Fig. 3. C5aR1 deficiency promotes ischemia-induced revascularization in vivo.

WT or C5ar1^−/− mice were subjected to hindlimb ischemia and analyzed after 2 weeks. a Revascularization of the hindlimbs after femoral artery ligation was visualized by laser Doppler fluximetry (LDI). We found increased revascularization in C5ar1^−/− animals. Data are shown as the mean ± SEM (n = 7 animals per group) and are displayed as the percentage of the perfusion in the contralateral control limb. *p < 0.05. b Representative LDI images of mouse hindlimbs after femoral artery ligation illustrate increased revascularization in C5ar1^−/− animals compared with WT controls. c Vessel density in the gastrocnemius muscle of the ischemic limbs was quantified by immunofluorescence staining. Vessels were visualized by isolectin B4 (IB4 in green, nuclei in blue), and images of whole-muscle sections were acquired as tile scans and analyzed. At 14 days after the induction of ischemia, C5ar1^−/− mice exhibited a significantly higher vessel density than WT controls. ×200 magnification, scale bars represent 200 µm. d Quantification of the IB4-positive area fraction in the muscle sections reveals higher vessel abundance in ischemic C5ar1^−/− hindlimbs. Data are shown as the mean ± SEM (n = 10 whole-muscle sections per group) and are displayed as the percentage of control. The IB4-positive area fraction in WT control hindlimbs represents 100%. *p < 0.001. e Furthermore, WT or C5ar1^−/− mice were subjected to hindlimb ischemia and platelets were depleted systemically by injection of platelet depleting serum starting on the first day post induction of ischemia. We could not detect significant differences (p < 0.05) in revascularization. Data are shown as the mean ± SEM (n = 7 animals per group) and are displayed as the percentage of the perfusion in the contralateral control limb. f Vessel density in the gastrocnemius muscle of the ischemic limbs was quantified by immunofluorescence staining as described in c. At 14 days after the induction of ischemia, WT and C5ar1^−/− mice with platelet depletion did not exhibit significant differences in vessel density. ×200 magnification, scale bars represent 200 µm. Image is representative of 10 whole-muscle sections per group analyzed. Two-way ANOVA with Bonferroni’s post hoc test in a, e. Two-sided Student’s t test in d.