Protein Z Exerts Pro-Angiogenic Effects and Upregulates CXCR4

<div><p>Objective</p><p>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.</p><p>Approach</p><p>Angiogenic potency of PZ was assessed in established <i>in vitro</i> 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.</p><p>Results</p><p><i>In vitro</i>, 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 <i>in vitro</i> 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.</p><p>Conclusions</p><p>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.</p></div

PZ upregulates CXCR4 surface expression on endothelial cells <i>in vitro</i> and mediates its angiogenic effects via CXCR4 <i>in vivo</i>.

<p>Flow cytometric analysis of CXCR4 surface expression on HUVECs after 8 and 24 hours of incubation with PZ (red line), displayed in representative histograms (<b>A</b> and <b>C</b>). Quantitative analysis (<b>B</b> and <b>D</b>) of CXCR4 expression. Stimulation with PZ resulted in an 1.4-fold (B) and almost 2-fold (D) increase of CXCR4 expression vs. unstimulated cells (ctrl, black line). Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks; p<0.05; $vs. ctrl; n = 4–8 independent experiments. ANOVA on ranks; p<0.05;$ vs. ctrl; n = 4–8 independent experiments. <b>E</b>, Representative images of confocal laser scanning microscopy of HUVECs stimulated for 8 or 24 hours with SDF-1 (50 ng/ml) or PZ (3 µg/ml), displaying an increased surface expression of CXCR4 after stimulation with both substances. <b>F</b>, Representative pictures of thermal imaging of mice hindlimbs. <b>G</b>, Quantitative summary of pad temperature differences pre OP, post OP and on POD 21. In both groups treated with AMD3100 no significant change in temperature difference on POD 21 was detectable. Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks repeated measures; * p<0.05 vs. pre OP; n = 3–6. <b>H</b>, Representative pictures of M. gastrocnemius after immunofluorescent staining for CD31 (red) or cell nuclei (DAPI, blue). <b>I</b>, Quantitative summary of enumbered CD31/DAPI double positive cells revealed no significant increase after induction of ischemia in both groups. Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks; n = 3–6.</p

PZ in ischemia-induced neovascularization <i>in vivo</i>.

<p><b>A</b>, Representative pictures of thermal imaging of mice hindlimbs. <b>B</b>, Quantitative summary of pad temperature differences. On POD 21 PZ^+/+ mice showed a significant reduction in temperature difference compared to immediately after induction of ischemia and significant lower temperature difference to PZ^−/− mice, which offered no changes. Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks repeated measures; * p<0.05 vs. pre OP, § p<0.05 vs. post OP, # p<0.05 vs. other genotype on POD 21, n = 11–16. <b>C</b>, After 21 days, PZ^−/− mice treated with AdV-PZ showed significant reduction in temperature difference in the ischemic hindlimb. Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks repeated measures; * p<0.05 vs. pre OP, § p<0.05 vs. post OP; n = 10. <b>D</b>, Representative pictures of M. gastrocnemius muscle tissue after immunofluorescent staining for CD31 (red) or cell nuclei (DAPI, blue). Intracardiac injection of an endothelial specific lectin confirmed functional perfusion of CD31/DAPI double positive capillaries (green). <b>E</b>, PZ^+/+ mice exhibited significant higher density of CD31/DAPI-double positive capillaries per square millimeter of ischemic M. gastrocnemius tissue compared to the non-ischemic PZ^+/+ muscle tissue. In muscle tissue of PZ^−/− mice, ischemia did not result in remarkable neovascularization. The number of CD31/DAPI double-positive cells in ischemic PZ^−/− mice M. gastrocnemius was even significantly lower compared to ischemic PZ^+/+ mice. Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks; & p<0.05 vs. non-ischemic, # p<0.05 vs. other genotype with ischemia; n = 11–16. <b>F</b>, After exposure of PZ^−/− mice to AdV-PZ, the compromised angiogenic phenotype of PZ^−/− mice after induction of ischemia was significantly reversed. Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks; & p<0.05 vs. non-ischemic, # p<0.05 vs. AdV-GFP with ischemia, n = 10.</p

PZ promotes the migration and capillary-like tube formation of endothelial cells <i>in vitro</i>.

<p><b>A</b>, Representative images of scratch-wound closures after 8 and 24 hours of incubation with SDF-1 or PZ, ctrl corresponds to untreated cells. 100-fold magnification. Exposure of endothelial cells to PZ (3 µg/ml) led to a significantly higher wound closure after 8 (<b>B</b>) and 24 hours (<b>C</b>) comparable to stimulation with SDF-1 (50 ng/ml). Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks; $p<0.05 vs. ctrl; n = 4–7 independent experiments. <b>D</b>, Representative images of tubular networks after 8 hours of incubation in matrigel angiogenesis assay in the presence of SDF-1 (50 ng/ml), PZ (3 µg/ml), PZ+PTX and PTX alone (100 ng/ml), ctrl are untreated cells. 100-fold magnification. <b>E</b>, HUVECs showed significantly enhanced formation of capillary-like tubular structures on Matrigel when incubated with SDF-1 and PZ compared with ctrl. Coincubation of endothelial cells with PZ and PTX abolished the PZ-mediated increase in tube formation, while incubation with PTX alone had no effect on tube formation. Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks;$ p<0.05 vs. ctrl; ß p<0.05 vs. PZ; n = 6 independent experiments.</p

Targeting PZ and CXCR4 with siRNA results in decreased migratory capacity and endothelial tube formation <i>in vitro</i>.

<p>PZ and CXCR4 knock-down in unstimulated HUVECs as well as CXCR4 knock-down in PZ and SDF-1 stimulated HUVECs resulted in migration capacity comparable to that of ctrl after both 8 (<b>A</b>) and 24 hours (<b>B</b>). Data are given as box plots indicating the median with the 25^th and 75^th percentiles. Ø corresponds to unstimulated cells. ANOVA on ranks; $p<0.05 vs. ctrl; n = 4 independent experiments. Representative images (<b>C</b>) and quantitative analysis (<b>D</b>) of capillary-like tube formation. HUVECs targeted with siRNA against PZ or CXCR4 showed significantly reduced capacity for capillary-like tube formation compared to ctrl. Addition of PZ or SDF-1 to CXCR4 siRNA targeted cells failed to restore endothelial tube formation. Data are given as box plots indicating the median with the 25^th and 75^th percentiles. Ø corresponds to unstimulated cells. ANOVA on ranks;$ p<0.05 vs. ctrl; n = 4 independent experiments.</p