Citrullinated and Malondialdehyde–Acetaldehyde-Modified Fibrinogen Activates Macrophages and Promotes Coronary Endothelial Cell Inflammation

Figures

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  Figure 1. Increased MAA expression, MAA-CIT co-localization, and perivascular fibrosis in RA-HF hearts. Cardiac tissues from non-RA HF and RA-HF patients (n = 3 each) were sliced and imaged. Representative images of 20× frame at the apex of left ventricle are shown. (A) Immunohistochemistry using anti-CIT and anti-MAA antibodies. CIT (green) and MAA (red) expression and co-localization (orange) were quantified with the Fiji plug-in. scale bar = 100 µm. (B) Trichrome staining. Collagen deposition (blue) was quantified with the Fiji plug-in. Data shown in graphs are mean ± standard deviation. Student’s t-test was performed and statistical differences between groups are shown: * p < 0.05, ** p < 0.01. White scale bar = 100 µm.

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  Figure 2. HCAEC differential gene expression after treatment with antigen-stimulated macrophage supernatant. Gene expression was compared with control HCAECs treated with MϕU-SNFIB 8 h post-stimulation. (A) Volcano plot showing MϕU-SNFIB-MAA-CIT vs. MϕU-SNFIB. Colored dots indicate significant changes (Adjusted p < 0.05). Genes in purple text are the top two inflammatory cytokines and leukocyte adhesion molecules. (B) Heat map showing log2 fold change in the top 30 differential expressed genes comparing MϕU-SNFIB-MAA, MϕU-SNFIB-CIT, and MϕU-SNFIB-MAA-CIT to Mϕ-SNFIB. (C) Summary interaction graph between enriched genes and pathways. (D) Top enriched canonical pathways comparing MϕU-SNFIB-MAA-CIT vs. MϕU-SNFIB. For panel (A–D), orange: positive z score (upregulation), blue: negative z score (downregulation).

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  Figure 3. HCAEC gene expression following direct antigen stimulation and indirect stimulation with macrophage supernatants. MCP1, IL6, ICAM1, and VCAM1 mRNA expression was measured by RT-PCR in HCAEC 8 h (panel (A–D)) and 24 h (panel (E–H)) after stimulation. Relative quantification (RQ) compared to media-cultured negative control cells was calculated and is shown on the y-axis. Two-way ANOVA was performed, and the statistical differences between direct and indirect stimulation are shown: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Statistical differences between CIT- and/or MAA-modified FIB compared with unmodified FIB are shown: ¶ p < 0.05; ¶¶ p < 0.01; ¶¶¶ p < 0.001; ¶¶¶¶ p < 0.0001.

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  Figure 4. HCAEC protein secretion following direct antigen stimulation and indirect stimulation with macrophage supernatants. MCP-1, IL-6, ICAM-1, and VCAM-1 protein concentrations in HCAEC supernatants were measured by ELISA 8 h (panel (A–D)) and 24 h (panel (E–H)) after stimulation. Two-way ANOVA was performed and the statistical differences between direct and indirect stimulation are shown: * p < 0.05; *** p < 0.001; **** p < 0.0001. Statistical differences between CIT- and/or MAA-modified FIB compared with unmodified FIB are shown: ¶ p < 0.05; ¶¶ p < 0.01; ¶¶¶¶ p < 0.0001.

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  Figure 5. FIB-MAA-CIT activates NF-κB and p38 pathways in macrophages. Representative images of phosphorylated and total NF-κB, p38, Erk1/2, and STAT1 are shown, with β-actin as a loading control. Band intensities were quantified, and the ratio between phosphorylated and total protein was calculated and graphed. One-way ANOVA was performed and the statistical differences between groups are shown: * p < 0.05; ** p < 0.01; **** p < 0.0001.

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  Figure 6. Inhibition of p38 and NF-κB reduces macrophage cytokine production. 48 h cytokine concentrations in MϕU-SN were measured by ELISA for (A) MCP-1, (B) IL-6, (C) TNF-α, and (D) IL-1β. 48 h cytokine concentrations in MϕP-SN were measured for (E) MCP-1, (F) IL-6, (G) TNF-α, and (H) IL-1β. One-way ANOVA was performed and the statistical differences between groups are shown: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. For panels (E–H), red circles represent female PBMC donors, and blue squares represent male PBMC donors.

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  Figure 7. HCAEC inflammatory gene expression following p38 and NF-κB inhibition of macrophages. HCAEC mRNA expression after 24 h stimulation with MϕU-SN was measured by RT-PCR for (A) IL6, (B) MCP1, (C) ICAM1, and (D) VCAM1. HCAEC mRNA expression after 24 h stimulation with MϕP-SN was measured for (E) IL6, (F) MCP1, (G) ICAM1, and (H) VCAM1. Relative quantification (RQ) compared to media-cultured negative control cells was calculated and is shown on the y-axis. One-way ANOVA was performed and the statistical differences between groups are shown: * p < 0.05; *** p < 0.001; **** p < 0.0001. For panels (E–H), red circles represent female PBMC donors, and blue squares represent male PBMC donors.

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  Figure 8. HCAEC inflammatory protein release following p38 and NF-κB inhibition of macrophages. HCAEC supernatant protein concentrations after 24 h stimulation with MϕU-SN were measured by ELISA for (A) IL-6, (B) MCP-1, (C) ICAM-1, and (D) VCAM-1. HCAEC supernatant protein concentrations after 24 h stimulation with MϕP-SN were measured for (E) IL-6, (F) MCP-1, (G) ICAM-1, and (H) VCAM-1. One-way ANOVA was performed and the statistical differences between groups are shown: * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001. For panels (E–H), red circles represent female PBMC donors, and blue squares represent male PBMC donors.