Figure 6.
C5aR1-induced inhibition of neovascularization by platelets depends on CXCL4 secretion and is distinct from platelet activation. (A-B) Murine endothelial cells (MHEC-5T) were incubated in wells on Matrigel with increasing concentrations of CXCL4 (0.5-10 μg/mL), and branching points of forming tubes as well as branching lengths were determined as indicated in “Materials and methods” (also refer to supplemental Figure 19). Data are displayed as the mean ± SEM (n = 7). ∗P < .05. (C-D) We generated C5aR1−/−CXCL4−/− double knockout mice. Platelets isolated from these animals were stimulated with ADP, and the expression of platelet activation markers CD62P or activated GPIIbIIIa were analyzed by flow cytometry. Data are shown as the mean ± SEM (n = 5) and are displayed as geometric MFI. ∗P < .05 in comparison to WT control. ns = no significant difference was measured. (E) Platelets from WT, C5aR1−/−, or C5aR1−/−CXCL4−/− mice were stimulated with C5a, and the supernatant was added to endothelial cells (MHEC-5T). In comparison to C5aR1−/− and C5aR1−/−CXCL4−/− platelet supernatant, coincubation with C5a-conditioned WT platelet supernatant led to lower endothelial migration. Double-deficient platelet supernatant did not differ significantly from C5aR1−/− platelet supernatant. Data represent mean ± SEM. n = 6. ∗P < .05. (F-G) MHEC-5T cells were incubated with the C5a-stimulated supernatant of platelets isolated from WT, C5aR1−/−, and C5aR1−/−CXCL4−/− mice. In 1 group, the supernatant of C5aR1−/− platelets was additionally supplemented with CXCL4 (2 μg/mL). C5a-stimulated WT platelet supernatant inhibited endothelial tube formation, which was not detectable in C5aR1−/− and C5aR1−/−CXCL4−/− platelets. Reconstitution with CXCL4 in the C5aR1−/− group led to a similar level of tube formation as in the control WT group. Data are displayed as the mean ± SEM (n = 4-5). ∗∗P < .01, ∗P < .05. (H-I) Similarly, MHEC-5T cells were incubated with the C5a-stimulated supernatant of platelets isolated from WT mice but additionally treated with a blocking antibody to the CXCL4 receptor, CXCR3. Data are displayed as the mean ± SEM (n = 7). ∗P < .05 in comparison to IgG control. (J-K) HLI was induced in C5aR1−/− mice or C5aR1−/−CXCL4−/− mice and a WT control group, which were generated as described in the “Methods.” Double knockout mice showed no increased revascularization in comparison to C5aR1−/− mice. Data are presented as the mean ± SEM (n = 5-7) and are displayed as a percentage of the perfusion in the contralateral control limb. n.s. = no significant difference could be observed compared with C5aR1−/− animals. (M) Shows representative LDI images of mouse hind limbs after femoral artery ligation and during revascularization over 14 days.

C5aR1-induced inhibition of neovascularization by platelets depends on CXCL4 secretion and is distinct from platelet activation. (A-B) Murine endothelial cells (MHEC-5T) were incubated in wells on Matrigel with increasing concentrations of CXCL4 (0.5-10 μg/mL), and branching points of forming tubes as well as branching lengths were determined as indicated in “Materials and methods” (also refer to supplemental Figure 19). Data are displayed as the mean ± SEM (n = 7). ∗P < .05. (C-D) We generated C5aR1−/−CXCL4−/− double knockout mice. Platelets isolated from these animals were stimulated with ADP, and the expression of platelet activation markers CD62P or activated GPIIbIIIa were analyzed by flow cytometry. Data are shown as the mean ± SEM (n = 5) and are displayed as geometric MFI. ∗P < .05 in comparison to WT control. ns = no significant difference was measured. (E) Platelets from WT, C5aR1−/−, or C5aR1−/−CXCL4−/− mice were stimulated with C5a, and the supernatant was added to endothelial cells (MHEC-5T). In comparison to C5aR1−/− and C5aR1−/−CXCL4−/− platelet supernatant, coincubation with C5a-conditioned WT platelet supernatant led to lower endothelial migration. Double-deficient platelet supernatant did not differ significantly from C5aR1−/− platelet supernatant. Data represent mean ± SEM. n = 6. ∗P < .05. (F-G) MHEC-5T cells were incubated with the C5a-stimulated supernatant of platelets isolated from WT, C5aR1−/−, and C5aR1−/−CXCL4−/− mice. In 1 group, the supernatant of C5aR1−/− platelets was additionally supplemented with CXCL4 (2 μg/mL). C5a-stimulated WT platelet supernatant inhibited endothelial tube formation, which was not detectable in C5aR1−/− and C5aR1−/−CXCL4−/− platelets. Reconstitution with CXCL4 in the C5aR1−/− group led to a similar level of tube formation as in the control WT group. Data are displayed as the mean ± SEM (n = 4-5). ∗∗P < .01, ∗P < .05. (H-I) Similarly, MHEC-5T cells were incubated with the C5a-stimulated supernatant of platelets isolated from WT mice but additionally treated with a blocking antibody to the CXCL4 receptor, CXCR3. Data are displayed as the mean ± SEM (n = 7). ∗P < .05 in comparison to IgG control. (J-K) HLI was induced in C5aR1−/− mice or C5aR1−/−CXCL4−/− mice and a WT control group, which were generated as described in the “Methods.” Double knockout mice showed no increased revascularization in comparison to C5aR1−/− mice. Data are presented as the mean ± SEM (n = 5-7) and are displayed as a percentage of the perfusion in the contralateral control limb. n.s. = no significant difference could be observed compared with C5aR1−/− animals. (M) Shows representative LDI images of mouse hind limbs after femoral artery ligation and during revascularization over 14 days.

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