCR4 antagonist AMD3100 supports the idea that PZ mediates angiogenesis via a G-protein coupled pathway and involves the SDF-1/CXCR4 axis. This is underlined by the fact that addition of the G-protein inhibitor PTX to PZ-stimulated endothelial cells abolished the effect of PZ on capillary-like tube formation.
The results of the current study reveal a role of PZ in ischemia-induced angiogenesis, which involves a G-protein coupled pathway and a raised surface expression of CXCR4. Our findings thereby extend the involvement of PZ from the coagulation cascade to a beneficial modulation of vascular homeostasis.
蛋白Z（PZ）是一种无催化活性的维生素K依赖型凝血因子。有证据表明PZ是人类外周动脉疾病发生的独立危险因素。然而，PZ在缺血驱动的血管生成和血管愈合过程中的作用迄今尚未阐明。
使用内皮细胞在既定的体外试验中评估PZ的血管生成能力。将PZ缺陷（PZ(-/-)）小鼠及其野生型同窝小鼠（PZ(+/+)）进行后肢缺血处理。此外，将PZ(-/-)小鼠暴露于表达PZ的腺病毒（AdV-PZ）或对照腺病毒（AdV-GFP）。在另一组动物中，将PZ(-/-)小鼠分别暴露于AdV-PZ和AdV-GFP，并分别与CXCR4拮抗剂AMD3100联合使用。
在体外，PZ刺激内皮细胞的迁移活性和毛细血管样管形成，其效果与基质细胞衍生因子-1（SDF-1）相当。PZ(-/-)小鼠缺血后肢的缺氧驱动的新生血管形成和再灌注减少，通过腺病毒基因转移可将其恢复至PZ(+/+)小鼠的水平。针对PZ的小干扰RNA（siRNA）消除了PZ对体外内皮细胞的刺激作用，并且在敲低CXCR4后PZ无法恢复降低的迁移能力。PZ刺激的内皮细胞上CXCR4的表面表达增加，以及在与CXCR4拮抗剂AMD3100联合处理后，AdV-PZ对PZ(-/-)小鼠的恢复作用被消除，这支持了PZ通过G蛋白偶联途径介导血管生成并涉及SDF-1/CXCR4轴的观点。向PZ刺激的内皮细胞中添加G蛋白抑制剂百日咳毒素（PTX）消除了PZ对毛细血管样管形成的作用，这一事实进一步证明了这一点。
本研究结果揭示了PZ在缺血诱导的血管生成中的作用，这涉及G蛋白偶联途径和CXCR4表面表达的增加。我们的发现从而将PZ的作用从凝血级联扩展到对血管稳态的有益调节。
