c5ar1 antagonist pmx205  (Cayman Chemical)

Journal: Nature communications

Article Title: The C5a/C5a receptor 1 axis controls tissue neovascularization through CXCL4 release from platelets.

doi: 10.1038/s41467-021-23499-w

Figure Lengend Snippet: 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.

Article Snippet: For some experiment, platelets were preincubated with the C5aR1 antagonist PMX205 (used at 15 μM for 30 min at 37 °C, Tocris) or control peptide.

Techniques: In Vivo, Ligation, Control, Staining, Injection

Journal: Nature communications

Article Title: The C5a/C5a receptor 1 axis controls tissue neovascularization through CXCL4 release from platelets.

doi: 10.1038/s41467-021-23499-w

Figure Lengend Snippet: Fig. 4 Platelet-specific C5a receptor deletion inhibits revascularization but does not alter platelet deposition in vivo. Pf4-cre+ C5ar1fl/flmice were generated as described in the “Methods” section. Hindlimb ischemia was induced, and Pf4-cre−C5ar1fl/flmice served as controls. a Platelet-specific C5a receptor 1 knockout mice showed increased revascularization. Data are presented as the mean ± SEM (n = 5 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 Pf4-cre+ C5ar1fl/flmice compared with littermate control animals. c Vessel density in the representative gastrocnemius muscle sections (vessel marker IB4 in green, nuclei in blue) of the ischemic limbs of Pf4-cre+ C5ar1fl/flshowed significantly higher vessel density than Pf4-cre−C5ar1fl/flcontrols. Scale bars represent 200 µm. d Quantification of the IB4-positive area fraction in the muscle sections reveals higher vessel abundance in ischemic Pf4-cre+ C5ar1fl/flhindlimbs 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 IB4-positive area fraction in Pf4-cre−C5ar1fl/flhindlimbs represents 100%. *p < 0.05. e By staining for CD42b (red), platelet deposition was quantified in whole-muscle sections of Pf4-cre+ C5ar1fl/flor of Pf4-cre−C5ar1fl/flmice, as described in the “Methods” section. No difference in the number of platelets per area or microthrombi size was detected. Displayed are representative images for both genotypes, IB4 staining (green) depicts vascular structures; DAPI (blue) depicts nuclei. Scale bars represent 100 µm. f CD42b-positive single platelets were quantified by size using automated digital image analysis as described in the “Methods” part in whole-muscle sections. There was no significant difference between Pf4-cre+ C5ar1fl/fland Pf4-cre−C5ar1fl/flmice both with respect of the number of platelets per muscle area as well as the size of the platelets/microthrombi. Data are shown as the mean ± SEM (n = 6 whole-muscle sections per group) and are displayed as the percentage of control. The readings in Pf4-cre−C5ar1fl/flhindlimbs represent 100% in both graphs. *p < 0.05. Two-way ANOVA with Bonferroni’s post hoc test in a. Two-sided Student’s t test in d, f.

Article Snippet: For some experiment, platelets were preincubated with the C5aR1 antagonist PMX205 (used at 15 μM for 30 min at 37 °C, Tocris) or control peptide.

Techniques: In Vivo, Generated, Knock-Out, Control, Ligation, Marker, Staining

Journal: Journal of Neurochemistry

Article Title: Complement C3a and C5a Receptors Are Presented in Mouse Sciatic and Human Sural Nerves and Selectively Modulate the Neuronal Function of Large‐Caliber Fibers in Mice

doi: 10.1111/jnc.70129

Figure Lengend Snippet: C5aR1 is mainly localized in unmyelinated fibers. Longitudinal and cross‐sections of human sural nerves were labeled for C5aR1 (red, A; green, D), Caspr (green, B), and NCAM (E, red). Double‐stained images are merged (C, F). Teased mouse sciatic nerve images of labeled C5aR1 (red, G, J) and Caspr (green, H). Double‐stained mouse sciatic fibers were merged (I). Peripherin (green, K) merged (L). Caspr‐contactin‐associated protein; NCAM‐neural cell adhesion molecule; n = 10 mice; Scale bar 10, 20 μM.

Article Snippet: The C5aR1 agonist BM213 (1 μM, Cat. No. HY‐145237, MedChemExpress), the C5aR1 antagonist PMX205 (1 μM, Cat. No. 13607, Cayman Chemicals, Ann Arbor, Michigan, USA) and the combination were applied at the same final concentrations as described above.

Techniques: Labeling, Staining

Journal: Journal of Neurochemistry

Article Title: Complement C3a and C5a Receptors Are Presented in Mouse Sciatic and Human Sural Nerves and Selectively Modulate the Neuronal Function of Large‐Caliber Fibers in Mice

doi: 10.1111/jnc.70129

Figure Lengend Snippet: mRNA and protein expression in the mouse sciatic nerve. Quantitative PCR (qPCR) analysis and western blotting were performed on sciatic nerves from 13‐week‐old mice ( n = 6 nerves). Intrinsic expression of the complement receptors and factors is seen (A). The outcomes are presented relative to hypoxanthine guanine phosphoribosyltransferase expression using the 2 −ΔCT method calculation method. Proteins C3aR and C5aR1 are present in mouse sciatic nerves ( n = 6 nerves) (B, C). HPRT‐hypoxanthine guanine phosphoribosyltransferase; ** p < 0.01.

Article Snippet: The C5aR1 agonist BM213 (1 μM, Cat. No. HY‐145237, MedChemExpress), the C5aR1 antagonist PMX205 (1 μM, Cat. No. 13607, Cayman Chemicals, Ann Arbor, Michigan, USA) and the combination were applied at the same final concentrations as described above.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot