Figure 3.
The platelet C5aR1 contributes to growth factor-mediated angiogenesis. In vivo Matrigel plug assay experiments were carried out as described in the “Materials and methods.” (A) We detected increased complement activation (C3b, red) in the angiogenic tissue, which colocalizes with vascular structures (IB4, green). (B-C) After 7 days, matrigels were explanted, and a single-cell suspension generated from the plugs was analyzed by flow cytometry. We observed increased presence of platelets (CD41+; B) and the presence of C5aR1 on platelets (CD41+CD45−C5aR1+; C). n = 7 to 8 ∗P < .05, ∗∗P < .01. (D-G) Isolated platelets were coinjected with bFGF into the Matrigel solution before implantation into mice. We observed an inhibition of neovascularization by platelet addition. (D) Panel D shows reduced macroscopic vascularization in explanted plugs, which was associated with reduced cellularity (E) (H and E staining) and reduced green signal (F) (IB4 staining for vessels). (G) n = 7 to 8 plugs were analyzed. Matrigels injected with vehicle control represent 100%. ∗P < .05 in comparison to control. (H) WT or C5aR1−/− mice were applied in the Matrigel plug in vivo assay. We observed increased angiogenesis in the absence of C5aR1. n = 8 to 11 plugs were analyzed. The group with WT mice represents 100%. ∗P < .05. When platelets were depleted systemically by injection of platelet-depleting serum, we could not detect significant differences in angiogenesis. Data are shown as the mean ± SEM (n = 7 animals per group) and are displayed as % of WT mice. ns = no statistically significant difference could be observed. (I) PF4-cre+C5aR1fl/fl mice were generated as described in “Methods.” (J) Platelet-specific C5a receptor 1 knockout mice showed increased revascularization. Data are presented as the mean ± SEM (n = 9) and are displayed as % of the perfusion in the contralateral control limb. ∗P < .05 in comparison to cre-negative WT mice. (K-L) MHEC-5T were coincubated with platelet releasate after C5a stimulation. C5a-stimulated WT platelet supernatant inhibited endothelial tube formation. Data are displayed as the mean ± SEM (n = 5). ∗P < .05. (L) Panel L shows representative images of these in vitro experiments.

The platelet C5aR1 contributes to growth factor-mediated angiogenesis. In vivo Matrigel plug assay experiments were carried out as described in the “Materials and methods.” (A) We detected increased complement activation (C3b, red) in the angiogenic tissue, which colocalizes with vascular structures (IB4, green). (B-C) After 7 days, matrigels were explanted, and a single-cell suspension generated from the plugs was analyzed by flow cytometry. We observed increased presence of platelets (CD41+; B) and the presence of C5aR1 on platelets (CD41+CD45−C5aR1+; C). n = 7 to 8 ∗P < .05, ∗∗P < .01. (D-G) Isolated platelets were coinjected with bFGF into the Matrigel solution before implantation into mice. We observed an inhibition of neovascularization by platelet addition. (D) Panel D shows reduced macroscopic vascularization in explanted plugs, which was associated with reduced cellularity (E) (H and E staining) and reduced green signal (F) (IB4 staining for vessels). (G) n = 7 to 8 plugs were analyzed. Matrigels injected with vehicle control represent 100%. ∗P < .05 in comparison to control. (H) WT or C5aR1−/− mice were applied in the Matrigel plug in vivo assay. We observed increased angiogenesis in the absence of C5aR1. n = 8 to 11 plugs were analyzed. The group with WT mice represents 100%. ∗P < .05. When platelets were depleted systemically by injection of platelet-depleting serum, we could not detect significant differences in angiogenesis. Data are shown as the mean ± SEM (n = 7 animals per group) and are displayed as % of WT mice. ns = no statistically significant difference could be observed. (I) PF4-cre+C5aR1fl/fl mice were generated as described in “Methods.” (J) Platelet-specific C5a receptor 1 knockout mice showed increased revascularization. Data are presented as the mean ± SEM (n = 9) and are displayed as % of the perfusion in the contralateral control limb. ∗P < .05 in comparison to cre-negative WT mice. (K-L) MHEC-5T were coincubated with platelet releasate after C5a stimulation. C5a-stimulated WT platelet supernatant inhibited endothelial tube formation. Data are displayed as the mean ± SEM (n = 5). ∗P < .05. (L) Panel L shows representative images of these in vitro experiments.

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