Protein Z Exerts Pro-Angiogenic Effects and Upregulates CXCR4

OBJECTIVE 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. APPROACH 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. RESULTS 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. CONCLUSIONS 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.