Fig. 5. C5aR1 deficiency on platelets induces increased collateral artery formation, capillarization, and pericyte coverage.

Platelet-specific C5aR1-deficient mice were subjected to hindlimb ischemia. To visualize collateral arteries, mice were perfused with a contrast agent at d9 after induction of ischemia and subjected to microCT analysis as described in the “Methods” section. a 3D reconstructions of arteries from Pf4-cre⁻ C5ar1^fl/fl mice showed collateral artery formation, while the main femoral artery (arrows) was no longer perfused. a, b A comparison of collateral artery formation in mice with C5aR1-deficient or -competent platelets shows increased collateralization in platelet-specific C5ar1-knockout mice. Images are representative of 3–4 mice, i.e., 3–4 vessel trees analyzed. c The size distribution of vessels within the ischemic hindlimbs of Pf4-cre⁺ C5ar1^fl/fl mice and Pf4-cre⁻ C5ar1^fl/fl mice was quantified within the microCT data in mouse hindlimbs proximal to the knee in the region of the adductor muscle (for details on methodology, please refer to the “Methods” section and Supplementary Fig. 8). Pf4-cre⁺ C5ar1^fl/fl mice displayed larger vessels in both the small vessel range as well as the medium-sized vessel range. Data are shown as overlaid single measurements of each of the 3–4 vessel trees analyzed and as mean ± 95% confidence interval of all vessels quantified within each size region (n = 3–4 animals, i.e., vessel trees per group). Data are stated in pixels/voxels and 1 voxel represents 21.6 µm. Furthermore, the mean vessel diameter shows a clear trend that vessels in the proximal part of the ischemic hindlimb are larger in Pf4-cre⁺ C5ar1^fl/fl mice compared to controls (right) going along with improved revascularization. d Characterization of arteries in the distal ischemic hindlimb gastrocnemius muscle of Pf4-cre⁺ C5ar1^fl/fl or Pf4-cre⁻ C5ar1^fl/fl mice reveal no significant differences in large artery size or abundance of arterioles (SMA in red, vessel marker IB4 in green). ×200 magnification, scale bars represent 200 µm, arrows mark large muscle arteries double positive for SMA and IB4. e Large distal muscle arteries were assessed by measuring the perimeter of the 5 largest arteries present in a whole-muscle section acquired by tile scanning at ×200 magnification (left). Furthermore, the area fraction in the muscle sections of SMA-positive vessels was quantified at ×200 magnification. Data are shown as the mean ± SEM (n = 10 whole-muscle sections per group) and are displayed as the percentage of control. The sum of artery perimeters of the five largest arteries per section or the area fraction of SMA staining in Pf4-cre⁻ C5ar1^fl/fl hindlimb muscle sections represent the 100% control. n.s. = no significant difference was observed. f Platelet-specific C5ar1-knockout mice displayed increased pericyte coverage of vessels in the ischemic muscle tissue at d14 after induction of ischemia. (pericyte marker NG2 in red, vessel marker IB4 in green). ×200 magnification, scale bars represent 200 µm. g Pericyte density was assessed by measuring the area fraction of NG2-positive staining in whole-muscle sections acquired by tile scanning at ×200 magnification (left). Furthermore, pericyte coverage was assessed by calculating the spatial colocalization of NG2 and IB4 staining. Data are shown as the mean ± SEM (n = 10 whole-muscle sections per group) and are displayed as the percentage of control. The area fraction of NG2 staining or the pericyte coverage in Pf4-cre⁻ C5ar1^fl/fl hindlimb muscle sections represent 100%. *p < 0.05 or <0.01. Two-sided Student’s t test in e, g.