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Figure 4 - Protein Z Exerts Pro-Angiogenic Effects and Upregulates CXCR4

Figure 4.  PZ upregulates CXCR4 surface expression on endothelial cells in vitro and mediates its angiogenic effects via CXCR4 in vivo.
Flow cytometric analysis of CXCR4 surface expression on HUVECs after 8 and 24 hours of incubation with PZ (red line), displayed in representative histograms (A and C). Quantitative analysis (B and D) 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 25th and 75th 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. E, 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. F, Representative pictures of thermal imaging of mice hindlimbs. G, 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 25th and 75th percentiles. ANOVA on ranks repeated measures; * p<0.05 vs. pre OP; n = 3–6. H, Representative pictures of M. gastrocnemius after immunofluorescent staining for CD31 (red) or cell nuclei (DAPI, blue). I, 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 25th and 75th percentiles. ANOVA on ranks; n = 3–6.
PZ upregulates CXCR4 surface expression on endothelial cells in vitro and mediates its angiogenic effects via CXCR4 in vivo. Flow cytometric analysis of CXCR4 surface expression on HUVECs after 8 and 24 hours of incubation with PZ (red line), displayed in representative histograms (A and C). Quantitative analysis (B and D) 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 25th and 75th 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. E, 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. F, Representative pictures of thermal imaging of mice hindlimbs. G, 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 25th and 75th percentiles. ANOVA on ranks repeated measures; * p<0.05 vs. pre OP; n = 3–6. H, Representative pictures of M. gastrocnemius after immunofluorescent staining for CD31 (red) or cell nuclei (DAPI, blue). I, 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 25th and 75th percentiles. ANOVA on ranks; n = 3–6.
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