Figure 2.
The platelet C5aR1 modulates ischemia-driven vessel growth. (A) WT, C5−/− or C5aR1−/− mice were subjected to HLI and analyzed after 2 weeks, and revascularization of the hindlimbs after femoral artery ligation was visualized by laser doppler fluximetry (LDI) under standardized conditions with the scale adapted to the experiment. Representative LDI images of mouse hind limbs after femoral artery ligation illustrate increased revascularization in C5aR1−/− animals compared with WT controls or C5−/− mice. (B) We found increased revascularization in C5aR1−/− animals in comparison to WT or C5−/− mice. Data are shown as the mean ± SEM (n = 5-8 animals per group) and are displayed as % of the perfusion in the contralateral control limb. ∗P < .05. (C) Representative images depicting capillaries (stained by IB4) and pericyte coverage (colocalization of IB4 and NG2) in the analyzed genotypes 14 days after induction of ischemia. (D) At day 14, there was no significant increase in capillary density in C5aR1−/− mice. Data represent the mean ± SEM. n = 9 to 12. (E) An increase in pericyte coverage was observable in C5aR1−/− mice compared with WT and C5−/− mice at day 14. Data represent mean ± SEM. n = 9 to 15. ∗∗P < .01, ∗∗∗P < .001. (F) Arteriolar abundance was highest in C5aR1−/− mice. There was an increase in arteries in ischemic hind limb muscle tissue compared with control tissue, which was significant for WT and C5aR1−/− mice but not for C5−/− mice. Data represent mean ± SEM. n = 6 to 9. ∗P < .05. (G) When WT or C5aR1−/− mice subjected to HLI were additionally treated with heparin, we observed no difference in revascularization anymore. Data are shown as the mean ± SEM (n = 6-8 animals per group) and are displayed as % of the perfusion in the contralateral control limb. (H) Furthermore, WT or C5aR1−/− mice were subjected to HLI, and platelets were depleted systemically by injection of platelet-depleting serum starting on the first day after induction of ischemia. This scheme induces persistent and pronounced platelet depletion, as verified previously.23 Vessel density in the gastrocnemius muscle of the ischemic limbs was quantified by immunofluorescence staining. The 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. Fourteen days after the induction of ischemia, C5aR1−/− mice additionally treated with platelet-depleting serum exhibited no significant difference in comparison to WT mice. Images 400× original magnification, scale bars represent 10 μm. (I) Quantification of vessel core numbers per area at day 14 in WT and C5aR1−/− mice, which received platelet-depleting serum over 2 weeks, revealed no significant difference. Data represent the mean ± SEM. n = 7 to 8. n.s. = no statistically significant difference was observed.

The platelet C5aR1 modulates ischemia-driven vessel growth. (A) WT, C5−/− or C5aR1−/− mice were subjected to HLI and analyzed after 2 weeks, and revascularization of the hindlimbs after femoral artery ligation was visualized by laser doppler fluximetry (LDI) under standardized conditions with the scale adapted to the experiment. Representative LDI images of mouse hind limbs after femoral artery ligation illustrate increased revascularization in C5aR1−/− animals compared with WT controls or C5−/− mice. (B) We found increased revascularization in C5aR1−/− animals in comparison to WT or C5−/− mice. Data are shown as the mean ± SEM (n = 5-8 animals per group) and are displayed as % of the perfusion in the contralateral control limb. ∗P < .05. (C) Representative images depicting capillaries (stained by IB4) and pericyte coverage (colocalization of IB4 and NG2) in the analyzed genotypes 14 days after induction of ischemia. (D) At day 14, there was no significant increase in capillary density in C5aR1−/− mice. Data represent the mean ± SEM. n = 9 to 12. (E) An increase in pericyte coverage was observable in C5aR1−/− mice compared with WT and C5−/− mice at day 14. Data represent mean ± SEM. n = 9 to 15. ∗∗P < .01, ∗∗∗P < .001. (F) Arteriolar abundance was highest in C5aR1−/− mice. There was an increase in arteries in ischemic hind limb muscle tissue compared with control tissue, which was significant for WT and C5aR1−/− mice but not for C5−/− mice. Data represent mean ± SEM. n = 6 to 9. ∗P < .05. (G) When WT or C5aR1−/− mice subjected to HLI were additionally treated with heparin, we observed no difference in revascularization anymore. Data are shown as the mean ± SEM (n = 6-8 animals per group) and are displayed as % of the perfusion in the contralateral control limb. (H) Furthermore, WT or C5aR1−/− mice were subjected to HLI, and platelets were depleted systemically by injection of platelet-depleting serum starting on the first day after induction of ischemia. This scheme induces persistent and pronounced platelet depletion, as verified previously.23 Vessel density in the gastrocnemius muscle of the ischemic limbs was quantified by immunofluorescence staining. The 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. Fourteen days after the induction of ischemia, C5aR1−/− mice additionally treated with platelet-depleting serum exhibited no significant difference in comparison to WT mice. Images 400× original magnification, scale bars represent 10 μm. (I) Quantification of vessel core numbers per area at day 14 in WT and C5aR1−/− mice, which received platelet-depleting serum over 2 weeks, revealed no significant difference. Data represent the mean ± SEM. n = 7 to 8. n.s. = no statistically significant difference was observed.

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