Journal: Molecular Therapy

Article Title: A Novel C5a-neutralizing Mirror-image ( l -)Aptamer Prevents Organ Failure and Improves Survival in Experimental Sepsis

doi: 10.1038/mt.2013.178

Figure Lengend Snippet: NOX-D20 binds to C5 but does not inhibit complement-mediated hemolysis. SPR measurement of NOX-D20 binding to human ( a ) C5a, ( b ) C5a(desArg), and ( c ) C5. Kinetic rate constants k a and k d are shown as mean ± SEM. Data are representative for at least three individual measurements. ( d ) Human serum pretreated with NOX-D20 (black squares) or C5-binding aptamer C5C6 (black triangles) was incubated with opsonized sheep erythrocytes. Hemolysis was quantified by photometric measurement of hemoglobin in the supernatant at 405 nm. Normalized data representative for three independent experiments is shown. RU, response units.

Article Snippet: Recombinant human and mouse C5a was from R&D Systems (Wiesbaden, Germany), recombinant human C5a(desArg) from Hycult Biotech (Beutelsbach, Germany), and human C5 purified from serum from Sigma-Aldrich (Taufkirchen, Germany).

Techniques: Binding Assay, Incubation

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: The expression of C5a and NETs in arterial thrombi. A ELISA. The concentrations of C5a in plasmas from patients with angina or STEMI ( n = 15 each). B ELISA. The concentrations of C5a in plasmas from C57 BL/6 mice with FeCl 3 -induced arterial thrombosis ( n = 12) compared with sham mice ( n = 11). C Immunofluorescence staining of cross-sections of the LCCA for CitH3 (red), Ly6g (green) and DAPI (blue) 6 h after FeCl 3 exposure. Scale bar = 75 µm. Data are presented as mean ± SD

Article Snippet: Recombinant human C5a (0.1 μM, Novoprotein, China) was used to stimulate the neutrophil-like cells for 2 h. Mitochondrial separation was performed using the Mitochondria Isolation Kit (Beyotime, China) according to the manufacturer’s instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: C5a promoted arterial thrombosis by triggering neutrophils to release NETs. A , B H&E staining of thrombus, cross-sections ( A ) and longitudinal sections ( B ). Scale bar = 100 µm. C Quantification of the thrombus size in cross-sections by H&E staining ( n = 5 each). D Thrombus weight ( n = 5 each). E The blood flow velocity in the LICA was dramatically decreased in the FeCl 3 -induced thrombus mice compared with the sham mice, and this change was reversed by PMX53. F Quantification of the blood flow velocity ( n = 4 each). G Immunofluorescence staining of cross-sections of the LCCA for CitH3 (red), Ly6g (green) and DAPI (blue) 6 h after FeCl 3 exposure. Thirty minutes before FeCl 3 exposure, PMX53 was administered by intraperitoneal injection. Scale bar = 75 µm. H Quantification of NET formation capacity shown as the ratio of NETs/neutrophil in LCCA cross-sections subjected to immunofluorescence staining (NaCl group = 3, PMX53 group = 4). I Representative images of immunofluorescence stainings of NET formation for DNA (SYTOX green), CitH3 (red), Ly6g(blue) in vitro after stimulation of C5a, and NET formation was attenuated by PMX53. Scale bar = 75 µm. J Quantification of NET formation capacity in vitro shown as the percentage of NET release, which was assessed by immunofluorescence staining ( n = 3 each). (K) NETosis was measured using a plate reader assay. NET release is expressed as percentage of total DNA ( n = 3 each). Data are presented as mean ± SD

Article Snippet: Recombinant human C5a (0.1 μM, Novoprotein, China) was used to stimulate the neutrophil-like cells for 2 h. Mitochondrial separation was performed using the Mitochondria Isolation Kit (Beyotime, China) according to the manufacturer’s instructions.

Techniques: Staining, Immunofluorescence, Injection, In Vitro

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: Promotion of NETosis by C5a was Mito-ROS-dependent. A Fluorescence images showing ROS production in mitochondria indicated by MitoSox Red dye. Scale bar = 100 µm. B Quantification of the production of Mito-ROS shown as MitoROS relative fluorescence (folds of the control group) ( n = 4 each). C Representative images of immunofluorescence staining for DNA (SYTOX green), CitH3 (red), Ly6g (blue) in vitro showing the presence of NETs. Scale bar = 75 µm. D Quantification of NET formation capacity shown as percentage of NET release in vitro, as assessed by immunofluorescence staining ( n = 3 each). E NETosis was measured using a plate reader assay, ( n = 3 each). Data are presented as mean ± SD

Article Snippet: Recombinant human C5a (0.1 μM, Novoprotein, China) was used to stimulate the neutrophil-like cells for 2 h. Mitochondrial separation was performed using the Mitochondria Isolation Kit (Beyotime, China) according to the manufacturer’s instructions.

Techniques: Fluorescence, Immunofluorescence, Staining, In Vitro

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: Interactions among C5a, mitochondrial STAT3 and NETs. A , B Western blot analysis was performed to test the expression levels of mitochondrial STAT3 and p-STAT3 (Ser 727 ) in neutrophil-like cells cocultured with C5a, compared with the control. VDAC, a marker of mitochondria, was used as a loading control for mitochondria ( n = 5 each). C NET release in response to buffer or AG490 was measured using a plate reader assay ( n = 3 each). D Representative images of immunofluorescence staining for DNA (SYTOX green), CitH3 (red) and Ly6g (blue) in vitro after stimulation with AG490 showing the presence of NETs. Scale bar = 100 µm. E Quantification of NET formation capacity shown as the percentage of NET release in vitro after stimulation with buffer or AG490, as assessed by immunofluorescence staining. ( n = 3 each). Data are presented as mean ± SD

Article Snippet: Recombinant human C5a (0.1 μM, Novoprotein, China) was used to stimulate the neutrophil-like cells for 2 h. Mitochondrial separation was performed using the Mitochondria Isolation Kit (Beyotime, China) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Marker, Immunofluorescence, Staining, In Vitro

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: Colivelin abolished C5a-induced NETosis in vitro. A Fluorescence images showing ROS production in mitochondria indicated by MitoSox Red dye. Colivelin abolished the elevation of Mito-ROS levels induced by C5a in vitro. Scale bar = 100 µm. B Quantification of the production of Mito-ROS showed as MitoROS relative fluorescence (folds of the control group) ( n = 4 each). C Representative images of immunofluorescence staining for DNA (SYTOX green), CitH3 (red), and Ly6g(blue) in vitro after stimulation with C5a showing NET formation. NET formation was attenuated by Colivelin. Scale bar = 75 µm. D Quantification of NET formation capacity shown as percentage of NET release in vitro, as assessed by immunofluorescence staining ( n = 3 each). E NETosis was measured using a plate reader assay ( n = 5 each). Data are presented as mean ± SD

Article Snippet: Recombinant human C5a (0.1 μM, Novoprotein, China) was used to stimulate the neutrophil-like cells for 2 h. Mitochondrial separation was performed using the Mitochondria Isolation Kit (Beyotime, China) according to the manufacturer’s instructions.

Techniques: In Vitro, Fluorescence, Immunofluorescence, Staining

Journal: Thrombosis Journal

Article Title: Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis

doi: 10.1186/s12959-022-00384-0

Figure Lengend Snippet: Visual summary: the effect of complement C5a in arterial thrombosis. In arterial thrombosis, C5a chemotactically attracts neutrophils to migrate towards the culprit site and triggers the release of NETs, which contribute to thrombosis by promoting coagulation and stabilizing clots. C5a-induced promotion of NET release is dependent on Mito-ROS production. C5a induces the Mito-ROS production by inhibiting mitochondrial STAT3 activity

Article Snippet: Recombinant human C5a (0.1 μM, Novoprotein, China) was used to stimulate the neutrophil-like cells for 2 h. Mitochondrial separation was performed using the Mitochondria Isolation Kit (Beyotime, China) according to the manufacturer’s instructions.

Techniques: Coagulation, Activity Assay

Journal: Experimental and therapeutic medicine

Article Title: The complement C3a-C3aR and C5a-C5aR pathways promote viability and inflammation of human retinal pigment epithelium cells by targeting NF-κB signaling.

doi: 10.3892/etm.2022.11420

Figure Lengend Snippet: Figure 3. Complement C3a and C5a aggravate inflammation in HRPE cells via the NF‑κB signaling pathway. HRPE cells were treated with recombinant human complement component C3a (2 µg/ml) or C5a (1 µg/ml) with or without NF‑κB inhibitor PDTC (10 µM), and the release of (A) TNF‑α, (B) IL‑1β, (C) IL‑6, (D) PGE2 and (E) IL‑10 was measured by ELISA. ***P<0.001 relative to control; ###P<0.001 relative to C3a or C5a treatment. PGE2, prostaglandin E2; HRPE, human retinal pigment epithelium.

Article Snippet: Following 24 h of treatment, the contents of various cytokines and other compounds were analyzed in HRPE cell supernatants and/or human vitreous humor were determined using the following ELISA kits in accordance with the manufac‐ turers' protocols: Tumor Necrosis Factor‐α Assay Kit (cat. no. H052‐1), Interleukin‐1β Assay Kit (cat. no. H002), Interleukin‐6 Assay Kit (cat. no. H007‐1‐1) and Interleukin‐10 Assay Kit (cat. no. H009‐1; all from Nanjing Jiancheng Bioengineering Institute); prostaglandin E2 (PGE2) ELISA Kit (cat. no. E‐EL‐0034c; Elabscience Biotechnology Co., Ltd.); Human Complement Fragment 3a ELISA Kit (cat no. CSB‐E08509h) and Human Complement Fragment 5a ELISA Kit (cat. no. CSB‐E08512h; both from Cusabio).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Control

Journal: Experimental and therapeutic medicine

Article Title: The complement C3a-C3aR and C5a-C5aR pathways promote viability and inflammation of human retinal pigment epithelium cells by targeting NF-κB signaling.

doi: 10.3892/etm.2022.11420

Figure Lengend Snippet: Figure 4. Complement C3aR and C5aR antagonist inhibit inflammation and NF‑κB signaling in HRPE cells challenged with complement C3a and C5a. HRPE cells were treated with recombinant human complement component C3a (2 µg/ml) and with or without C3aR antagonist SB290157 (20 µM), and the release of (A) TNF‑α, (B) IL‑1β, (C) IL‑6, (D) PGE2 and (E) IL‑10 was determined by ELISA. HRPE cells were treated with recombinant human complement component C5a (1 µg/ml) and with or without C5aR antagonist CCX168 (2 µM), and the release of (F) TNF‑α, (G) IL‑1β, (H) IL‑6, (I) PGE2 and (J) IL‑10 was determined by ELISA. (K) The phosphorylation of NF‑κB and expression of NF‑κB in HRPE cells treated with recombinant human complement component C3a (2 µg/ml) and with or without C3aR antagonist SB290157 (20 µM) were determined by western blot. (L) The phosphorylation of NF‑κB and expression of NF‑κB in HRPE cells treated with recombinant human complement component C5a (1 µg/ml) and with or without C5aR antagonist CCX168 (2 µM) were determined by western blot. ***P<0.001 relative to control; ###P<0.001 relative to C3a or C5a treatment. p‑NF‑κB, phosphorylated NF‑κB; C5aR, C5a receptor; HRPE, human retinal pigment epithelium; CCX, CCX168; SB, SB290157.

Article Snippet: Following 24 h of treatment, the contents of various cytokines and other compounds were analyzed in HRPE cell supernatants and/or human vitreous humor were determined using the following ELISA kits in accordance with the manufac‐ turers' protocols: Tumor Necrosis Factor‐α Assay Kit (cat. no. H052‐1), Interleukin‐1β Assay Kit (cat. no. H002), Interleukin‐6 Assay Kit (cat. no. H007‐1‐1) and Interleukin‐10 Assay Kit (cat. no. H009‐1; all from Nanjing Jiancheng Bioengineering Institute); prostaglandin E2 (PGE2) ELISA Kit (cat. no. E‐EL‐0034c; Elabscience Biotechnology Co., Ltd.); Human Complement Fragment 3a ELISA Kit (cat no. CSB‐E08509h) and Human Complement Fragment 5a ELISA Kit (cat. no. CSB‐E08512h; both from Cusabio).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Expressing, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: The “C3aR Antagonist” SB290157 is a Partial C5aR2 Agonist

doi: 10.3389/fphar.2020.591398

Figure Lengend Snippet: SB290157 potently activates C3aR-mediated ERK signaling in transfected CHO cells. (A) Agonism testing of SB290157 on CHO-C3aR cells. Serum-starved CHO-C3aR cells (50,000/well) were stimulated with respective concentrations of SB290157 or purified human C3a for 10 min before being lysed. (B) Agonism testing of SB290157 on CHO-C5aR1 cells. Serum-starved CHO-C5aR1 cells (50,000/well) were stimulated with the respective ligands at the indicated concentrations for 10 min before being lysed. (C) Antagonism testing of SB290157 on CHO-C5aR1 cells. Serum-starved CHO-C5aR1 cells (50,000/well) were pre-treated with SB290157 (10 µM) or vehicle (0.1% DMSO) for 30 min before being stimulated with 0.3 nM of C5a for 10 min and then lysed. The phospho-ERK1/2 content in the cell lysate was measured and normalised to the maximum C3a-induced (for A ) or 0.3 nM C5a-induced (for B , C ) levels before being combined. Data represent mean ± S.E.M. of triplicate measurements from 3 to 6 independent experiments (n = 3–6).

Article Snippet: Recombinant human C5a and recombinant mouse C5a were purchased from Sino Biological (Beijing, China).

Techniques: Transfection, Purification

Journal: Frontiers in Pharmacology

Article Title: The “C3aR Antagonist” SB290157 is a Partial C5aR2 Agonist

doi: 10.3389/fphar.2020.591398

Figure Lengend Snippet: SB290157 induces C5aR2-mediated β -arrestin 2 recruitment in transfected HEK293 cells. HEK293 cells were transiently transfected using C5aR2-Venus and β -arrestin 2-Rluc8 BRET pairs for 24 h and seeded (100,000/well) overnight. Filtered light emissions between 460–485 nm (Rluc8) and 520–545 nm (Venus) were continually monitored for 90 min with SB290157, P32, C5a or vehicle added at the 0 min time point. Data represent (A) the time course of ligand-induced BRET ratios (Venus/Rluc8 emission ratio) caused by the respective ligands at the indicated concentrations, (B) the corresponding concentration-response curves for SB290157 and P32 at 40 min post ligand addition, and (C) the ligand-induced BRET ratios (efficacy) of SB290157, P32 or C5a at 40 min post ligand addition. Data represent the mean ± S.E.M. of triplicate measurements from 3 to 5 independent experiments (n = 3–5).

Article Snippet: Recombinant human C5a and recombinant mouse C5a were purchased from Sino Biological (Beijing, China).

Techniques: Transfection, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: The “C3aR Antagonist” SB290157 is a Partial C5aR2 Agonist

doi: 10.3389/fphar.2020.591398

Figure Lengend Snippet: C5aR2 activation dampens C5a-induced ERK signaling in human monocyte-derived macrophages and mouse bone marrow-derive macrophages. HMDMs (A) or BMDMs (B,C) were serum-starved overnight and pre-treated with SB290157 (50 µM) or P32 (100 µM) and the corresponding solvent-only control (Vehicle) for 30 min, prior to stimulation with recombinant human C5a (10 min, for A) or recombinant mouse C5a (5 min, for B and C) . The phospho-ERK1/2 content in the cell lysate was measured and normalised to the maximum vehicle-treated C5a-induced levels before being combined. Data represent mean ± S.E.M. of triplicate measurements using cells from 3-6 independent donors (n = 3–6) for HMDMs, or 4 mice (n = 4) for BMDMs. Two-way ANOVA with Sidak's post hoc test. * p < 0.05, ** p < 0.01, **** p < 0.0001, P32 or SB290157 pre-treated vs. control-treated cells stimulated by respective concentrations of C5a.

Article Snippet: Recombinant human C5a and recombinant mouse C5a were purchased from Sino Biological (Beijing, China).

Techniques: Activation Assay, Derivative Assay, Recombinant