c5a antagonist pmx53  (MedChemExpress)

Journal: Frontiers in Immunology

Article Title: Dysregulated complement activation during acute myocardial infarction leads to endothelial glycocalyx degradation and endothelial dysfunction via the C5a:C5a-Receptor1 axis

doi: 10.3389/fimmu.2024.1426526

Figure Lengend Snippet: C5a Concentrations and eGC Components Are Increased After Myocardial Infarction and Alter Endothelial Function in STEMI Patients. (A) Serum levels of C5a, syndecan-1, heparan sulfate, and hyaluronan (measured by enzyme-linked immunosorbent assay, ELISA) as well as nitric oxide (NO) concentrations (measured chemiluminescence based). (B) Correlation of C5a concentrations in ST-elevation myocardial infarction (STEMI) patient sera vs. syndecan-1, heparan sulfate, and hyaluronan as well as of endothelial glycocalyx (eGC) height, days until discharge from hospital, and NO concentration (calculated without data from LOW group). Rho (r) and p-values (p) shown for correlations. Groups: LOW: STEMI patient sera of the lowest C5a concentration quartile; HIGH: STEMI patient sera of the top C5a-concentration quartile; CTR: healthy donors. p-values: ****p<0.0001; **p<0.01; *p<0.05.

Article Snippet: In further experiments, HUVEC were stimulated with 50 ng/mL of C5a with or without the C5a-Receptor-1 antagonist (C5aRA) PMX53 (Sigma Aldrich, Hamburg, Germany; catalog number 533683), according to the manufacturer’s instructions [concentration 1 µg/mL (1:1000)] for 24 h. In addition, the endothelial cells were stimulated with 10% patient (STEMI) or healthy donor (CTR) sera (instead of FCS) for 24 h either with or without C5aRA (PMX53).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Dysregulated complement activation during acute myocardial infarction leads to endothelial glycocalyx degradation and endothelial dysfunction via the C5a:C5a-Receptor1 axis

doi: 10.3389/fimmu.2024.1426526

Figure Lengend Snippet: C5a Incrementally Changes the Nanomechanical Properties of the Endothelial Surface Layers in a Concentration-Dependent Manner (A–C) Statistical analysis of atomic force microscopy (AFM) nanoindentation measurements of human umbilical vein endothelial cells (HUVEC) monolayers. Data showing mean ± SD of (A) cortical stiffness, (B) endothelial glycocalyx (eGC) height and (C) eGC stiffness. Each dot represents a single cell measurement (8 force-distance curves per dot; N=6). (D–F) Representative fluorescence images and statistical fluorescence intensity analyses. (D) Phalloidin-tetramethylrhodamine–stained (red: F-actin) and (E) Wheat germ agglutinin (WGA)-stained (green; representing unspecific eGC components) HUVEC monolayers. Graphs showing the measured fluorescence intensity in arbitrary units. (F) Syndecan-1 antibody-stained HUVEC monolayers (green: syndecan-1; blue: cell nucleus) after stimulation. Graphs showing the amount of syndecan-1 per cell relative to the control group. ( D–F : N=5; scale bars: 50 µm). Groups: LOW: ST-elevation myocardial infarction (STEMI) patient sera of the lowest C5a concentration quartile; HIGH: STEMI patient sera of the top C5a -concentration quartile; CTR: healthy donors. p-values: ****p<0.0001; ***p<0.001; **p<0.01.

Article Snippet: In further experiments, HUVEC were stimulated with 50 ng/mL of C5a with or without the C5a-Receptor-1 antagonist (C5aRA) PMX53 (Sigma Aldrich, Hamburg, Germany; catalog number 533683), according to the manufacturer’s instructions [concentration 1 µg/mL (1:1000)] for 24 h. In addition, the endothelial cells were stimulated with 10% patient (STEMI) or healthy donor (CTR) sera (instead of FCS) for 24 h either with or without C5aRA (PMX53).

Techniques: Concentration Assay, Microscopy, Fluorescence, Staining, Control

Journal: Frontiers in Immunology

Article Title: Dysregulated complement activation during acute myocardial infarction leads to endothelial glycocalyx degradation and endothelial dysfunction via the C5a:C5a-Receptor1 axis

doi: 10.3389/fimmu.2024.1426526

Figure Lengend Snippet: C5a stimulation leads to cortical stiffening and eGC degradation. Different concentrations of C5a as well as different stimulation durations were tested for their influence on cortical stiffness measured via atomic force microscopy (AFM) nanoindentation technique. (A) Statistical analysis of AFM measurements of human umbilical vein endothelial cells (HUVEC) monolayers. Data showing mean ± SD of cortical stiffness after stimulation with different concentrations of C5a (0, 1, 10, 50 und 100 ng/mL; N=5). (B) Statistical analysis of AFM measurements of HUVEC monolayers showing mean ± SD of cortical stiffness after stimulation with 50 ng/mL of C5a using different stimulation durations (0, 30, 60, and 120 min and 24 h; N=5). (C) Statistical analysis of AFM measurements of HUVEC monolayers showing mean ± SD of cortical stiffness after stimulation with 100 ng/mL of C5a using different stimulation durations (0, 30, 60, and 120 min and 24 h; N=5). (D–F) Statistical analysis of AFM nanoindentation measurements. Data showing mean ± SD of (D) cortical stiffness, (E) eGC height, and (F) eGC stiffness after stimulating HUVEC monolayers with 50 ng/mL of C5a for 24 h (N=5). (G–I) Representative fluorescence images and statistical fluorescence intensity analyses. (G) Phalloidin-tetramethylrhodamine-stained (red: F-actin) and (H) Wheat germ agglutinin (WGA)-stained (green; representing unspecific eGC components) HUVEC monolayers after stimulation as described. Graphs showing the measured fluorescence intensity in arbitrary units. (I) Syndecan-1 Antibody-stained HUVEC monolayers (green: syndecan-1; blue: cell nucleus) after stimulation. Graphs showing the amount of syndecan-1 per cell relative to the control group. ( G–I : N=6; scale bars: 50 µm). p-values: ****p<0.0001; ***p<0.001.

Article Snippet: In further experiments, HUVEC were stimulated with 50 ng/mL of C5a with or without the C5a-Receptor-1 antagonist (C5aRA) PMX53 (Sigma Aldrich, Hamburg, Germany; catalog number 533683), according to the manufacturer’s instructions [concentration 1 µg/mL (1:1000)] for 24 h. In addition, the endothelial cells were stimulated with 10% patient (STEMI) or healthy donor (CTR) sera (instead of FCS) for 24 h either with or without C5aRA (PMX53).

Techniques: Microscopy, Fluorescence, Staining, Control

Journal: Frontiers in Immunology

Article Title: Dysregulated complement activation during acute myocardial infarction leads to endothelial glycocalyx degradation and endothelial dysfunction via the C5a:C5a-Receptor1 axis

doi: 10.3389/fimmu.2024.1426526

Figure Lengend Snippet: C5a-receptor-1 antagonist (PMX53) reduces impairment of the vascular surface after AMI and C5a stimulation. (A–C) Statistical analysis of atomic force microscopy (AFM) nanoindentation measurements of human umbilical vein endothelial cells (HUVEC) monolayers. Data showing mean ± SD of (A) cortical stiffness, (B) endothelial glycocalyx (eGC) height, and (C) eGC stiffness of CTR, C5a, and C5a+RA groups. Each dot represents a single cell measurement (8 force-distance curves per dot; N=5) as described in detail in the methods section. (D–F) Statistical analysis of AFM nanoindentation measurements of HUVEC monolayers. Data showing mean ± SD of (D) cortical stiffness, (E) eGC height, and (F) eGC stiffness of CTR, STEMI, and STEMI+RA groups. Each dot represents a single cell measurement (8 force-distance curves per dot; N=5) as described in detail in the methods section. (G–I) Representative fluorescence images and statistical fluorescence intensity analyses. (G) Phalloidin-tetramethylrhodamine-stained (red: F-actin) and (H) Wheat germ agglutinin (WGA)-stained (green; representing unspecific eGC components) HUVEC monolayers after stimulation as described. Graphs showing the measured fluorescence intensity in arbitrary units. (I) Syndecan-1 antibody-stained HUVEC monolayers (green: syndecan-1; blue: cell nucleus) after stimulation. Graphs showing the amount of syndecan-1 per cell relative to the control group. ( G–I : N=5; scale bars: 50 µm). (J) Expression levels of C5a-Receptor1 genes via quantitative PCR after stimulation of HUVEC. Gene-specific mRNA expression was measured using the ΔΔCt method relative to expression of ribosomal protein L28 (endogenous control) and normalized to unstimulated controls (N=3). (K) Small GTPases activation analysis of RhoA from confluent HUVEC. Bar graphs show raw optical density (O.D.) measured with an absorbance wavelength of 490 nm (N=3). (L) Small GTPases activation analysis of Rac1 from confluent HUVEC after treatment. Bar graphs show raw optical density (O.D.) measured with an absorbance wavelength of 490 nm (N=3). Groups: CTR: stimulation with standard cell culture media; C5a: cell culture media + 50 ng/mL of C5a; C5a+RA: cell culture media + 50 ng/mL of C5a + C5a-Receptor antagonist (PMX53; 1:1000). Serum groups: CTR: stimulation with cell culture media + 10% Serum of healthy donors; STEMI: cell culture media + 10% serum of STEMI patients (final C5a concentration 8.7 ng/mL); STEMI+RA: cell culture media + 10% serum of STEMI patients + C5a-Receptor antagonist (PMX53; 1:1000). p-values: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05.

Article Snippet: In further experiments, HUVEC were stimulated with 50 ng/mL of C5a with or without the C5a-Receptor-1 antagonist (C5aRA) PMX53 (Sigma Aldrich, Hamburg, Germany; catalog number 533683), according to the manufacturer’s instructions [concentration 1 µg/mL (1:1000)] for 24 h. In addition, the endothelial cells were stimulated with 10% patient (STEMI) or healthy donor (CTR) sera (instead of FCS) for 24 h either with or without C5aRA (PMX53).

Techniques: Microscopy, Fluorescence, Staining, Control, Expressing, Real-time Polymerase Chain Reaction, Activation Assay, Cell Culture, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Dysregulated complement activation during acute myocardial infarction leads to endothelial glycocalyx degradation and endothelial dysfunction via the C5a:C5a-Receptor1 axis

doi: 10.3389/fimmu.2024.1426526

Figure Lengend Snippet: The influence of C5a and STEMI on eGC condition and cortical stiffness are attenuated in a mouse model for C5a-receptor-1-knockout. (A–C) Statistical analysis of atomic force microscopy (AFM) nanoindentation measurements of living endothelial cells on isolated mouse aorta preparations. Aortas of wild-type (WT; N=6) and C5a-Receptor-1-knock-out (KO; C5aR1 –/– ; N=6) mice were harvested and made available for AFM measurements as described in detail in the methods section. Groups: Preparations of WT and KO aortas were stimulated with either cell culture media (control group, CTR), cell culture media + 50 ng/mL of C5a (C5a group), or cell culture media + 10% STEMI serum (STEMI group). Data showing mean ± SD of (A) cortical stiffness, (B) eGC height, and (C) eGC stiffness of single cell measurements of living endothelial cells on isolated mouse aorta preparations (8 force-distance curves per single cell; n=50 cells per mouse; N=6 mice per group). (D) Correlation of WT endothelial glycocalyx (eGC) height vs. WT cortical stiffness and KO eGC height vs. KO cortical stiffness (CTR and stimulation with STEMI serum). (E) Correlation of WT eGC height vs. WT eGC stiffness and KO eGC height vs. KO eGC stiffness (CTR and stimulation with STEMI serum). Rho (r) and p-values (p) shown for correlations. (F) Sprouting angiogenesis aortic ring assay of C5 -/- (B6(Cg)Tg(Ins2-GP)zbz) and mice double deficient for C5aR1 and CXCL4 (C5aR1 -/ -CXCL4- /- ) (on days 3, 4, 5, 6 and 7). (G) Tube formation assay of MHEC-5T cells after C5a stimulation. p-values: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant.

Article Snippet: In further experiments, HUVEC were stimulated with 50 ng/mL of C5a with or without the C5a-Receptor-1 antagonist (C5aRA) PMX53 (Sigma Aldrich, Hamburg, Germany; catalog number 533683), according to the manufacturer’s instructions [concentration 1 µg/mL (1:1000)] for 24 h. In addition, the endothelial cells were stimulated with 10% patient (STEMI) or healthy donor (CTR) sera (instead of FCS) for 24 h either with or without C5aRA (PMX53).

Techniques: Knock-Out, Microscopy, Isolation, Cell Culture, Control, Aortic Ring Assay, Tube Formation Assay

Journal: Frontiers in Immunology

Article Title: Dysregulated complement activation during acute myocardial infarction leads to endothelial glycocalyx degradation and endothelial dysfunction via the C5a:C5a-Receptor1 axis

doi: 10.3389/fimmu.2024.1426526

Figure Lengend Snippet: Endothelial function is improved after treatment with C5aRA. (A) Data showing mean ± SD of monocyte adhesion forces to human umbilical vein endothelial cells (HUVEC) monolayers measured via atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) of control (CTR), C5a, and C5a+RA groups (N=5). (B) Mean ± SD of adhesion energy between monocyte and HUVEC monolayer (energy needed to separate monocyte from HUVEC) of CTR, C5a, and C5a+RA groups (N=5) measured via AFM-based SCFS. (C) Exemplary force-distance curve of SCFS. Red circle indicates the maximum adhesion force between monocyte and HUVEC monolayer. Gray area representing the total adhesion energy calculated as the area under the curve (AUC). (D) Data showing mean ± SD of monocyte adhesion forces to HUVEC monolayers measured via AFM-based SCFS of CTR, ST-elevation myocardial infarction (STEMI), and STEMI+RA groups (N=5). (E) Mean ± SD of adhesion energy between monocyte and HUVEC monolayer (energy needed to separate monocyte from HUVEC) of CTR, STEMI, and STEMI+RA groups (N=5) measured via AFM-based SCFS. (F) Exemplary picture of AFM cantilever with mounted monocyte (white arrow) before adhesion measurements (scale bar: 25 µm). (G) Representative fluorescence images of human monocytes (green: CD14-labeled monocytes; indicated by white arrow) adhesive to HUVEC monolayer of CTR, C5a, and C5a+RA groups (blue: HUVEC nuclei; indicated by white dashed box). Statistical analysis showing mean ± SD of adherent monocytes per region of interest (ROI) quantified via monocyte-wash-away assay with previous CD14-staining (CTR, C5a, and C5a+RA groups; N=6; scale bar: 40 µm). (H) Representative fluorescence images of human monocytes (green: CD14 labeled monocytes) adhesive to HUVEC monolayer of CTR, STEMI, and STEMI+RA groups (blue: HUVEC nuclei). Statistical analysis showing mean ± SD of adherent monocytes per region of interest (ROI) quantified via monocyte-wash-away assay with previous CD14-staining (CTR, STEMI, and STEMI+RA groups; N=6; scale bar: 40 µm). (I) Statistical analysis of nitric oxide (NO) concentrations in cell culture media supernatant. NO products were measured by chemiluminescence-based via NOA-280i for CTR, C5a, and C5a+RA groups (N=3) as well as for CTR, STEMI, and STEMI+RA groups (N=3). (J) Statistical evaluation of wound healing assays for CTR, C5a, and C5a+RA groups (N=7). Data showing the 24-h growth rate (µm/h) as described in the methods section as well as wound closure in % (*: CTR vs. C5a). (K) Statistical evaluation of wound healing assays for CTR, STEMI, and STEMI+RA groups (N=7). Data showing the 24-h growth rate (µm/h) as described in the methods section as well as wound closure in % (*: CTR vs. STEMI; +: CTR vs. STEMI+RA; #: STEMI vs. STEMI+RA). Groups: CTR: stimulation with standard cell culture media; C5a: cell culture media + 50 ng/mL of C5a; C5a+RA: cell culture media + 50 ng/mL of C5a + C5a-Receptor antagonist (PMX53; 1:1000). Serum groups: CTR: stimulation with cell culture media + 10 % serum of healthy donors STEMI: cell culture media + 10 % serum of STEMI patients; STEMI+RA: cell culture media + 10 % serum of STEMI patients + C5a-Receptor antagonist (PMX53; 1:1000). p-values: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ++p<0.01; +p<0.05; #p<0.05.

Article Snippet: In further experiments, HUVEC were stimulated with 50 ng/mL of C5a with or without the C5a-Receptor-1 antagonist (C5aRA) PMX53 (Sigma Aldrich, Hamburg, Germany; catalog number 533683), according to the manufacturer’s instructions [concentration 1 µg/mL (1:1000)] for 24 h. In addition, the endothelial cells were stimulated with 10% patient (STEMI) or healthy donor (CTR) sera (instead of FCS) for 24 h either with or without C5aRA (PMX53).

Techniques: Microscopy, Force Spectroscopy, Control, Fluorescence, Labeling, Adhesive, Staining, Cell Culture

Journal: The FASEB Journal

Article Title: Inhibition of biomaterial-induced complement activation attenuates the inflammatory host response to implantation

doi: 10.1096/fj.12-225888

Figure Lengend Snippet: C5aR signaling is involved in shaping the host immune response after deep-tissue implantation of PP mesh. A) Image of a PP mesh on the day of implantation into the peritoneal space (D0) and on the day of its retrieval (D5). B, C) Levels of granulocytes (B) and macrophages (C) isolated from the tissue surrounding the PP mesh, 5 d after implantation in the peritoneal space of C5aR−/− and C5aR+/+ mice, as assessed by flow cytometry (n=7/strain). D, E) Effect of C5aR antagonism on granulocyte (D) and macrophage (E) accumulation around the implanted mesh in WT mice treated with either C5aRa or an inactive analog (control C5aRa) (n=4/treatment). F, G) Histological analysis of the meshes retrieved from WT mice treated with either C5aRa or inactive control compound. Cell density in the stroma (F) and mesh interface (G) was evaluated in H&E-stained sections (n=4/treatment); cell density is expressed as described for Fig. 3B, C. H) Immunofluorescent staining for C3 cleavage products in tissue sections retrieved from WT mice 5 d after implantation. Bright green staining indicates the presence of deposited C3c fragments at the interface. Incubation of slides with PBS, instead of anti-C3c Ab, was used as a negative control. *P < 0.05; unpaired t test.

Article Snippet: For the complement inhibition studies, blood samples were preincubated with 20 μM of the compstatin analog Cp20 (AcI[CVW(Me)QDWSarAHRC]mI-NH 2 ), linear inactive peptide (IAVVQDWGHHRAT-NH 2 ; ref. 21 , 22 ), 10 μM of the C5a receptor antagonist PMX53 (AcF-[OPdChaWR]; synthesized by GL Biochem, Shanghai, China; ref. 23 ), or its inactive control [AcdChaPWFRO-NH 2 ; synthesized in house], for 5 min prior to biomaterial addition.

Techniques: Isolation, Flow Cytometry, Control, Staining, Incubation, Negative Control